Indian Patents
Title of Invention

"A DIKETOPIPERAZINE"
Abstract Compounds represented by the following structure (I) are disclosed: as are methods for making such compounds, wherein said methods comprise reacting a diacyldiketopiperazine with a first aldehyde to produce an intermediate compound; and reacting the intermediate compound with a second aldehyde to produce the class of compounds with the generic structure, where the first aldehyde and the second aldehydes are selected from the group consisting of an oxazolecarboxaldeyhyde, imidazolecarboxaldehyde, a benzaldehyde, imidazolecarboxaldehyde derivatives, and benzaldehyde derivatives, thereby forming the above compound wherein R1, R1', R1, R2, R3, R4, R5, and R6, X1 and X2, Y, Z, Z1, Z2, Z3, and Z4 may each be separately defined in a manner consistent with the accompanying description. Compositions and methods for treating vascular proliferation are also disclosed.
Full Text Related Applications This application is a continuation-in-part of U.S. Patent Appli

ation No.

10/632,531 filed on August 1, 2003, and claims priority to U.S. Provisional Application No. 60/542,073 filed February 4, 2004 and to U.S. Provisional Application No. 60/624,262 filed November 1, 2004, all of which are entitled DEHYDROPHENYLAHISTNS AND ANALOGS THEREOF AND THE SYNTHESIS OF DEHYDROPHENYLAHISlfeNS AND ANALOGS THEREOF; and each of which is incorporated herein by reference in its entirety.
Background of the Invention Field of the Invention
The present invention relates to compounds and methods of synthetic preparation in the fields of chemistry and medicine. More specifically, the presenf invention relates to compounds and procedures for making compounds useful in the treatmenf of cancer and the treatment of fungal infections.
Brief Description of the Related Art
. It is thought that a single, universal cellular mechanism controls the
regulation of the eukaryotic cell cycle process. See, e.g., Hartwell, L.H. et al, Science
ij
(1989), 246: 629-34. It is also known that when an abnormality arises in the control
mechanism of the cell cycle, cancer or an immune disorder may occur. Accordingly, as is
also known, antitumor agents and immune suppressors may be among the substances that
regulate the cell cycle. Thus, new methods for producing eukaryotic cell cycle inhibitors are
needed as antitumor and immune-enhancing compounds, and should be useful in the
treatment of human cancer as chemotherapeutic, anti-tumor agents. See, e.g., Rob|rge, M. et
a/., Cancer Res. (1994), 54,6115-21. '
" Fungi, especially pathogenic fungi and related infections, represent an
increasing clinical challenge. Existing antifungal agents are of limited efficacy and toxicity,

and the development and/or discovery of strains of pathogenic fungi that are resistant to drugs
currentty fcteifcMe or under developmentt. By way of example, fangi that are pathogenic^
humans ii|cll«^tfp^*ip^ kefyr, C.
krusei and C. galbrata; Aspergillus spp. including A. fumigatus and A. flavus; Cryptococcus
neoformaks\ Blastomyces spp. including Blastomyces dermatitidis; Pneumocystis carinii;
I : •' Coccidioiifes immitis; Basidiobolus ranarum', Conidiobolus spp.; Histoplasma capsulatum;
Rhizopus jfepp. including R. oryzae and R. microsporus; Cunninghamella spp.; Rhizomucor

spp.; Par^coccidioides brasiliensis; Pseudallescheria boydii; Rhinosporidium seeberi; and Sporothrtk schenckii (Kwon-Chung, KJ. & Bennett, J.E. 1992 Medical Mycology, Lea and
Febiger, !v|alvern, PA).
ii [0005} Recently, it has been reported that tryprostatins A and B (which are
diketopiperazines consisting of proline and isoprenylated tryptophan residues), and five other stracturallV-related diketopiperazines, inhibited cell cycle progression in the M phase, see Cui, C. et'fyl, 1996 J Antibiotics 49:527-33; Cui, C. et al 1996 J Antibiotics 49:53,4^40, and that these compounds also affect the microtubule assembly, see Usui, T. et al. 1998 Biochem /333:543J48; Kondon, M. et al. 1998 J Antibiotics 51:801-04. Furthermore, natural and synthetic compounds have been reported to inhibit mitosis, thus inhibit the eukaryotic cell cycle, by) binding to the colchicine binding-site (CLC-site) on tubulin, which is a macromol|cule that consists of two 50 kDa subunits (a- and (3-tubulin) and is the major constituent of microtubules. See, e.g., Iwasaki, S., 1993 MedRes Rev ISilSS-igj^Hbmel, E. 1996 Medt Res Rev 16:207-31; Weisenberg, R.C. et al., 1969 Biochemistiy 7:4466-79. Microtubules are thought to be involved in several essential cell functions, such as axonal
I
transport, icell motility and determination of cell morphology. Therefore, inhibitors of microtubule function may have broad biological activity, and be applicable to medicinal and
t;
agrochemi|;al purposes. It is also possible that colchicine (CLC)-site ligands such as CLC, steganacinl see Kupchan, S.M. et al., 1973 J Am Chem Soc 95:1335-36, podophyllotoxin, see Sackett, D|L., 1993 Pharmacol Ther 59:163-228, and combretastatins, see Pettit, G.R. et al, 1995 J M$d Chem 38:166-67, may prove to be valuable as eukaryotic cell cycle inhibitors and, thus, may be useful as chemotherapeutic agents.

Although diketopiperazine-type metabolites have been isolated from various fungi as myeotoxins, see Horak R.M. et al, i981'\i/(dyrCA*fe' Cbmttt 1263467; Ali M. et al, 1898 Toxicology Letters 48:235-41, or as secondary jxietafeoliies, ^eeSmed|gaard J. et al, 1996 J Microbiol Meth 25:5-17, little is known about the specific structure of the
ij
diketopiperazine-type metabolites or their derivatives and their antitumor . activity, particularly in vivo. Not only have these compounds been isolated as mycoioxins, the chemical synthesis of one type of diketopiperazine-type metabolite, phenylaMstii|, has been described by Hayashi et al. in J. Org. Cheat. (2000) 65, page 8402. In the ar| one such
i;
diketopiperazine-type metabolite derivative, dehydrophenylahistin, has been prepared by enzymatic dehydrogenation of its parent phenylahistin. With the incidences of caicer on the rise, there exists a particular need for chemically producing a class of substantially purified diketopiperazine-type metabolite-derivatives having animal cell-specific pro|liferation-inhibiting activity and high antitumor activity and selectivity. There is therefore a particular need for an efficient method of synthetically producing substantially purified, and structurally and biologically characterized, diketopiperazine-type metabolite-derivatives. j
•-J4K Also, PCT Publication WO/0153290 (July 26, 2001) describes a non-
synthetic method of producing dehydrophenylahistin by exposing phenylahistm or a particular phenylahistin analog to a dehydrogenase obtained from Streptomyces alb^lus.
Summary of the Invention ;
Compounds, and methods for the synthetic manufacture of compounds, are disclosed for a class of compounds having the structure of Formula (I):

FV ^1" V ^4
\ T p-> ^4 '

;n N
.. K2
Y
Rl z

(I)
The disclosed compounds have the structure of Formula (I) wherein:

[0010J RI, R4, and Re, are each separately selected from the group cti^liting of a hydrogen fatom, a halogeti atom, and saturated C1-C24 alkyl, unsaturated C1-CJA alkenyl, cycloalky| cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl| amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, bydroxy, carboxy, -CO-O-R7, cyano, alkylthio, halogenated alkyi including polyhalogfsnated alkyl, halogenated carbonyl, and carbonyl -CCO-R7, wherein R7 is selected from a hydrogen atom, a halogen atom, and saturated C1-C24 alky], unsaturated C1-624 alkenyl, cfycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups;
[Of 11] RI' and RI" are independently selected from the group consisting of a
hydrogen atom, a halogen atom, and saturated Ci-Cz4 alkyl, unsaturated C]-Ca4 alkenyl,
cycloalkylf cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted
heteroarylj amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted
phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, halogenated alkyl including
polyhalog«nated alkyl, halogenated carbonyl, and carbonyl -CCO-R7, wherein R7 +Is' selected
from a hydrogen atom, a halogen atom, and saturated Q-C24 alkyl, unsaturated Ci-Cz4
alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and
substituted] phenyi groups; "
[0012] R.2, RS, and RS are each separately selected from the group consisting of a hydrogen a^om, a halogen atom, and saturated C1-Ci2 alkyl, unsaturated C1-C|2 alkenyl, acyl, cycloalkyl,} alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl gribups;
[0013] X1 and X2 are separately selected from the group consisting of an oxygen
atom, a ni^ogen atom, and a sulfur atom, each either unsubstituted or substituted with a RS
group, as defined above;
• u Y is selected from the group consisting of a nitrogen atom, a fubstituted
nitrogen atom with a Rs group from above, an oxygen; atom, a sulfur atom, a. oxidized sulfur
atom, a methylene group and a substituted methylene group; I
r n is an integer equal to zero, one or two; i
Z, for each separate n, if non-zero, and Z\, Zz, Zs and Z| are each
separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxlrgen atom;
and f
_ t the dashed bonds may be either single or double bonds; ?
; with the proviso that, in a particular compound, if R1, R1', R2, R3, R4 and
R5 are each a hydrogen atom, then it is not true that X1 and X2 are each an oxygee atom and
R6 is either3,3-dimethylbutyl-l-ene or a hydrogen atom. I
* The methods comprise the steps of: I
> 0 reacting a diacyldiketopiperazine with a first aldehyde to produce an
intermediate compound; and -
* M reacting said intermediate compound with a second aldehyde fo produce
said class of compounds with said generic structure, wherein f
:„, said first aldehyde and said second aldehydes are selected rromj the group
consisting of an oxazolecarboxaldeyhyde, imidazolecarboxaldehyde, a benfcaldehyde,
imidazolecarboxaldehyde derivatives, and benzaldehyde derivatives, thereby forming a
compound wherein |
IUM&. % The disclosed compounds have the structure of Formula (I) wherein: •!w>»j R1, R4, and R6, are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated C)-Ci( alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, Substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and Substituted phenyl groups, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R7, wherein R7 |s selected from a hydrogen atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated C1-Cz4 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, leteroaryl,

substitute! heteroaryl, amino, substituted amino, nitro, azido, substituted nitrO'plKninyl, and substitute! Pnenyl groups;
[0025] RI' and RI" are independently selected from the group consisting of a hydrogen |atom, a halogen atom, and saturated C1-C24 alkyl, unsaturated O^Gui alkenyl, cycloalky| cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl substituted

heteroaryl

amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted

phenyl grimps, hydroxy, carboxy, -CO-O-R7, cyano, alkylthio, halogenated alkyl including polyhalog mated alkyl, halogenated carbonyl, and carbonyl -CCO-R7, wherein ISlWselected from a hydrogen atom, a halogen atom, and saturated C|-C24 alkyl, unsaturatiid C1-C24 alkenyl, %cloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substitute^ heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups;
[0026] R2, R3, and RS are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-Ciz alkyl, unsaturated Ci-Cn alkenyl, acyl, cycloalkyljj alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
[0(|27] Xj and Xa are separately selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each either unsubstituted or substituted with a RS group, as 4efined above;
[OflpS] Y is selected from the group consisting of a nitrogen atom, a substituted nitrogen a^om with a RS group from above, an oxygen atom, a sulfur atom, a oxidized sulfur atom, a mdjthylene group and a substituted methylene group;
[OQfZ9] n is an integer equal to zero, one or two;
JL
[OOfJO] Z, for each separate n, if non-zero, and Zi, Zz, Z,} and 2* are each
separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
and |
[OOpl] the dashed bonds may be either single or double bonds.
[OOfJ2] hi preferred embodiments of the compound and method, the imidazolec^rboxaldehyde is 5-(l,l-dimethyl-2-ethyl)imidazole-4-carboxaldehyde and the

benzaldeayde comprises a single methoxy group. Additional preferred embodmients of the
compounds described herein include compounds having a t-butyl group, a dimetfioxy group,
a chloro- group, and a methylthiophen group, and methods of making such compounds, as
well as the compounds described in Tables 2, 3 and 4, as well as methods of mjaking such
compounds.: I
[0033] Also disclosed are methods and materials for treating neoplastjc tissue or preventing cancers or infection by a pathogenic fungus. These methods and materials are
particularly well suited for treatment of mammalian subjects, more particularly humans, and
j involve administering to the subject a dehydrophenylahistin or its analog. The method
comprises administering to the subject a composition comprising an effective aijtitumor or
antirungal amount of a dehydrophenylahistin or its analog. :;
[0034] Further embodiments relate to methods for treating a condition in an animal, which methods can include administering to the animal a compound as described herein in an amount that is effective to reduce vascular proliferation or in an amount that is effective to reduce vascular density. Exemplary conditions include neoplasms, such as .cancers, as well as other conditions associated with or which rely upon vascijlarization, including for example, immune and non-immune inflammation, rheumatoid arthrigs, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, reti^opathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasiaj rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, and the like. I In some embodiments, the disease is not cancer.
[0035] Other embodiments relate to methods of inducing vascular collapse in an animal. The methods can include treating said animal with a therapeuticaUy effective amount of a compound of the Formula (I) as described herein, for example. The theripeutically effective amount of said compound can cause tubulin depolymerization in the vascqlature.
[0036] Preferably the animal can be a human. Preferably the disease| can be a
tumor, a diabetic retinopathy, an age-related macular degeneration, and the likej In some
aspects the disease is not cancer or cancer can be specifically excluded from the methods and
uses. Preferably, the compound is KPU-02. ?
8

[OCJ37] Still further embodiments relate to pharmaceutical compositions for treating otf preventing vascular proliferation comprising a pharmaceutically effective amount of a compound disclosed herein together with a pharmaceutically acceptable carrier therefor. The vascu|ar proliferation can be a symptom of a disease, for example, cancer, age-related
macular degeneration and diabetic retinopathy. .
I
(OCJJ38) Some embodiments relate to methods of preferentially targeting tumor vasculaturf; over non-tumor tissue vasculature. The methods can include the step of administering to an animal, preferably a human, a compound having the structure of Formula (I) as described herein. The non-tumor tissue can be, for example, skin, muscle, brain, kidney, he^rt, spleen, gut, and the like. The tumor vasculature can be preferentially targeted over non-tjimor tissue vasculature, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%jand 90%.
[OQJ39] Other embodiments relate to methods of preferentially targeting tumor vasculaturf over non-tumor tissue vasculature, which methods can include administering to an animalj an agent that preferentially targets tumor vasculature over non-tumor tissue vasculaturf.
[OQS40] Further embodiments relate to uses of a compound having the structure of Formula (|) in the preparation of a medicament for the treatment of a condition associated with increased vasculature or which relies upon vasculature. In some aspects the condition can be cancer, while in others, cancers particular types or all cancers are specifically excluded. ; The condition can be any other that is associated with hypervascularization, associated jjwith vasculature or which relies upon vasculature. Examples include immune and non-immune inflammation, rheumatoid arthritis, chronic articular rheumatism, psoriasis, diabetic jfetinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, comeal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, and the like.
Brief Description of the Drawings
[OOrtl] The accompanying drawings, which are incorporated in and form part of the specififcation, merely illustrate certain preferred embodiments of the present invention. Together \\fith the remainder of the specification, they are meant to serve to explain preferred

modes of making certain compounds of the invention to those of skilled in the jftrt. In the drawings:
[0042] Figure 1 illustrates a reaction scheme for producing dehydroph^iylahistins
by reacting ^a diacyldiketopiperazine 1 with an imidazolecarboxaldeheyde 2 t$> yield an
intermediate compound 3 which is reacted with a benzaldehyde 4 to jbroduce a
dehydrophenylahistJn. |
[0043] Figure 2 depicts the HPLC profile of the synthetic crude
dehydrophenylahistin. >
[0044] Figure 3 illustrates a reaction scheme for producing dehydrophepylahistins
by reacting a diacyldiketopiperazine 1 with a benzaldehyde 4 to yield an intermediate
compound 17 which is reacted with an imidazolecarboxaldeheyde 15 to produce a
dehydrophenylahistin. j!
[0045] Figure 4 depicts the HPLC profiles of the crude synthetic tBu-
dehyrophenylahistin produced from Route A and from Route B. j
[0046] Figure 5 illustrates two modification strategies for dehydroPLHjfor potent
cytotoxic activity. i
[0047] Figure 6 depicts the putative active conformation of dehydroJJLH at the
phenyl moiety. |
[0048] Figure 7 depicts Cytochrome P450 metabolism of phenylahistin. |
[0049] Figure 8 illustrates the Z-E migration of tBu-dehydroPLH. [0050] Figure 9 depicts the synthesis and prodrug image of aiyl-E-tBu-
dehydroPLH. [0051] Figure 10 depicts the temperature gradient of 3-Z-Benzylidene-l-
dimethylallyl> lH-imidazol-4"-Z-ylmethylene]-piperazine-2,5-dione. [0052] Figure 11 depicts the temperature gradient of 3-Z-benzyiidene-|»-(5"-tert-
butyl- l//-imidazol-4"-Z-ylmemylene)-piperazine-2,5-dione. ,;
[0053] Figure 12 depicts the effect of KPU-2, KPU-35 and t-butyl-phenylahistin in comparison to colchicine and taxol on HuVEC monolayer permeability to FITC-Bextran.
[0054] Figure 13 depicts the effect of KPU-2 alone and in combination inith CPT-11 on estimated rumor growth in the HT-29 Human Colon Tumor Xenograft model IO

[0065] Figure 14 depicts the effect of KPU-2 alone and in combination with CPT-11 on the height of tumors excised at autopsy in individual mice in the HT-29 Human Colon Tumor Xe|ograft model.
[00(56] Figure 15 depicts the effect of KPU-2 alone and in combination with CPT-11 on estimated tumor growth in the HT-29 Human Colon Tumor Xenograft model.
[OOJI57] Figure 16 depicts the effect of KPU-2 alone and in combination with CPT-11 on the height of tumors excised at autopsy in individual mice in the HT-29 Human Colon Tumor Xenograft model.
[0058] Figure 17 depicts the effects of: A. KPU-2, B. KPU-35 and C. t-butyl-phenylahistin alone and in combination with CPT-11 on estimated tumor growth hi the HT-29 human |*>lon tamor xenograft model.
[00^9] Figure 18 depicts the effects of A. KPU-2, B. KPU-35 and C. t-butyl-phenylahisfin alone and in combination with CPT-11 on the weight of tumors excised at autopsy in Individual mice in the HT-29 Human Colon Tumor Xenograft model.
i
[OOJ60] Figure 19 depicts the effects of KPU-2 alone and in combination with CPT-11 on tumor growth in the HT-29 human colon tumor xenograft model: comparison of three studies.
[0061] Figure 20 depicts the effects of KPU-2 alone and in combination with CPT-11 oji final tumor weights in the HT-29 human colon tumor xenograft model: comparison of three studies.
[08f>2] Figure 21 depicts the effects of KPU-2 alone or in combination with Taxotere [OOJS3] Figure 22 depicts the effects of A. KPU-2, B. KPU-35 and C. t-butyl-phenylahisiin alone and in combination with Taxotere on the estimated tumor growth based on observations made during the in-life portion of the DU-145 Human Prostate Tumor Xenograft ^lodel.
[00|54] Figure 23 depicts the effects of KPU-2 alone and in combination with Taxotere ojn the individual excised tumor weights at autopsy in the DU-145 Human Prostate Tumor Xenograft Model.
II

[0065] Figure 24 depicts the effects of KPU-35 alone and in combination with
Taxotere on the individual excised tumor weights at autopsy in the DU-145 Hum|n Prostate
Tumor Xenograft Model. [0066] Figure 25 depicts the effects of A. KPU-2, B. KPU-35 and JC. t-butyl-phenylahistin alone and in combination with Taxotere in MCF-7 Human Breast Tumor Xenograft model.
[0067] Figure 26 depicts the effects of KPU-2 alone and in combustion with Taxotere on estimated tumor growth in the A549 Human Lung Tumor Xenograft model.
[0068] Figure 27 depicts the effects of KPU-2 alone and in combination with
Taxotere on the excised tumor weights at autopsy in the A549 Human Lufeg Tumor
Xenograft model. I
[0069] Figure 28 depicts the effects of KPU-2 alone and in combination with
Paclitaxel on estimated tumor weight in the murine mammary fat pad implanted j^DDA-231
Human Breast Tumor model. ;
[0070] Figure 29 depicts effects of A. KPU-2, B. KPU-35 and (Jl. t-butyl-
phenylahistin alone and in combination with Paclitaxel in the Murine Melanomi B16 FIO
Metastatic Tumor Model. [0071] Figure 30 depicts effects of KPU-35 and KPU-02 on tumor vasfulature in
the dorsal skinfold chamber of Figure 30. I
[0072] Figure 31 depicts effect of KPU-02 in combination with CPT|ll on the
estimated tumor weight in the HT-29 human colon tumor xenograft model. j
[0073] Figure 32 depicts effect of KPU-02 in combination with CPT-|l 1 on the
excised tumor weight in the HT-29 human colon tumor xenograft model. |
[0074] Figure 33 depicts rapid tubulin depolymerization in HuVEC cells induced
by KPU-02 and KPU-35. j
[0075] Figure 34 depicts effect of KPU-02 on monolayer permeability |in HuVEC
cells. I
[0076] Figure 35 depicts the effect of KPU-02 on tumor blood flow in the P22 rat sarcoma model using the 125I-IAP technique.

[0(f77I Figure 36 depicts the effect of KPU-02 15 mg/kg IP (expressed as % vehicle control) an blood flow in different tissues 1 and 24 hours post-dose.
[OCJ78] Figure 37 depicts the tumor necrosis induced by KPU-02 7.5 and 15.0
'• f
mg/kg IP i the P22 rat sarcoma model
[OCf79] Figure 38 lists the activity of various tBu-dehydro-PLH derivatives at HT-29 cells.
[OflisO} Figure 39 depicts 3D QSAR (CoMFA) analysis of tBu-dehydro-PLH derivatives.
[00(81] Figure 40 depicts X-ray crystallographic analysis of tBu-dehydro-PLH derivative^.
[0082] Figure 41 depicts the biologically activity of various phenylahistin derivative^ compared to colchicine.
[0083] Figure 42 depicts the effect on cell cycle progression of HeLa cells by tBu-dehydro-PJLH (KPU-2) and KPU-35.
[OCte4] Figure 43 depicts the effect of dehydro-PLH and tBu-dehydro-PLH (KPU-2) on drugfsensitive and drug-resistant tumor cell lines as compared to paclitaxel.
[O085] Figure 44A depicts turbidity spectra of microtubule protein polymerization in the presence of DMSO drug vehicle (0), 1.25 uM (n), 2.5 uM (—), and 5 uM (o) KPU-02.
[0086] Figure 44B depicts turbidity spectra of microtubule protein polymerization in the pres^snce of DMSO drug vehicle (0), 1.25 uM (D), 2.5 u,M (—), and 5 uM (o) CA4.
[0087] Figure 44C depicts turbidity spectra of microtubule protein polymerization in the presence of DMSO drug vehicle (0), 1.25 uM (a), 2.5 uM (—), and 5 uM (o) CLC.
[OO88] Figure 45 depicts inhibition of MT in the absence or presence of a range of KPU-02 (4),CA4 (n), and colchicine (0) concentrations.
[00(89] Figure 46A depicts frequency histograms of mean microtubule lengths in vitro at steady state in the presence of KPU-02.
[OOJ90] Figure 46B depicts frequency histograms of mean microtubule lengths in vitro at steady state in the presence of CA4.
[00191] Figure 46C depicts frequency histograms of mean microtubule lengths in vitro at steady state in the presence of CLC.

[0092] Figure 47A depicts electron micrographs of MAP-rich n|icrotubules
formed in vitro at steady state in the presence of KPU-02. |
[0093] Figure 47B depicts electron micrographs of MAP-rich n|icrotubules
formed in vitro at steady state in the presence of CA4. !
[0094] Figure 47C depicts electron micrographs of MAP-rich m^crotubules
formed in vitro at steady state in the presence of CLC. i
[0095] Figure 48 depicts a graphical summary of MT length



at steady
state in the presence of KPU-02, CA4, and colchicine. »
[0096] Figure 49A depicts fluorescence emission spectra of tubiflin in the
presence of increasing KPU-02. •
[0097] Figure 49B depicts a fit to fluorescence emission maxima at J487 run to
obtain the Kd of tubulin for KPU-02. The inset depicts residuals. j
[0098] Figure 49C depicts double reciprocal transformation of the bind ng data.
>n assay of
[0099] Figure 50 depicts the graphical results of a competitive inhibit*
colchicine binding to tubulin with various concentrations of [3H]CLC in the absc nee (0), or
presence of 10 uM KPU-02 (o) or 10 \iM CA4 (n). ;
[0100] Figure 51 depicts log [compound] response curves for mitotic progression
inhibition by KPU-02, CA4, and CLC in MCF7 cells cultured in the presence of KpU-02 (o),
CA4 (n), and colchicine (0). ;
[0101] Figure 52 depicts immunofluorescence microscopy images of h|CF7 cells,
a-d: Tubulin in control—(a) Tubulin in control, (b) KPU-02, (c) CA4, and (d) CLC treated
cells; e-h: DNA in control—(e) DNA in control, (f) KPU-02, (g) CA4, and (h) CfLC treated
cells. ,;
[0102] Figure 53A depicts immunofluorescence microscopy images | of MCF7
cells treated with KPU-02 }
[0103] Figure 53B depicts immunofluorescence microscopy images 1 of MCF7 cells treated with CA4.
[0104] Figure 53C depicts immunofluorescence microscopy images!of MCF7
cells treated with CLC. I

[0105] Figure 54A depicts hnmunofluorescence microscopy images of MCF7 cells treated with KPU-02
[OJ06] Figure 54B depicts immimofluorescence microscopy images of MCF7 cells treated with CA4.
[0|07J Figure 54C depicts immimofluorescence microscopy images of MCF7 cells treated with CLC.
[0108] In certain Figures, compounds are identified using an alternative designation. A complete chart to convert these alternative designations is as follows:

A designation Designation used herein
N »I-2350 (-)-phenylahistin
N »I-2352 KPU-01
N N.2353 KPU-03
N 1-2354 KPU-04
N »I-2355 KPU-05
N J-2356 KPU-06
N 1-2357 KPU-07
N 1-2358 KPU-02
N 1-2359 KPU-08
N 1-2360 KPU-09
N 1-2361 KPU-10
N 1-2362 KPU-11
N 1-2363 KPU-12
N 1-2364 KPU-13
N. 1-2365 KPU-14
N. 1-2366 KPU-15
H 1-2367 KPU-16
H 1-2368 KPU-17
N3 1-2369 KPU-18
Nl 1-2370 KPU-19
Ni 1-2371 KPU-21
Nl 1-2372 KPU-22
N] 1-2373 KPU-23
NJ 1-2374 KPU-24
Nl 1-2375 KPU-25
Nl 1-2376 KPU-28
N] 1-2377 KPU-26
N! 1-2378 BCPU-27
NJ 1-2379 KPU-29
N] 1-2380 KPU-20
N] 1-2381 KPU-30
Nl 1-2382 KPU-31
NJ 1-2383 KPU-32
Nl 1-2384 KPU-33
N^ 1-2385 KPU-34
15

Alternative designation Designation used herein
NPI-2386 KPU-35
NPI-2387 KPU-36
NPI-2388 KPU-37
NPI-2389 KPU-38
NPI-2390 KPU-39
NPI-2391 KPU-40
NPI-2392 KPU-41
NPI-2393 KPU-42
NPI-2394 KPU-43
NPI-2395 KPU-44
NPI-2396 KPU-45
NPI-2397 KPU-46
NPI-2398 KPU-47
NH-2399 KPU-48
NPI-2400 KPU-49
NPI-2401 KPU-50
NPI-2402 KPU-51
NPI-2403 KPU-52
NPI-2404 KPU-53
NPI-2405 KPU-54
NPI-2406 KPU-55
NPI-2407 KPU-56
NPI-2408 KPU-57
NPI-2409 KPU-58
NPI-2410 KPU-59
NPI-2411 KPU-60
NPI-2412 KPU-61
NPI-2413 KPU-62
NPI-2414 KPU-63
NPI-2415 KPU-64
NPI-2416 KPU-65
NPI-2417 KPU-66
NPI-2418 KPU-67
NPI-2419 KPU-68
NPI-2420 KPU-69
NPI-2421 KPU-70
NPI-2422 KPU-71
NPI-2423 KPU-72
NPI-2424 KPU-73
NPI-2425 KPU-74
NPI-2426 KPU-75
NPI-2427 KPU-76
NPI-2428 KPU-77
NPI-2429 KPU-79
NPI-2430 KPU-80
NP1-2431 KPU-81
NPI-2432 KPU-82

A tentative designation Designation used herein
N 1-2433 KPU-83
N 1-2434 KPU-84
N •1-2435 KPU-86
N 1-2436 KPU-87
N 1-2437 KPU-88
N 1-2438 KPU-89
N 1-2439 KPU-90
N 1-2440 KPU-9I
N 1-2441 KPU-92
N 1-2442 KPU-80
N 1-2455 KPU-94
N 1-2456 KPU-95
N 1-2457 K?U-96
N 1-2458 KPU-97
Ml 1-2459 KPU-98
N 1-2460 t-butyl phenylahistm
Detailed Description of the Preferred Embodiment
[Ott)9] Each reference cited herein, including the U.S. patents cited herein, is to be considered incorporated by reference in its entirety into this specification, to the full extent permissiblf! by law. U.S. Patent Application No. 10/632,531, and PCT Application No. PCTUS03J24232, both filed on August 1, 2003, and both entitled, "DEHYD^OPHENYLAHISTINS AND ANALOGS THEREOF AND THE SYNTHESIS OF DEHYpROPHENYLAHISTINS AND ANALOGS THEREOF," are incorporated herein by reference in their entireties.
[01110] The disclosure provides methods for the synthetic preparation of compound!, including novel compounds, including dehydrophenylahistin and dehydrophjbnylahistin analogs, and provides methods for producing pharmaceutically acceptablej cell cycle inhibitors, antitumor agents and antifungal agents in relatively high yield, whepin said compounds and/or their derivatives are among the active ingredients in
i:
these cell (cycle inhibitors, antitumor agents and antifungal agents. Other objects include providing |iovel compounds not obtainable by currently available, non-synthetic methods. It is also ar^j object to provide a method of treating cancer, particularly human cancer, comprising the step of administering an effective tumor-growth inhibiting amount of a member o| a class of new anti-tumor compounds. This invention also provides a method for preventing) or treating a pathogenic fungus in a subject which involves administering to the
n

subject an effective anti-fungal amount of a member of a class of new (anti-fungal compounds, e.g., administering a dehydrophenylahistb or its analog in an ajnount and manner which provides the intended antifimgal effect. In the preferred embodiment of the compounds and methods of making and using such compounds disclosed herein, but not
1
necessarily in all embodiments of the present invention, these objectives are met. ;
[0111] Disclosed herein, also, are compounds, and methods of producing a class
of compounds, wherein the compounds are represented by Formula (I): f


Rr R

(D
[0112] wherein:
[01.13] RI, R4, and Re, are each separately selected from the group consisting of a
| hydrogen atom, a halogen atom, and saturated Ci-Cz4 alkyl, unsaturated Ci-C|4 alkenyl,
cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, iubstituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and iubstituted
phenyl groups, hydroxy, carboxy, -CO-O-Ry, cyano, alkylthio, halogenated alkyl including
I polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R?, wherein R? |s selected
from a hydrogen atom, a halogen atom, and saturated Ci-Ca4 alkyl, unsaturated CrC24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, |ieteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and
!i
substituted phenyl groups;
[0114] RI' and RI" are independently selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-Cz4 alkyl, unsaturated Ci-C^4 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-Ry, cyano, alkylthio, halogenated alkyl I including
\8

polyhalog^nated alkyl, halogenated carbonyl, and carbonyl -CCO-R?, wherein R? is selected from a hydrogen atom, a halogen atom, and saturated Ci-Cz4 alkyl, unsaturated Ci-C24 alkenyl, cj^cloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,
j
substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substitute^ phenyi groups;
[0|15] R, RI' and RI" are either covalently bound to one another or are not covalentlyjbound to one another;
[0|16] Ra, Rs, and Rs are each separately selected from the group consisting of a hydrogen |tom, a halogen atom, and saturated C|-Cu alkyl, unsaturated C\-Cn alkenyl, acyl, cycloalkyl| alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
[0^17] Xi and Xz are separately selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each either unsubstituted or substituted with a RS group, as defined above;
[OJ18] Y is selected from the group consisting of a nitrogen atom, a substituted nitrogen a|om with a Rs group from above, an oxygen atom, a sulfur atom, a oxidized sulfur
atom, a mfthylene group and a substituted methylene group;
I [0119] n is an integer equal to zero, one or two;
[0120] 2, for each separate n, if non-zero, and Zi, Za, £3 and Z* are each separately {{selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
1!
and
[0121] the dashed bonds may be either single or double bonds.
[OJJ22] The method comprises a method of producing compounds of Formula (I) by the stepjp of:
[0123] reacting a diacyldiketopiperazine with a first aldehyde to produce an
f:
intermediate compound; and
[01JJ24] reacting said intermediate compound with a second aldehyde to produce said class ^f compounds with said generic structure, wherein

[0125] said first aldehyde and said second aldehydes are selected fronjt the group consisting of an oxazolecarboxaldeyhyde, imidazolecarboxaldehyde, a be^zaldehyde, imidazolecarboxaldehyde derivatives, and benzaldehyde derivatives, thereby Jforming a compound of Formula (I) wherein
[0126] RI, R4, and Rs, are each separately selected from the group consisting of a
hydrogen atom, a halogen atom, and saturated Ci-Ca4 alkyl, unsaturated Q-cL alkenyl,
cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, Substituted
heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted
phenyl groups, hydroxy, carboxy, -CO-O-R?, cyano, alkylthio, halogenated alkyl including
polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R/, wherein R/jjis selected
from a hydrogen atom, a halogen atom, and saturated Ci-Cj4 alkyl, unsaturated Q-C24
alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, jheteroaryl,
substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, pjbenyl, and
substituted phenyl groups; j
[0127] RI' and RI" are independently is selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-C24 alkyl, unsaturated C|-C$4 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, Substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R/, cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R?, wherein R? |s selected from a hydrogen atom, a halogen atom, and saturated Q-C24 alkyl, irasatura&d Ci-C24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, ^eteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, ptenyl, and substituted phenyl groups;
[0128] R2, RS, and R$ are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-Cn alkyl, unsaturated Ci-C]2 alkfenyl, acyl,
IT
cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted ieteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
2-0

[OIfZ9] X| and Xj are separately selected from the group consisting of an oxygen atom, a nifrogen atom and a sulfur, atom, and
[01J30] Y is selected from the group consisting of a nitrogen atom, a substituted
I nitrogen atjom with a Rs group from above, an oxygen atom, a sulfur atom, a oxidized sulfur
atom, a mothylene group and a substituted methylene group;
[OS31] Z, for each separate n, if non-zero, and Z\, Za, Z$ and Z separately Selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
and I
[0^2] the dashed bonds may be either single or double bonds.
[Olt33] Also provided are pharmaceutically acceptable salts and pro-drug esters of the compound of Formulae (I) and (IT) and provides methods of synthesizing such compoundl by the methods disclosed herein.
[01J34] The term "pro-drug ester," especially when referring to a pro-drug ester of the compound of Formula (I) synthesized by the methods disclosed herein, refers to a chemical perivatrve of roe compound that is rapidly transformed in vivo to yield the compound} for example, by-hydrolysis in blood or inside tissues. The term "pro-drug ester" refers to cjjerivatives of the compounds disclosed herein formed by the addition of any of several esljbr-forming groups that are hydrolyzed under physiological conditions. Examples of pro-drujg ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymlsthyl, as well as other such groups known in the art, including a (5-R-2-oxo-l,3-dioxolen~4|-yl)memyl group. Other examples of pro-drug ester groups can be found in, for example, t". Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S. Syiaposium Series, American Chemical Society (1975); and "Bioreversible Carriers in Drug EJesign: Theory and Application", edited by E. E. Roche, Pergamon Press: New York, 14-J21 (1987) (providing examples of esters useful as prodrugs for compounds containingjcarboxyl groups).
[01135] The term "pro-drug ester," as used herein, also refers to a chemical derivative {of the compound that is rapidly transformed in vivo to yield the compound, for example, fey hydrolysis in blood. The term "pro-drug ester" refers to derivatives of the compound^ disclosed herein formed by the addition of any of several ester-forming groups

that are hydrotyzed under physiological conditions. Examples of pro-drug e^fter groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl aod methoxymethyl| as well as other such groups known in the art, including a (5-R-2-oxo-l,3-dioxolen-4-yl)methyl group. Other examples of pro-drug ester groups can be found in, for example, T. Higubhi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S. Symposium Series, American Chemical Society (1975); and "Bioreversible Carriers in Drug Design: "fheory and Application", edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987)j(providing examples of esters useful as prodrugs for compounds containing carboxyl groups), f
[0136] The term "pharmaceutically acceptable salt," especially when re|erring to a
pharmaceutically acceptable salt of the compound of Formula (T) synthesized by flfe methods
disclosed herein, refers to any pharmaceutically acceptable salts of a compound, and
preferably refers to an acid addition salt of a compound. Preferred ex|unples of
pharmaceutically acceptable salt are the alkali metal salts (sodium or potassium), the alkaline
earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or from
pharmaceutically . acceptable organic amines, for example Ci-C? ajkylamine,
cyclohexylamine, triethanolamine, ethylenediamine or tris-(hydroxymemyl)-aminbmethane.
With respect to compounds synthesized by the method that are basic amines, th^ preferred
examples of pharmaceutically acceptable salts are acid addition salts of pharmaceutically
acceptable inorganic or organic acids, for example, hydrohalic, sulfuric, phosphoric acid or
aliphatic or aromatic carboxylic or sulfonic acid, for example acetic, succinic, laqtic, malic,
tartaric, citric, ascorbic, nicotinic, methanesulfonic, p-toluensulfonic or naphthalejnesulfonic
acid. [0137] The term "pharmaceutically acceptable salt," as used herein, alsp refers to any pharmaceutically acceptable salts of a compound, and preferably refers tf> an acid addition salt of a compound. Preferred examples of pharmaceutically acceptable sjalt are the alkali metal salts (sodium or potassium), the alkaline earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or from pharmaceutically |cceptable organic amines, for example Ci-C? alkylamine, cyclohexylamine, triethqaiolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane. With respect to compounds that are basic amines, the preferred examples of pharmaceutically acceptable salts are acifl addition
•2L2-

salts of pHarmaceutically acceptable inorganic or organic acids, for example, hydrohalic, sulfuric, phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid, for example acetic, su|cinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, p-toluensulltinic or naphthalenesulfonic acid.
[0188] Preferred pharmaceutical compositions disclosed herein include pharmaceutically acceptable salts and pro-drug esters of the compound of Formula (I) synthesized by the method disclosed herein. Accordingly, if the manufacture of pharmaceutical formulations involves intimate mixing of the pharmaceutical excipients and the active ingredient in its salt form, then it is preferred to use pharmaceutical excipients which are son-basic, that is, either acidic or neutral excipients.
(01JJJ9J In preferred embodiments of the methods of the compounds disclosed herein, a relatively rigid, planar pseudo three-ring structure may be formed. To stabilize such a relative!^ rigid, planar pseudo three-ring structure, Ra may preferably be chosen to be hydrogen, i
[OlftO] In other preferable embodiments of the compounds and methods described herein, n uj equal to zero or one, more preferable one, and Z2, Zs, and Z4, and each separately selected from an oxygen atom, a nitrogen atom, and a carbon atom, more preferable at one least one df Z%, Z$, and TLt, being a carbon atom, and most preferable at least two of TLi, Zs, and Z4bei^ a carbon atom. All Z's may simultaneous be carbon atoms.
2-3
[Olfll] Still other preferred embodiments of the methods and compositions disclosed hjerein imvolve compounds having the structures of Formulae (la) and (Ib), below:



[0142] wherein the variable groups are as defined herein.
[0143] The term "halogen atom," as used herein, means any one ol| the radio-
stable atoms of column 7 of the Periodic Table of the Elements, i.e., fluorindL chlorine,
bromine, or iodine, with fluorine and chlorine being preferred. ;
[0144] The term "alkyl," as used herein, means any unbranched orj branched,
substituted or unsubstituted, saturated hydrocarbon, with Q-Ce unbranched, I saturated,
?
unsubstituted hydrocarbons being preferred, with methyl, ethyl, iosbutyl, and tert-|utyl being most preferred. Among the substituted, saturated hydrocarbons, d-Ce mono- ajpd di- and per-halogen substituted saturated hydrocarbons and ammo-substituted hydrocarbons are preferred, with perfluromethyl, perchloromethyl, perfluoro-tert-butyl, and perchlor^-tert-butyl being the most preferred. The term "substituted" has its ordinary meaning, a$ found in numerous contemporary patents from the related art. See, for example, U.S. Fjatent Nos. 6,583,143, 6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443; and J6,350,759. Specifically, the definition of substituted is as broad as that provided in U.S. patent No. 6,583,143, which defines the term substituted as any groups such as alkyl, aryl| arylalkyl, heteroaryl, heteroarylalkyl, heterocycle and heterocyclealkyl, wherein at least on^ hydrogen
atom is replaced with a substituent. The term "substituted" is also as broad as the! definition provided in U.S. Patent No. 6,509,331, which defines the term "substituted alkyl" ijjuch that it refers to an alkyl group, preferably of from 1 to 10 carbon atoms, having fixim 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting bfalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cy^loalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, amiioacyloxy,
oxyacylamino, cyano, halogen, .hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol,
i thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, he|erocyclic,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —So-substituted a|cyl, --SO-aryl, -SO-heteroaryl, --SO2-alkyl, -SO2-substituted alkyl, -SOz-aryl and -SOa-Ieteroaryl. The other above-listed patents also provide standard definitions for the term "substituted" that are well-understood by those of skill in the art. The term "cycloalkyl" refers toi any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the |ing. The

term "acyllj refers to alkyl or aryl groups derived from an oxoacid, with an acetyl group being preferred. I
t
[0145] The term "alkenyl," as used herein, means any unbranched or branched,
s
substituted i or unsubstituted, unsaturated hydrocarbon including polyunsaturated hydrocarbons, wi* Cj-Ce unbranched, mono-unsaturated and di-unsaturated, unsubstituted hydrocarbons being preferred, and mono-unsaturated, di-halogen substituted hydrocarbons being mos| preferred. In the RI and Rt positions, of the compound of structure (I) a z-isoprenyl ^loiety is particularly preferred. The term "cycloalkenyl" refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.
101J16] The terms "aryl," "substituted aryl," "heteroaryl," and "substituted heteroaryl,| as used herein, refer to aromatic hydrocarbon rings, preferably having five, six,
or seven atjjjms, and most preferably having six atoms comprising the ring. "Heteroaryl" and
I "substitute^ hetefoaryl," refer to aromatic hydrocarbon rings in which at least one
heteroaton% e.g., oxygen, sulfur, or nitrogen atom, is in the ring along with at least one carbon aftnjb.
[Olf7] The term "alkoxy" refers to any unbranched, or branched, substituted or unsubstitutpd, saturated or unsaturated ether, with Cj-Ce unbranched, saturated, unsubstituted ethers bei|g preferred, with methoxy being preferred, and also with dimethyl, diethyl, methyl-iso|>utyl, and methyl-tert-butyl ethers also being preferred. The term "cycloalkoxy" refers to iny non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.
[Ol|»8] The terms "purified," "substantially purified," and "isolated" as used herein ref^- to the compound being free of other, dissimilar compounds with which the compoundfis normally associated in its natural state, so that the compound of the invention comprises fit least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, tjjy weight, of a given sample.
(Ol|l9] The compound of Formula (I) may be chemically synthesized or produced from reagjjents known and available in the art. For example, modifications of diacyldikefjbpiperazine (diacetyldiketopiperazine) have been described, for example, by Loughlin & al, 2000 Bioorg Med Chem Lett 10:91 or by Brocchini et al in WO 95/21832.

The diacyldiketopiperazine (diacetyldiketopiperazine) may be prepared, for |xample, by
diacetyJation of inexpensive 2,5-piperazinedione (TCI Cat. No. G0100, 25 g) with sodium
acetate and sodium anhydride. The diacetyl structure of the activated deketopifljerazine can
be replaced with other acyl groups, to include carbamates such as Boc (t-butoxy^arbonyl), Z
(benzoyloxycarbonyl). ,
[0150] The imidazolecarboxaldehyde may be prepared, for exampldL according the procedure disclosed in Hayashi et al, 2000 J Organic Chem 65: 8402 as depicted below:


THF.-70'C 88%

pyrtdnM- QTs O
86%

reflux C14Cjj'C) SB%



4MNaOH
»•
EtOH.rt.
aa%

COCb
CHzCla,rl

EtOCOCHtffOOH
BuU I
(»-
THF.-7D to litre 32%



0 O

SOjCb
CHCb, Wlux 77%

0 0

HaO
rBfIu^1*[rc 46%

DIBM.HJJ
L.
Trtuena.-Sjl1 69% »


MnO2
acetone, rt
95%

0 0 CHCb, rafllM
18

HzNCHO,
00 / reflUH 145°C
21% (2 step)

blEIALH
TolfjenEj -50'C 66%
UWH4
IHF, rt



OH
NH
Mn02

95%

15

The synthetic method disclosed herein may be preferably performed in the presence of cesium carbonate as a base in DMF and in a deoxygenated atmosphere. The inert atmospher| circumvents the probable oxidation of activated a-cafbon atoms of the diketopipetazine ring during the treatment with cesium carbonate (see below) as reported, for example, |y Watanabe et al, 18th International Congress of Heterocyclic Chemistry in Yokohama! Japan (30 July 2001), Abstract, page 225.



jAirJ Cs salt
DMF Air-oxidation of Activated Carbonyl Compounds with Cesium Salts
[Olf>3] Other embodiments of the synthetic method involve modifications to the compound! used in or otherwise involved in the synthesis of compounds represented by Formula (I|. Such derivatives may include modifications to the phenyl ring, introduction of other aromatic ring systems, position of the aromatic ring, alterations to the imidazole ring system an Phymaceutical Compositions
[Qlt>4] The present invention also encompasses the compounds disclosed herein, optionally sand preferably produced by the methods disclosed herein, in pharmaceutical compositions comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration, which have a pharmaceutically effective amount of the products disclosed |bove in a pharmaceutically acceptable earner or diluent. Acceptable carriers or diluents fof therapeutic use are well Icnown in the pharmaceutical art, and are described, for example, i$ Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the
2-T

pharmaceutical composition. For example, sodium benzoate, ascorbic acid and jesters of p-
hydroxybenzoic acid may be added as preservatives. In addition, antioxjdants and
suspending agents may be used. |
[0155] The dehydrophenylahistin or dehydrophenylahistin analog compositions may be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for | injectable administration; patches for transdermal administration, and sub-dermal deposits af d the like. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions,
ii
solid forms suitable for solution or suspension in liquid prior to injection or infi|sion, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitlpl, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, human serum albuinin and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor
amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering igents, and
*
the like. If desired, absorption enhancing preparations (for example, liposomef), may be
utilized. I
[0156] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.^ Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organip oils such as soybean, grapefruit or almond oils, or synthetic fatty acid esters, such as ethw oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substaqces which
1
increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, lorbitol, or
!
dextran. Optionally, the suspension may also contain suitable stabilizers or ijents that increase the solubility of the compounds to allow for the preparation of highly concentrated
solutions.
I
[0157] Pharmaceutical preparations for oral use may be obtained by jjsombining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired,! to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugarsj including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize

starch, wh|at starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypi^ylmelfayl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If jdesired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidon*, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided v^tti suitable coatings. For this purpose, concentrated sugar solutions may be used, which vasty optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycel, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent fixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. For mis purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talci polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be adiled to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Such formulations can be made using methods known in the art (see, for example, U.S. Patent Nos. 5,733,888 (injectable compositions); 5,726,181 fpoorly water soluble compounds); 5,707,641 (therapeutically active proteins or peptides); |,667,8®9 (lipophilic agents); 5,576,012 (sohibilizing polymeric agents); 5,707,615 (anti-viral I formulations); 5,683,676 (particulate medicaments); 5,654,286 (topical formulations); 5,688,529 (oral suspensions); 5,445,829 (extended release formulations); 5i653,987 (liquid formulations); 5,641,515 (controlled release formulations) and 5,601,845 (spheroid formulations).
[Olf>8] Further disclosed herein are various pharmaceutical compositions well known in| the pharmaceutical art for uses that include intraocular, intranasal, and intraauricular delivery. Pharmaceutical formulations include aqueous ophthalmic solutions of the active compounds in water-soluble form, such as eyedrops, or in gellan gum (Shedden et al., 200fl Chn Ther 23(3):440-50) or hydrogels (Mayer et al., 1996 Ophthalmologica 210:101-3); ophthalmic ointments; ophthalmic suspensions, such as microparticulates, drug-containing small polymeric particles that are suspended in a liquid carrier medium (Joshi, A., 1994 J O4il Pharmacol 10:29-45), lipid-soluble formulations (Aim et al., 1989 Prog Clin Biol Res 3f 2:447-58), and microspheres (Mordenti, 1999 Toxicol Sci 52:101-6); and ocular

inserts. Such suitable pharmaceutical formulations are most often and preferably formulated to be sterile, isotonic and buffered for stability and comfort. Pharmaceutical compositions may also include drops and sprays often prepared to simulate in many resf ects nasal secretions to ensure maintenance of normal ciliary action. As disclosed in Remington's Pharmaceutical Sciences (Mack Publishing, 18th Edition), and well-known to thosp skilled in the art, suitable formulations are most often and preferably isotonic, slightly ^uffered to
maintain a pH of 5.5 to 6.5, and most often and preferably include antimicrobial preservatives
ii
and appropriate drug stabilizers. Pharmaceutical formulations for intraauricul^r delivery
3,
include suspensions and ointments for topical application in the ear. Common solvents for
such aural formulations include glycerin and water. I
[0159] When used as a cell cycle inhibitor, a mmor-growth-inhibitmg, or a
f
fungus-growth-inhibiting compound, the compound of Formula (I) can be administered by either oral or a non-oral pathways. When administered orally, it can be administered in capsule, tablet, granule, spray, syrup, or other such form. When administered nojji-orally, it can be administered as an aqueous suspension, an oily preparation or the like orf as a drip,
|
suppository, salve, ointment or the like, when administered via injection oi| infusion,
subcutaneously, intreperitoneally, intravenously, intramuscularly, or the like, similarly, it
may be administered topically, rectally, or vaginally, as deemed appropriate by mqse of skill
in the art for bringing the compound into optimal contact with a tumor, thus inhibiting the
growth of the tumor. Local administration at the site of the tumor is also contemplated, either
before or after tumor resection, as are controlled release formulations, depot formulations,
and infusion pump delivery. I
Methods of Administration ;!
[0160] The present invention also encompasses methods for making and for administering the disclosed chemical compounds and the disclosed pharmaceutical compositions. Such disclosed methods include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathwaj^, which
: administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like; administration via in|ection or

infusion, s|bcutaneously, intraperitoneally, intravenously, intramuscularly, intradennally, or the like; j as well as (c) administration topically, (d) administration rectally, or (e) administration vaginally, as deemed appropriate by those of skill in the art for bringing the compoundf into contact with living tissue; and (f) administration via controlled released formulations, depot formulations, and infusion pump delivery. As further examples of such modes of |idmmistration and as further disclosure of modes of administration, disclosed herein are fvarious methods for administration of the disclosed chemical compounds and pharmaceutical compositions including modes of administration through intraocular, intranasal, and intraauricular pathways.
[01^1] The pharmaceutically effective amount of the dehydrophenylahistin or dehydroph|nylahistin analog composition required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a f desired effect, but will depend on such factors as weight, diet, concurrent medication* and other factors which those skilled in the medical arts will recognize.
[OIJ52] In practicing the methods, the products or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. For example, as disclosed herein, the compounds disclosed herein are effective in the treatment j&f cancer when used in combination with other actives, specifically other chemother^peutics, for example biologies and the specific chemotherapeutics CPT-11, Taxotene (docataxel) and paclitaxel. The compounds disclosed herein are also effective in the treatment of cancer when used in combination with other actives, including anti-vascular agents, anti-angiogenenic agents, such as Erbuitux (Imclone/bristol-Myers) and Iressa (AstraZen
subcutaneously, intramuscularly, colonically, rectalty, vaginally, nasally or intoapfsritoneally,
employing a variety of dosage forms. Such methods may also be applied to testii|g chemical
activity in vivo. I
(0163) As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will varyj depending upon the age, weight and mammalian species treated, the particular compounds} employed, and the specific use for which these compounds are employed. The deternfination of effective dosage levels, that is the dosage levels necessary to achieve the desiredfresult, can be accomplished by one skilled hi the art using routine pharmacological methods. I Typically, human clinical applications of products are commenced at lower dosage levels, vfith dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods. j
[0164] hi non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired |ffect is no longer achieved or adverse side effects disappear. The dosage may range broadly, jdepending upon the desired affects and the therapeutic indication. Typically, dosages may tjje between about 10 microgram/kg and 100 mg/kg body weight, preferably between fbout 100 microgram/kg and 10 mg/kg body weight Alternatively dosages may be |ased and calculated upon the surface area of the patient, as understood by those of skill In the art.
|
Administration may be oral on an every third day, every other day, daily, twice daili, or thrice
daily basis. jj
P
[0165] The exact formulation, route of administration and dosage canjbe chosen by the individual physician in view of the patient's condition. See for example, Fintl et al., in The Pharmacological Basis of Therapeutics, 1975. It should be noted that thej attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would als|o know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the! route of
32-

administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program cdbiparable to that discussed above may be used in veterinary medicine.
[01JB6] Depending on the specific conditions being treated, such agents may be formulated! and administered systemically or locally. A variety of techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing! Co., Baston, PA (1990). Suitable administration routes may include oral, rectal, transdermajjl, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventrieular, intravenous, via infusion, intraperitoneal, intranasal, or intraocular injections. •
[01JS7] For injection or infusion, the agents may be formulated hi aqueous solutions, jlfor example, in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants f appropriate to the barrier to be permeated are used in the formulation. Such penetrantsfare generally known in the art. Use of pharmaceutically acceptable carriers to formulate JBie compounds herein disclosed for the practice of the invention into dosages suitable fojjr systemic administration is within the scope of the invention. With proper choice Of carrier! and suitable manufacturing practice, the compositions disclosed herein, in particular,}those formulated as solutions, may be administered parenterally, such as by intravenous injection or infusion. The compounds can be formulated readily using pharmaceijtically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to fce treated.
[0168] Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. All molecules present inf an aqueous solution at the time of liposome formation are incorporated into the

aqueous interior. The liposomal contents are both protected from the exte&al micro-
environment and, because liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may
be directly administered intracellularly. i
[0169] Determination of the effective amounts is well within the capability of
|
those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharm|ceuticaUy. The preparations formulated for oral administration may be in the form of tablelp, dragees, capsules, or solutions. The pharmaceutical compositions may be manufactured i^ a manner that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
[0170] Compounds disclosed herein can be evaluated for efficacy a$d toxicity using known methods. For example, the toxicology of a particular compound, or pf a subset of the compounds, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The
I
results of such studies are often predictive of toxicity in animals, such as mammae, or more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular compound may be established using several art recognized methods, such as in vitro methods, animal models, or human clinical trials. Art-recogniz4d in vitro
models exist for nearly every class of condition, including the conditions abated by the
!
compounds disclosed herein, including cancer, cardiovascular disease and various fungal
infections. Similarly, acceptable animal models may be used to establish efficacy of
chemicals to treat such conditions. When selecting a model to determine efficacy, ihe skilled
artisan can be guided by the state of the art to choose an appropriate model, dose, a$d route of
administration, and regime. Of course, human clinical trials can also be used to determine the
efficacy of a compound in humans. -3 +

[0|71J When used as an anti-cancer agent, or a tumor-growth-inhibiting compoun4 the compounds disclosed herein may be administered by either oral or a non-oral pathways.! When administered orally, it can be administered in capsule, tablet, granule, spray, syrup, or jother such form. When administered non-orally, it can be administered as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the likf, when administered via injection or infusion, subcutaneous!/, intreperitoneally, intraveno^ry, inframuscularly, intradermally, or the like. Similarly, it may be administered topically, fectally, or vaginally, as deemed appropriate by those of skill in the art for bringing the compound into optimal contact with a tumor, thus inhibiting the growth of the tumor. Local adnjinistration at the site of the tumor or oilier disease condition is also contemplated, either befdre or alter tumor resection, or as part of an art-recognized treatment of the disease condition.} Controlled release formulations, depot formulations, and infusion pump delivery are similarly contemplated.
[OJ72J When used as an anti-cancer agent or an anti-tumor agent, may be orally or non-orall)| administered to a human patient in the amount of about .0007 mg/day to about 7,000 ragJday of the active ingredient, and more preferably about 0.07 mg/day to about 70 mg/day of? the active ingredient at, preferably, one time per day or, less preferably, over two to about tfn times per day. Alternatively and also preferably, the compound may preferably be administered in the stated amounts continuously by, for example, an intravenous drip. Thus, for |i patient weighing 70 kilograms, the preferred daily dose of the active anti-tumor ingredientfwould be about 0.0007 mg/kg/day to about 35 mg/kg/day including 1.0 mg/kg/day and 0.5 rcjg/kg/day, and more preferable, from 0.007 mg/kg/day to about 0.050 mg/kg/day, including 0.035 mg/kg/day. Nonetheless, as will be understood by those of skill in the art, in certain situations it may be necessary to administer the anti-tumor compound in amounts that
?
excess, o^j even far exceed, the above-stated, preferred dosage range to effectively and aggressively treat particularly advanced or lethal tumors.
[0|73] When used as an antifungal agent the preferable amount of the dehydrophfenylahistin or its analog effective in the treatment or prevention of a particular fungal palhogen will depend in part on the characteristics of the fungus and the extent of infection, and can be determined by standard clinical techniques. In vitro or in vivo assays

may optionally be employed to help identity optimal dosage ranges. Effective dojjtes may be extrapolated from dose-response curves derived from in vitro analysis or prefe -ably from animal models. The precise dosage level should be determined by the attending p tysician or other health care provider and will depend upon well known factors, includir g route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the infection; the use (or not) of concomitant therapie .
[0174] The effective dose of the dehydrophenylaMstin or its analog wi 1 typically
be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about l|> mg/kg of
mammalian body weight per day, administered in single or multiple doses. Generally, the
compound may be administered to patients in need of such treatment in a daily do|e range of
about 1 to about 2000 mg per patient. [0175] To formulate the dosage including the compounds disclosed |erein as a tumor-growth-inhibiting compound, known surface active agents, excipients, Smoothing
\agents, suspension agents and pharmaceutically acceptable film-forming substances and coating assistants, and the like may be used. Preferably alcohols, esters, sulfateti aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, ipagnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, softium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may tie used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methyiacetate-methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalajtes and the
like may be used as suspension agents. In addition to the foregoing preferred ingredients,
1
sweeteners, fragrances, colorants, preservatives and the like maybe added to the adininistered formulation of the compound, particularly when the compound is to be administerefl orally.
[0176] The compositions disclosed herein in a pharmaceutical compositions may also comprise a pharmaceutically acceptable carrier. Such compositions may be prepared for storage and for subsequent administration. Acceptable carriers or diluents for therapeutic use

are well k|K>wn in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). For example, such compositions may be formulated and used as tablets, capsules or solutions for oral administration; suppositories for rectal or vaginal administration; sterile solutions or suspensions for injectable administration. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions. Suitable excipients include, but are not limited toj saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochlo^de, aad the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (for example, Bposomes), may be utilized.
[0^77] The pharmaceutically effective amount of the composition required as a dose will {depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent
i>
medicatiof and other factors which those skilled in the medical arts will recognize.
J
[0|78J The products or compositions, as described above, may be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. Specifically, the disclosed compounds products may be used alone or in combination with other cheqaotherapeutics or biologies, including antibodies, for the treatment of cancer, or in combination with other anti-infective for the treatment of fungal infection. These products or compositions can be utilized in vivo or in vitro. The useful dosages and the most useful modes of administration will vary depending upon the age, weight and animal treated, the particular | compounds employed, and the specific use for which these composition or compositions are employed. The magnitude of a dose in the management or treatment for a particular disorder will vary with the severity of the condition to be treated and to the route of administration, and depending on the disease conditions and then* severity, the compositions may be formulated and administered either systemically or locally. A variety of techniques

for formulation and administration may be found in Remington's Pharmaceutical Sciences,
18th ed., Mack Publishing Co., Easton, PA (1990). I
[0179] To formulate the compounds of Formula (I), preferably synthetically
produced according to the methods disclosed herein, as a cell cycle iahibito^ a tumor-
growth-inhibiting, or an antifiragal compound, known surface active agents, jjexcipients,
smoothing agents, suspension agents and pharmaceutically acceptable fi|m-forming
substances and coating assistants, and the like may be used. Preferably alcohols, esters,
sulfated aliphatic alcohols, and the like may be used as surface active agenlj^; sucrose,
glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, inagnesium
aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium
carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium cartjjoxymethyl
cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and
the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya
may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of
a carbohydrate such as cellulose or sugar, or methyiacetate-methacrylate copofymer as a
derivative of polyvinyl may be used as suspension agents; and plasticizers su(fli as ester
phthalates and .the like may be used as suspension agents, hi addition to thd foregoing
preferred ingredients, sweeteners, fragrances, colorants, preservatives and the li|:e may be
added to the administered formulation of the compound produced by the method, {Jarticularly
when the compound is to be administered orally. I
[0180] The cell cycle inhibitors, the antitumor agents, and the antifuijgal agents that may be produced by the method may be orally or non-orally administered t| a human patient in the amount of about 0.001 mg/kg/day to about 10,000 mg/kg/day oftthe active ingredient, and more preferably about 0.1 mg/kg/day to about 100 mg/kg/day ofjjthe active ingredient at, preferably, once every three days on a cyclic basis, once every other day, one time per day, twice per day, or less preferably, over two to about ten time^ per day. Alternatively and also preferably, the compound produced by the method may preferably be administered in the stated amounts continuously by, for example, an intravenous dkp. Thus, for the example of a patient weighing 70 kilograms, the preferred daily dose of Jthe active anti-tumor ingredient would be about 0.07 mg/day to about 700 grams/day, land more
38

preferable} 7 mg/day to about 7 grams/day. Nonetheless, as will be understood by those of skill hi t$e art, in certain situations it may be necessary to administer the anti-tumor compound produced by the method in amounts that excess, or even far exceed, the above-stated, preened dosage range to effectively and aggressively treat particularly advanced or lethal tumors.
[Of&l] In the case of using the cell cycle inhibitor produced by methods as a biochemical test reagent, the compound produced by methods of the invention inhibits the progression of the cell cycle when it is dissolved in an organic solvent or hydrous organic solvent aifd it is directly applied to any of various cultured cell systems. Usable organic solvents include, for example, methanol, methylsulfoxide, and the like. The formulation can, for example, be a powder, granular or other solid inhibitor, or a liquid inhibitor prepared using an organic solvent or a hydrous organic solvent. While a preferred concentration of the compound produced by the method of the invention for use as a cell cycle inhibitor is generally |n the range of about 1 to about 100 ug/ml, the most appropriate use amount varies depending on the type of cultured cell system and the purpose of use, as will be appreciated by person* of primary skill in the art. Also, in certain applications it may be necessary or preferred to persons of ordinary skill in the art to use an amount outside the foregoing range.
[0182] From a pharmaceutical perspective, certain embodiments provide methods for preverjting or treating fungal infections and/or a pathogenic fungus in a subject, involve administering to the subject a composition including a.dehydrophenylahistin or its analog, for example, Administering the dehydrophenylahistin or its analog in an amount and manner which provides the intended antifungal effect.
[Of 83] Other embodiments include the treatment or prevention of infection in a patient byfa pathogenic fungus such as those listed above or referred to below.
[Oj84] Another embodiment relates to the treatment or prevention of infection in a patient by a pathogenic fungus which is resistant to one or more other antifungal agents, especiallyfan agent other than dehydrophenylahistin or its analog, including e.g. amphotericin B or analogs or derivatives thereof (including 14(s)-hydroxyamphotericin B methyl ester, the hydrazidejj of amphotericin B with l-amino-4-methylpiperazine, and other derivatives) or other pol^ene macrolide antibiotics, including, e.g., nystatin, candicidin, pimaricin and

,1
natamycin; flucytosine; griseofulvin; echinocandins or aureobasidins, including naturally occurring and semi-synthetic analogs; dihydrobenzo[a]napthacenequinones; fnucleoside peptide antifongals including the polyoxins and nikkomycins; allylamines such ps naftifine and other squalene epoxidase inhibitors; and azoles, imidazoles and triazoles si|ch as, e.g., clotrimazole, miconazole, ketoconazole, econazole, butoconazole, oxiconazole, terconazole,
i|
itraconazole or fluconazole and the like. For additional conventional antifungalj| agents and new agents under development, see e.g. Turner and Rodriguez, 1996 Current Pharmaceutical Design, 2:209-224. Another embodiment involves the treatment or prevention of Infection in a patient by a pathogenic fungus in cases in which the patient is allergic tol otherwise intolerant of, or nonresponsive to one or more other antifungal agents or in whon| the use of other antifungal agents is otherwise contra-indicated. Those other antifungal agents include,
J!
among others, those antifungal agents disclosed above and elsewhere herein.
[0185] hi the foregoing methods for treatment or prevention, a
dehydrophenylahistin or its analog, is administered to the subject in an effective! antifungal
amount. |
[0186] Other embodiments relate to the treatment or prevention of infection by a pathogenic fungus in a patient by administration of a dehydrophenylahistin or its analog, in
3
conjunction with the administration of one or more other antifungal agents, including for example, any of the previously mentioned agents or types of agents (e.g. in combination with treatment with amphotericin B, preferably in a lipid or liposome formulation; an azole or triazole such as fluconazole, for example; an auleobasidin;
I
dihydrobenzo[alnapthacenequinone; or an echinocardin) as well as with a, different
dehydrophenylahistin or its analog. f
[0187] The dehydrophenylahistin or its analog may be administered before, after
or at the same time the other antifungal agent is administered. In certain embodiments, the
combination therapy will permit the use of reduced amounts of one or both | antifungal
components, relative to the amount used if used alone. :
[0188] Still other embodiments relate to administration of a dehydrophenylahistin or its analog to a subject for the treatment or prevention of infection by a pathogenic fungus, where the subject is immunosuppressed or immunocompromised, e.g. as the result of genetic

disorder, ^isease such as diabetes or HIV or other infection, chemotherapy or radiation treatment for cancer or other disease, or drug- or otherwise induced immtmosuppression in connection} with tissue or organ transplantation or the treatment of an autoimmune disorder. Where thei patient is being or will be treated with an immunosuppressive agent, e.g., in connection! with a tissue or organ transplantation, a dehydrophenylahistin or its analog may be co-adtn|nistered with the immunosuppressive agent(s) to treat or prevent a pathogenic fungal infection.
[01JB9) Another aspect of this invention is the treatment or prevention of infection by a pathogenic fungus in a patient infected, or suspected of being infected, with HIV, by administraf on of an antifungal dehydrophenylahistin or its analog, in conjunction with the administration of one or more anti-HIV therapeutics (including e.g. HIV protease inhibitors, reverse transcriptase inhibitors or anti-viral agents). The dehydrophenylahistin or its analog may be aAninistered before, after or at the same time as administration of the anti-HIV agent(s). [OljpO] Another aspect of this, invention is the treatment or prevention of infection by a pathogenic fungus in a patient by administration of an antifungal dehydrophenylahistin or its analog, in conjunction with the administration of one or more other antibiotic compounds, especially one or more antibacterial agents, preferably in an effective amount and regiment to treat or prevent bacterial infection. Again, the dehydrophenylahistin or its analog m&jf be administered before, after or at the same time as administration of the other agent(s).
[01|>1] Pathogenic fungal infections which may be treated or prevented by the disclosed methods include, among others, Aspergillosis, including invasive pulmonary aspergillosfs; Blastomycosis, including profound or rapidly progressive infections and blastomycfijsis in tie central nervous system; Candidiasis, including retrograde candidiasis of the urinarjjr tract, e.g. in patients with kidney stones, urinary tract obstruction, renal transplantation or poorly controlled diabetes mellitus; Coccidioidomycosis, including chronic disease vfhich does not respond well to other chemotherapy; Cryptococcosis; Histopolasfnosis; Mucormycosis, including e.g. craniofacial mucomrycosis and pulmonary mucormyct>sis; Paracoccidioidomycosis; and Sporotrichosis. It should be noted that

administration of a composition comprising an antifunga] amount of one! or more
dehydrophenylahistin or its analogs may be particularly useful for treating or pifeventing a
pathogenic fungal infection in a mammalian subject where the fungus is resistant to one or
more other antifungal therapies, or where the use of one or more other antifungal therapies is
contraindicated, e.g., as mentioned above. jj
[0192] Antifungal pharmaceutical compositions containing at least onelantifungal
I
dehydrophenylahistin or its analog, are also provided for use hi practicing the} disclosed methods. Those pharmaceutical compositions may be packaged together with an appropriate
package insert containing, inter alia, directions and information relating to their jantifungal
use. Pharmaceutical compositions are also provided which contain onejj or more
•j
dehydrophenylahistin or its analog together with a second antifungal agent. I
Methods of Treating Fungal Infections I
[0193] Certain embodiments disclosed herein relate to methods for treating or preventing a pathogenic fungal infection, including for example AspergillosisJ including invasive pulmonary aspergillosis; Blastomycosis, including profound or rapidly progressive infections and blastomycosis in the central nervous system; Candidiasis, including retrograde candidiasis of the urinary tract, e.g. hi patients with kidney stones, urinary tract obstruction, renal transplantaion or poorly controlled diabetes mellitus; Coccidioidomycosis,| including
j
chronic disease which does not respond well to other chemotherapy; Cryptbcoccosis; Histopolasmosis; Mucormycosis, including e.g. craniofacial mucormycosis and pulmonary mucormycosis; Paracoccidioidomycosis; and Sporotrichosis. The methods majjy involve administering at least one antifungal dehydrophenylahistin or its analog, as described above, to a human subject such that the fungal infection is treated or prevented, in certain embodiments the dehydrophenylahistin or its analog may be administered in conjunction with administration of one or more non-dehydrophenylahistin or its analog antifungal a|ents such as amphotericin B, or an imidazole or triazole agent such as those mentioned previously.
[0194] The pathogenic fungal infection may be topical, e.g., caused l|y, among other organisms, species of Candida, Trichophyton, Microspontm or Epiderindbhyton or mucosal, e.g., caused by Candida albicans (e.g. thrush and vaginal candidiasis). The
t:
infection may be systemic, e.g., caused by Candida albicans, Cryptococcus ndpformans,

Aspergillu^fumigatus, Coccidiodes, Paracocciciodes, Histoplasma or Blastomyces spp. The infection ^iay also involve eumycotic mycetoma, chromoblastomycosis, cryptococcal meningitits| or phycomycosis.
[Olj>5] Further embodiments relate to methods for treating or preventing a pathogenicf fungal infection selected from the group consisting of Candida spp. including C. albicans, t
[01JP6] Further embodiments relate to methods for treating or preventing a pathogenic) fungal infection which is resistant to other antifungal therapy, including pathogenic! fungal infections which are resistant to one or more antifungal agents mentioned elsewhere herein such as amphotcricin B, flucytosine, one of the imidazoles or triazoles (including je.g. fluconazole, ketoconazole, itraconazole and the other previously mentioned
examples).; The methods may involve administering to the patient one or more antifungal
i1
dehydroph^nylahistin or its analog, in an amount and dosing regimen such that a fungal infection resistant to another antifungal therapy in the subject is treated or prevented.
[Olf 7] Further embodiments relate to methods for treating or preventing a pathogenic| fungal infection in a patient who is allergic to, intolerant of or not responsive to another antifungal therapy or in whom the use of other antifungal agents is otherwise contra-indicated, |ncludmg one or more other antifungal agents mentioned elsewhere herein such as amphoteriqtin B, flucytosine, one of the imidazoles or triazoles (including e.g. fluconazole, ketoconazdjle, itraconazole and the other previously mentioned examples). The methods may involve administering to such patient one or more antifungal dehydrophenylahistin or its analog, in |tn amount such that a fungal infection is treated or prevented.

Packaged Dehydrophenylahistin or its analogs
(0198] Certain embodiments relate to packaged dehydrophenylahitin or its analogs, preferably packaged nonimmunosuppressive antifungal dehydrophenylaiistin or its analogs, which term is intended to include at least one dehydrophenylahistin or its analog, as described above, packaged with instructions for administering the dehydrophenylahistin or its analog(s) as an antifungal agent without causing a untoward immunosuppresAve effects
F
within a human subject. In some embodiments, the non-immunosuppressivejj antifungal dehydrophenylahistin or its analog is a member of one of the preferred subsets of 4ompounds described above. The dehydrophenylahistin or its analog can be packaged aloile with the instructions or can be packaged with another dehydrophenylahistin or its analog, Iraparnycin or another ingredient or additive, e.g., one or more of the ingredients of the phainaceutical compositions. The package can contain one or more containers filled with one or fiore of the
ingredients of the phan-naceutical compositions. Optionally associated ivith such
I containers) can be a notice in the form prescribed by a governmental agency regjulating the
manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. jj
I
(0199] The following non-limiting examples are meant to describe the preferred
methods using certain preferred embodiments. Variations in the details of thej particular
methods employed and in the precise chemical compositions obtained will undopbtedly be
appreciated by those of skill in the art ;
EXAMPLE 1 ,;
A. Synthesis of Dehvdropjienylahistin ;
[0200] Dehydrophenylahistin was synthesized by condensation acconjjing to the
following basic reaction scheme, as shown in Figure 1: '•,





[021)1] 25.0 g of global 2,5-piperazinedione 1 [2,5-piperazinedione (Aldrich G640-6), 2J5.0 g, 0.218 mol] in 100 mL of acetic anhydride (Ac2O) was mixed with sodium
i
acetate (N|OAc) (17.96 g, .0218 mol). The mixture was heated at 110°C for 8 h using a double co^ed condenser under an Ar atmosphere. After AcjO was removed by evaporation, the residui was dissolved in AcOEt, washed with 10% citric acid, 10% NaHCOs and saturated If aCl (three times each), dried over NajSO/t, and concentrated in vacuo. The residue was triturdjted with ether to form a solid. This solid was recrystallized from EtOAc with ether-hexafie to afford 26.4 g (61%) of N,N'-diacetyl-2,5-piperazinedione 1.
piperazineflione 2
[02J)2] To a solution of 5-(l,l-dimethyl-2-propenyl)imidazole-4-carboxaldehyde (100 mg, (|.609 mmol) in DMF (2 mL) was added compound 1 (241 mg, 1 .22 mmol) and the solution wjas repeatedly evacuated in a short time to remove oxygen and flushed with Ar, followed fy the addition of Cs2COj (198 mg, 0.609 mmol) and the evacuation-flushing process w|s repeated again. The resultant mixture was stirred for 5 h at room temperature. After the sfolvent was removed by evaporation, the residue was dissolved in the mixture of EtOAc an| 10% NaaCOj, and the organic phase was washed with 10% NaaCOa again and saturated iffaCl for three times, dried over NaaSCU and concentrated in vacuo. The residual oil was purified by column chromatography on silica using CHCla-MeOH (100:0 to 50:1) as an eluant tj> give 60 mg (33 %) of a pale yellow solid 2. Dehydrop|envlahigtin
[0^03] To a solution of 2 (30 mg, 0.099 mmol) in DMF (0.8 mL) was added benzaldeh^de (5 1 uL, 0.496 mmol, 5 eq) and the solution was repeatedly evacuated in a short time to refnove oxygen and flushed with Ar, followed by the addition of CsaCOa (53 mg, 0.149 mmol, 1.5 eq) and the evacuation-flushing process was repeated again. The resultant mixture w|is heated for 2.5 h at 80°C. (The temperature must be increased slowly. Rapid heating increases the production of E-isomer at the benzylidene moiety.) After the solvent was removed by evaporation, the residue was dissolved in EtOAc, washed with water for two

i!
times and saturated Nad for three times, dried over Na2SC>4 and concentrated in \acuo. On TLC using CHCU-MeOH (10:1), you can observe a spot with bright grien-yellow luminescence at 365 nm UV. The purity of this crude product was more than J75% from HPLC analysis. The resulting residue was dissolved in 90% MeOH aq and jjapplied to reverse-phase HPLC column (YMC-Pack, ODS-AM, 20 x 250 mm) and eluted usiig a linear gradient from 70 to 74% MeOH in water over 16 min at a flow rate of 12 mL/m n, and the

desired fraction was collected and concentrated by evaporation to give a 19.7 mj

(60%) of

yellow colored dehydrophenylahistin. The HPLC profile of the synth ;
dehydrophenylahistin is depicted in Figure 2. [0204] In the purification of dehydrophenylahistin, as shown in Figure J4, a major peak was the desired Z- form compound of dehydrophenylahistin. The formatiojii of an E-isomer was observed as a minor component (about 10%), which was eluted as a |nore polar peak than Z-isomer. As other minor peaks, the reduced Z- and jE-compounds, injwhich the dimethylallyl part of dehydrophenylahistin was reduced, was also observed. The! formation of these reduced compounds was due to the aldehyde 2 with a reduced impurity, |vhich was generated during the reduction of with DIBAL-H and was not separated in the subsequent
f
process.
[0205] These minor compounds could be removed by preparative HPLC purification, afforded dehydrophenylahistin with the Z-configuration at the benzy^dene part in a 60% yield (20% yield in two steps) with more than 95% purity. The compounds with E-
ij
configuration at the imidazole side of the diketopiperazine ring were not observed in this
HPLC chart, suggesting that the first reaction from compound 1 to 3 in Figufre 1 is Z-
selective. B. Chemical Characteristics: jj
[0206] The above dehydrophenylahistin compound is a pale yellow jjsolid. Its structure is confirmed by standard NMR analyses.

EXAMPLE 2 Synthesis and Physical Characterization of tBu-dehvdrophenvlahistin Derivatives
[02Jb7] Structural derivatives of dehydrophenylahistin were synthesized according to the folding reaction schemes to produce tBu-dehydrophenylahistin. Synthesis by Route A (see Figjjure 1) is similar in certain respects to the synthesis of the dehydrophenylahistin synthesize! as in Example 1.
Route A





T^^0
tCm
15
C82CQ3/DMF 33% (1st)

16

50%

tBu-dehydroPLH






Route B

1.2%

tBu-dehydroPLH

Route A:
[0208] N,N'-diacethyl-2,5-piperazinedione 1 was prepared as in Example 1. lH-AceWl-3-(fZ)-145-tert-butvl-l/f4-imidazolvl1methvlidenen-2.5-pipeffazinedionefl6')


H


[0209] To a solution of 5-tert-butylimidazole-4-carboxaldehyde IS (3JJ02 g, 19.8. mmol) in DMF (30 mL) was added compound 1 (5.89 g, 29.72 mmol) and the s
int to give
column chromatography on silica using CHCls-MeOH (100:0 to 50:1) as an ehi
1.90 g (33 %) of a pale yellow solid 16. 1H NMR (270 MHz, CDC13) 6 12. if* (d, br-s,
1H), 9.22 (br-s, 1H), 7.57 (s, 1H), 7.18, (s, 1H), 4.47 (s, 2H), 2.65 (s, 3H|, 1.47 (s,
9H).
2) t-Bu-dehvdrophenvlam'stin


!H


[0210] To a solution of l-Acetyl-3-{(Z)-l-[5-tert-bbtyl-ltf-4-imidazoIyI]methylidene}]-2,5-piperazmedione (16) (11 mg, 0.038 mmol) in DMIf (1.0 mL) was added benzaldehyde (19 fiL, 0.19 mmol, 5 eq) and the solution was repeatedlylevacuated
in a short time to remove oxygen and flushed with Ar, followed by the addition
(43 mg, 0.132 mmol, 3.5 eq) and the evacuation-flushing process was repeated a^ain. The
resultant mixture was heated for 2.5 h at 80°C. After the solvent was removed by

evaporation, the residue was dissolved in EtOAc, washed with water for two times and saturated ^JaCI for three times, dried over NaaSO4 and concentrated in vacuo. The resulting residue w|s dissolved in 90% MeOH aq and applied to reverse-phase HPLC column (YMC-Pack, OD|-AM, 20 x 250 mm) and eluted using a linear gradient from 70 to 74% MeOH in water ovef 16 min at a flow rate of 12 mL/min, and the desired fraction was collected and concentrated by evaporation to give a 6.4 mg (50%) of yellow colored tert-butyl-dehydroph^ylahistin. *H NMR (270 MHz, CDC13) 6 12.34 br-s, 1H), 9.18 (br-s, 1H), 8.09 (s, ill), 7.59 (s, 1H), 7.31 - 7.49 (m, 5H), 7.01 s, 2H), 1.46 (s, 9H).
[02111] The dehydrophenylahistin reaction to produce tBu-dehydrophenylahistin is identical t| Example 1.
[0212] The total yield of the tBu-dehydrophenylahistin recovered was 16.5%. Route B: ;
[0213] N,N'-diacethyl-2,5-piperazinedione 1 was prepared as in Example 1. 1) l-Acet4-3-f(Z)-benzvlidenel 1-2.5-piperazinedione (17)

O
[02fl4] To a solution of benzaldehyde 4 (0.54 g, 5.05. mmol) in DMF (5 mL) was added confpound 1 (2.0 g, 10.1 mmol) and the solution was repeatedly evacuated hi a short time to reqfiove oxygen and flushed with Ar, followed by the addition of CsaCOa (1.65 g, 5.05 mmol) and the evacuation-flushing process was repeated again. The resultant mixture was stirred forj 3.5 h at room temperature. After the solvent was removed by evaporation, the residue w^s dissolved in the mixture of EtOAc and 10% NaaCOa, and the organic phase was washed wfh 10% Na2COs again and saturated NaCl for three tiines, dried over NaaSO4 and concentrated in vacuo. The residual solid was recrystalized from MeOH-ether to obtain a off-white gjolid of 17; yield 1.95 g (79%).

2) t-Bu-dehvdroDhenvlahistin


IH


[0215] To a solution of l-Acetyl-3-[(2)-benzylidenel]-2,5-piperazm|dione (17)
I
and concentrated in vacuo. The resulting residue was dissolved in 90% and applied to reverse-phase HPLC column (YMG-Pack, ODS-AM, 20 x 25(
(48 mg, 0.197 mmol) in DMF (1.0 mL) was added 5-tert-butylimidazole-4-carbpxaldehyde 15 (30 mg, 0.197 mmol) and the solution was repeatedly evacuated in a short tim| to remove oxygen and flushed with Ar, followed by the addition of CsaCOs (96 mg, 0.296 mmol) and the evacuation-flushing process was repeated again. The resultant mixture was h MeOH aq mm) and
eluted using a linear gradient from 70 to 74% MeOH in water over 16 min at a 1 low rate of
j 12. mL/min, and the desired fraction was collected and concentrated by evaporatidb to give a
0.8 mg (1.2%) of yellow colored tert-butyl-dehydrophenylahistin. j
[0216] The total yield of the tBu-dehydrophenylahistin recovered was ti.9%.
i [0217] The HPLC profile of the crude synthetic tBu-dehyrophenylalistin from
Route A and from Route B is depicted hi Figure 4. j
[0218] Two other tBu-dehydrophenylahistin derivatives were sjynthesized according to the method of Route A. In the synthesis of the additional tBu-
y
dehydrophenylahistin derivatives, modifications to the benzaldehyde compound 4 4vere made.
[0219] Figure 4 illustrates the similarities of the HPLC profiles (Colujbn: YMC-Pack ODS-AM (20 x 250mm); Gradient: 65% to 75% in a methanol-water sy4em for 20 min, then 10 min in a 100% methanol system; Flow rate: 12mL/min; O.D. 230 nnjf) from the synthesized dehydrophenylahistin of Example 1 (Fig 2) and the above exemp|ified tBu-dehydrophenylahistin compound produced by Route A.
[0220] The sequence of introduction of the aldehydes is a relevant to thfe yield and is therefore aspect of the synthesis. An analogue of dehydrophenylahistin was synthesized, as

a control of model, wherein the dimethylallyl group was changed to the tert-butyl group with a similar static hindrance at the 5-position of the imidazole ring.
[02JJ1] The synthesis of this "tert-butyl (tBu)-dehydrophenylahistin" using "Route A" was as Ishown above: Particularly, the sequence of introduction of the aldehyde exactly follows th^ dehydrophenylahistin synthesis, and exhibited a total yield of 16.5% tBu-dehydroph|nylahistin. This yield was similar to that of dehydrophenylahistin (20%). Using "Route B"j where the sequence of introduction of the aldehydes is opposite that of Route "A" for the dehydrophenylahistin synthesis, only a trace amount of the desired tBu-dehydroPLH was obtained with a total yield of 0.9%, although in the introduction of first benzaldehyde 4 gave a 76^o yield of the intermediate compound 17. This result indicated that it may be difficult tojj introduce the highly bulky imidazole-4-carboxaldehydes 15 with a substituting group having a quaternary-carbon on the adjacent 5-position at the imidazole ring into the intermediate compound 17, suggesting that the sequence for introduction of aldehydes is an important aspect for obtaining a high yield of dehydrophenylahistin or an analog of dehydroph|nylahistin employing the synthesis disclosed herein:
|
[02JZ2] From the HPLC analysis of the final crude products, as shown in Figure 4, a very higl| content of tBu-dehydrophenylahistin and small amount of by-product formations were obscured in the crude sample of Route A (left). However, a relatively smaller amount of the desifed tBu-dehydrophenylahistin and several other by-products were observed in the sample obtained using Route B (right).

EXAMPLE 3
Alternative. Larger-Scale Synthesis of Dehvdrophenvlahistin and Analog
Synthesis of 3-Z-Benzylidene-6-[5"-(l,l-dimethylattyl)~lH-imidazol-4 "-Z-piperazine-2,5-dione [Dehydrophenylahistin] (I)
OH 0
e,f
o o



iW :;
o o




NH
• r ?H r-v.
vo o

W

[02|J3] Reagents: a) LDA, CH3CHO; b) Tos-Cl, pyridine; c) DBU; d) NaOH; e) C2C1202; tf KOOCCH2COOEt, BuLi; g) SO2C12; h) H2NCHO, H2O; i) LiAlU,; j) MnOz; k) l,4-diacetj|[-piperazine-2,5-dione, Cs2COs; 1) benzaldehyde,
3-Hydroyy\2,2-dimethyl-butyric acid methyl ester


^
LDA OH O
0
(02)24] A solution of LDA in heptane/THF/ethylbenzene (2 M, 196 ml, 0.39 mol) was added funder argon to a solution of methyl isobutyrate (45 ml, 0.39 mol) in THF (270 ml) at -60° and the resultant mixture was stirred for 30 min. A solution of acetaldehyde (27 ml, 0.48 mol) fin THF (45 ml), precooled to -60°, was added slowly and the resulting solution stirred for fa further 30 min. Saturated ammonium chloride (50 ml) was added and the solution wjjis allowed to warm to room temperature. The reaction mixture was extracted with ethyl acetafe, and the extracts were washed with HCI (2 M), sodium bicarbonate, then brine. The organic layer was dried over magnesium sulfate, filtered, then evaporated to give a clear oil (52.6 gj). Distillation 76-82°/30 mmHg gave pure 3-hydroxy-2,2-drmethyl-butyric acid methyl estfr (42.3 g, 74%). (Burk et al., J. Am. Chem. Soc., 117:4423-4424 (1995)).
(024J 'H NMR (400 MHz, CDC13) 5 1.15 (d, /= 6.2 Hz, 3H); 1.17 (s, 6H); 2.66
(d, J= 6.2 pz, 1H, -OH); 3.71 (s, 3H, -OMe~); 3.87 (app quintet, J= 6.4 Hz, 1H, H3).
2,2-Dimettiyl-3-(toluene-4-sulfonyloxy)-butyric acid methyl ester


'N.
OH 0 Pyridine
53

{0226] To a cooled (0°) solution of 3-hydroxy-2,2-diniethyl-butyric |cid methyl
jj
ester (52.0 g, 0.36 mol) in pyridine (100 ml) was added gradually, _p-toluene sulfo^yl chloride (69.0 g, 0.36 mol). The mixture was allowed to warm to room temperature and jwas stirred for 60 h. The reaction was again cooled in ice and was acidified by addition of |IC1 (2 M).
I
The resultant solution was extracted with ethyl acetate, the extracts were washed with HCI,
ii
then brine, dried and evaporated to give an oil which formed a white precif itate upon standing. This mixture .was dissolved in the minimum amount of ethyl acetate ani men light petroleum was added to afford a white precipitate which was collected and washedj with more light petroleum. The filtrate was partially evaporated and a second crop of crystals was collected and added to the first to afford 2,2-dimethyl-3-(toluene-4-sulfonyloxy)-tij,ityric acid methyl ester (81.2 g, 76%).
102271 'H NMR (400 NMz, CDC13) 8 1.12 (s, 3H); 1.13 (s, 3H); 1.24J(d, / = 6.4
Hz, 3H); 2.45 (s, 3H, -PhMe) 3.58 (s, 3H, -OMe)\ 4.94 (quartet, J= 6.4 Hz, 1H, H$), 7.33 (d,
Jr=8.0Hz,2H), 7.78(d,y=8.0Hz,2H). j
[0228] Evaporation of the final filtrate afforded additional crude 2,2-4ime1:hyl-3-
(toluene-4-sulfonyloxy)-butyric acid methyl ester (19.0 g, 18%). v j
s
2,2-Dimethyl-but-3-enoic acid methyl ester i



DBU

[0229] A solution of 2,2-dimethyl-3-(toluene-4-sulfonyloxy)-butyric a|;id methyl ester (18.06 g, 0.06 mol) in DBU (15 ml) was heated at 140-160° for 3.5 h. The rr|ixture was allowed to cool to room temperature and was then diluted with ether. The mixture was washed with HCI (1 M), sodium bicarbonate, then brine. The ethereal layer was- dried and partially evaporated to give a concentrated solution of 2,2-dimethyl-but-3-enoic ajbid methyl ester (10 g). (Savu and Katzenellenbogen, J. Org. Chem, 46:239-250 (1981)j. Further

evaporaticjb was avoided due to the volatility of the product (bp 102°). (Tsaconas e; al., Aust. J. Chem., p:435-437 (2000)).
102*» 'H NMR (400 NMz, CDC13) 81.31 (s, 6H); 3.68 (s, 3H); 5.06 (d, J = 17. 1
Hz, 1H, -^H^CHj); 5.11 (d, J = 10.7 Hz, 1H, -CH**CH2); 6.03 (dd, J= 17.1, 10.7 Hz, 1H, -
2,2-Dimet$yl-but-3-enoic acid





[Oj 31] The above ethereal solution of 2,2-dimethyl-but-3-enoic acid methyl ester (10 g) wait diluted with ethanol (25 ml), sodium hydroxide (4 M, 22 ml) was added and the mixture wfas stirred overnight. The solution was partially evaporated to remove the ethanol and the resultant mixture was added to HC1 (1M, 100 ml). The product was extracted with ethyl acetate and the extracts were dried and evaporated to give 2,2-dimethyl-but-3-enoic acid (6.01 g, s|% 2 steps). (Hayashi et al., /. Org. Chem., 65:8402-8405 (2000).
l°^2} 'H NMR (400 MHz, CDC13) 5 1.33 (s, 6H); 5.11 (d, J= 10.8 Hz, 1H, -
CH~CH2% 5.15 (d, J = 17.2 Hz, 1H, ~CH=CH2); 6.05 (dd, J = 17.2, 10.8 Hz, 1H, -
[0|33] Monoethyl hydrogen malonate (Wierenga and Skulnick, "Aliphatic and Aromatic j (3-keto Esters fiom Monoethyl Malonate: Ethyl 2-Butyrylacetate," Organic Syntheses pollective Volume 7, 213).





[0|34] Ethyl potassium malonate (25.0 g, 0.15 mol) was suspended in water (15.6 ml) and cooled in an ice bath. Concentrated HC1 (12.5 ml) was added dropwise over 30 min,
-5.5

then the mixture was stirred for a further 10 min. The precipitate was filtered, t|en washed
twice with ether. The filtrate was separated and the aqueous phase was extractedjwith ether.
| The combined ethereal solutions were dried (MgSO-O and evaporated to affordl as an oil,
monoethyl hydrogen malonate (19.2 g, 99%) which was dried under vacuum o\ ernight (or 50°/1 mm for 1 h) prior to use.

4,4-Dimethyl-3-oxo-hex-5-enoic acid ethyl ester

!)C2O,a,/DMF
OH 2 * V *v
- x-v
o o

[0235] Oxalyl chloride (3.83 ml, 43.9 mmol) was added dropwise to a |x>oled (0°) solution of 2,2-dimethyl-but-3-enoic acid (5.0 g, 43.9 mmol) and DMF (1 drop) iroanhydrous dichloromethane (25 ml). The mixture was stirred for 1 h at 0°, then for 16 Ih at room temperature. Fractional distillation (12P/760 mmHg) afforded 2,2-diraethyl-l|ut-3-enoyl
';
chloride (4.1 g, 71%). j
[0236] Monoethyl hydrogen malonate (7.2 g, 0.05 mol) and bipy|idyl (few milligrams) were dissolved in THF (90 ml) and the system was flushed with nitn >gen. The solution was cooled to -70°, then BuLi (2.5 M in hexanes, 37 ml, 0.09 mol) was adtied. After

5 ml) was
the addition of only ~10 ml of BuLi the solution turned pink and additional THF (
required to enable magnetic stirring. The cooling bath was removed and the remaining BuLi was added, the temperature was allowed to reach -10°, upon which the solution turned colorless. The mixture was again cooled to -60° and a solution of 2,2-dimethyl-b|ut-3-enoyl
chloride (4.1 g, 0.03 mol) in THF (12 ml) was added dropwise. After addition wa| complete
| the mixture was allowed to warm to 0° and stir for 3 h, then it was added to a 1:1 mixture of
ether/1 M HC1 (260 ml) at 0° and stirred for a further 1.5 h. The organic layer wasj removed,
1 .washed with HC1 (1 M), sodium bicarbonate solution, brine then dried and evaporated to give
4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (5.6 g, 98%). (Hayashi et al., J. O^g. Chem., 65:8402-8405 (2000). Distillation with a Kugelrohr oven (160°/1 mmHg) afforded pure material.

'H NMR (400 MHz, CDC13) 8 1.26 (s, 6H); 1.27 (t, J = 6.9 Hz, 3H, CH2CH3)\ 3.51 (s, 2H); 4.18 (q, J = 6.9 Hz, 2H, -CH2CH3); 5.20 (d, /= 17.7 Hz, 1H, CH=CH3\, 5.21 (d, J= 9.6 Hz, 1H, -CH=CH2); 5.89 (dd, 7= 17.7, 9.6 Hz, 1H, -CH=CH2).
2-Chloro^t, 4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester





[0|38] Sulfuryl chloride (0.84 ml, 10.4 mmol) was added to a cooled (0°) solution of 4,4-din|ethyl-3-oxo-hex-5-enoic acid ethyl ester (1.83 g, 9.93 mmol) in chloroform (7 ml). The resulfmg mixture was allowed to warm to room temperature and stir for 30 min, after which it ^ras heated under reflux for 2 h. After cooling to room temperature the reaction mixture vifas diluted with chloroform, then was washed with sodium bicarbonate, water then brine. Tii|e organic phase was dried and evaporated to afford, as a brown oil, 2-chloro-4,4-dimethyl-|-oxo-hex-5-enoic acid ethyl ester (2.01 g, 93%). (Hayashi et al., /. Org. Chem., 65:8402-4*05 (2000).
{OX¥} 'H NMR (400 MHz, CDC13) 8 1 .28 (t, J = 7.0 Hz, 3H, -CH2CH3); 1 .33 (s,
3H); 1.34 j(s, 3H); 4.24 (q, /= 7.0 Hz, 2H, -CH2CH3); 5.19 (s, 1H; 5.28 (d, J= 16.9 Hz, 1H, -5.29 (d, J = 10.9 Hz, 1H, -CH=CH2); 5.96 (dd, J = 16.9, 10.9 Hz, 1H, -
LC/MS tR = 8.45 (2 1 9.3 [M(Cl37)+Hf [024 1 ] This material was reacted without further purification. 5-(lJ-Diifyethyl-allyl)~3H-imidazole-4-carboxylic acid ethyl ester




O OH


[0342] A suspension of 2-chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (19.4 g, 0|)9 mol) and water (1.94 ml, 0.11 mol) in formamide (36.8 ml) was shaken briefly, then dispensed into 15 x 18 ml vials. The vials were sealed and heated at 150° for 5 h. After

cooling to room temperature, the vials' contents were combined and extracted e|diaustively
with chloroform. The extracts were dried and evaporated to afford a concentratediformamide
solution (14.7 g). This was added to a silica column (7 cm diameter, 11 cm height).packed in
1% MeOH/1% Et3N in chloroform. Elution of the column with 2 L of this mixture followed
by 2 L of 2% MeOH/1% EtsN in chloroform afforded, in the early fractions, a j compound
suspected of being 5-(l,l-dimethyl-allyl)-oxazole-4-carboxylic acid ethyl ester (1J3 g. 7%).
[0243] HPLC (214nm) tg = 8.68 (50.4%) min. 102441 'H NMR (400 MHz, CDC13) 8 1.40 (t, / = 7.2 Hz, 3H, -CHjC^s); 1.54 (s,
6H); 4.38 (t, J = 7.2 Hz, 2H, -CftCffj); 5.03 (d, J= 17.4 Hz, 1H, -CH=CH2); fj.02 (d, / =
10.4 Hz, 1H, -CH=*CH2); 6.26 (dd, /= 17.4,10.4 Hz, 1H, -CH=CH2)\ 7.83 (s, 1H)|
[0245] LCMS tR = 8.00 (210.1 [M+Hf, 361.1 [2M+HJ*) min. ;
[0246] Recovered from later fractions was the desired 5-(l,l-dJmethyf-allyl)-3H-
imidazole-4-carboxylic acid ethyl ester (3.13 g, 17%). (Hayashi et al., /. O^g. Chem.,
65:8402-8405 (2000)). j
[0247] HPLC (214nm)/* = 5.52 (96.0%) min. {
102481 'H NMR (400 MHz, CDC13) 8 1.38 (t, J = 7.0 Hz, 3H); 1.57 (si 6H); 4.35
(q, J = 7.0 Hz, 2H); 5.04-5.14 (m, 2H, -CH=CH2); 6.28 (dd, J = 18.0, 10.4 Hz, 1H, -
CH=CH2); 7.52 (s, 1H). j
[0249] LC/MS tR = 5.30 (209.1 [M+H]+, 417.2 [2M+H]"1) min. j
[0250] Additional 5-(l,l-dimethyl-allyl)-3H-imidazole-4-carboxylic |cid ethyl ester was also recovered from the column (3.59 g, 19%) which was of lower purity but still
sufficient for further reaction. I [0251] Another byproduct isolated from a similar reaction (smaller scale) by
further elution of the column with 5% MeOH/1% Et3N hi chloroform was a ;ompound
suspected of being 5-(l,l-dimethyl-altyl)-3H-imidazole-4-carboxylic acid (0.27 g, i %).
[0252] HPLC (245nm) ft = 5.14 (68.9%) min. 102531 'H NMR (400 MHz, CD3OD) 6 1.45 (s, 6H); 4.97 (d, J = 10.6; Hz, 1H, -
CH=CH2-); 5.01 (d, J = 17.7 Hz, 1H, -CH=CH2)\ 6.28 (dd, / = 17.7, 10.6 bz, 1H, -
; 7.68 (s, 1H). j
[0254] LCMS tR = 4.72 (181.0 [M+H]+, 361.1 pM+H]"*) min.
[5~(lJ-D^nethyl-allyl)-3H-imidazol-4-yl]-methanol

OH


N=
[0|55] A solution of 5-(l,l-dimethyl-allyl)-3H-imidazole-4-carboxylic acid ethyl ester (3.13 g, 15.0 mmol) in THF (60 ml) was added dropwise to a suspension of lithium aluminium hydride (95% suspension, 1.00 g, 25.0 mmol) in THF (40 ml) and the mixture was stirred at room temperature for 4 h. Water was added until the evolution of gas ceased, the mixture was stirred for 10 min, then was filtered through a sintered funnel. The precipitate! was washed with THF, then with methanol, the filtrate and washings were combined^ evaporated, then freeze-dried to afford [5-(l,l-dimethyl-allyl)-3H-unidazol-4-yl]-methanol |2.56 g, 102%). Residual water was removed by azeotroping with chloroform prior to further fraction. (See Hayashi et al, J. Org. Chem., 65:8402-8405 (2000)).
[0$S6] HPLC (240nm) ft = 3.94 (56.8%) min.
•"f71 'H NMR (400 MHz, CD3OD) 5 1.43 (s, 6H); 4.57 (s, 2H); 5.01 (d, J =
10.5 Hz, ^H, -CH*=CH2y, 5.03 (d, J= 17.7 Hz, 1H, -Cff=Cft); 6.10 (dd, J = 17.7, 10.5 Hz, 1H, -C/NC26); 7.46 (s, 1H).
[Oi58] LC/MS tK = 3.77 (167.3 [M+H]4) min. 5-(l, l-DiTfyethyl-aUyl)-3H-imidazole-4-carbaldehyde
OH />
MnO,

[0|59] Manganese dioxide (20 g, 0.23 mol) was added to a solution of [5-(l,l-dimethyl-|llyl)-3H-imidazol-4-yl]-methanol (2.56 g, 0.02 mol) in acetone (300 ml) and the resulting fixture was stirred at room temperature for 5 h. The mixture was filtered through filter pap|r and the residue was washed with acetone. The filtrate and washings were combined land evaporated to afford 5-(l,l-dimethyl-allyl)-3H-imidazole-4~carbaldehyde (1.82 g, 51%). fHayashi etal., J. Org. Chem., 65:8402-8405 (2000)).
[0|60] HPLC (240nm) fc = 4.08 (91.5%) min.

!026IJ 'H NMR (400 MHz, CDC13) S 1.56 (s, 6H); 5.16 (d, /= 10.6JHz, 1H,
CH=CH2); 5.19 (d, J = 17.3.Hz, 1H, CH=CH2); 6.22 (dd, /- 17.3, 10.6 Hz, 1H, -jaf-7.75 (s, 1H), 10.02 (s, JH, HCO).
[0262] LC/MS tR = 3.75 (165.2 [M+Hft min.
l-Acetyl-3-[5 '-(l,l-dimethyl-allyl)-lH-imidazol-4 '-Z-ylmethylene]-piperazine-2,5-$ione


o
'/


[0263] To a solution of 5-(l,l-dimethyl-allyl)-3H-imidazole-4-carbalde|yde (1.78
g, 0.01 mol) in DMF (35 ml) was added l,4-diacetyl-piperazine-2,5-dione (8.59 g,jo.04 mol)
and the mixture was evacuated, then flushed with argon. The evacuation-flushuf g process
was repeated a further two times, then cesium carbonate (3.53 g, 0.01 mol) was ac|ded. The
evacuation-flushing process was repeated a further three times, then the resultant mixture was
heated at 45° for 5 h. The reaction mixture was partially evaporated (heating ijjnder high
vacuum) until a small volume remained and the resultant solution was added dropwjise to ice-
water (50 ml). The yellow precipitate was collected, washed with water, then free4e-dried to
afford l-acetyl-3-[5'-(l,l-dimethyl-allyl)-lH-imidazol-4'-ylmethylene]-piperazinef2,5-dione
(1.18g, 36%). (Hayashi, Personal Communication (2001)). ^
[0264} HPLC(214nm)to = 6.01 (72.6%) min.
102651 'H NMR (400 MHz, CDC13) 8 1.53 (s, 6H); 2.64 (s, 3H); 4.4J7 (s, 2H);
5.19 (d, /= 17.3 Hz, 1H, -CH=CH2); 5.23 (d, /= 10.7 Hz, 1H, -CH^CHfr 6.0^ (dd, J = 17.3, 10.7 Hz, 1H, -Cff=C#2); 7.16 (s, 1H), 7.59 (s, 1H), 9.47 (bs, 1H); 12. H (bs, 1H) [observed -2% l,4-diacetyl-piperazine-2,5-dione contamination 8 2.59 (s, 6H); 4.6(j| (s, 4H).]
[0266] LC/MS fo = 6.65 (303.3 [M+tff , 605.5 [2M+H]*) min. (n.b( different
system used). '

3-Z-Ber^^ene-6-f5"-(lJ-dimethylanyl)-m4midazol-4"-Z-ylmethyleneJ-piperazine-2,5-dione



o y^^ . o
[Oi67] To a solution of l-acetyl-3-[5Xl>dimemyl-allyl)-lH-imidazol-4'-ylmethyle^e]-piperazine-2,5-dione (2.91 g, 9.62 mmol) in DMF (70 ml) was added benzaldeh^de (4.89 ml, 48.1 mmol) and the solution was evacuated, then flushed with Argon. The evacuation-flushing process was repeated a further two times, then cesium carbonate (4.70 g, 1^4 mmol) was added. The evacuation-flushing process was repeated a further three times, the^ the resultant mixture was heated under the temperature gradient ad shown below.
[0368] After a total time of 5 h the reaction was allowed to cool to room temperature and the mixture was added to ice-cold water (500 ml). The precipitate was collected, gashed with water (300 ml), then freeze-dried to afford a yellow solid (2.80 g). This material was dissolved in chloroform (250 ml) filtered through filter paper and evaporate^ to azeotrope remaining water. The residual yellow precipitate (2.70 g, HPLC (214nm) |R = 7.26 (93.6%) min.) was partially dissolved in chloroform (20 ml), the suspension was sonicated for 5 min, then the solid was collected and air dried to afford 3-Z-benzylidei|e-6-[5"-(l,l-dimemylallyl)-l^-irmdazol-4"-Z-ylmethylene]-piperazine-2,5-dione (1.82 g, 54%) (Hayashi, Personal Communication (2001)), m.p. 239-240° (dec.).
(0^69] HPLC (214nm)to = 6.80 (1.92) min, 7.33 (95.01%).
'H NMR (400 MHz, CDC13) 5 1.53 (s, 6H); 5.18 (d, J= 17.6 Hz, 1H, -5.21 (d, J = 11.0 Hz, 1H, -CH=CH2); 6.06 (dd, J = 17.6, 11.0 Hz, 1H, -6.99 (s, 1H, -C-C=CH); 7.00 (s, 1H, -C-C=C#); 7.30-7.50 (m, 5 x ArH); 7.60 (s, H2"); 8.01 (bs, NH); 9.31 (bs, NH); 12.30 (bs, NH).
(0271] LC/MS tR = 6.22 (349.3 [M+H]*, E isomer), 6.73 (349.5 [M+Hf, 697.4 [2M+H]+, JZ isomer) min.
[0172] ESMS m/z 349.5 [M+H]+, 390.3 [M+CRtCN]"1".

[0273] Evaporation of the chloroform solution gave additional 3TZ-ben^ylidene-6-[5"-( 1,1-dimethylaUyl)-1/f-imidazoM
[0274] HPLC (214nm)to = 7.29 (84.5%) miu. j
3-E-Benzylidene-6-[5"-(l, 1 -dime%lallyl)-lH-imidazol-4"-Z-ylraethylene]-piper4zine-2,5-
dione




o


[0275] Preparative HPLC purification of a crude sample of material sjynthesized
as above afforded the geometric isomer 3-E-Berizylidene-6-[5"-(l,l-dimethyJallyl)-lH-
imidazol-4"-Z-ylmethylene]-piperazine-2,5-dione (1.7 mg). «
[0276] HPLC (214nm) tR = 6.75 (87.79) min.
102771 'H NMR (400 MHz, CDCb) 6 1.52 (s, 6H); 5.19 (d, J = 20j8 Hz, IH,
CH=CH2); 5.22 (d, J= 14.0 Hz, IH, CH=CH2); 6.05 (dd, J= 18.0, 10.4 Hz, IH, pH=CH2); 6.33 (s, IH, C-C=CH); 6.90-7.65 (m, 7H).
[0278] ESMS m/z 349.5 [M+H]'1", 390.4
62.

Synthesis I of 3-Z-Benzylidene-6-(5"-tert-butyl-lH-imidazol-4"-Z-ylmethylene)-pipera2ine-2,5-dione JJ2)
o o



O 0

.OH




o o.

[02f79] Reagents: g) SO2C12; h) HaNCHO, H2O; I)LiAlH4; j) MnO2; k) 1,4-diacetyl-p^3erazine-2,5-dione, Cs2CO3j 1) benzaldehyde,
2-Chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester


o

ci

[02J80] Sulfuryl chloride (14.0 ml, 0.17 mol) was added to a cooled (0°) solution of ethyl pfvaloylacetate (27.17 g, 0.16 mol) in chloroform (100 ml). The resulting mixture was allowed to warm to room temperature and was stirred for 30 min, after which it was heated under reflux for 2.5 h. After cooling to room temperature, the reaction mixture was diluted wvfh chloroform, then washed with sodium bicarbonate, water then brine.

[0281] The organic phase was dried and evaporated to afford, as a c|ear oil, 2-
chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (33.1 g, 102%). (Duifmt et al.,
"Aminoalkylimidazoles and Process for their Production." Patent No. GB 1341^75 (Great
Britain, 1973)). [0282] HPLC (214nm) tK = 8.80 (92.9%) min.
102831 'H NMR (400 MHz, CDC13) 8 1.27 (s, 9H); 1.29 (t, / « 7.2 H^ 3H); 4.27
(q, J= 7.2 Hz, 2H); 5.22 (s, 1H). »
102841 13C NMR (100 MHz, CDC13) 8 13.8,26.3, 45.1, 54.5, 62.9, 165J1,203.6.
1;
5-tert-Butyl-3H-imidazole-4-carboxylic acid ethyl ester i




o


°VOH

[0285] A solution of 2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl jester (25.0 g, 0.12 mol) in formamide (47.5 ml) and water (2.5 ml) was shaken, then dispensed into 15 x 8 ml vials. All vials were sealed and then heated at 150° for 3.5 h. The vials werejallowed to cool to room temperature, then water (20 ml) was added and the mixture was exhaustively extracted with chloroform. The chloroform was removed to give a concentrated formamide solution (22.2 g) which was added to a flash silica column (6 cm diameter, 12 fan height) packed in 1% MeOH/1% EtsN in chloroform. Elution of the column with 2 J L of this mixture followed by 1 L of 2% MeOH/1% Et3N in chloroform gave, in the early factions, a product suspected of being 5-tert-butyl-oxazole-4-carboxylic acid ethyl ester (6.3 g, 26%).
[0286] HPLC (214nm) fe - 8.77 min. 102871 'H NMR (400 MHz, CDC13) 8 1.41 (t, J = 7.2 Hz, 3H); 1.43 (sj 9H); 4.40
(q, J = 7.2 Hz, 2H); 7.81 (s, 1H). i
102881 13C ^y^ (10Q MRz> CDC13) 8 14.1, 28.8, 32.5, 61.3, 136.9, 14^.9, 156.4,
158.3. (
[0289] ESMSm/zl98.3 [M+Hf, 239.3 [M+CH4CN]+. ]
[0290] LC/MS ts = 7.97 (198.1 [M+H]*) min.

[0391J Recovered from later fractions was 5-tert-butyl-3H-imidazole-4-carboxyJic acid ethyl lester (6.20 g, 26%). (Durant et al, "Aminoalkylimidazoles and Process for their Productio4." Patent No. GB1341375 (Great Britain, 1973)).
[02f92] HPLC (214nm) t» = 5.41 (93.7%) min.
IOMN 'H NMR (400 MHz, CDC13) 8 1.38 (t, /== 7.0 Hz, 3H); 1 .47 (s, 9H); 4.36
(q,V= 7.2fHz, 2H); 7.54 (s, 1H).
toz^I I3C NMR (100 MHz> CDC13) 8 13.7, 28.8, 32.0, 59.8, 124.2, 133.3, 149.2,
162.6.
[02J95] ESMS m/z 197.3 [M+H]+, 238.3 [M+C^CN]"1".
[03196] Further elution of the column with 1L of 5% MeOh/1% Et3N gave a compound suspected of being 5-tert-butyl-3H-imidazole-4-carboxylic acid (0.50 g, 2%).
[02^7] HPLC (245nm) tR = 4.68 (83.1%) min.
'H NMR (400 MHz, CD3OD) 8 1.36 (s, 9H); 7.69 (s, 1H). 'H NMR (400 MHz, CDC13) 8 1 .37 (s, 9H); 7.74 (s, 1 H). 'HNMR (400 MHz, CD3SO) 8 1.28 (s, 9H); 7.68 (s, 1H).
[03^)1] ESMS m/z 1692 [M+Hf , 210.4 [M+CH4CNf .


NH
(5-tert-Butyl-3H-imidazol-4-yl)-methanol

UAIH4

[0302] A solution of 5-tert-butyl-3-imidazole-4-carboxylic acid ethyl ester (3.30 g, 16.8 mjnol) in THF (60 ml) was added dropwise to a suspension of lithium aluminium hydride (9J5% suspension, 0.89 g, 22.2 mmol) in THF (40 ml) and the mixture was stirred at room temf erature for 3 h. Water was added until the evolution of gas ceased, the mixture was stirref for 10 min, then was filtered through a sintered funnel. The precipitate was washed v|th THF, then with methanol, the filtrate and washings were combined and evaporated. The residue was freeze-dried overnight to afford, as a white solid (5-tert-butyl-

3H-imidazol-4-yl)-methanoI (2.71 g, 105%). (Durant et al., "Aminoalkylimid^oles and Process for their Production." Patent No. GB1341375 (Great Britain, 1973)).
[0303] HPLC(240nm)^ = 3.70 (67.4%) min.
103041 'H NMR (400 MHz, CD3OD) 8 1.36 (s, 9H); 4.62 (s, 2H); 7.43 (|, 1H).
|03°* • 13C NMR (100 MHz, CD3OD) 831.1, 33.0, 57.9, 131.4, 133.9, 1^0.8.
[0306] LC/MS b - 3.41 (155.2 [M+H]4) min.
[0307] This material was used without further purification. 5-tert-Butyl-3H-imidazole-4-carbaldehyde

.OH
MnO2
^
\ r"
N=/ N=/ ,
[0308] Manganese dioxide (30 g, 0.35 mol) was added to a hetefogeneous
solution of (5-tert-butyl-3H-imidazol-4-yl)-methanol (4.97 g, 0.03 mol) in acetonq (700 ml)
and the resulting mixture was stirred at room temperature for 4 h. The mixture wps filtered
through a pad of Celite and the pad was washed with acetone. The filtrate and wasttings were
combined and evaporated. The residue was triturated with ether to afford, as a colorless
solid, 5-tert-butylr3H-imidazole-4-carbaldehyde (2.50 g, 51%). (Hayashi, j Personal
Communication (2000)). i
[0309] HPLC (240nm) ts = 3.71 (89.3%) min.
[03101 1H NMR (400 MHz, CDC13) 8 1.48 (s, 9H); 7.67 (s, 1H); 10.06 0,1H).
[0311] LC/MS tR = 3.38 (153.2 [M+H]=) min.
[0312] Evaporation of the filtrate from the trituration gave additional 5-|tert-butyl-3H-imidazole-4-carbaldehyde (1.88 g, 38%).



l-Acetyl-3\(5 '-tert-butyl-lH-imdazol-4 '-Z-ylmethylene)-piperazine-2,5-dione
O
O O
[03J13] To a solution of 5-tert-butyl-3#-irmdazole-4-carbaldehyde (2.50 g, 164.4 mmol) in |j)MF (50 ml) was added l,4-diacetyl-piperazine-2,5-dione (6.50 g, 32.8 mmol) and the solution was evacuated, then flushed with argon. The evacuation-flushing process was repeated a!further two times, then cesium carbonate (5.35 g, 16.4 mmol) was added. The evacuation-flushing process was repeated a further three times, then the resultant mixture was stirred at rjbom temperature for 5 h. The reaction mixture was partially evaporated (heat and high vacuiim) until a small volume remained and the resultant solution was added dropwise to water (|00 ml). The yellow precipitate was collected, then freeze-dried to afford 1-acetyl-3-(5'-tert-l|utyl-lH-imidazol-4'-Z-yhnethylene)-piperazine-2,5-dione (2.24 g, 47%). (Hayashi, Personal Communication (2000)).
[03JM] HPLC (214nm) ** = 5.54 (94.4%) min.
r°31^ 'H NMR (400 MHz, CDC13) 5 1.47 (s, 9H); 2.65 (s, 3H), 4.47 (s, 2H);
7.19 (s, 1HJ); 7.57 (s, 1H), 9.26 (s, 1H), 12.14 (s, 1H).
t031^ 13C NMR (100 MHz, CDC13+CD3OD) 5 27.3, 30.8, 32.1, 46.5, 110.0,
123.2, 131j4,1332, 141.7, 160.7,162.8,173.0
[03JI7] LC/MS tR = 5.16 (291.2 [M+Hf, 581.6 [2M+H]*) min. 3-Z-Benzytodene-6-(5 "-tert-butyl-lH-imidazol-4 "-Z-ylmethylene)-piperazine-2,5-dione




O


[0^18] To a solution of l-acetyl-3-(5'-tert-butyl-lH-imidazol-4'-Z-ylmethylene)-piperazine|2,5-dione (2.43 g, 8.37 mmol) in DMF (55 ml) was added benzaldehyde (4.26 ml, 41.9 mmoj) and the solution was evacuated, then flushed with nitrogen. The evacuation-

flushing process was repeated a further two times, then cesium carbonate (4.09 g, |2.6 mmol) was added. The evacuation-flushing process was repeated a further three times, then the resultant mixture was heated under the temperature gradient as shown below. After a total time of 5 h the reaction was allowed to cool to room temperature and the mixture {was added to ice-cold water (400 ml). The precipitate was collected, washed with water, then freeze-dried to afford a yellow solid (2.57 g, HPLC (214nm) fc = 6.83 (83.1%) min.). Th|s material was dissolved in chlorofonn (100 ml) and evaporated to azeotrope remaining watei, resulting in a brown oil. This was dissolved in chloroform (20 ml) and cooled in ice. After |>0 min the yellow precipitate was collected and air-dried to afford 3-Z-benzylidene-6-(5'-tertJbutyl-l/f-imidazol-4"-Z-ylmethylene)-piperazine-2,5-dione (1.59 g, 56%). (Hayashi,* Personal Communication (2000)).
[03191 HPLC (214nm) fe = 6.38 (2.1%), 6.80 (95.2) min,
|0320) 'H NMR (400 MHz, CDC13) 8 1.46 (s, 9H); 7.01 (s, 1H, -C-C=fH); 7.03
(s, 1H, -C-C=C#); 7.30-7.50 (m, 5H, Ar); 7.60 (s, 1H); 8.09 (bs, NH); 9.51 (bs, 1|H); 12.40 (bs, NH).
[0321] LC/MS b - 5.84 (337.4 [M+H]+, E isomer), 6.25 (337.4 [M+|jf, 673.4
[2M+H]*, Z isomer) min. .
[0322] ESMS m/z 337.3 [M+H]+, 378.1 [M+CHiCNf. [
[0323] Evaporation of the chloroform solution gave additional 3-Z-benzylidene-6-
(5"-tert-butyl-lJfir-hnidazol-4"-Z-ylmethylene)-piperazine-2,5-dione (0.82 g, 29%|. HPLC
(214nm) tR = 6.82 (70.6%) min. . I
General Experimental
[0324] Sodium bicarbonate refers to a 5% solution. [0325] Organic solvents were dried over sodium sulfate unless otherwise stated.
Analytical Conditions
t
NMR Conditions
103261 'H NMR (400 MHz) analysis was performed on a Varian Inova |jnity 400 MHz NMR machine. Samples were run in deuterated chloroform containing 0|1% TMS (unless otherwise specified). Chemical shifts (ppm) are referenced relative to IMS (0.00

ppm) or CfeOH at 3.30 ppm for samples run CDsOD. Coupling constants are expressed in
hertz (Hz)|
Analytical HPLC Conditions
[03|27J System 6 conditions:
[02J28] RP-HPLC was done on a Rainin Microsorb-MV CIS (5 urn, 100A) 50 x 4.6 mm column.
[03f29J Buffer A: 0.1% aqueous TFA
[0430] Buffer B: 0.1% TFA in 90% aqueous MeCN
[03JJ311 Gradient: 0 -100% Buffer B over 11 min
[03J32J Flow rate: 1.5mL/min LCMS Conditions
[0333] LCMS were run on a Perkin-Elmer Sciex API-100 instrument.
[0334] LC conditions:
[03;35] Reverse Phase HPLC analysis
[03|36) Column: Monitor 5 |om CIS 50x4.6 mm
[03J37] Solvent A: 0.1% TFA in water
[0338] Solvent B: 0.085% TFA in 90% aqueous MeCN
[03(39] Gradient: 0-100% B over 11.0 min
[0340] Flow rate: 1.5mL/min
[03>41] Wavelength: 214nm
[02|42] MS conditions:
[03)43] Ion Source: lonspray
[0344] Detection: Ion counting
[03J45] Flow rate to the mass spectrometer: 300 uL/min after split from column (1|5 mL/min). ESMS Conditions
[0346] ESMS was done on a Perkin Elmer/Sciex-API m LC/MS/MS using an electrospr4y inlet.
[02(47] Solvent: 0.1% AcOH in 60% aqueous MeCN
[0348] Flow rate: 25 uL/min

(0349] lonspray: 5000V
[0350] Orifice plate: 55V
[0351] Acquisition time: 2.30 min
[0352] Scan range: 100-1000 amu/z
[0353] Scan step size: 0.2 amu/z Preparative RP-HPLC Purification Conditions
[0354] Reverse phase HPLC purification was carried out using Nebullt with the Waters XterraMS column (19x50 mm, 5 |itm, CIS) using the following conditions:
[0355] Solvent A: 0.1% aqueous TFA
[0356] Solvent B: 0.1% TFA in 90% aqueous MeCN
[0357] Gradient: 5-95% B over 4 min
[0358] Flow rate: 20 mL/min
[0359] Wavelength: 214 ran
[0360] Abbreviations are as follows: br s: broad singlet; BuLi: n-butyl lithium; d:
doublet; DBU: l,8-diazabicyclo[5.4.0]undec-7-ene; ESMS: electrospray mass spebtrometry,
HCI: hydrochloric acid; HPLC: high performance liquid chromatography; LCMS: liquid
chromatography mass spectrometry; LD: lithium diisopropylamide; M+: moleculkr ion; m:
multiplet; MeCN: acetonitrile; M: mass spectrometry, MW: molecular weight; NMji: nuclear
magnetic resonance; q: quartet; s: singlet; : triplet; IR: retention time; TFA: trifljioroacetic
acid; THF: tetrahydrofuran ' (
Detailed Procedure for the Synthesis ofDehydrophenylahistin




0 CS2C03/DMF
1

_L_>
Cs2CO3/DMF

l-Acetyl-4{(^0-l-[5-(l,l-dimethyl-2-propenyl)-lfl-4-imidazolyl]methylidene}]-2,5-piperazin^dione (2)

[03|i>l] To a solution of 5-(l,l-dimethyl-2-propenyl)imidazole-4-carboxaldehyde (100 mg, 01609 mmol) in DMF (2 mL) was added compound 1 (241 mg, 1.22 mmol) and the solution wfts repeatedly evacuated in a short time to remove oxygen and flushed with AT, followed l}y the addition of CsaCOs (198 mg. 0.609 mmol) and the evacuation-flushing process w*s repeated again. The removal of oxygen is preferred because such removal is believed toi decrease oxidation of alpha-carbon at the position 6 of the diketopiperazine ring. The resultant mixture was stirred for 5 h at room temperature. After the solvent was removed by evaporation, the residue was dissolved in the mixture of EtOAc and 10% Na2CO3, and the organic phpe was washed with 10% Na2COs again and saturated NaCl for three times, dried over Naa(fO3 and concentrated in vacuo. The residual oil was purified by column chromatoglaphy on silica using CHCh-MeOH (100:0 to 50:1) as an eluant to give 60 mg (33%) of afpale yellow solid 2. Dehydroplienylahistin
[0^62] To a solution of 2 (30 mg, 0.099 mmol) in DMF (0.8 mL) was added benzaldelr|de (51 \iL, 0.496 mmol, 5 eq) and the solution was repeatedly evacuated in a short time to remove oxygen and flushed with Ar, followed by the addition of CsaCOs (53 mg, 0.149 mm$>l, 1.5 eq) and the evacuation-flushing process was repeated again. The resultant mixture wfes heated for 2.5 h at 80°C. (The temperature must be increased slowly. Rapid heating increases the production of E-isomer at the benzylidene.moiety.) After the solvent was renwvijed by evaporation, the residue was dissolved in EtOAc, washed with water for two times and isaturated NaCl for three times, dried over NaaSCU and concentrated in vacuo. On TLC using CHCU-MeOH (10:1), you can observe a spot with bright green-yellow luminescence at 365 nm UV. The purity of this crude product was more than 75% from

HPLC analysis. The resulting residue was dissolved in 90% MeOH aq and {applied to reverse-phase HPLC column (YMC-Pack, ODS-AM, 20 x 250 mm) and eluted usi tig a linear gradient from 70 to 74% MeOH in water over 16 min at a flow rate of 12 mL/m n, and the desired fraction,was collected and concentrated by evaporation to give a 19.7 ing (60%), although the yields are not optimized for each step,, of yellow colored dehydrophei ylahistin.
EXAMPLE 4 j
Biological Characteristics of Dehvdrophenylahistin and Dehvdrophenvlahistin Analogs
A. Biological Evaluation I
[03631 The biological characteristics of synthesized tBu-dehydrophinylahistin and dehydrophenylahistin were evaluated in both HT29 human colon cells, land PC-3 prostatic adenocarcinoma cells.
[0364] HT-29 (ATCC HTB-38) a human colorectal adenocarcii|oma was maintained in McCoy's complete medium (McCoy's 5A medium with L-glutamine and 25mM HEPES supplemented with 10% FBS, ImM Na pyruvate, IX NEAA| 2mM L-
glutamine, and Pen/Strep at lOOIU/ml and 100ng/ml, respectively). PC-3 .(ATJJCC CRL-
t
1435), a human prostate adenocarcinoma, was maintained in F12K complete medium (F12K medium supplemented with 10% FBS; 2mM Glutamine; 1% HEPES; and Pe|i/Strep at
lOOIU/ml and lOO^ig/ml, respectively). Cell lines were cultured at 37 °C, 5% CC)| in a 95%
i humidified incubator.
[0365] For tumor cytotoxicity assays HT-29 or PC-3 cells were seedej^ at 5,000
cells/well in 90 ul complete media into a Corning 3904 black-walled, clear-bot|om tissue
culture plate and the plate were incubated overnight to allow cells to establish an phase growth. 20 mM stock solutions of dehydrophenylahistin and tBu-dehydrophanylahistin
were prepared in 100% DMSO and stored at -20 °C. 10X concentrated serial dilutions of the
two compounds were prepared in appropriate culture medium for final concentrations ranging
from 2.0 x 10"5 M to 2.0 x 10"'° M. Ten jil volumes of the 10X serial dilutions werjb added to
the test wells in triplicate and the plates returned to the incubator for 48 hours. JThe final
concentration of DMSO was 0.25% in all samples. I
[0366] Following 48 hours of drug exposure 10 p.1 of 0.2 mg/ml j resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg2+, Ca2+ free PBS was added tof each well
"72-

and the plaftes were returned to the incubator for 3-4 hours. The plates were removed and resazurin fluorescence was measured using 530 nm excitation and 590 nm emission filters in a Fusion fiuorimeter (Packard Instruments). Resazurin dye without cells was used to determine |he background, which was subtracted from the data for all experimental wells. The data were analyzed using Prism software (GraphPad Software). The data were normalized! to the average of the cells treated with media only (100% cell growth) and ECso values wer| determined using a standard sigmoidal dose response curve fitting algorithm.
[03f7] As indicated in Table 1 below, tBu-dehydrophenylahistin demonstrates about a 4-t|mes greater cytotoxic activity in comparison with dehydrophenylahistin. Table 1. Cytotoxic Effect of dehydrophenylahistin and derivative.

O


Dehydrophenylahistin

tBu-dehydrophenylahlstin


EC50 (nM)
cell APLH tBu-APLH
HT29 48 13
1 PC-3 [0368] See also Figure 41 for additional data at HT-29, PC-3, and P-388 cells. B. Structure and Activity Study of Dehvdrophenvlabistin Derivatives
[03|69] The cytotoxic effects of phenylahistin, dehydrophenylahistin and various derivatives! of dehydrophenylahistin were examined in P388 murine leukemia cells, HT-29 human cot>n cells, and PC-3 prostatic adenocarcinoma cells.
[($70] As explained above, HT-29 a human colorectal adenocarcinoma was maintained in McCoys complete medium (McCoys 5A medium with L-glutamine and 25mM HJPES supplemented with 10% FBS, ImM Na pyruvate, IX NEAA, 2mM L-glutamine| and Pen/Strep at lOOIU/ml and 100(J.g/ml, respectively). PC-3, a human prostate

adenocaroinoma, was maintained in F12K complete medium .(F12K medium supplemented with 10% FBS; 2mM Glutamine; 1% HEPES; and Pen/Strep at lOOIU/ml and J100ng/ml, respectively). Cell lines were cultured at 37 °C, 5% CO2 in a 95% humidified incubator.
[03711 For tumor cytotoxicity assays HT-29 or PC-3 cells were seedefl at 5,000 cellsAvell in 90 }d complete media into a Corning 3904 black-walled, clear-bot|om tissue culture plates and the plates were incubated overnight to allow cells to establish an|l enter log phase growth. 20 mM stock solutions of dehydrophenylahistin and tBu-dehydropr4nylahistin were prepared in 100% DMSO and stored at -20 °C. 10X concentrated serial dilutions of the two compounds were prepared in appropriate culture medium for final concentrations ranging from 2.0 x 10"5 M to 2.0 x 10~10 M. Ten ul volumes of the 10X serial dilutions we^e added to
the test wells in triplicate and the plates returned to the incubator for 48 hours. The final
concentration of DMSO was 0.25% in all samples.
[0372] Following 48 hours of drug exposure lOul of 0.2 mg/mlj resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg2+, Ca2+ free PBS was added tcj each well and the plates were returned to the incubator for 3-4 hours. The plates were removed and resazurin fluorescence was measured using 530 nm excitation and 590 nm emissicjjn filters in a Fusion fluorimeter (Packard Instruments). Resazurin dye without cells was used to determine the background, which was subtracted from the data for all experimental wells. The data were analyzed using Prism software (GraphPad Software). The ijdata were normalized to the average of the cells treated with media only (100% cell growthf and ECso values were determined using a standard sigmoidal dose response curve fitting algorithm.
[0373] ECso and ICso values of phenylahistin, dehydrophenylafistin and dehydrophenylahistin derivatives are summarized in Table 2 below.

Table 2. SAR study of phenvlahistin or dehvdrophenylahlstin and of dehvdrophenvlahistin derivatives




PC-3
COMPOUNDS
(-)-Phen^ahistiBi

STRUCTURE

EC50 (nM)
HT-29
n.t.
1600

ICso(nM)
P-388
833 ±153 (n=5)




0
KPf-1

48

4.7

36+12.8 (n=5)




KPlr-2

13

31.8 + 5.0 (n=5)




KP-6 tBu-APL»-p-OMe

>2000

n.t.

9333 ± 5457 (n=3)




OMe
KP-8 tBu-APLt-o-OMe

89

315 ±137 (n=4)




KPJJ-9 : tBu-APLlJ-M-OMe

31

20.8 + 68 (n=4)



Colc^icine

208±68 (n=4)

[0374] Modifications to the phenyl ring have a significant effect of th| cytotoxic activities. In comparison with the activity of tBu-dehydrophenylahistin (#6), the jactivity of the methoxy group at the meta-position (KPU-9) exhibited the highest activity tha^ the other derivatives with an JCjo of 20.&B.3 nM.in P388 cells. The KPU-9 derivative als4 exhibited
|
cytotoxicity in HT-29 cells (EC5o 31 nM). Dehydrophenylahistin, tBu-dehydroph|nylahis1in
(KPU-2) and the KPU-9 derivative all exhibited cytotoxicity in P388 cells. j
C. Structure and Activity Study of Additional Dehvdrophenvlahistin Derivativij
[0375] The cytotoxic effects of phenylahistin, dehydrophenylahistin aid various additional derivatives of dehydrophenylahistin were examined in HT-29 human oolon cells and PC-3 prostatic adenocarcinoma cells using the methodology described above. •
Table 3. SAR study of phenylahistin. dehvdropbenvlahistin and of additional
dehvdrophenvlahistin derivatives !





n.t
16
COMPOUNDS
(-)-Phenylahistin

STRUCTURE

SALT FORM

M.W.
350.41




KPU-1 APLH

348.40

4.7




KPU-2 tBu-APLH

336.39

13




KPU-6 tBu-APLH-p-OMe

366.41

n.t.


OMe
MPO
COMPUNDS
KPf-8 tBu-APLlf-o-OMe

STRUCTURE

SALT FORM

M.W.
366.41

HT-29 PC-3
89




KPTt-9 tBu-APLEJ-m-OMe

366.41

31




OMe
KPTJ-14
tBu-APllH-2,3-dioile

TFA

396.44 510.46 (-fTFA)

610

96%



KPUf-12
tBu-APlk-2,4-
diO*e

OMe

396.44

4980




OMe
OMe
KPll-10
tBu-APHH-2,5-
diOMe

396.44

1350




OMe
MeO
KPUf-15
tBu-APljH-2,6-
diOliie

TFA

396.44 510.46 (+TFA)

4430

96%




KPUM3
tBu-APlk-3,4-
diOMe

396.44

2130

11


Me
COMPOUNDS
KPU-16
tBu-APLH-3,5-
diOMe

STRUCTURE

SALT FORM

M.W.
396.44

HT-
4;

>(nM)
PC-3
82%




OMe
KPU-11
tfiu-APLH-3,4,5-triOMe

426.47




OMe
KPU-17
tBu-APLH-2,3,4-triOMe

TFA

426.47 540.49 (+TFA)

40^0

94%




KPU-18 tBu-APLH-o-Cl

TFA

370.83 484.86 (+TFA)

44

100




KPU-19 tBu-APLH-m-Cl

TFA

370.83 484.86 (+TFA)

98%




KPU-20 tBu-APLH-p-Cl

TFA

370.83 484.86 (+TFA)




KPU-21
tBu-APLH-2Cl-5-NO2

TFA

415.83 529.85 (+TFA)

5

100


COMPcluNDS
KPUl-22
tBu-APlk-3,4-
methylenp-dioxy

STRUCTURE

SALT FORM
TFA

M.W.
380.40 494.42 (+TFA)

HT-29 PC-3
82
95%




O
KHJ|23 tBu-APLH-2-OH-
3-Ofle (o-vaniHin)

TFA

382.41 496.44 (+TFA)

5870

86%




OMe
KRjf-24
tBu-A|>LH-
cyclized-|3-MeO

TFA

364.40 487.42 (+TFA)

7040

100




KPl|-25
tBu-AI^H-4-
pyri|iyl

TFA

337.38 565.42 (+2TFA

544

98%




KPUJ-28
tBu-Al|LH-2-
pyrijlyl

TFA

337.38
565.42
(+2TFA

>20 MM

99%




KPl-26 tBu-APLW-2-furyl

TFA

326.35 440.37 (+TFA)

600

88%




KPl|-27
tBu-APL|l-5-Me-2-th^snyl

TFA

356.44 470.47 (+TFA)

80

97%



r29 PC-3
4
COMPOUNDS
KPU-29
tBu-APLH-3-Me-2-thienyl

STRUCTURE

SALT FORM
TFA

M.W.
356.44 470.47 (+TFA)

81%

[0376] Additional cytotoxicity assays were performed as described alfove under
this example, using Resazurin fluorescence as an indicator of cell viability. The results are
shown below in Table 3.1. ',
Table 3.1. Study of henvlahistin. dehvdrohenylahistin and of additional
dehydrophenylahistin derivatives
NPI Structure Chemical name ECso(M) |% cytotox n
Mean SD pMean SD
2350 0^ (-HS)-Halimide, (-)-(S)-Phenylahistin, (-)-(S)-PLH 3.94E-07 9.49E-08 | 87 2 5
2352 0 Dehydrophenylahistin, delta-PLH, KPU-1 4.26E-08 1.08E-08 86 2 5
2354 °^s§> KPU-4, D,L-bislactim-PLH >2E-05 NA 4 NA 2
2355 cn^ KPU-5, D,L-monolactim-PLH >2E-05 . NA 13 NA 2
2356 I^Yr^w N-\ KPU-6 6.57E-06 NA ! 63 NA 2
2357 V KPU-7 8.25E-06 NA I 63 NA 2
2358 O^ «LxJp« t-butyl-delta-PLH, KPU-2 1.49E-08 3.77E-09 I 86 2 5
'ft.

10

















-a
.
T3 7s
TO
Is)
03 i.

4>-6-05
CB
S. 01

1!
C)55 fl
(D

W^
a>
\v
?

10^. Ol
or
I
en

;!
ls>i.
r
is
CD
s
O9

I
Fo

09

7s
TJ

03
00

co

co

CO

IV)

2371 O ClH/*"? "f'rVS H"^^Y"H ^fH>C'££HI ° Nc^ KPU-21, tBu-delta-PLH-2-CI-5-NO2 5.34E-08 4.85E-09 i I 86 3 3
2372 /•"•N HN'r*^Tf^!i'o\ KPU-22, tBu-delta-Pm-3,4-methylene-dtoxy 8.73E-08 1.10E-08 !i 84 4 3
2373 I 1"/=»N HH'^^f^f^»f'M*™^OU« XJ >v% l KPU-23, tBu-delta-PLH-2-OH-3-OMe (o-vanillin) 5.36E-06 NA | 79 NA 2
2374 I M./*y "V'rY^i V^Sr ~~V ^te ** KPU-24, tBu-delta-PLH-cyclized-3-MeO 6.92E-06 MA 1 58 NA 2
2375 i/«»N HtfW**)"VkA^ U* H-°te 5 KPU-25, tBu-defta-PLH-4-pyridyl 5.48E-07 NA i 82 NA 2
2376 8 HI/*? "I1 rY"l^^A^A^IW N^1vi^ s KPU-28, tBu-delta-PLH-2-pyridyl >2E-05 NA | 39 NA 2
2377 Hju^ro KPU-26, tBu-delta-PLH-2-furyl 7.12E-07 NA I1 80 NA 2
2378 sHN^ HirY'^;VM*HNy»«kX>H *-»^4". KPU-27, tBu-delta-PLH-5-Me-2-thienyl 8.52E-08 1.35E-08 .[i 85 3 3
2379 9HtTt Hl?'ri;8>r^iNH«. H.°i^, 8 KPU-29, tBu-delta-PLH-3-Me-2-thienyl 4.70E-08 1.30E-08 1 J86 3 3
2380 ^ra-•TTA KPU-20; t-butyl-delta-PLH-p-CI •*• TfA salt (MW: 484) 7.09E-07 NA \ j 81 NA 2
2381 i r^Hu'"'!1 **? UBY°^vA^sjH ^1H»cie 8 KPU-30, tBu-defta-RLH-2,3-methytendioxy; TFA salt (MW:494.42) 5.20E-07 NA ( 82 NA 2
2382 i/^N HN-VY^*y*>L^ 1^1•vV 8 KPU-31, tBu-delta-PLH-3-pyridyl; 2TFA salt (MW 565.42) 1.03E-07 1.16E-08 J86 3 3
82.

; H^oWS KPU-32, tBu-defta-
' -X*^5 PLH-o-Me; TFA salt
2383 1 (MW: 464.44) 4.58E-08 1.15E-08 86 3 3
; g KPU-33, tBu-delta-
i w/^lf Mlif'r*Y**i\*«kskxNH "~i^ PLH-3-Me-2-pyridyl;
I Hje'Lu ° °"> 2TFA salt (MW:
2384 1 ^ifl*1. 579.45) >2E-05 NA 37 NA 2
KPU-34, tBu-delta-
jr^^Y^ F PLH-4-F; TFA salt
2385 ; ^ei^H. (MW:468.40) 5.01 E-07 8.48E-08 81 5 3
? •S^'-V-^Aw
: ^Cj wCj^JO1*1 KPU-35, tBu-delta-
j i)';^ PLH-m-F; TFA salt
2386 HiB«5H) (MW:468.40) 1.31E-08 5.32E-09 85 2 5
i 8 FH, KPU-36, tBu-delta-
UI/"W HIJ T*X1*S>.|H %*k((«k,,NH IW PLH-5-Me-4-im;
1 / | 2TFA salt
2387 I "* fi^i (MW:584.47) 2.35E-06 NA 84 NA 2
? 1 1
i m/*"? "TrYS y*k«*S.'NH e^ KPU-37, tBu-delta-
PLH-o-F, TFA salt
2388 | vi^H, (MW: 468.40) 3.00E-08 1.65E-08 86 3 3
i wO1 T'Or'tr011' KPU-38, tBu-delta-
2389 j ^O" PLH-m-Me; TFA salt (MW: 464.44) 4.67E-08 9.64E-09 86 3 3
; Hoo6na KPU-39, tBu-delta-
T^^^j OHj PLH-p-Me; TFA Salt
2390 VJ^H, (MW: 464.44) 4.83E-07 6.15E-08 84 4 3
-aaica KPU-40, tBu-delta-
o^^ii * PLH-p-Br; TFA Salt
2391 . "> 61^. (MW: 529.31) 7.14E-07 NA 82 NA 2
H3jAcrair KPU-41, tBu-delta-
^fva^T PLH-m-Bn TFA Salt
2392 "•"ll^ (MW: 529.31) 3.11E-08 7.29E-09 87 3 3
A-|j LJ|J'"V-^*V_eSl»UM 7 Y r r^ o rwi ^l ^L uu ^fc./ KPU-42, tBu-delta-
/^"^g PLH-3-thienyl; TFA
2393 ii^1! Salt (MW: 456.44) 5.58E-08 6.23E-09 86 3 3
-O^PCL KPU-43, tBu-defta-
4^*6^ X^V%N PLH-p-CN; TFA Salt
2394 ^ertfj (MW: 475.42) >2E-05 NA 14 NA 2

•cmjftr* KPU-44, tBu-delta-
' "^ PLH-m-EtO; TFASalt
2395 (MW: 494.46) 4.59E-08 1.02E-08 I 85 4 3
if"*1 KPU-45, tBu-delta- !
Ho[T^rYS PLH-2,4.6-TriOMe;
xf^*0 CH. "" TFA Salt (MW: j
2396 HjC-J^H, 540.49) >2E-05 NA i 2 NA 2
V0"
""CllXjCTj KPU-46, tBu-delta-
Jr^ii PLH-0-N02; TFA
2397 "^tfcH. Salt (MW: 495.41) 5.03E-08 1.24E-08 S 86 4 3
m/*1? H'li'T'VY^o'^y^vAJlH l^J KPU-47, tBu-delta- I
PLH-m-NO2; TFA ;
2398 H'°'S%H, Salt (MW: 495.41) 4.47E-08 1.19E-08 I 86 3 3
K*- * Va * KPU-48, tBu-delta- I
JL& 2* PLH-P-NO2; TFA
2399 •° ii^H, Salt (MW: 495.41) >2E-05 NA ) 23 NA 2
-x6^yN KPU-49, tBu-delta-
••/T^lf PLH-m-CN; TFASaH i
2400 ^"fi^H, (MW: 475.42) 3.56E-08 1.40E-08 i 85 3 3
hK^!!O^Tj KPU-50, tBu-delta-
JT^if PLH-o-Br; TFA Salt
2401 ^"ii^i (MW: 529.31) 4.05E-08 1.09E-08 J86 3 3
-xAra0" KPU-51, tBu-delta-
Jr^^s PLH-m-OH; TFASalt
2402 ^"ii^' (MW: 466.41) 3.64E-07 1.86E-07 I 86 2 4
°»w*°" KPU-52, tBu-delta-
HK*^ ^? iLTj PLH-2-NO2-5-CI;
^^1)1 i TFA salt (MW:
2403 H>°'S%H, 529.85) 9.97E-08 2.63E-08 i 86 2 5
"CoVtS KPU-53, tBu-delta-
jT^^d PLH-o-OH; TFA Salt
2404 ^°oi%H> (MW: 466.41) 6.10E-06 1.23E-06 I 66 10 3
i T KPU-54, tBu-delta- j
PLH-2-OH-5-OMe;
xf^ii J^ TFA Salt (MW:
2405 * gjCHj, HjC 496.44) >2E-05 NA i 25 12 4
«*»j HN"*^S^^SV«**^,r"-j;j -1 jjtt-j IL / KPU-55, tBu-delta-
2406 "^iT* PLH-3-furanyl; TFA Salt (MW: 440.37) 3.59E-08 1.03E-08 I 86 2 5

; »njyfi KPU-56, tBu-delta-
Jr^if iT PLH-2-OH-5-Br; TFA
2407 j "> ie Salt (MW: 545.31) >2E-05 NA 13 6 3
i KPU-57, tBu-delta-
M/^f ¥ IT IT"VtvA^NM «V-s0 PLH-3-OH-4-OMe;
1 Tf ?; H,c-Xeu ° ^ TFA Salt (MW;
2408 496.44) 7.48E-06 8.62E-07 66 4 3
9?" KPU-58, tBu-delta-
• HOCI^O«OL PLH-2-OH-4-OMe;
JT^IT SH, TFA Salt (MW:
2409 "»Cg^H, 496.44) >2E-05 NA 8 10 4
: H^AnS KPU-59, tBu-delta-
jT^jf ii PLH-2-OH-5-CI; TFA
2410 "1° 4i|H, Salt (MW: 500.86) >2E-05 NA 37 17 3
J?^0 KPU-60, tBu-defta-
"OOO^-^"0"" PLH-5-Me-2-furanyl;
2411 ; ^ Cl^a TFA Salt (MW: 454.40) 5.72E-08 2.00E-08 85 2 5
g KPU-61, tBu-detta-
HOUX>^' PLH-5-CI-2-thionyl;
2412 vifT* TFA Salt (MW: 490.88) 1.65E-07 3.32E-08 85 3 3
"Cc6rt> KPU-62, tBu-delta-
4^*8 PLH-2-thionyl; TFA
2413 H,p ^H, Salt (MW: 456.44) 2.74E-08 6.29E-09 88 2 3
jfpH, KPU-63, tBu-defta-
HN^JJjTj^lL/ PLH-N-Me-2-pyrrole;
^T^^s'^ TFA Salt
2414 ; """T^ (MW:453.42) 1.17E-07 3.14E-08 88 3 3
: -p^Vcx01 KPU-64, tBu-delta-
4^*6 61 PLH-3,5-diCI; TFA
2415 • ' ^C6I%HJ Satt(MW 519.30) 9.49E-08 5.05E-09 87 3 3
? l i
HOL^GSTO^'F KPU-65, tBu-delta-
4^*8^ PLH-m-CF3; TFA Salt
2416 H,C g^H, (MW 518.41) 4.40E-08 7.40E-09 87 2 3
X JO KPU-66, tBu-delta-
'KX^OsTTj PLH-1 -napthalene;
xf"^!^ TFA Salt (MW 500.47
2417 , ^"S^. ) 1.49E-08 2.12E-09 88 2 3
[ j?^^ KPU-67, tBu-delta-
i; ""».-^BL ^L UU I j«L _J PLH-2-napthatene;
2418 1 H'ci^, TFA Salt (MW 500.47) 1.73E-06 NA 83 NA 2

-aJr^r0 KPU-68, tBu-delta- ~1
2419 *»•£/* PLH-2,3-diCI; TFA Salt (MW 51 9.30) 2.99E-08 4.46E-09 88 2 3
-ojsjrxr- KPU-69, tBu-delta-
A*-". PLH-m-Vinyl; TFA
2420 Salt (MW 476.45) 1.70E-08 1.52E-09 88 2 3
°xAno KPU-70, tBu-defta-
4^i! PLH-oxazole; TFA
2421 VJ^H, Salt (MW 451 .40) 2.22E-08 3.28E-09 i 88 2 3
f^tSyCft}>l'T*!#SV"'!%l HjC J^_ JL -Jx. NH IL J KPU-71, tBu-delta- PLH-2-Hydroxybenzyl;
CH, 0 TFA Salt (MW ;
2422 495.91) >2E-05 NA {14 NA 2
I-..S KPU-72, tBu-delta-
HN^JXTX^-^ PLH-4-Br-2-thionyl;
j^^g" » TFA Salt (MW
2423 K>° fi^H, 484.49) 1.71E-07 1.86E-08 I 86 3 3
JL^,3 KPU-73, tBu-delta- I
HN^J J^ jjj «_/""cH, PLH-5-Et-2-thionyl;
xjC^ir TFA Salt (MW
2424 l="? H"Co5rt!rBr KPU-74, tBu-deKa- I
Jr^s" PLH-5-Br-2-furyl; TFA i
2425 vfi^ Salt (MW 51 9.27) 3.05E-08 8.71 E-09 J87 3 3
h»/^| J^ JJ^ * y^CH. KPU-75, tBu-delta- :i
2426 ^T» PLH-5-Et-2-furyl; TFA Salt (MW 468.43) 2.42E-07 MA 1 86 NA 2
-oArt>ci KPU-76, tBu-delta-
J^*6 PLH-5-CI-2-furyl; TFA
2427 ^"fi^ Salt (MW 474.82) 3.82E-08 8.88E-09 187 2 3
H^N H.1^ KPU-77, tBu-defta-
sjT^& \ PLH-2-F-5-I; TFA Salt
2428 HiC^H, (MW 594.30) 4.17E-08 7.78E-09 | 87 2 3
o j-01^ KPU-79, tBu-delta-
^^^fi^vS PLH-2-(Methylthio);
Ji~*l^ TFA Saft (MW
2429 Wb^t 496.50) 2.86E-08 3.59E-09 i87 2 3
/=N HN"^r»#YV'0'VFH y*k^-1f-(*1 v*' f KPU-80, tBu-delta- \
H'°6i^M» PLH-m-OCF3; TFA
2430 Salt (MW ) 1.43E-08 4.93E-09 j 87 2 3

t

N)

ro




















KPU-83, tBu-delta PLH-2-OH-5-IBU; TFA Salt (MW
KPU-81, tBu-delta-PLH-2-F5-OMe; TFA Salt )
KPU-92, tBu-delta PLH-2-N02Me; TFA Salt
KPU-91, tBu-delta-PLH-2,6-di-Me; TFA
KPU-87, tBu-delta-PLH-2-F-5-Me; TFA Salt
KPU-90, tBu-delta-PLH-2,3-di-Me; TFA Salt
KPU-82, PLH-4-F Salt (M
tBu-delta OMe; TF
-3-O
Go

COT3;*

9° CO 10
S
en
§
nn

5 §

CO TJX
-
.
H CD
5?

to

g
m 6
CO CO
en m
_x O

COTJP': n> r- TJ
** 1C
-1
5

00

00
S
m

NJ
S S
CD



23

8?

00

§



to

ro



10

to

CO

O)

Co

to

CO

CO

ro

to

CO

2455 ? ^wT? Hi?"VYr°'CH'""yA^jA^lilH 6^-V r KPU-94, tBu-delta-PLH-2-NH2-3-OMe; TFA Salt 3.49E-07 NA j 87 NA 2
2456 HjC-gcH, ° OH> KPU-95, tBu-delta-PLH-3-OMe-2-furyl; TFA Salt 1.25E-07 3.68E-08 I 88 2 3
2457 Ht/881!1 ^"rVS""yJ^A^NH «vXrV-* KPU-96, tBu-delta-PLH-2-NH2; TFA Salt 4.32E-07 NA | 86 NA 2
2458 i/*N Hw^ILJ. u^4". KPU-97, tBu-delta-PLH-m-NH2; TFA Salt 3.10E-08 1.71E-09 I 87 3 3
2459 H,oT.EH, H0' OH KPU-98, tBu-defta-PLH-3-B(OH)2-2-thienyl; TFA Salt 2.53E-08 4.31E-09 88 3 3
2460 °^ (-MBu-PLH 2.55E-07 1.24E-07 I 87 2 5
EXAMPLE 5 '
Other Dehvdrophenvlahistin Analogs
i
A. Modifications for the synthesis of dehydrophenvlahistin derivatives
[0377] Other derivatives of dehydrophenylahistin are synthesized Rising the
foregoing techniques alone or in conjunction with other well known organic j synthesis
techniques.
[0378] Modifications to the diacyldiketopiperazine and the first ar|d second
aldehydes involved in the synthesis method vary according to the desired derivative to
i
produce. Derivatives are synthesized that:
A) modify the phenyl ring and/or introduce other aromatic ring systems,
B) alter the position of the aromatic ring,
C) alter the imidazole aromatic ring system, and/or
D) modify the 5-position on the imidazole ring.
[0379] The figure below depicts regions of the dehydrophenylahistin Qompound modified to produce derivatives of dehydrophenylahistin. Non-limiting examples of

modifications are disclosed, and based on this disclosure would be understood by those of skill in theSart.




A 1) Modification of the phenyl ring besed on the structure of known anti-tubulin compounds Alkyl, Halogen, Alkoxy, Acetyl, Sutfonamide, Amino, Hydroxyl, Nitro, etc.


Combretastatln A-4 Colchtelne
2) Introduction of other aromatic ring systems

HjNOjS' MoL Wt: 385.85
E707D 0.15 ug/mL P-388 Phase II






B Position of the aromatic ring
C Crfange to the other ring systems


^

etc

D Further modification of the 5-positin on the imidazole ring
HK J _ HN
3.

[0297] Expanding on the above modifications to the dehydrophinylahistin compound, derivatives of the compound may include the following substitutions at the phenyl ring (A): -CF3, -SO2NH2 (-SO2NR]R2), -SO3H, -CONH2 (-CONR^ -COOH,
etc. Other ring systems (C) may also include the following: ;
ii
V-


B. Examples of synthesized, dehvdrophenvlabistin derivatives
[0380] Additional examples of synthesized dehydrophenylahistin deriNJatives are disclosed in the Table 4.
Table 4. Additional synthesized derivatives of dehvdrophenvlahistin 't
SALT FORM
j
STRUCTURE
M.W.
COMPOUNDS


KPU-20 tBu-APLH-p-Cl

TFA

1370.83 J484.86 t+TFA)




O
KPU-30 tBu-APLH-2,3-methylendioxy

TFA

Q 80.40
5494.42 t+TFA)




KPU-31 tBu-APLH-3-pyridyl

2TFA

337.38
J565.42 (|t-2TFA)




KPU-32 tBu-APLH-o-Me

TFA

350.41
H64.44 (+TFA)

COMPOUNDS

STRUCTURE

SALT FORM

M.W.




Me
: KPU-33 tBu-API|H-3-Me-2-pyridyl

2TFA

351.40
579.45 (+2TFA)




KPU-34 tBto-APLH-4-F

TFA

354.38
468.40 (+TFA)




KPU-35 tBii-APLH-m-F

TFA

354.38
468.40 (+TFA)




KPU-36 tBu-/5ffLH-5-Me~4-im

2TFA

356.42
584.47 (+2TFA)




KPU-37 t^u-APLH-o-F

TFA

354.38
468.40 (+TFA)




KPU-38 tBi|-APLH-m-Me

TFA

350.41
464.44 (+TFA)




KPU-39 tB^-APLH-p-Me

TFA

350.41
464.44 (+TFA)

COMPOUNDS

STRUCTURE

SALT FORM

M.W.




KPU-40 tBu-APLH-p-Br

TFA

\ 41 5.28
1529.31 K+TFA)




KPU-41 tBu-APLH-m-Br

TFA

1415.28
S 529.31 i^-TFA)





KPU-42 tBu-APLH-3-thienyl

TFA

1342.42
1456.44 JC+TFA)




KPU-43 tBu-APLH-p-CN

TFA

1361.40
H75.42 (+TFA)




KPU-44 tBu-APLH-m-EtO

TFA

{380.44
J494.46 j[+TFA)




OMe
KPU-45 tBu-APLH-2,4,6-TriOMe

TFA

H26.47
1540.49 (+TFA)




KPU-46 tBu-APLH-o-NO2

TFA

381.39
3495.41 ^-TFA)

COMPOUNDS

STRUCTURE

SALT FORM

M.W.




i KPU-47 tBu-^PLH-m-NO2

TFA

381.39
495.41 (+TFA)




iKPU-48 tBu|APLH-p-NO2

TFA

381.39
495.41 (+TFA)




KPU-49 tBu|APLH-m-CN

TFA

361.40
475.42 (+TFA)




LPU-50 tBtf-APLH-o-Br

TFA

415.28
529.31 (+TFA)




KPU-51 tBiMAPLH-m-OH

TFA

352.39
466.41 (+TFA)




ci
iKPU-52 tBu-A^LH-2-NO2-5-Cl

TFA

415.83
529.85 (+TFA)





>KPU-53 tBuiAPLH-o-OH

TFA

352.39
466.41 (+TFA)

COMPOUNDS

STRUCTURE

SALT FORM

M.W.




KPU-54 tBu-APLH-2-OH-5-OMe

TFA

1382.41 J496.44




KPU-55 tBu-APLH-3-furanyl

TFA

1326.35
J440.37 S(+TFA)




Br
KPU-56 iBu-APLH-2-OH-5-Br

TFA

H31.28
{545.31 i(+TFA)




KPU-57 tfiu-APLH-2-OH-4-OMe

TFA

!382.41
1496.44 ^f-TFA)




KPU-58 tBu-APLH-2-OH-4-OMe

TFA

3382.41
1496.44 ](+TFA)




O
Cl
KPU-59 tBu-APLH-2-OH-5-Cl

TFA

386.83 j(+TFA)
500.86




KPU-60 tBu-APLH-5-Me-2-foranyl

NH

TFA

J340.38
1454.40 JC+TFA)

CC$MPOUNDS

STRUCTURE

SALT FORM

M.W.




2KPU-61 tBu-AP$H-5-Cl-2-thionyl

TFA

376.86
490.88 (+TFA)




iKPU-62 tBu-4PLH-2-thionyl

TFA

342.42
456.44 (+TFA)




KPU-63 tBu-APLp-N-Me-2-pyrrole

TFA

339.39
453.42 (+TFA)




IKPU-64
tBu-^PLH-3,5-diCl

TFA

405.27




KPU-65
tBu4APLH-m-CF3

TFA

404.39




KPU-66
tBu-APtH-1-Naphthalene

TFA

386.44




KPU-67
tBu-APlH-2-Naphthaleaie

TFA

386.44

COMPOUNDS

STRUCTURE

SM.T FORM

M.W.




KPU-68
TBu-APLH-2,3-diCl

TFA

405.27




KPU-69
TBu-APLH-m-Vinyl

TFA

362.42




KPU-77
TBu-APLH-2-F-5-I

TFA

480.28




KPU-79
tBu-APLH-2-(Methylthio)

TFA

; 368.45




KPU-80
TBu-APLH-m-OCF3

TFA

|420.38




O OMe
KPU-81
TBu-APLH-2-F-5-OMe

TFA

384.38




KPU-82
TBu-APLH-4-F-3-OMe

TFA

084.38

CjOMPOTNDS

STRUCTURE

SALT FORM

M.W.




KPU-83
TBu-£PLH-2-OH-5-tBii

TFA

408.39





NH
KPU-84
TBu-APLH-cyclohexane

TFA

341.39




KPU-86
TBufAPLH-2-Me-3-F

TFA

368.39




KPU-87
TBu|APLH-2-F-5-Me
KPU-88
TBi%APLH-2-Cl-6-F

O

TFA
TFA

368.39
388.83




KFU-89
TB4APLH-2,5-di-F

TFA

372.38




KPU-90
TBu^PLH-2,3-di-Me

TFA

364.38




O
KPU-91
TBu-|iPLH-2,6-di-Me

TFA

364.38


COMPOUNDS
KPU-92
tBu-APLH-2-NO2-3-OMe

STRUCTURE

SM.T FORM
TFA


M.W.
411.39




KPU-93
TBu-APLH-2,5-diMe

TFA

1 364.38




KPTJ-94
tBu-APLH-2-NH2-3-OMe

TFA

|381.399




NHj
KPU-96
TBu-APLH-2-NH2
KPU-97
TBu-APLH-m-NH2

TFA
TFA

1351.41
351.41

C. Evaluation of dehvdropheflylahistin derivatives
[0381] Evaluation of derivatives described above is assessed according to the methods described in Example 3. Additional evaluation of the derivatives are extended to specific activities such as determining the inhibiting effect on cell proliferation, thej effects on a specific cellular mechanism (i.e. microtuble function), effects on cell cycle progression, evaluating in vitro anti-tumor activity against cancer cell lines, etc. Some evaluation method protocols are given below.

V) Qell Proliferation Inhibiting Effect of Dehvdrophenvlahistin And Its Analogs [•382] Into each well of a 96-well microtiter plate, 100^1 of A-549 cells derived from human hag cancer prepared to 10s cells/ml in a culture medium obtained by adding 10% bo'fine fetus serum to EMEM culture medium (Nissui Seiyaku Co., Ltd.) having antitumofr effect against A-549 cells derived from human lung cancer are placed. Methanol solution f the derivative obtained by the above-listed examples are added to the wells of the uppermost row, specimens are diluted by the half-log dilution method and added, and the plate is incubated in a carbon dioxide gas incubator at 37°C for 48 hours. The result is added in lots |of lOfil with MTT reagent (3-(4,5-dimethyl-2-fliiazole)-2,5-diphenyl-2H-tetra bromide)J!(lmg/ml • PBS), followed by incubation in a carbon dioxide gas incubator at 37 °C for 6 ho^rs. The culture medium is discarded and the crystal of produced in the cells are dissolved in 100)jl/well of dimethylsulfoxide. Absorption of 595 nm light is then measured with a mjcroplate reader. By comparing the light absorptions of the untreated cells to that of cells treafted with a specimen of a known concentration, the specimen concentration that inhibitedjcell proliferation 50% (ICso) is calculated.
2} Cell Cycle Inhibiting Activity of Dehvdrophenvlahistin And Its Analogs [0383] Cell strain A431 is derived from human lung cancer. EMEM culture medium Containing 10% bovine fetal serum and 1% MEM nonessential amino acid solution (SIGMAJM2025) is used to incubate A431 cells at 37 °C in an incubator saturated with 5% carbon dioxide gas and water vapor. The refined specimen of dehydrophenylahistin obtained by the methods above is added to the cells in the log-growth phase and progression of the cell cycle is analyzed by flow cytometer and microscopic observation.
[0884] The effect on cell cycle progression of HeLa cells is depicted in Figure 42.
EXAMPLE 6 Stmofrure-Aetivitv Relationship Of Synthesized Dehvdrophenvlahistin rDehydroPLIT)
Derivatives
1) Overview in derivative syntheses
[dbsSJ Many, but not all, of the derivatives of dehydroPLH disclosed herein include ope, two, or three modifications at the phenyl ring (Figure 5 below). The derivatives were syrrifhesized by the methods described above. As shown in Table 5, certain compounds

showed more potent cytotoxic activity than dehydroPLH and ffiu-dehydroPLHl The most potent compound exhibiting an EC50 value of 3 nM was KPU-90. This value vfas 16-times and 4-times higher than that of dehydroPLH and tBu-dehydroPLH, respectively. These derivatives have mono-substitution at the o- or m-position of the phenyl ring with the halogen atoms such as fluorine and chlorine atoms or the methyl, vinyl or metHoxy group. Derivatives with substitutions to heteroaryl structures such as the npahthalene, th|ophene and furan rings also elicited a potent activity.. KPU-35, 42, 69, 80 and 81 also shqwed higher activity than tBu-dehydroPLH.
Tables. Synthetic potent dehydroPLH derivatives

Compound

Structure

ECso (nM)




KPU-9

31




KPU-35

10




KPU-18

42




KPU-19

20




KPU-38

45

100

Compound
KPU-37

Structure
0 F

ECso (nM)
21




KPU-41

31





KPU-29

Me

44




O OMe
KPU-16

42




KPU-32

O Me

42






KPU-42

54





KPU-46

O NO2

44





KPU-44

43

\0

Compound
tBu-APLH (KPU-2)


o
Structure

ECso (nM)
13




o
KPU-69

16




KPU-80

13




OMe
KPU-81

19


KPU-90


DehydroPL H (KPU-1)

48

2) Introduction of the methoxy groups to the phenyl ring
(0386] Colchicine recognizes the same binding site on p-tubulin: as PLH. Colchicine has four characteristic methoxy groups on its A and B rings. Aj series of substitutions with the single or multiple methoxy groups was performed and the j results of cytotoxic activity are shown in Table 6.
102.

Table 6._ Effect of the methoxv group substitution on the proliferation of HT-29 cells

Compound

Structure

EC50 (nM)





DehydroPLH (KPU-1)

48




tBuAPLH (KPU-2)

13





KPU-8
KPU-9

o

O OMe

89
31





KPU-6

o

6730

O OMe



O OMe
KPU-10

1350




KPU-12

O OMe

4980


OMe
Compound
KPU-13
KPU-14

Structure
o

>Me

EC50 (nM)i
2130
610




OMe
MeO
O
KPU-15

4430




OMe
OMe
KPU-16

42




OMe
KPU-24

7040

[0387] The result demonstrated that substitutions at the w- or o-positioft increased cytotoxic activity against HT-29 cells. KPU-9 and 16 showed high activity. The methoxy-derivatives with triple substitution (KPU-11, 17 and 45) also showed activity. Thfc structure of KPU-24 was assigned by MASS analysis.
3) Modification with the electron-withdrawing groups
[0388] To study more expanded structure-activity relationship on the ptyenyl ring, a series of different functional groups were introduced, which include both! electron-withdrawing and -donating groups. The result of «ytotoxiciry against HT-29 cells is shown in Tables 7 and 8, respectively.

[03^9] Substitution at the o- or m-position effectively increased activity. These results wa|e well consistent with the case of the methoxy group. Table 7. I|gect of the electron-withdrawing group on proliferation of HT-29 cells

Compound

Structure

EC5o (nM)




KPU-18

42




KPU-19

20




KPU-20

545




KPU-21

51




CI
KPU-52

110




KPU-37

21




KPU-35

10

Compound

Structure

ECso (nM)




KPU-34

466




KPU-50

38



Br
NH
NH

KPU-41
KPU-40

NH
!r°v
/=N NH
-/\ o
O
NH.

Br

31
623




NH
KPU-46


NH
O NO,

44




NO,
/=*N NH
KPU-47

NH.

40




KPU-48


O
O

N02

Compound

Structure

ECso (nM)





KPU-49

28



KPU-43

TsjbleS. Effect of the electron-donating group on proliferation of HT-29 cells


Compound
KPU-8

Structure
O OMe

ECso (»M)
89





KPU-9

31




KPU-6
KPU-44

o

6730
43

/O?

Compound

Structure

EC50 (nM)





KPU-30

477





KPU-22

82




KPU-32

O Me

42





KPU-38

45




KPU-39

460




KPU-53

20 |4,M





KPU-51

617

Compound

Structure

EC5o (nM)




KPU-23

5870




O
KPU-58

>20nM




OMe
KPU-54

>20nM




KPU-57

>20nM

(03|90] The present disclosure is not bound by or limited to any particular scientific $ieory. Nonetheless, it is appreciated that persons of skill in the art may interpret the resultsjjpresented herein to suggest that a relatively smaller functional group, affecting less steric hindrance, may be preferred to elicit more potent activity, and slightly large groups such as t^e ethoxy group (when compared to the methoxy group) or the Br atom (when compared fto the Cl atom) may affect steric hindrance unfavorable to interaction with, for example, ?the tubulin binding site. Moreover, because the electrical property of these substituenis did not affect the activity, it is suggested that these relatively small substituents do not directly interact with the binding site of p-tubulin, but restrict the conformation of dehydroPJ|H suitable for the binding. Or, as another possible hypothesis, the hydrophobic property niay be a more important factor at the binding site for o- or m-position on P-tubulin, since introduction of the hydrophilic hydroxyl group, which can form the hydrogen bonding as a hydrogen-donor, drastically decreased the activity.

[0391] As shown in Table 9, the effect of the substituents in the cytotojijic activity at the o-position may be ordered* as in the case of m-position, as shown in Tabl4 10. The compounds having effective functional groups, which showed higher activity than tBu-dehydroPLH, may also be further modified. And since the migration of the stereochemistry from Z to E under the visible light irradiation was observed, substituents that decrease the electron density in the conjugated double bonds may contribute to the reduction jof Z to E migration by the light, results in more physicochemically stable structures. Tempejrarure can also effect this migration.
[0392] Modification at two parts of the ring can be preferred for the defelopment of potent but also biologically stable compounds. The phenyl ring of phenyjahistin is oxidized by cytochrome P-450. Double modification that reduces the electron density of the phenyl ring may therefore be effective to avoid P-450 oxidation. Thus, the combination of the small electron withdrawing group such as the fluorine atom to the element that can increase the activity such as -OMe, -Me, -Cl, -F and Br, may result in more jjotent and biologically stable drug compounds.
Table 9. Summary of modification at the o-position

Compound

Structure

ECso(nM)




KPU-2

48




OMe
KPU-8

89




O
KPU-37

21

\0

Compound

Structure
O Cl

ECso (nM)




KPU-18

NH

42



KPU-50


O
O Br

38

Q N02


NH
KPU-46

NH.

44




NH
NH
KPU-32

NH.

42



NH

KPU-53

NH

O OH

>20|jM

Table 10. Summary of modification at the gt-position

Compound

Structure

ECso (nM)





KPU-2

48




KPU-9

31

III

Compound

Structure

ECso (nM)





KPU-35

10




KPU-19

20





KPU-41

31




o
KPU-47

40




KPU-38

45




KPU-51

617




KPU-49

28

Compound

Structure

ECso (nM)




KPU-44

43

4) Substitution of the phenyl ring to aryl-heterocycles
[0&93] The phenyl ring may also be replaced by heteroaryl groups. The result of such replacements in terms of the cytotoxic activity is shown in Table 11. Since the arylic nitrogen |toms can form a hydrogen bonding with a NH group of the diketopiperazine ring and restrict the conformation of the molecule between pyridine and diketopiperazine rings to an uniplanar structure, the active conformation of dehydroPLH would be required a certain level of d|hedral angle formed by the steric repulsion between an amide hydrogen atom of the diketopipiprazine ring and an o-hydrogen atom of the phenyl ring (Figure 6). Table 1|. Effect of the replacement with the heteroarvl ring on proliferation of HT-29 cells

Compound

Structure

EC5o (nM)


KPU-28


KPU-31

96




KPU-25

544

Compound

Structure

ECso(nM)



O Me
KPU-33



KPU-26

600





KPU-60

O

71





KPU-42

O

54





KPU-27

80





KPU-29

44





CI
KPU-61

1 -V.

184

Compound

Structure

EC50 (nM)




KPU-36

2790




0
KPU-63

105

[03J>4] Replacing the phenyl ring with a smaller furan or thiophene ring, for example, KHJ-29 or -42, exhibited activity. The phenyl ring can be changed to other aromatic slhicture while maintaining the potent activity.
5) Metabolism of phenylahistin
[03f>5] In the recent his study, (±)-phenylahistin was treated with rat hepatic microsome or human hepatic P450s. In human case at least seven metabolites were detected, and two off them, i.e., PI and P3, were major metabolites, represented more than 60% of the recovered Metabolites.
[0396] Since there is no exo-olefin structure in tBu-dehydroPLH, present synthesized derivatives have no oxidization like PI and P4. However, oxidizations such as P3 and P5S are formed during the hepatic metabolism. Various derivatives, which prevent such metatfolism, are effective to avoid P450 oxidization at the phenyl ring. The imidazole ring can al|o be modified to avoid the unfavorable oxidation.
6) Physicochemical stability of dehydroPLH
[03&7] The physicochemical stability is one of the unfavorable problems of dehydroPLJH. In phenylahistin, since there is no additional olefin structure at the benzyl part, there is not such problem. However, in dehydroPLH, the benzylidene moiety can be easily activated, |>robably with the visible light, and the Z to E migration frequently occurs due to the existence of longer conjugation of the double bond. This migration occurred even under normal rocjhi light, hi the cytotoxic assay, some of the compounds migrate to £-form during the incubation, almough this migration probably equilibrates at the 1 : 1 ratio in the case of
115

dehydroPLH. This migration can be controlled. The Z to E migration is alsof known in combretastatin A4, a same type of tubulin inhibitor, and a few studies for impjjoving this problem were reported.
7) Prqdrug Synthesis
[0398] The E-form may also be used as a prodrug of dehydroPLH orf of one or more of its analogs, including those analogs described herein. One of thefundesired properties of anti-tubulm drugs involves its low selectivity between tumor and intact tissues, although these drugs belong to one of the molecular target therapies. This causesjundesired side effects. However, if the compounds functions selectively only in tumor tissue^, negative side effects of anti-microtubule drugs can be reduced. Since the dehydroPLH (Z|form) can be produced from its £-isomer by visible light irradiation, the .E-fbrm is administered and photo irradiation is performed only at the tumor site, then only, the tumor is dajmaged by photo-produced Z-form and the adverse effect to the intact tissues is reduced.
[0399] The 2?-fbrm can be protected chemically by the addition of a Jbulky but biodegradable acyl group, which is introduced into the diketopiperazine ring as a prodrug. This acyl group can be cleaved by the protease in the body. Therefore, the ajpylated-E-compound is maintained before administration, then after administration it is chained to the real E-form, which can migrate to the bioactive Z-form by the local photo irradiatioji.
[0400] The synthetic scheme of this acyl-E-form of tBu-dehyd|:oPLH is summarized in Figure 9.
EXAMPLE 7
Pharmaceutical Formulations of the Synthesized Dehvdrophenvlahistins I 1) Formulations Administered Intravenously, bv Drip. Infection. Infusion or Tt|e Like
[0401] Vials containing 5 g of powdered glucose are each added aseptjjcally with 10 mg of a compound synthesized by the method and sealed. After being charged with nitrogen, helium or other inert gas, the vials are stored in a cool, dark place. Befcfe use, the contents are dissolved in ethanol and added to 100 ml of a 0.85% physiological jjsalt water solution. The resultant solution is administered as a method of inhibiting the gfowth of a cancerous tumor in a human diagnosed as having such a tumor at between approx|mately 10 ml/day to approximately 1000 ml/day, intravenously, by drip, or via a subcutaneous or
M
H*

intraperitofieal injection, as deemed appropriate by those of ordinary skill in the art.
2) Formulation to be Administered Orally Or The Like
[0402] A mixture obtained by thoroughly blending 1 g of a compound synthesized by the method, 98 g of lactose and 1 g of hydroxypropyl cellulose is formed into granules by any conventional method. The granules are thoroughly dried and sifted to obtain a granule preparatio^ suitable for packaging in bottles or by heat sealing. The resultant granule preparations are orally administered at between approximately lOOml/day to approximately 1000 ml/d^y, depending on the symptoms, as deemed appropriate by those of ordinary skill in the art of treating cancerous tumors in humans.
3) Formulation to be Administered Topically
[04)3] Administration to an individual of an effective amount of the compound can also b$ accomplished topically by administering the compound(s) directly to the affected area of thefj skin of the individual. For this purpose, the compound administered or applied is in the fornj of a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, gly^erol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral o^ls. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Other materials such as anti-oxidants, hurnectants, viscosity stabilizers^ and similar agents may be added as necessary. Percutaneous penetration enhancers»such as Azone may also be included. In addition, in certain instances, it is expected that the compound may be disposed within devices placed upon, in, or under the skin. Suc|i devices include patches, implants, and injections which release the compound into the skfn, by either passive or active release mechanisms.
EXAMPLES \ In v^ro Pharmacology of KPU-2. KPU-35 and t-butvl phenvlahistin
[04>4] The in vitro efficacy studies performed with KPU-2, KPU-35 and t-butyl phenylahi^tin included: A) a panel of six tumor cell lines, B) studies in multidrug-resistant tumor cellf, and C) studies to determine the mechanism of action.

A). Study of KPU-2. KPU-35 and t-butvl phenvlahistinin a panel of six tumor cfol lines
[0405] The following cell lines (source in parentheses) were used: HT|9 (human colon tumor;. ATCC; HTB-38), PCS (human prostate tumor; ATCC; CRL-1435), ljfflDA-MB-231 (human breast tumor; ATCC; HTB-26), NCI-H292 (human non-small cell li|ng tumor; ATCC; CRL-1848), OVCAR-3 (human ovarian tumor; ATCC; HTB-161), B16-FJO (murine melanoma; ATCC; CRL-6475) and CCD-27sk (normal human fibroblast; AT^C; CRL-1475). Cells were maintained at subconfluent densities in their respective culture n|edia.
[0406] Cytotoxicity assays were performed as described above in E|cample 4,
using Resazurin fluorescence as an indicator of cell viability. ;
[0407] The disclosed compounds are effective agents against a variety off different
and distinct tumor cell lines. Specifically, for example, KPU-2 and KPU-35 ^ere most
i effective on the HT-29 tumor cell line, both in terms of potency (active in the low aanomolar
range) and efficacy (most responsive in terms of the maximum cytotoxic effec^j); t-butyl-
• ! phenylahistin exhibited its greatest potency against the PC-3 tumor cell line, although the
greatest efficacy was displayed against the HT-29 cell line; KPU-2 and KPlf-35 were generally 10-40 fold more potent than t-butyl-phenylahistin whereas the efficacy A*|as similar for all three compounds in the different tumor cell lines; the HT-29, PC-3, MD.4-MB-231 and NCI-H292 tumor cell lines all responded similarly to the NPI compounds, whereas the B16-F10 appeared to be somewhat less sensitive, t-butyl-phenylahistin displayedja marked differential between normal fibroblasts and the tumor cell lines, with a ratio ranging from >20 - >100, except for the OVCAR-3 cell line.

Table 12 jkctivitv of KFU-2. KPU-3S and t-butvl phenvlahistin in the Tumor Panel Screen

Cell-Line KPU-2 KPU-35 t-butyl-phenylahistin
Mean SD n Mean SD n Mean SD n
Hf -29 Cojon ICSOiiM % Cytotoxicity 9.8 82.5 2.4 5.3 44 8.2 81.3 2.0 4.0 4 4 420 88 473 0.2 3 3
PC-3 Prostate ICSOJiM % Cyjjotoxicity 13.4 60.3 0.7 2.1 4 4 13.2 56.8 2.5 1.0 4 4 174 59.5 - 2 2
MDA-MB-231 Breast ICSOnM % Cytotoxicity 13.8 56.7 1.97.2 33 9.7 59.3 4.2 5.6 4 4 387 65.5 - 2 2
NCI-H293 Lung IC50 pM % Cytotoxicity 17.5 70.5 1.1 2.9 4 4 15.9 68.5 1.1 2.9 4 4 384 65 1945 3 3
OVCAR-I Ovary ICSOfiM % Cytotoxicity >20,00 0 45.8 3.0 4 4 >20,000 39 2.2 44 >20,00037 - 2 2
B16-F10pelanoma ICSOpM % Cytotoxicity 37.1 71.8 26.3 2.5 4 ' 4 32.3 72.0 19.9 2.2 4 4 736 74 '650 2 3 3
CCD-27s| Fibroblasf IC50 fiM % Cytotoxicity 9.2 64.3 2.9 2.4 44 7.4 60.8 2.6 1.9 4 4 >20,000 45 - 2 2
B). Studies in Drue Resistant Cell Lines
[0408] One of the major challenges in the use of chemotherapeutic agents in clinical oncology is the development of resistance to the drug effect by the tumor cells. There are several mechanisms for the development of resistance, each of which will have differential! effects on chemotherapeutic drugs. These mechanisms include increased expression lofATP-dependent efflux pumps such as the P-glycoprotein encoded \syMDRl or the multidirug-resistance associated protein 1 encoded by MRP1. Reduced drug uptake, alteration qjf the drug's target, increasing repair of drug-induced DNA damage, alteration of the apopto^c pathway and the activation of cytochrome P450 enzymes are other examples of mechanises by which cancer cells become resistant to anticancer drugs. The selected

compounds were studied in three different cell lines that exhibit two different mechanisms of resistance; the overexpression of the P-glycoprotein and altered topoisomerase II activity.
1) Human Uterine Sarcoma Tumor Cell Line Pair: MES-SA (Taxol Senffitive) and MES-SA/Dx5 (Taxol Resistant).
[0409] This cell line expresses elevated mdr-1 mRNA and P-glycojirotein (an extrusion pump mechanism). Pretreatment with cyclosporin-A (CsA) blocks P-g^coprotein and reinstates activity in the resistant cell line for those compounds for which the resistance is due to elevated P-glycoprotein.
[0410] As can be seen from Table 13, KPU-2, and KPU-35 haveij the same potency in the resistant cell line as in the sensitive line and the potency |>f t-butyl-phenylahistin was only slightly reduced. Cyclosporin A (GsA) pretreatment did n|>t alter the potency of the selected compounds. In contrast, taxol was virtually inactive in | the MES-SA/DX5 resistant cell line, whereas this compound was very potent in the sensitivfe cell line. CsA treatment restored the sensitivity to taxol of the MES-SA/DX5 cell line. iThe MES-SA/DX5 cell line also showed reduced susceptibility to etoposide (60 fold), doxoAibicin (34 fold) and mitoxantrone (20 fold).
[0411] These data indicate that the effects of KPU-2, KPU-35 aifd t-butyl-phenylahistin are not susceptible to the taxol-related resistance mechanism (p-gWfcoprotein) in this cell line, and that cross-resistance from taxol does not occur to theae selected compounds in this model.
120

Table 13 Activity of KPU-2. KPU-3S. t-butvl-pheavlahistin and Taxol in MES-SA Taxol Sensitivf and MES-SA/DXS Taxol Resistant Human Uterine Sarcoma Tumor Cell Lines

Compound Study MES-SA Sensitive MES-SA/DX5 Resistant
No CsA CsA Pretreat No CsA CsA Pretreat
ICSO uM ICSO nM Ratio No CsA ICSO nM RatioMES-SA ICSO nM RatioNo CsA
KPU-2 ! 8.5 19.4 27.4 1.4 10.5 21.7 1.2 1.1 37.8 1.74
Study I i Study II
KPU-35 Study I Study iq 6.6 3.9 2.0 0.5 5.2 2.5 0.8 0.6 6.7 2.7
t-butyl-phenylahistiTi Study I Study n^ 144122 162 1.3 825 694 5.7 4.3 622 0,9
Taxol ^ Study! Study II j Study III 4.4 13.3 7.3 7.6 2.8 0.6 0.4. >20,000 »100 >24,000 >455 »8 >3000 40 2.0 • «0.25 «0.001
[0412] See also the additional data presented in Figure 43.
2") Human jAcute Promyelocvtic Leukemia Cell Line Pair: HL-60 (Mitoxantrone-Sensitive') andHL-6Q|MX-2 flVfttoxantrone-Resistanf)
[04|3] This cell line is considered to have atypical drug resistance properties with altered topoisomerase n catalytic activity without overexpression of P-glycoprotein.
[0414] As can be seen in Table 14, these results indicate that the potencies of the selected nqfvel compounds are very similar in the sensitive and resistant HL-60 cell lines. In contrast, Mitoxantrone loses efficacy by a factor of 24-fold in the resistant HL-60/MX-2 cell line.
[045] Thus, KPU-2, KPU-35 and t-butyl-phenylahistin are not susceptible to the same resistance mechanisms as Mitoxantrone in this cell line, and there is no cross-resistance from Mitoxantrone to these selected novel compounds in this model.

Table 14. Activity of KPU-2. KPU-35. t-butvl-phenvlahistJn and Mitoxantrone infthe HL-60 Human Acute Promvelocvtic Leukemia Tumor Sensitive and Resistant Cell Life Pair

Compound HL-60 Sensitive HL-60 Resistant
ICSOnM IC50 nM Ratio to Sensitive
KPU-2 6.4 8.17 1.28
KPU-35 9.2 7.3 0.79
t-butyl-phenylahistin 255 175 0.69
Mitoxantrone 202 4870 24.1
3>. Human Breast Carcinoma Cell Line Pair: MCF-7 (Taxol Sensitive') and MJSF-7/ADR (Taxol Resistant)
[0416] This study involved KPU-2 in comparison to taxol. KPU-2 defnonstrated similar potencies in both the sensitive and resistant members of this cell line pair. Jn contrast, taxol was virtually inactive in the resistant cell line whereas there was low inane-molar potency in the sensitive cell line (Table 15).
[0417] These studies confirm in a different human tumor cell line! that taxol
resistance does not transfer to KPU-2. '(
Table 15. Activity of KPU-2 and Taxol in the MCF-7 Human Breast Carcinoma jgensitive
and Resistant Cell Line Pair i

Compound MCF-7 Sensitive MCF-7/ADR Resistant |
IC50 nM 1C 50 nM Ratio to Sensitive
KPU-2 39.6 27.4 0.69
Taxol 2.6 »100 »38
C) Studies of the Mechanism of Action
1). Action on Microtubule Function -,
[0418] Human umbilical vein endothelial cells (HuVEC from Cam&rex) were
used in this study, for evaluating the effects of KPU-2 and t-butyl-phenyiahistin in
comparison to colchicine and taxol on tubulin by staining for a-tubulin. i
[0419] Thirty minutes exposure to KPU-2, t-butyl-phenylahistin or colchicine (all at 2 uM) induced microtubule depolymerization as was indicated by the laclf: of intact microtubule structure in contrast to that observed in the DMSO Control and cell jimembrane blebbing (a clear indication of apoptosis) in the HuVEC cells, whereas taxol did toot induce

microtubu|e depolymerization under these conditions. Colchicine is a known microtubule depolyme^zang agent whereas taxol is a tubulin stabilizing agent. Similar results were obtained ^hen CCD-27sk cells were exposed to KPU-2 or colchicine.
2). Induction of Apoptosis
[0420] Apoptosis and its dysregulation play an important role in oncology, the selective induction of the programmed cell death cycle in tumor cells is the goal of many chemotherftpeutic drug discovery programs. This induction of apoptosis can be demonstrated by different methods including the characteristic cell membrane blebbing, DNA fragmentaf on, hyperphosphorylation of the antiapoptotic factor Bcl-2, activation of the caspase cascade and cleavage of poly (ADP ribose) polymerase (PARP).
[0421] The characteristic signs of apoptotic cell death include cell membrane blebbing, fisrupttoh of nuclei, cell shrinkage and condensation and finally cell death, very distinctive! from necrotic cell death. KPU-2 induced the typical morphological changes associated! with early stages of apoptosis in human prostate tumor cells. A similar finding was also cjear in the treatment of HuVEC cells with KPU-2.
3\ DNA
[0422] A late stage characteristic of apoptosis is intemucleosomal DNA cleavage that result^ in a distinctive ladder pattern that can be visualized by gel electrophoresis. This approach ^as used to study the effect of KPU-2 on DNA laddering in Jurkat cells (human T cell leukemia line) in comparison to halimide and dehydrophenylahistin (KPU-1). KPU-2 induced DNA laddering at the 1 nM concentration whereas halimide and KPU-1 were much less potentl
4). Activation of the Caspase Cascade
[04J3] Several enzymes in the caspase cascade are activated during apoptosis, including jCaspase-3, -8 and-9. The activity of Caspase-3 was monitored in Jurkat cells following ^eatment with KPU-2, KPU-35 and t-butyl-phenylahistin.
[04§24] The results indicate that caspase-3 was activated in a dose-dependent manner byftreatment with all three compounds in a manner similar to halimide. The caspase-

3 activation occurred over a similar concentration range as for the ICSOs for cyt|>toxicity in
the Jurkat cell line (Table 16). Table 16 Cvtotoxicitv of KPU-2. KPU-35 and t-butvl-pheavlahistni ia JurkaiCells

NPI Compound Cytotoxicity i
Potency IC50 nM Efficacy %CeIH)eath!
KPU-2 11 94 I
KPU-35 5 93 1
t-butyl-phenylahistin 165 93 j
Mitoxantrone 41 99 i
5X Cleavage of PoMADP-ribose') Polvmerase (PARF) in Jurkat Cells
[0425] In order to assess the ability of these compounds to induce apoptosis in Jurkat cells, cleavage of poly(ADP-ribose) polymerase (PARP) was monitored. JPARP is a 116 kDa nuclear protein that is one of the main intracellular targets of Caspafee-3. The
cleavage of PARP generates a stable 89 kDa product, and this process cans be easily
I
monitored by western blotting. Cleavage of PARP by caspases is one of the hallmarks of
I
apoptosis, and as such serves as an excellent marker for this process. KPU-2 jit 100 nM
induced cleavage of PARP in Jurkat cells 10 hours after exposure of the c^lls to the compound. KPU-2 appeared to be more active than either halimide or KPU-1.
6). Enhanced Vascular Permeability in HuVEC Cells
[0426] Compounds that depolymerize microtubules (e.g. combretasjtatin A-4-phosphate, ZD6126) have been shown to induce vascular collapse in tumors in fz'vo. This vascular collapse is preceded by a rapid induction of vascular cell permeability initially to electrolytes and soon after to large molecules. The enhanced permeability of HuV^C cells to a fluorescent-labeled dextran is used as a proxy assay for vascular collapse.
[0427] KPU-2, KPU-35 and t-butyl-phenylahistin all rapidly (withi|i 1 hour)
induced significant HuVEC monolayer permeability, to an extent similar to colch|cine. The
microtubule stabilizing agent taxol was inactive in this assay (Figure 12). j
7). Profile in A Broad Kinase Screen i
[0428] KPU-2 was initially screened at a concentration of 10 pM in a p&nel of 60 different kinases; the ATP concentration was 10 uM. Four kinases were inhibitedfby greater

than 50%f in the primary screen and the ICSO's determined in secondary screening are presented sin Table 17. All of the IC50 values are in the low micromolar range, which indicates that inhibition of these kinases is not related to the low nanomolar activities observed lor tumor cell cytotoxicity.
Table 17. Activity of KPU-2 against Selected Kinases

Kinase IC50 (nM)
CDKl/Cyj ilin B (human) 10.1
c-RAF(hi man) 8.9
INKS Crafl 6.8
Lyn (moui fe) 11.1
EXAMPLE 9
In vivo Pharmacology
[0429] Preliminary studies with KPU-2 were performed using the MX-1 (breast) and HT-29 (colon) xenograft models and the P-388 murine leukemia tumor model, in the mouse. Other tumor models selected on the basis of activity in the in vitro tumor panel were the DU-1J&5 (prostate), MCF-7 (breast), and the A549 (hrag) cell lines. The human pancreatic* tumor (MiaPaCa-2) was also included. The novel compounds were studied as monother^jpy and in combination with a clinically-used chemotherapeutic agent. The doses of the selected novel compounds were determined from the acute tolerability testing (Maximally Tolerated Dose, MTD) and were adjusted if necessary during each study. The doses of t|e clinically-used chemotherapeutic agents were selected on the basis of historical studies. '
[O^SO] KPU-2 was the first compound to be studied in these five tumor models. Followingjj the initial results from this study, all three compounds were compared hi the HT-29 humani colon tumor, the DU-145 human prostate and the MCF-7 human breast tumor xenograft fnodels.
{0431J The above models all use the subcutaneous xenograft implantation technique pnd are potentially subject to selective effects of a compound on the subcutaneous vasculaturje producing a magnified (or apparent) antirurnor activity. In order to circumvent mis possibility, two other tumor models have been incorporated in the research. One of these is the observation of lung metastases following the intravenous injection of B16-F10 mouse

melanoma tumor cells. The other model is the implantation of MDA-231 huipan breast
tumor cells in the mouse mammary fat pad. While this latter model is a xenograft |nodel, the
subcutaneous vasculature does not play a role. Methyls I), Xenograft..Models =
[0432] Animals used were (exceptions are indicated for individual studies):
female nude mice (nu/nu) between 5 and 6 weeks of age (~20g, Harlan); group sizf was 9-10
mice per group unless otherwise indicated. 1
[0433] Cell lines used for tumor implantation were: HT-29 human colon tumor,
MCF-7 human breast tumor, A549 human non.small cell lung tumor, MiaPaC^-2 human
pancreas tumor; DU-145 human prostate tumor. r
[0434] Selected novel compounds were administered as monotherajly via the
intraperitoneal (i.p.) route at the doses indicated for the individual study; for the combination
studies the selected reference chemotherapy agents were injected 15-30 min p^ior to the
compound. [0435] Vehicles used in these studies were: 12.5% DMSO, 5% Crem^phor and
82.5% peanut oil for the selected novel compounds; (1:3) Polysorbate 80:13% qthanol for
taxotere; (1:1) Cremaphonethanol for paclitaxel; for CPT-11 each mL of solutionjcontained
20 mg of irinotecan hydrochloride, 45 mg of sorbitol NF powder, and 0.9 mg of tactic acid,
the pH being adjusted to 7.4 with NaOH or HC1. Saline dilutions are used to achieve the
injection concentrations used for the reference compounds. «
HT-29 fluroan Colon Tumor Model I
[0436] Animals were implanted subcutaneously (s.c.) by trocar with fragments of HT-29 tumors harvested from s.c. growing tumors in nude mice hosts. When the |umor size reached 5 mm x 5 mm (about 10-17 days) the animals were matched into treatment and control groups. Mice were weighed twice weekly and tumor measurements wer4 obtained using calipers twice weekly, starting on Day 1. The tumor measurements were converted to estimated mg tumor weight using the formula (W2xL)/2. When the estimated tunior weight of the control group reached an average of 1000 mg the mice were weighed, sacrificed and the tumor removed. The tumors were weighed and the mean tumor weight per group was

calculated! and the tumor growth inhibition (TGI) was determined for each group (100% minus the (change in the mean treated tumor weight/the change in the mean control tumor weight xllOO.
[0437] to this model unless otherwise noted for the individual study, the selected novel compounds were injected intraperitoneally every third day for 15 days [1, 4, 8, 11 and 15 (q3dx5)]; CPT-11 was administered intraperitoneally on days 1, 8 and 15 (qwx3). MCF-7 firman Bieast Tumor Model
[0438] Female nude mice (~20 g) were implanted s.c. with 21-day release estrogen (p.25 rag) pellets 24 hours prior to the s.c. implantation with MCF-7 tumor fragments {(harvested from s.c. tumors in nude mice hosts). The study then proceeded as described for the HT-29 model, using taxotere as the standard chemotherapy agent.
[0439] In this model unless otherwise noted for the individual study, the novel compounds were injected via the intraperitoneal route daily on Days 1-5, inclusive (qdx5); taxotere w£s administered intravenously on Days 1, 3 and 5 (qodx3). A549 Hunfran Lung Tumor Model
[0440] Animals were implanted s.c. by trocar with fragments of A549 tumors harvested from s.c. growing tumors in nude mice hosts. When the tumor size reached 5 mm x 5 mm (about 10-17 days) the animals were matched into treatment and control groups. The rest of thefstudy proceeded as described for the HT-29 model, using taxotere and CPT-11 as the standaijd chemotherapy agents.
[0441] In this model unless otherwise noted for the individual study, the tested compounds were administered via the intraperitoneal route on a q3dx5 dose schedule for the CPT-11 combination or on a qdxS dose regimen for the combination with taxotere; CPT-11 was administered via the intraperitoneal route on a qwx3 schedule; taxotere was administered intravenously on a qodx3 dose regimen. MiaPaCa-j Human Pancreas Tumor Model
[0442] Animals were implanted s.c. by trocar with fragments of MiaPaCa-2 tumors harvested from s.c. growing tumors in nude mice hosts. When the tumor size reached 5 mm x 3 mm (about 10-17 days) the animals were matched into treatment and control

groups. The rest of the study proceeded as described for the HT-29 model, using jgemcitabine
as the standard chemotherapy agent. t
[0443] In this model unless otherwise noted for the individual j study, test compounds were administered every third day via the intraperitoneal route on l|ays 1, 4, 7,
i
10 and 15 (q3dx5); gemcitabine was administered via the intraperitoneal route op Days 1, 4,
7 and 10 (q3dx4). i
DU-145 Human Prostate Tumor Model i
[0444] Male mice were implanted s.c. by trocar with fragments of DU|l45 tumors
harvested from s.c. growing tumors in nude male mice hosts. When the tumors reached ~ 5
mm x 5 mm ( at about 13-17 days) the animals were matched into treatment j^nd control
groups. The remainder of the study proceeded as for the HT-29 model, using taxjatere as the
standard chemotherapy agent. I
[0445] In this model unless otherwise noted for the individual jstudy, test
compounds were administered via the intraperitoneal route on Days 1, 3, 5, 8 and 11 (q3dx5);
taxotere was administered intravenously on Days 1,3 and 5 (q2dx3). |
2). Non Subcutaneous Implantation Tumor Models I
[0446] The animals used were: female nude mice (»«/««) (MDA-231 study) or B6D2F1 (B16-F10 studies) mice between 5 and 6 weeks of age (~20g, Harlan);|group size was 10 mice per group unless otherwise indicated.
[0447] The cell lines used were: MDA-MB-231 human breast tumor arjd B16-F10 murine melanoma cells.
[0448] NPI compounds were administered as monotherapy via the inniperitoneal
route at the doses indicated for the individual study; for the combination studies tfee selected
reference chemotherapy agents were injected 15-30 min prior to the NPI compound!
MDA-231 Human Breast Tumor ;
[0449] Female nude mice were injected in the mammary fat pad yrith 2xl06 MDA-231 cells harvested from in vitro cell culture. When the tumor size reachedjs mm x 5 mm (about 14-28 days) the animals were matched into treatment and control groups. The
:!
study then proceeded as described for the HT-29 model, using paclitaxel as th£ standard chemotherapy agent.

|0450] In this model unless otherwise noted for the individual study, the test compounds were administered via the intraperitoneal route on Days 1, 4, 8, 11 and 15 (q3dx5); pjaclitaxel was administered via the intraperitoneal route on Days 1-5 (qdx5). B16-F10 ifietastatic Murine Melanoma Model
[0451} Mice received B16-F10 cells (prepared from an in vitro cell culture of B16-F10 c|ells) by the iv route on Day 0. On Day 1 mice were randomized into treatment and control groups and treatment commenced. Mice were weighed twice weekly, starting on Day 1. All mi|;e are sacrificed on Day 16, the lungs removed, weighed and the surface colonies counted. Results are expressed as mean colonies of treated mice/mean colonies of control mice (T/C) x 100%). The metastasis growth inhibition (MGI) is this number subtracted from 100%. Paplitaxel was the standard chemotherapy agent used in this study.
[0452] In this model unless otherwise noted for the individual study, the test compounds were administered via the intraperitoneal route on Days 1-5 (qdx5); paclitaxel was administered intravenously on Days l-5(qdx5).
[0453] When appropriate (n > 3), results are presented as means ± SEM. Statistical analysis of studies with several groups was performed using ANOVA with Neuman-Ifeuls post test, unless otherwise indicated. A one-tailed t-test was also used based on the hypothesis that the compound or drug, or the combination, would reduce tumor growth.
Results
Studies in the HT-29 Human Colon Tumor Xenoeraft Model
1. In Kivo| Evaluation of KPU-2 +/- CFT-11 in the HT-29 Human Colon Tumor Xenoeraft Model i
[0454] This study assessed changes in dosage strength and dosing regimen for KPU-2 al [O^jSS] KPU-2 was administered at doses of 7.5 mg/kg ip daily for five days (qdx5), 3.f 5 mg/kg ip bid for five days, 7.5 mg/kg ip every second day for 10 days (qodxS) and 7.5 m$/kg ip every third day for 15 days (q3dx5). The combination of CTP-11 with NPI-2358 at a dose of 7.5 mg/kg ip q3dx5 resulted in a significantly greater effect than for either

compound alone, which lasted for the duration of the study (Figure 13). These observations during the in-life portion of the study were confirmed.by the mean group final tumor weights at autopsy for which only the combination group exhibited a statistically significant lower tumor weight than controls. In addition the difference between the mean tumor weights of the combination therapy and CPT-11 monotherapy groups was statistically significant (Figure 14). When the individual final tumor weights at autopsy are examined the greater effect of cothetapy is clear (Figure 14). The TGI of cotherapy was 78% as compared toJ38.9% for CPT-11 alone. The TGI for the combined therapy group exceeds the NCI criterion of 58% for a positive result. 2. Study of KPU-2 +/- Standard Chemotherapy vs. Five Human Tumor XenoRraft yodels
[0456] This study consists of five different arms, each with its ow^ protocol, timing, dosing regimen and reference compound. Each arm will be considered jwithin the presentation of the particular tumor model.
[0457] The aim of the HT-29 arm of the study was to investigate a sligfitly higher dose of KPU-2 (10 mg/kg ip q3dx5) in the HT-29 human colon tumor xenograft model as compared to those used in the study described above, in which a marked synergy was observed between KPU-2 (7.5 mg/kg ip q3dx5) and CPT-11 (100 mg/kg ip qwx3).
[0458] As can be observed in Figure 15, the combination of KPU-2 a|d CPT-11 in this model resulted in a marked synergy in the inhibition of tumor growth, with- the tumor growth being almost completely inhibited up to Treatment Day 29 in the combination therapy group. The combined therapy maintained efficacy and the estimated tumor grovWi for this group was significantly lower than for either monotherapy group. Accordingly, administration of KPU-2 and CPT-11 inhibited tumor growth and is an effective janti-tumor treatment.
[0459] The observations of the in-life portion of the study (estimated tumor growth, Figure 15) are supported by measurement of the weights of the tumors jexcised at autopsy (Figure 16). The tumor weights for the combination group was significantly less than the Controls (p [0460] When the individual final tumor weights are considered (F^g 16), the tumor size for the combination group was generally smaller than for the other treated or

pontrol groups. The TGI of the combination group was 65.8%, indicating a positive effect by the NCI cr|terion, while monotherapy did not reach the NCI criterion of TGI > 58%. 3. Study ffi activity of gPU-2. KPU-35 and t-butyl-phenvlahistin in the HT-29 Human Colon Tumor Xe|ograftaStqdv
104J61] The results of this study are presented in Figure 17 and Table 18. The combination therapy groups all indicated a marked synergy between the novel compounds and CPT-|1. The individual tumor weights demonstrate the effectiveness of the cotherapy treatment ^Figure 18). In each case the TGI for the combination group surpasses the NCI criterion fifr a positive effect, whereas the TGI for CPT-11 monotherapy did not reach this level. ;

Table 18. Summary of Studies Performed in the HT-29 Human Colon Tumor Model

Study Description NPI-Compound Chemotherapeutic Agent Combination Comments
Number Status Endpoint Number, mg/kgip Result TGI % Name, Dose Result TGI % Results TGI % Exceed NCI • CriterioD
2164 TGI KPU-2 7.5qdx5 7.5 q3dx5 No Effect No Effect CPT-11 100 ip 39* 78**;# Combination Synergy
2288 TGI KPU-2 10-+7.5 q3dx5 No Effect CPT-11 100 ip qwx3 36.5* 65.8** Combination Synergy SeeText
2139 TGI KPU-2 7.5 q3dx5 No Effect CPT-11 100 ip qwx3 32.7 80.7**,# Combination Synergy
2139 TGI KPU-35 10-»7.5 q3dx5 No Effect CPT-11 100 ip qwx3 32.7 83.3**,## Combination Synergy 1+ Day 13 1+ Day 27
2139 TGI t-butyl-phenylahistin 30 q3dx5 No Effect CPT-11 lOOip qwx3 32.7 77.7 *,# Combination Synergy
*p
4. Summary of the Effects of KPU-2,KPU-35 and t-butvl-phenvlahistin in Combination with CPT-11 infthe HT-29 Human Colon Tumor Xenograft Model
[0 58% for a positive effect. The results generated in the three studies are very comparable for both the inUife observations (Figure 19) and for the weights of the tumors excised at autopsy (Figure 20).
Studies in the DU-145 Human Prostate Tumor Xenograft Model (0463] Two studies have been completed with this model: the first study involved KPU-2 alojjae and in combination with taxotere; the second study compared KPU-2, KPU-35 and t-butyl-phenytahistin alone and in combination with taxotere.
1. Effect 4f KPU-2 in Combination with Taxotere ui the DU-145 Human Prostate Tumor
Xenograft Model
[0464] As can be seen from the data obtained during the in-life portion of this study (Fig|21), tbe most effective treatment of the DU-145 human prostate tumor was the combined fherapy of KPU-2 plus taxotere. The treatment effect was most pronounced at the beginning jof the study and appeared to be reduced as the study progressed. From treatment Days 20 -27, the combination therapy did provide an apparent TGI that exceeded the NCI criterion (|TGI > 58%), and the estimated tumor weight of the combined therapy was significant|y less than for either monotherapy.
2. Activit| of KfU-2. KPU-35 and t-butvl-phenvlahistin Alone or in Combination with
Taxotere i| the DU-145 Human Prostate Xenograft Model
[04(65] Based on the data obtained with KPU-2 in combination with taxotere in the Study\ described above a second study comparing KPU-2 to KPU-35 and t-butyl-phenylahistin alone and in combination with taxotere was initiated.
[04166] The observations made during the in-life portion of this study indicate that the combination of either KPU-2 or KPU-35 with taxotere has a greater reduction on tumor growth th|n for taxotere alone (Figure 22). The tumor growth was almost completely blocked b)| KPU-35 in combination with taxotere.

[0467] The excised tumor weights at autopsy confirmed the observations made during the in-life segment of the study. The combination of either KPU-2 (Figure 23) or KPU-35 (Figure 24) with taxotere was significantly more effective than taxote|e alone in blocking tumor growth , in the case of KPU-35, three often mice showed evidence| for tumor shrinkage. The tumor growth inhibition indices indicated a marked inhibition of tumor
growth for KPU-2 (group mean = 74.1 %) and an almost total block for KPU-35 (droop mean
i = 92.5%). Taxotere alone did not reach the NCI established criterion for a pos|tive effect
(TGA> 58%). I
5. Studies in the MCF-7 Human Breast Tumor Xenograft Model »
[0468] This study compared the effects of KPU-2, KPU-35 atfd t-butyl-phenylahistin in the MCF-7 human breast tumor xenograft model. The doges of the compounds were administered on Days 1,2, 3, 4, and 7; Taxotere was administered on Days 1,3 and 7.
[0469] The selected novel compounds have early onset, statistically significant
effects when used in combination with taxotere in this model, apparently almost Completely
blocking estimated tumor growth (Figure 25). Of the three compounds, KPU-2 appeared to
be the most effective, with t-butyl-phenylahistin also exhibiting a significant poteaitiation of
taxotere. *
6. Studies in the A549 Human Non Small Cell Lung Tumor Xeaograft Model i
[0470] The in-life observations during this study (Figure 26) indicated that the combination of KPU-2 (7.5 mg/kg ip, qdx5) with taxotere resulted in a marked inhibition of
•(
tumor growth as compared to the Control or either monotherapy group. This wasjjconfirmed
by the autopsy tumor weights, as the mean of the cotherapy group was significant^ less than
r
that of taxotere alone or the Control group (Figure 27). The cotherapy group turnfor weights form a cluster of low tumor weights, indicating the consistency of the effect. I
[0471] When the tumor growth index is calculated, the cotherapy groupjhad a TGI of 74.4% as compared to the control group well in excess of the NCI criterion finf a positive effect (TGI > 58%). Taxotere alone had a TGI of 26.1 %.

7. Studies |n the MDA-231 Human Breast Tumor Orthotopic Xcnoeraft Model
[0472J This model involves the placement of the human tumor tissue into the mouse mammary fat pad, a surrogate of the natural environment. In this manner the possibility! of a Positive effect due to a specific action on the subcutaneous vascular bed- is avoided. (This study compared the effect of KPU-2 (7.5 mg/kg ip, q3dx5) alone and in combination with paclitaxel (16 mg/kg ip, qdx5).
[0^73] Three weeks into the study there was a significant inhibition of tumor growth in the combination therapy group, a highly significant effect. This effect appeared to be more marked than for taxotere alone (Figure 28).
8. Studies in the Murine Melanoma B16 F10 Metastatic Tumor Model
[0474] This study examined the effect of KPU-2, KPU-35 and t-butyl-phenylahisjtin alone and in combination with paclitaxel on the number of metastases appearing on the surface of the lung 16 days after the intravenous injection of B16 F10 melanoma! cells to the mouse. This model is not a xenograft model; however, it does not involve a ^igh degree of vascularization into the tumor mass.
[0 EXAMPLE 10 Assays For Activity Against Pathogenic Fungi
[04J76J Comparative activity of a dehydrophenylahistin or its analog against a pathogenic fungus, relative to known antiftmgal compounds recited above, for use in determini% the dehydrophenylahistin or its analog's AF/IS value is measured directly against the fungall organism, e.g. by raicrotiter plate adaptation of the NCCLS broth macrodilution method described in Dtagn Micro and Infect Diseases 21:129-133 (1995). Antifungal activity cafn also be determined in whole-animal models of fungal infection. For instance, one may efnploy the steroid-treated mouse model of pulmonary mucormycosis (Goldaill, L.Z. & Sugar, ^..M. 1994 J Antimicrob Chemother 33:369-372). By way of illustration, in such
155

studies, a number of animals are given no dehydrophenylahistin or its analog, various doses of dehydrophenylahistin or its analog (and/or combinations with one or more otheil antifungal
F
agents), or a positive control (e.g. Amphotericin B), respectively, beginning before, at the
time of, or subsequent to infection with the fungus. Animals may be treated oncje every 24
hours with the selected dose of dehydrophenylahistin or its analog, positive |;ontrol, or
vehicle only. Treatment is continued for a predetermined number of days, e.g. up ^a ten days.
Animals are observed for some time after the treatment period, e.g. for a total of th ree weeks,
with mortality being assessed daily. Models can involve systemic, pulmonary, vaginal and
other models of infection with or without other treatments (e.g. treatment witfe steroids)
designed to mimic a human subject susceptible to infection. 1
[0477] To further illustrate, one method for determining the in vivo fierapeutic
efficacies (ED50, e.g. expressed in mg dehydrophenylahistin or its analog/kg subject), is a
rodent model system. For example, a mouse is infected with, the fungal pathogen (such as by
intravenous infection with approximately 10 times the 50% lethal dose of the pathogen (106
C. albicans cells /mouse). Immediately after the fungal infection, dehydrophenylahistin
compounds are given to the mouse at a predetermined dosed volume. The ED50 isjjcalculated
by the method of Van der Waerden (Arch Exp Pathol Pharmakol 195:389-412, 1)940) from
the survival rate recorded on 20th day post-infection. Generally, untreated control animals
die 7 to 1 3 days post-infection. I
[0478] In another illustrative embodiment, C. albicans Wisconsin (C^S) and C. tropicalis (Cl 12), grown on Sabouraud dextrose agar (SDA) slants for 48 h at|28°C, are
i
suspended in saline and adjusted to 46% transmission.at 550 ran on a spectrophotometer. The inoculum is further adjusted by hemacytometer and confirmed by plate counts to be approximately 1 or 5 x 107 CFU/ml. CF-1 mice are infected by injection 1 or 5 £ 106 CPU into the tail vein. Antifungal agents are administered intravenously or subcuta|ieously in ethanolrwater (10:90), 4 h post infection and once daily thereafter for 3 or 4 ifiore days. Survival is monitored daily. The EDso can be defined as that dose which allowSs for 50% survival of mice.

EXAMPLE 11
i Evaluating Antimicotic Activity
[04J79] Benzimidazoles and griseoiulvin are anti-tubulin agents capable of binding to fungal jpicrotubules. Once bound, these compounds interfere with cell division and intracellul^r transport in sensitive organisms, resulting in cell death. Commercially, benzimidaloles are used as fungicidal agents in veterinary medicine and plant disease control. 4 wide variety of fungal species, including Botrytis cinerea, Beauveria bassiana, Helmintho^porium solani, Saccharomyces cerevisiae and Aspergillus are susceptible to these molecules.! Toxieity concerns and increasing drug resistance, however, have negatively impacted tfceir usage. Qriseofulvin is used clinically to treat ringworm infections of the skin, hair and |iails, caused by Trichophyton sp., Microsporum sp., and Epidermophyton floccosumj. Its antifungal spectrum, however, is restricted to this class of fungal organisms. Genotoxidfty is also a significant side effect. Terbinafine, while an alternative first-line treatment, Hs more costly. Further, clinical resistance recently has been observed in Trichopkyjpn rubrum (the major causative agent for all dermatophyte infections).
[0480] In Candida albicans, microtubule/microfilament formation is affected where cell| are exposed to the microtubule inhibitors nocodazole and chloropropham. These results further validate the exploration of cytoskeleton inhibitors as effective antimycotic agents. Accordingly, several of the compounds disclosed herein were evaluated for antimycotii; activity.
[0481] Specifically, disclosed compounds were evaluated alongside commercially available microtubulin inhibitors as well as recognized antifungal agents. The test compound| and controls used in this study: (-)-Phenylahistin, KPU-1, KPU-2, KPU-11 and KPU-17, |CPU-35, t-butyl phenylahistin, Colchicine (commercial microtubulin inhibitor tested versfis 3 Candida isolates), Benomyl (commercial microtubulin inhibitor tested versus 3 Candida isolates), Griseofulvin (commercial microtubulin inhibitor and antibiotic control for testing| versus 6 dermatophyte isolates), Amphotericin B (antibiotic control for testing versus 3 (§andida isolates), Itraconazole (antibiotic control for testing versus 2 Aspergillus isolates).

[0482] Microorganisms against which these compounds were testecf included:
Candida albicans, Candida glabrata, Aspergillus fianigatus, Trichophyto^ rubrum,
Trichophyton mentagrophytes, Epidermophyton floccosum. With the exception glabrata (one isolate), two isolates of each species were tested. !
[0483] Antirungal susceptibility testing was accomplished according to the methods outlined in the National Committee for Clinical Laboratory Standards, M38-A "Reference Method for Broth Dilution Antirungal Susceptibility Testing of jConidium-Forming Filamentous Fungi; Approved Standard." This includes testing in RPMlJl640 with glutamine and without bicarbonate, an inoculum size of 0.4 - 5 x io4, and incubation at 30 or 35°C for 48 hours. The minimum inhibitory concentration (MIC) was defined asjjthe lowest concentration that resulted in an 80% reduction in turbidity as compared to £J drug-free control tube. Drug concentrations were 0.03-16 ug/ml for the investigational compounds, 0.015-8 fig/ml for itraconazole and griseofulvin.
[0484] The minimum inhibitory concentration (MIC) at which a compound prevented the growth of the target microorganism was assessed according to th| modified version of the NCCLS protocol. Minimum inhibitory concentrations (MIC) were determined
I
at the first 24-hour interval where growth could be determined in the drug-free control tube. The defined MIC was the lowest concentration that exhibited an 80% reduction fn turbidity as compared to the growth control. The minimum lethal concentration OflLC) was determined by plating 0.1 ul from the MIC concentration and each concentration above the
MIC. The MLC was called at the first concentration that exhibited five or fewer colonies of
j fungal growth representing a 99.95% kill. When a MIC was obtained, a minimum! fungicidal
concentration (MFC) was determined to assess the fungistatic/fungicidal nature of the compound. This procedure entails diluting drug-treated cell samples (removed from test wells containing compound at and above the MIC) to compound concentrations significantly below the inhibitory concentration and depositing them on agar plates. The coinpound is scored as fungistatic if the cells are able to resume growth and fungicidal if no ijegrowth is possible because the compound had killed the organisms.

[0485] Compounds disclosed herein were shown to be effective against two Trichopk$on species, T. rubrum is the principal causative agent for human dermatophytic infections and would be the key organism to target in the development of a clinical agent.
{0486] Compounds KPU-2, KPU-11 and KPU-17, KPU-35 & t-butylpheniiahistiB were equivalent in potency or in some cases more potent than griseorulvin, a current, standard pharmaceutical agent used for treating dermatophytic infectionsjj
[0487] Compounds (-)-Phenylahistin and KPU-1 were significantly less potent than the o|her compounds when tested versus T. rubrum and weaker but more comparable to the others j/ersus the sensitive T. mentagrophytes isolate.
(0488] In those instances when an MFC could be determined, the results indicate that these pompounds are fungistatic in nature (see Tables 19 and 20).

Table 19. Antifungal Activity of Dehydrophenylahistins and Analogs Thereof

Compound MICs and MFCs, fig/ml
C. albicans 90028 C albicans 10231 C glabrata A. fumigatus isolate #1 A. fumigatus isolate #2
MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC
(-)-Phenylahistin >70 ND** >70* ND >70 ND >16 ND >16 ND
KPU-1 >68* ND >68 ND >68 ND >16 ND >16 ND
KPU-2 >32 ND >32 ND >32 ND >16 ND >16 ND
KPU-1 1 and KPU-17 >32 ND >32 ND >32 ND >16 ND 0.06 >16
KPU-35 >32 ND >32 ND >32 ND >16 ND t-butyl phenylahistin >32 ND >32 ND >32 ND >16 ND amphotericin B 0.5 0.5 0.5 0.5 1 1 ND ND ND ND
griseofiilvin ND ND ND ND ND ND ND ND 0.5 ND
itraconazole ND ND ND ND ND ND 1 ND ND ND
colchicine >128 ND >128 ND >128 ND ND ND ND ND
benomyl 64 >512 64 >512 64 >512 ND ND ND ND

Table 20. Antifungal Activity of Dehydrophenylahistins and Analogs Thereof

Compound -™ - - — - -•-• ™ ~ "•—- —•- • imciuuTMFC* M#mT' — •"'""" ' "~'"~'-' ~ "; " "
r. rubrum isolate #1 r. rubrum isolate #2 T. mentagrophytes isolate #1 J. mentagrophytes isolate #2 E.floccosum isolate #1 E.floccosum isolate #2
MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MFC
NPI2350 >16 ND 0.16 >16 16 >16 >16 ND >16 ND >16 ND
NPI2352 >16 ND 025 >16 4 >16 >16 ND >16 ND >16 ND
NPE2358 >16 ND 16 >16 ND >16 ND >16 ND
NH2362 0.06 >16 O.03 16 >16 ND >16 ND >16 ND
NPI2386 16 >16 ND >16 ND >16 ND
NPI2460 16 >1.6 ND >16 ND >16 ND
amphotericin B ND ND ND ND ND ND ND ND ND ND ND ND
griseofulvin 0.5 ND O.015 ND 1 ND 2 ND 2 ND 4 ND
itraconazole ND ND ND ND ND ND ND ND ND ND ND ND
colchicine ND ND ND ND ND ND ND ND ND ND ND ND
benomyl ND ND ND ND ND ND ND ND ND ND ND ND

EXAMPLE 12
Evaluating vascular targeting activity
(0489] Tumors and neoplastic conditions can be treated using the Compounds disclosed herein. The occlusion of the blood supply in tumors with vascular targeting agents (VTAs) induces regression of the tumors. The compounds disclosed herein, inclupngNPU-02 and KPU-35, for example, can be as VTAs. Many VTAs exhibit their vasculaf effects by interacting at the colchicine-binding site on microtubules. This interaction f induces a characteristic, rapid collapse and occlusion of established vasculature in the tumor and therefore compromises the integrity of existing vessels leading to necrosis.
[0490] Vascular collapse can occur, for example, within 30-60 |minutes of exposure to the VTA and involves changing the shape of the immature and proliferating, but
1
not the quiescent and mature, endothelial cells in the central portion of the tumor. This differential effect on vascular cells provides a rationale for the selective effects on the tumor due to the higher percentage of proliferating immature endothelial cells in the tumor blood vessels versus normal blood vessels. VTAs can be classified into three overlapping spectra
'!
of activity: (1) potent vascular and cytotoxic effects, (2) potent vascular with wea|c cytotoxic
effects, and (3) potent cytotoxic with weak vascular effects.
In Vivo Vascular Targeting Activity of KPU-02 and KPU-35 '[0491] Animal models are essential to investigate new therapies ijhat inhibit
tumor-induced angiogenesis, target the established tumor vasculature, and inhibit tumor
growth. ' [0492] A murine syngeneic "pseudo-orthotopic" breast cancer model yas used to address these issues. Torres Filho et al., Microvascular Research (1995)49, 212-^26, which is incorporated herein by reference in its entirety. To create the "pseudo-orthotojjjic milieu," the coverslip of a dorsal skinflap chamber was removed and small pieces of mamihary fatpad from donor mice were implanted into the chamber. On top of the fatpad graft, tumor spheroids containing N202 mammary tumor cells transduced with Histone (^2B)-green fluorescent protein (GFP) were applied. The use of H2B-QFP transduced cells} allows for visualizing tumor growth and monitoring mitosis and apoptosis.

[0493J Fluorescence video microscopy allows for the relatively non-invasive study of tujmor microcirculation in conscious mice. This model can provide data regarding the effects jjof compounds on tumor vasculature, tumor growth, mitosis and apoptosis, and is useful to fxamine the activity of compounds either alone or in combination with other therapeutic^. Utilizing this model, KPU-02 and KPU-35 were shown to induce a rapid vascular c [04f>4] On day 12 of tumor growth, mice were treated i.v. with a 2-minute infusion of 5 mg/kg KPU-35, a 5 minute i.v. infusion of 10 mg/kg KPU-02, or bolus of vehicle (1|% solntol (w/w) + 2% DMSO in water). On day 13, 5-minute infusions of 10 mg/kg KPfM)2, KPU-35 or vehicle were administered. Treatments with KPU-02 or KPU-35 were well folerated. Mice were observed for two additional days. Tumor area, blood flow rate, and fascular density within and surrounding the tumor were visualized. Real-time observations were recorded at various time-points using still photos and video microscopy.
[0495] This study demonstrates the rapid collapse of the central vasculature after the single |.v. treatment with either KPU-02 or KPU-35. The changes in vascular functions resulted ina significant central tumor necrosis, without an observed effect on the vasculature in the surrounding fat pad or skin (Figure 30). These observations support the selectivity and specificity|of KPU-02 and KPU-35, which both individually can disrupt established tumor vasculaturf. In Vivo Activity of KPU-02 in Human Tumor Xenogrqfts
[0496] When KPU-02 was administered with CPT-11 (Mnotecan), Taxotere or Paclitaxel^ marked antitumor activity was seen in the human colon (HT-29), breast (MCF-7; MDA-MBJpl) and lung (A549) tumor xenograft models (Table 21). The effect of KPU-02 in the HT-J29 model was robust, reproducible in three studies, and showed a dose-dependent effect i.e.,|7.5 mg/kg was statistically greater than 2.5 mg/kg (Figures 32, 33). In vitro Activity of KPU-02 and KPU-35 in HuVEC cells
[0^97] The above-described in vivo effects of KPU-02 and KPU-35 on tumor vasculaturp were supported by the in vitro effects of the same compounds in HuVEC cells. Human umbilical vein endothelial cells are considered a good in vitro model of tumor

endothelium, which is considered "immature". Tumor endothelium lacks supporting vascular
mural cells and is increasingly reliant on microtubule network for integrity off the tumor
vasculature. Therefore, disruption of the microtubule network tamos causep vascular
collapse.
KPU-02 induces rapid tubulin depolymerization in HuVEC cells.
[0498] Human umbilical vein endothelial cells (HuVECs; Cambrex CC2519A) were maintained at subconfluent densities in EGM-2 (Cambrex) media. The |cells were cultured in an incubator at 37°C in 5% COa and 95% humidified air. For tubulin staining assays, HuVEC cells were seeded at a density of 3xl04 cells/ml in EGM-2 on tissue culture compatible coverslips (Fisher). The plates were returned to the incubator for 2 day*.
[0499] Stock (20 mM) solutions of the test compounds were prepare^ in 100%
i DMSO. 400X concentrated dilutions of the compounds were prepared in 100% DilSO. 5 jil
volumes of the dilutions were added to individual wells resulting in a final concentration of 200 nM. The final concentration of DMSO was 0.25% in all samples. The plates were returned to the incubator for 30 minutes. HuVEC cells were treated for 30 min with 200 nM KPU-02 or KPU-35.
[0500] The cells were rinsed in dPBS before fixation in 10%(v/v) neutral buffered formalin for 10 minutes at room temperature. Following fixation, a-tubulin wasj visualized by indirect immunofluorescence. Specifically, the cells were permeabilized ini|0.2%(v/v) triton X-100/dPBS for 10 minutes. The cells were washed prior to transferring thelcoverslips to a humidified chamber, the coverslips were blocked for two hours in antibOdy buffer (2%(w/v) BSA/ 0.1%(v/v) Tween 20/ dPBS). The coverslips were incubated wijh 50 \i\ of 0.1 g/ml mouse a-tubulin (Molecular Probes) in antibody buffer for 1 hour before washing and incubation with 50 \d of 1 ng/ml goat anti-mouse FFTC (Jackson ImmuiioResearch Laboratories) for one hour in the dark. Finally, the cells were washed and treated with 2 ug/ml DAPI (Molecular Probes) for 10 minutes before rinsing in H2O and mounting with Vectashield (Vector Labs) mounting media. The cells were imaged using ja 60x oil
immersion objective on an upright microscope (Olympus BX51). The images we|-e digitally
i
captured using a CCD camera and Magnafire 2.0 software (Olympus). Post image (processing was performed in Photoshop Elements 2.0 (Adobe) and in Microsoft Powerpoint.
IHM

[05^1] Figure 33 shows that KPU-02 and KPU-35 rapidly induce tubulin depolymer^ation in HuVEC cells. KPU-02 induces dose dependent monolayerpermeability in HuVEC cells.
[05|2] Human umbilical vein endothelial cells (HuVECs; Cambrex CC2519A) were maintained at subconfluent densities in EGM-2 (Cambrex) media. The cells were cultured njj an incubator at 37°C in 5% COa and 95% humidified air. For monolayer permeability assays, HuVEC cells were seeded at IxlO5 cells/ ml in EGM-2 media on FibronectinUoated 3.0 pm Fluoroblok inserts (Becton Dickinson) in 24-well plates. The plates were| returned to the incubator for 4 days to allow the cells to reach confluency.
[0503] Stock solutions (20 mM) of the test compounds were prepared in 100% DMSO. l(fX concentrated serial dilutions of the compounds were prepared in EGM-2. 10 n\ volumes of the serial dilations were added to the test inserts in duplicate resulting in final concentrations ranging from 2 (JM to 2 nM. The final concentration of DMSO was 0.25% in
!i
all sampled The cells were treated with 2 nM-2 |jM KPU-02 for 15 minutes.
[OSJJM] FrrC-Dextran (50 mg/ml) in dPBS (38.2 kDa; Sigma) was diluted 2.5 fold in EGM-2| 10 (jl of FTTC-Dextran was added to each insert. The final concentration of FrrC-Dex^n was 1 mg/ml. The plates were returned to the incubator and 30 minutes later the fluorescence of the lower chambers of the 24 well plates was read using a Fusion fluorimeteij (Packard Bioscience) with Xex = 485 run and Xem = 530 nm filters.
fO^)51 Figure 34 shows that KPU-02 is able to induce monolayer permeability in a dose dependent manner. The results shown in Figure 34 represent the mean ± S.D. of three independent experiments.
Blood Flcfy in the P22 Rat Sarcoma Model with I2SI-IAP
[05JD6] Tumor blood flow was assessed in a model using a quantitative 125I-iodoantipyjine (IAP) technique in rats bearing a P22 rat sarcoma. KPU-02 (15 rag/kg, IP) markedly ^nd selectively reduced tumor blood flow to 23% of vehicle at 1 hour after administration; blood flow remained markedly reduced 24 hours later (59% vehicle). In contrast, b|ood flow in non-tumor tissues was affected to a much lesser extent at 1 hour (see Figure 35)j

[0507] The reduction in blood flow at 24 hours post-dose was mojfe variable
between tissues for KPU-02 compared to vehicle, as shown in Figure 36. The bld flow to
the tumor was the most affected, other tissues exhibited a small.reduction in tflood flow,
skeletal muscle blood flow appeared to be increased at 24 hours post-dose. ]
[0508] The effects of KPU-02 observed at 1 hour appear to be longer lasting and
more selective for tumor blood flow than that previously reported for CA4P usinjjg the same
technique. I
[0509] In an experiment with the P22 rat sarcoma model, it was demonstrated that KPU-02 7.5 and 15 mg/kg IP (n=2 per dose) produced a dose-dependent tumor necjrosis by 24 hours post-dose, with the highest dose resulting in an almost total necrosis of thjje tumor as shown in Figure 37. All tumors in the KPU-02-treated rats showed evidence 4f necrosis, whereas tumors in vehicle-treated rats did not. The VTAs that have entered int^ the clinic (e.g., CA4P, ZD6126, AVE8062) show similar qualitative effects on tumor blood using the IAP methodology (or similar technology) to demonstrate reduced blood flow in |he P22 rat sarcoma tumor and in humans using the dce-MRI technique. See Stevenson JP| Rosen M, Sun W, Gallagher M, Haller DO, Vaughn D, et al.r "Phase I trial of the antivas|ailar agent combretastatin A4 phosphate on a 5-day schedule to patients with cancer, raagneti* resonance imaging evidence for altered tumor blood flow," J Clin Oncol 2003;21(2|):4428-38; Evelhoch JL, LoRusso PM, He Z, DelProposto Z, Polin L, Corbett TH, et al., "Magnetic resonance imaging measurements of the response of murine and human tumors to the vascular-targeting agent ZD6126," Clin Cancer Res 2004; 10(11):3650-7; and Gadgeel SM,
LoRusso PM, Wozniak AJ, Wheeler C. "A dose-escalation study of the novQl vascular-
I
targeting agent, ZD6126, in patients with solid tumors," Proc Am Soc §lin Oncol 2002;21:abstract 438; each of which is hereby incorporated by reference in its entirety.
Combination Therapy wim Microtabule Targeting Agents j
[0510] The findings that VTAs selectively damage the vasculature infthe central part of the tumor versus the periphery, which recovers functionality, support psing these agents in combination with chemotherapeutics (Taxol, Vinblastine and Cisplatirf), radiation and angiogenesis inhibitors directed against VEGF and EGF. The new |VTAs will

supplemen^ rather than supplant these therapies and should provide for greater autitumor
activities, i
Treatment frf other conditions
[OS|l) In addition to cancer, other diseases may be treated using the VTAs disclosed hjerein. Conditions include other neoplasms, retinopathies, and any other condition or disease |hat reMes upon blood supply, preferably blood supply from new vasculature in order to rerfiain viable and/or proliferate.
[05|2] Many conditions are associated with excessive or inappropriate vasculaturd Examples of conditions associated with excessive vasculature include inflammatory disorders such as immune and non-immune inflammation, rheumatoid arthritis, chronic articular rheumatism and psoriasis; disorders associated with inappropriate or inopportune invasion of vessels such as diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, cornea! graft rejection, retrolental fibroplasia| rubeosis, capillary proliferation in atherosclerotic plaques and osteoporosis; and cancer associated disorders, including for example, solid tumors, tumor metastases, blood bom tumops such as leukemias, angiofibromas, Kaposi .sarcoma, benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, as well as ot|er cancers which require vascularization to support tumor growth. Additional examples ff vasculature-dependent diseases include, for example, Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints and wound granulation. Furthermore, excessive vasculature is also associated with clinical problems Is part of biological and mechanical'implants (tissue/organ implants, stents, etc.). The instant compounds and compositions can be used to target vasculature, in preferably to preferentially target disease vasculature over non disease tissue vasculature, and thus the compound)^ and compositions can be used in the treatment of such conditions. Other diseases
in which Vascularization plays a role, and to which the instant compounds and compositions
[• can be usejjl, are Mown by those of skill in the art.
[0913] Examples of retinopathies include age-related macular degeneration (ARMD), {diabetic retinopathy, and the like. Pathological angiogenesis is a major contributing factor to a number of retinopathies that collectively are major cause of blindness

in the developed world. Kahn and Killer Am J Ophthalmol (1974) 78, 58-67k which is incorporated herein by reference in its entirety. For example, retinal and disk neovascularization occurs in 30-50% of patients with diabetic retinopathy for me re than 20 years. Yanko et al Retina (2003) 23, 518-522, which is incorporated herein by n ference in
its entirety. Furthermore, subretinal neovascularization is a serious complication i i ~10% of
i patients with macular degeneration. Ferris et al Arch Ophthalmol (1984), 102, 1640-1642,
which is incorporated herein by reference in its entirety. jj
[0514] Vascular targeting agents such as Combretastatin A-4 (CA-4) Jhave been shown to cause the disruption, of neovessels in non-neoplastic tissue. Griggs jet al Br J
Cancer (2001) 84, 832-835, which is incorporated herein by reference in its entirety.
'i. Additionally, CA-4P was shown to inhibit the retinal neovascularization that ocqurs during
proliferative retinopathy. Griggs et al Am J Path (2002) 160, 1097-1103, which is incorporated herein by reference in its entirely. Finally, CA-4P Phosphate was da nonstrated to suppress the development of VEGF induced retinal neovascularization and Inhibit the

development and/ or cause partial regression of choroidal neovascularization. N Invest Ophthalmology & Visual Sci (2003) 44, 3650-3655, which is incorporate

imbu et al herein by

reference in its entirety. The compounds disclosed herein can be used to treat ret nopathies.
For example, the methodologies of Griggs (2001 and 2002) and Nambu are usj^d to treat
i retinopathies. Furthermore, the compounds and compositions disclosed herein canbe used to
treat such retinopathies by applying the compounds and/or compositions to the target area in an effective amount for reducing vascular density and/or vascular proliferation.
I.'-.
148

Table 21. Effect of KPU-02 in Combination with Chemotherapy in Human Tumor Xenograft Models
Tumor Dose Reference Tumor Growth Inhibition (%) Tumor Regression (#/totaI)
Model KPtf-02 Chemotherap*
(# Studies) (mg/kg ip)
Colon 7.5 CPT-11
HT-29 Days Days 1, 8, 1
(3) 1,4,8,11,15
Breast Taxotere
MCF-7 7.5 Days
(2) qdx5 1,3,5
Breast 7.5 Paclitaxel
MDA-231 Days qdx5
(1) 1,4,8,11,15
Lung Taxotere
A549 7.5 Days
(1) qdx5 1,3,5
Agent
Reference
4/30
0/30
37+3
79+8
0/20
3/20
12; 58
26; 81
0/10
1/10
53
71
0/10
0/10
26
74

EXAMPLE 13
Structure-Activitv Relationship
[0515] The effect of activity from various modifications on the pheiyl ring of
tBu-dehydroPLH is illustrated by the data in Figure 38. It is apparent that substitution with
relatively hydrophobic and smaller functional groups at the m- or o- position irfcreased or
maintained the cytotoxic activity at HT-29 cells while substitutions at the j|- position
decreased activity. While not being bound to any particular theory, this data Suggests a
rigorous recognition of the phenyl ring by tubulin. [0516] 3D-QSAR (CoMFA) analysis (see Figure 39) also supports th| existence of sterically favorable fields at m- and o- positions and sterically unfavorable fields exist at the p- position. X-ray crystal analysis (see Figure 40) indicates that the conformation of potent derivatives require a certain amount of dihedral angle between the phenyl rjjng and the pseudo-tricyclic cor template formed by DKP and imidazole rings. Thus, modification with the appropriate conformational restriction of the phenyl ring may elicit potent actrvjity. While not being bound to any particular theory, it may be that the binding mode of PLH perivatives at the chochicine binding site of tubulin is different from mat of colchicines and; its known homologues.
EXAMPLE 14
In Vitro Action on Microtubules Purification of Microtubule Protein and Tubulin
[0517] Microtubule protein (MTP) was prepared as previously described (Farrell KW and Wilson L. (1987) Tubulin-colchicine complexes differentially poisofi opposite microtubule ends. Biochemistry 23(16):3741-8, which is incorporated herein by reference in its entirety). MTP preparations consisting of 70% tubuiin and 30% microtubulej-associated proteins (MAPs) were isolated from bovine brain by three cycles of warm polymerization and cold deporymerization in PEM100 (100 mM 1-4 piperazinediethansulfonic acidi (Pipes), 1 mM MgSO4) 1 mM EGTA, pH 6.8) and 1 mM GTP. MTP was drop-froze|i in liquid nitrogen and stored at -70°C until use. Tubulin was purified from microtubule|protein by phosphocellulose chromatography (PC-rubulin) and stored in PEM50 (50 mM P^jes, 1 mM

MgSO4, 1 fnM EGTA, pH 6.8). Protein concentration was determined by a Bradford assay (Sigma Chjpmicals, St. Louis, MO) using bovine serum albumin as the standard (Bradford, 1976).
Test Agents
[0518] Stock solutions of KPU-02 were prepared at a concentration of 20 mM in DMSO. s|tock solutions of Combretastatin A4 (National Cancer Institute, Bethesda, MD) (CA4) wasj prepared at a concentration of 5 mM in DMSO. Colchicine (Sigma Chemicals, St. Louis, Jv4O) (CLC) was prepared at a concentration of 3 mM in water. All agents were shielded t^am ambient light with amber Eppendorf tubes. Serial dilutions were made in DMSO an^|/or PEM50 to the desired concentrations.
>(
Determination of Steady-State Microtnbule Polymer Mass
[050) MTP (2 rag/ml) was polymerized into microtubules in the presence of a range of flrug concentrations in PEM100 containing 1 mM GTP and a final DMSO concentration of 0.5%. Samples were monitored by light scattering at 350 nm at 37°C for 75 minutes.
[0520] Polymerization reactions were centrifuged and the microtubule protein concentrations in the supernatant, a measure of the soluble tubulin at steady state, and the
ij
pellet, a measure of the microtubule polymer, were used to calculate the inhibition of polymerization. After incubation, polymerized microtubules were separated and sedimented from unp^lymerized MTP by centrifugation (150,000 x g, 45 minutes, 37°C). The supernatant was removed, and the microtubule pellets were depolymerized in deionized HjO (24 hours, jp°C) before protein determination by the Bradford assay.
i;
[0521] The percent inhibition was calculated in two ways and the values obtained from the tjivo ways were compared. In one way, a ratio of the microtubule protein in the pellet, drug to no drug, was calculated. Another ratio of microtubule protein in the pellet to the supernatant, drug to no drug, was also calculated. The numbers wete in close agreement and the feigner values were used because they were subject to less variance and experimental perturbation.
161

Microtubule Mean Length Distributions
[0522] Transmission electron microscopy was used to determine the nteair lerigth distribution of microtubules in the absence or presence of tested agent. At 75 njjmutes and prior to sedimentation, 10 ul aliquots from the polymer mass experiments werjb fixed by dilution into 290 ul PEMlOO-buffered 0.2% glutaraldehyde. Thirty microliteifs of fixed sample was settled onto formvar-coated 150 ICG mesh electron microscope g$ds for 90 seconds. Excess sample was wicked off with Whatman filter paper. Thirty mi|roliters of cytochrome C (1 nag/ml) was applied for 30 seconds to enhance protofilament resolution and facilitate negative staining. Uranyl acetate (1.5%) was applied for 20 seconds andjthe excess was wicked off. Grids were viewed in a Jeol electron microscope-1200 EX11 at |OOOX and 30,OOOX magnification. The Zeiss MOPffl was used to determine microtubiile length distributions and mean lengths for at least 100 microtubules per sample.
CLC Competition Assays
[0523] PC-tubulin (0.2 mg/ml) was incubated in PEM50 with 1 mMf GTP, 1% DMSO, 10 uM of tested agent and 7-25 uM [3H] CLC for 120 minutesj at 37°C. Measurement of [3H] CLC binding was followed by DEAE-cellulose filter-binding assay as described previously (Wilson, 1970). This method depends on the adsorption oi| tubulin to filter paper impregnated with DEAE-cellulose. Whatman DE81 filter paper was pife-wet with PEM50 prior to sample application. The total 100 ul reaction volume was applied^ to 2.5 cm disks of filter paper, over parafilm, on ice. The paper disks were washed by imfnersion in five successive 50 ml changes of PEM50, 5 mm/wash, 4°C, to remove alf unbound
j|
colchicine. The paper disks with adhering tubulin-bound colchicine were thqjn counted directly in a scintillation vial containing 2 ml of Beckman Coulter Ready Protein solution
(Fullerton, CA). All of the disks were washed together. Negligible binding of unbound CLC
I
to the paper disks occurred in controls, either in the presence of absence of tubulin.,
5
[0524] The K; values were calculated by linear regression of a doublet reciprocal plot of the experimental data in Microsoft Excel. The Km value of tubulin for CL(| under the experimental conditions was first determined, with x intercept equal to -I/Km. £4 BpP, Km in

the presence of drug, was determined experimentally. The K( was determined using the relationship Kro app = a Km, and for competitive inhibition a =• Km(l + [I]/Ki).
i; '
Fluorescence Spectroscopy
[OSES] Fluorescence measurements were performed using a Perkin-Elmer LS50B spectroflu [OS26] The bis-ANS fluorophore probes the hydrophobic surface of proteins and a change in Intensity of the bis-ANS fluorescence signal is a result of a change in the solvent accessible (surface area of a protein. If there is some conformational change that changes the tubulin-bis|-ANS interaction upon ligand binding, then bis-ANS can be used to report binding.
[05f27] PC-tubulin (0.2 mg/ml) was incubated with 0 - 30 nM KPU-02 at 25°C for 20 miijutes. Bis-ANS (25 uM) was then added and relative fluorescence intensities of samples wfare measured at 25°C within 15 minutes. Buffer blank spectra were collected and showed tl|at KPU-02 plus bis-ANS produced negligible fluorescence in the experimental wavelengt^ range.
[0528] The Ka was determined by fitting experimental data in Sigmaplot and Microsoft j Excel using the equation F = ((-Fmax x L)/(Ka 4- L)) + FO where F is the fluorescence intensity of bis-ANS-tubulin in the presence of total ligand concentration L, Fmax is the biljj-ANS fluorescence intensity of fully liganded tubulin, and FO is bis-ANS fluorescence in the absence of drag. Fmax was determined by plotting l/(Fo-F) versus 1/L and extrapolating to 1/L = 0. The fraction of binding sites- B occupied by KPU-02 was determined using the following relationship: B = (Fo-F)/(Fo-Fmax). The concentration of tree ligand wa| determined with Lfree = L - B[C] in which [C] is the molar concentration of ligand-binflmg sites, assuming a single binding site per tubulin dimer.

Inhibition of microtubule polymerization by KPU-02
[0529J KPU-02, CA4, and CLC were assayed for their ability tof alter the
polymerization of MAP-rich tubulin (MTP) (2 mg/ml) in a cell-free system in vi'frci Initially,
(-'" -I inhibition of polymerization was assayed using phosphocellulose-purified, m|crotubule-
associated protein-free tubulin (data not shown). KPU-02 was a more poten| inhibitor
towards MTs assembled with glycerol and DMSO seeds as compared to MTs assembled in
the presence of MAPs that copurify with tubulin. Although microtubule polymer in the
absence of stabilizing MAPs did not reach steady state over a 2-hour period, thjjese assays
demonstrated that KPU-02 interacts directly with purified tubulin and that it does njjat exert its
primary effect through a MAP. !
[0530] KPU-02 and CA4 inhibited MT polymerization more powerfully than
CLC as measured by light scattering (Figure 44) and sedimentation analysis Ofigure 45).
i MTP (2 mg/ml) was polymerized into microtubules in the presence of a ranie of drug
concentrations and allowed to reach steady state as monitored by light scattering |tt 350 nm. Figure 41 depicts turbidity spectra of microtubule protein polymerization in the presence of DMSO drug vehicle (0), 1.25 uM (o), 2.5 uM (—), and 5 uM (o) NPI-2358 (a), CfVl (b) and CLC (c).KPU-02 and CA4 inhibited MT polymerization with comparable potencies. Figure
rl
45 depicts inhibition of microtubule polymerization in the absence or presence off a range of
KPU-02 (o),CA4 (a), and colchicine (0) concentrations. The total polymer map after 75
H
" deviation
values from three experiments. The concentration at which polymerization was rambited 50% (ICso), is 2.4 ± 0.4 uM for KPU-02,2.2 ± 0.3 uM for CA4, and 7.6 ± 2.4 uM for CLC (Table 22). (Variances obtained by statistical analysis are reported as standard devialkm values
I
unless stated otherwise). At concentrations over the ICso for in vitro polymerizatiofi of MAP-rich tubulin, MTP displays aggregation kinetics, suggesting that KPU-02 and CAf sequester protein to prevent microtubule assembly.

Table 22. Microtubule polymerization inhibition concentrations.

Compound 'olymer Mass n
A.ve. IC50 ± sd
MM)
KPU-02 2.4 ± 0.4 4
CA4 2.2 ±0.3 3
CLC 7.6 ± 2.4 3
[05JJ51] As shown in Figure 44, all three of the tested agents produced a concentration dependent inhibition of the extent of microtubule polymerization from 1.25-5 uM. Therijs are two important differences to note among the spectra. First, the initial rate of increase ii| absorbance over time decreases with increasing drug concentration (Figure 44A and 44B). 1 The spectra indicate that there is a lag period for MT formation in the presence of KPU-02 gbd CA4. Drugs that significantly and rapidly reduce the soluble, assembly-
! • •
competen^Jpool of tubulin would decrease the initial rate of polymerization. In contrast, the initial rat^j of polymerization is unchanged at all concentrations of CLC (Figure 44C). Second, h|TP in the presence of KPU-02 or CA4 does not reach steady state at high drug
i!
concentra^ons (above 5 uM), as shown by the absorbance values that increase linearly with time (Figi^e 44A and 44B). In contrast, MTP in the presence of CLC reaches steady state at high drug (Concentrations (Figure 44C).
[0532] The amount of drug required to inhibit polymerization by 50% (ICso) was determine*! from the analysis of the linear relationship between the decrease in microtubule polymer sjpdimented by centrifugation with the increase in drug concentration (Figure 45). The errorj bars in Figure 45 represent standard deviation values from at least three independent experiments.
Decrease |n mean microtubule length measured by transmission electron microscopy
[0|33] Transmission electron microscopy was performed on agent-microtubule polymerization reactions to describe the polymer formed at steady state and to evaluate conclusions drawn from the light scattering spectra. KPU-02, CA4, and CLC all decreased

the lengths of the miciotubules formed at steady state. MTs were progressively siorter with
increasing drug concentration (Figures 46, 47 and 48). Figure 46 depicts f frequency
histograms of mean microtubule lengths in vitro at steady state in the presence of|(A) KPU-
02, (B) CA4, and (C) CLC. The Zeiss MOPIH was used to determine microtulfule length
distributions and mean lengths. At least 100 microtubules per drug concentrltion were
counted. Figure 47 depicts electron microscopy used to record microtubules in tf e absence
or presence of tested compounds. At 75 minutes, samples from polymer mass experiments
were fixed and stained and viewed in a Jeol electron mioroscope-1200 EXllf at 2000x
magnification. Representative electron micrographs of MAP-rich microtubules {formed in
vitro at steady state in the presence of (A) KPU-02, (b) CA4, and (C) CLC. Sc|le bar, 10
uM. Figure 48 depicts a graphical summary, of MT length decrease at steady a|ate in the
presence of KPU-02, CA4, and colchicine. Black bars, 1.25 uM, and shaded bais, 2.5 uM
drug. In the presence of KPU-02, and CA4, MTs are progressively shorter with Increasing
drug concentration, until the drug concentration at which MTP displays aggregation kinetics
as detected by turbidity, and no MTs are observed. Error bars are standard deviation values
from the measurement of at least 100 microtubules. :
[0534] KPU-02, CA4 and CLC did not affect MT nucleation. The jbumerous,
[i
short microtubules formed in the polymerization reactions evidence that the presence of
KPU-02, CA4, or CLC does not affect nucleation. If nucleation were affected, t|ien fewer,
longer microtubules, as opposed to numerous, shorter microtubules would have been
observed in drug-treated versus control samples. >
[0535] KPU-02 and CA4 were comparably potent in decreasing the aierage MT length. At 1.25 uM, the lowest drug concentration analyzed by electron mieroscopjk KPU-02 and CA4 decreased mean MT length by approximately 70%, and CLC by 40% (Figbre 48).
[0536] At drug concentrations over the ICso for in vitro n|icrotubule
ii
polymerization, microtubules are not observed by electron microscopy for KPU-0^ and CA4. In contrast, microtubules were observed by electron microscopy for all concenjtrations of CLC assayed. At concentrations over the ICso, microtubule protein in the presence of KPU-02 and CA4 displays aggregation kinetics, characterized by a linear increasje in light absorbance over time (Figure 44A and 44B), whereas in the presence of CLC, ligh| scattering
1*56

polymer reaches steady state (Figure 44C). Despite the observation that MTP with KPU-02 or CA4 increases absorbance at 350nm over time, drug-specific protein aggregates were not observed. Fluorescence Spectroscopy
[0^97] Tubulin (0.2 mg/ml) was incubated with a range of KPU-02 concentrations for 20 minutes at 25°C in PEM50 and 2 mM GTP. KPU-02 quenched bis-
i
ANS fluorescence in a concentration-dependent manner (Figure 49A). For KPU-02 and tubulin as measured by non-linear regression analysis of bis-ANS fluorescence intensity at the emissifra maximum, Kj = 10 ± 1.6 uM (standard error) (Figure 49B). The double reciprocal plot of the binding data, assuming a single binding site for KPU-02 per tubulin dimer, yielded a dissociation constant of 6.4 uM (Figure 49C). The two different K fluorescence. Figure 49B depicts fluorescence emission maxima at 487 nm fit to obtain thefKd of tubulin for KPU-02, 10 uM, standard deviation 1.6 uM. Inset, residuals. Figure 49 Competitive Inhibition of CLC Binding
[0338] KPU-02 and CA4 competitively inhibited CLC binding to tubulin (Figure 50). Figure 50 depicts the results of an inhibition assay where phosphocellulose-purified tubulin (0|2 mg/ml) was incubated with various concentrations of [3H]CLC in the absence (0), or presence of 10 uM KPU-02 (o) or 10 uM CA4 (a). Tubulin-CLC Km was 11 ± 4.4 uM and inhibition constants for KPU-02 and CA4 were 3.2 ± 1.7 uM and 2.4 ± 0.3 uM, respective|y. Constants were calculated from three independent experiments. The colchicinej-tubulin binding reaction is time and temperature dependent and the binding dissociation constant is Kj = 0.1-1 uM, depending on the conditions of the assay (Wilson L and Meza I. (1973) The mechanism of action of colchicine. Colchicine binding properties of

sea urchin sperm tail outer doublet tubulin. Journal of Cell Biology 58(3):709-1^, which is
] incorporated herein by reference in its entirety). Under Hie test conditions, the Kn| of tubulin
for CLC is 11 ± 4.4 uM. The Km may be considered the overall Kd of tubulin for CLC, however, due to the time-dependence of CLC binding, the Km is greater than tife reported values for the Kd. The Kj for KPU-02 and CA4 was 3.2 ± 1.7 uM and 2.4 jjb 0.3 uM, respectively. The K, is defined as the amount of drug required to inhibit CLC pinding by 50% and it is based on the amount of radioactive CLC bound to tubulin. The Ks isja measure of the drugs' ability to compete with CLC; it is not a direct measurement of d|ug-tubulin binding dissociation because of the method in which binding affinity is reported.
Results ,
At all concentrations of CLC assayed, MAP-rich tubulin reached steadj| state. In contrast, at higher KPU-02 or CA4 drug concentrations, MAP-rich tubuli^ did not
polymerize to steady state and microtubules were not observed by electron microscopy. KPU-02 and CA4 effectively decreased the concentration of available tubulin. Th|s decrease in the pool of soluble tubulin increased the MT critical concentration and \ prevented polymerization. The stoichiometric amounts of KPU-02 and CA4 required to decrease in vitro polymer mass coupled with the data that microtubule protein did not reach steady state above those concentrations, suggesting that KPU-02 and CA4 act by a sequestering mechanism in which soluble tubulin is bound and prevented from polymerization.
Observations by electron microscopy on the steady state, MAP-rich microtubules formed in the presence of the tested agents were consistent with the proposed mechanism that KPU-02 and CA4 sequester tubulin. There was a concentration-dependent decrease in the average microtubule length in the presence of KPU-02, CA4, and CLC. In the j|resence of KPU-02 and CA4, there was a drug concentration dependent decrease in the initial rate of polymerization, indicating that these drugs reduce the tubulin available for elongation. This decrease in the initial polymerization rate was not seen with CLC due to its slow Association with tubulin. Furthermore, microtubules were formed at CLC concentrations over its ICso for polymerization, but microtubules were not formed at KPU-02 or CA4 concentrations over their ICso for polymerization. While not being bound by any particular tfieory, the
re*

concentration of soluble tubulin bound by KPU-02 or CA4 must be under the critical concentration required for tubulin polymerization to proceed.
[OS^9] Binding studies indicated that tubulin has a lower affinity for KPU-02 than it has for (|LC. Inhibition of CLC binding to tubulin by KPU-02 and CA4 occurred within a 20-minute (incubation period, indicating that KPU-02 and CA4 association with tubulin approached equilibrium relatively faster than for CLC (data not shown). KPU-02 competitively inhtrited CLC binding to tubulin at a site overlapping with the CLC-binding site, consiltent with studies characterizing phenylahistin (halunide) (Kanoh K, Kohno S, Kataka LJTakahashi J and Uno I. (1999) (-)-Phenylahistin arrests cells hi mitosis by inhibiting ijtubulin polymerization. The Journal of Antibiotics 52(2): 134-141, which is incorporated herein by reference in its entirety). CA4, a structural analog of CLC, also competitivfsly inhibited CLC binding. Without being bound to any particular theory, it appears th|rt despite sharing a tubulin binding region with CLC, KPU-02 and CA4 interact with tubul|n and inhibit microtubules by a mechanism distinct from that of CLC.
EXAMPLE 15
In Vivo Action on Microtubules Cell culture studies
[0940] MCF7 human breast carcinoma cells (American Type Culture Collection, Manassas,[ VA) stably transfected with GFP-alpha-tubulin (Clontech, Palo Alto, CA) were cultured ixjj Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 0.1% pentHllin/streptornycin and nonessential ammo acids (Sigma) in 250-ml tissue culture flasks or i-fS-mm six-well plates (doubling time, 29 hours) at 37°C in 5% CO2. Cells were incubated fvith KPU-02, CA4, or CLC, prepared as described in Example 14, by replacing the original rr|edium with an equal volume of medium containing the required concentration of tested age|t or DMSO vehicle, and incubation was continued at 37°C for 20 hours.
Mitotic Progression
[0541] The fraction of cells in mitosis at a given drug concentration (mitotic index) wa| determined in the breast cancer cell line MCF7. Cells were plated at a density of

3 x 104 cells/ml in six-well plates. After 24 hours, cells were incubated in thejjabsence or presence of drug over a range of concentrations (1 nM to 1 uM) for 20 hours, llledia were collected and cells were rinsed with versene (137 mM Nad, 2.7 mM KC1, 1.5 ml|l KH2PO4,
8.1 mM Na2PO4, and 0.5 mM EDTA), detached with trypsin, and added back to t|e media to
I ensure that floating and poorly attached mitotic cells were included in the analysis. Cells
were fixed with 10% formalin in PBS overnight at 37°C, permeabilized in methtaiol for 10
minutes, and stained with 4,6-diamidino-phenylindole (DAPI) to visualize nuclfi. Stained
,1
cells were spread on coversh'ps in Vectashield mounting media (Burlingame, CA)iand sealed onto slides with nail polish. Fluorescence microscopy was used to determine mitcjjtic indices. Results were the mean and standard deviation of 4-7 experiments in which 300J cells were counted for each concentration. The ICso was the drug concentration that experimentally induced 50% of the maximal mitotic accumulation at 20 hours.
Immunofluorescence microscopy
J0542] Cells were prepared as for mitotic progression, except that fcells were
seeded onto poly-L-lysine (50 fig/ml, Sigma) treated coverslips. On the day of staining, cells
were rinsed in PBS and fixed in 10% formalin overnight at 37°C. Cells were rinsed in PBS,
permeabilized in methanol at -20°C, and hydrated witih PBS. Coverslips were treated with
20% normal goat serum in PBS/ BSA (1%) for 1 h at room temperature, pells were
incubated in a mouse monoclonal cocktail of anti-alpha- and beta-tubulin, DM1A /DMIB
diluted in PBS/BSA for 1 hour at room temperature, then stained with FTTC-tonjugated
secondary antibody and DAPI. Coverslips were mounted using Prolong antiibde media
(Molecular Probes, Eugene OR), j
!
Preparation of cells for analysis of microtubule dynamics ,
[0543] Cells were prepared as for mitotic progression, except that to promote cell spreading, cells were seeded onto glass coverslips that had been pretreated with po|y-L-lysine (50 ug/ml, Sigma) for 2 hours, followed by laminin and fibronectin (10 jig/ml, S^gma) for 1 hour at 37°C. Cells were incubated with drug or DMSO for 20 hours and serq|m-starved. Before analysis, coverslips were transferred to recording rnedia (culture media lading phenol

red and soflium bicarbonate buffered with 25 mM HEPES and supplemented with 3.5 g/L sucrose). IFo prevent photobleaching, Oxyrase (30 ul/ml, Oxyrase Inc., Mansfield, OH) was added to thjje recording media immediately before sealing cells in a double coversHp-enclosed chamber. I
Time-lapse microscopy and image acquisition
[OS|W] Microtubules were observed using a Nikon Eclipse E800 fluorescence microscop4 with apian apochromat 1.4 N.A. x 100 objective lens. The stage was enclosed in a Pyrex bo|t and maintained at 36 ± 1°C by a forced air heating system. Thirty images of each cell T$ete acquired at 4-s intervals using a Photometries CoolSNAP HQ digital camera (Tucson, 4?) driven by Metamorph software (Universal Imaging, Media, PA) at 10 MHz, with a 300 bis exposure time, a gain of 2, and 2x2 binning to enhance brightness.
Analysis of microtubule dynamics
[05ft5] The positions of the plus ends of microtubules over time were tracked using the j^etamerph Track Points application exported to Microsoft Excel and analyzed using Rea| Time Measurement software. The lengths of individual microtubules were graphed asj a function of time. Individual growth and shortening rates were determined by linear regression. Changes of > 0.5 utn between two points were considered to be growth or shortening! events, and changes of [0546] The time-based catastrophe frequency for each microtubule was calculated by dividing the number of catastrophes per microtubule by the time spent in growth or attenuation. The time-based rescue frequency per microtubule was calculated by dividing the total number of rescues per microtubule by the time spent shortening. The distance-based catastrophf and rescue frequencies were calculated similarly by dividing the number of transitionsfby the length grown or shortened, respectively. Microtubules that were visible for Ul

as the length grown and shortened divided by the total life span of the rnlcrotubule. Microtobules that were visible for > 0.3 min were included in the dynamicity analysis.
Cell cycle progression blocked at prometaphase •
[0547] The concentration range for KPU-02, CA4 and CLC over 4hich cells accumulate in mitosis were determined. After 20 hours, 60-70% of cells were inhibited at prometaphase, compared to 30-40% of cells at metaphase in studies on MT depcflymerizers such as the vmca alkaloids and 2-methoxyestradiol, and MT stabilizers sucl| as taxol,
epothilone B, and discodermolide (Jordan MA (2002) Mechanism of action of antitumor
I
drugs that interact with microtubules and tubulin. Current Medicinal Chemistry - Anti-Cancer Agents 2: 1-17, which is incorporated herein by reference in its entirety).! The drug concentration necessary for 50% maximal mitotic block (ICso) was evaluated between 1 nM
and 1 uM drug (Figure 51). Figure 51 depicts log [Drug] response curves |br mitotic
i! progression inhibition by KPU-02, CA4, and.CLC. MCF7 cells were cultured in tijje presence
of NPI-2358 (o), CA4 (a), and colchicine (0). To evaluate mitotic indices, MCF7|cells were plated at a density of 3 x 104 cells/ml in six-well plates. After 24 hours, cells werdj incubated in the absence or presence of drug over a range of concentrations (1 nM to 1 j|M) for 20 hours. Cells were fixed and stained with DAPI to visualize nuclei. Fluorescence i|iicroscopy
was used to determine mitotic indices. Results are the mean and standard deviation of three
F
or four experiments in which 300 cells were counted for each drug concentration. The
i
mitotic block IC50 for KPU-02 was 17.4 ± 1.2 nM, CA4 was 5.4 ± 0.7 nM, and CMP was 23.8 ±3.1nM(Table23).
Table 23. Inhibition of mitotic progression.

Compound Mitotic block Ave. ICSO ± sd (nM) n
KPU-02 17.4+1.2 4
CA4 5.4 ±0.7 3
CLC 23.8 ±3.1 4

[05JJI8] Most MT-targeting agents block mitosis at the metaphase to anaphase transition, f Mitotic block at the metaphase to anaphase transition is associated with suppressioi of MT dynamics. Without being bound to any particular theory, the earlier prometaphfse block, together with the depletion of MT polymer, suggests a distinct mechanism* of action for KPU-02 as compared with other MT depolymerizing drugs, e.g., vinblastinej which at low concentrations stabilize MT dynamics.
Depolymetization of the mitotic spindle and the interphase array MTs
[0549] KPU-02, CA4, and CLC were observed to be potent microtubule depolymer|zers in MCF7 cells. Although mitotic spindle microtubules are more susceptible to depolynferization and/or inhibition of polymerization than interphase array microtubules, both mic|otubule populations were affected (Figure 52). Figure 52 depicts immunofluorescence microscopy images of MCF7 cells. Interphase arrays are relatively more stab$ to depolymerization by KPU-02, CA4 and CLC than mitotic spindles. Cells were prepared and seeded as for mitotic progression and treated with the mitotic block ICso for each dfug for 20 hours. Cells were incubated in a mouse monoclonal cocktail of anti-alpha- an| beta-tubulin, DMJA/DM1B then stained with FITC-conjugated secondary antibody a|id DAPI. a-d, Tubulin in control (a), KPU-02 (b), CA4 (c), and CLC (d) treated cells, and f h, DMA in control (e), KPU-02 (f), CA4 (g), and CLC (h) treated cells. Narrow arrows indicate mitotic spindle polymer and mitotic chromosomes and thicker arrows indicate interphase arrays and nuclei.
[05|;0] At the IC25 for mitotic block, KPU-02 dramatically altered spindle morphology. Figure 53A-C depicts immunofluorescence microscopy images of MCF7 cells treated wil|i KPU-02 (A), CA4 (B), and CLC (C) for 20 hours. Mitotic spindle destruction with increasing drug concentration. 1-4, Alpha and beta tubulin in control (1), a concentration of K/JS for mitotic block (2), the ICso for mitotic block (3), and twice the ICso for mitoticj block (4); 5-8, corresponding images of DNA for the adjoining panels. There were no njprmal, bipolar spindles at me ICas for KPU-02 or CA4 (Figures 53A and B). Compounds-treated cells had monopolar or bipolar spindles with uncongressed chromosomes. In contrast! normal bipolar spindles persist at the ICas for CLC (Figure 53C). At the ICso for
fte

KPU-02, 75% of the mitotic cells contained asters or foci of tubulin, and the remaining cells
had no detectable mitotic polymer. In the presence of CLC, half of the cells wjere bipolar
with uncongressed chromosomes and the remaining half were monopolar. At concentrations
of twice the ICso for mitotic block, there was little detectable MT polymer in ntitotic cells
treated with KPU-02, CA4, or CLC. j
[0551] Microtubule interphase arrays were more resistant to depol] merization

than mitotic spindles for all of the compounds examined (Figure 52). However, a

qualitative

decrease in polymer was observed in a dose-dependent manner for all three i ompounds
(Figure 54 A-C). Figure 54A-C depicts immunofluorescence microscopy images of MCF7
!
cells treated with KPU-02 (a), CA4 (b), and CLC (c) for 20 hours. Interphase MT
I
depolymerization with increasing drug concentration. 1-4, Alpha and beta tubuliri in control (1), a concentration of ICis for mitotic block (2), the ICso for mitotic block (3), 4nd 2XICso for mitotic block (4); 5-8, corresponding images of DNA for the adjoining panels.
Presumably, tubulin is sequestered in these interphase cells, despite the presence of
! intracellular stabilizing MAPs, just as MAP-rich tubulin is sequestered in in vzflto polymer
1
mass assays. ,
Lack of suppression or modulation of MT dynamic instability in living MCFTJcells
[0552] KPU-02, as well as CA4, did not have a measurable effect on Mtr dynamic instability at concentrations effecting 25% (Table 24) or 50% (Table 25) of fhj» maximal mitotic block in MCF7 cells. Without being bound by any particular theory, Jthese data suggest that the antiproliferative mechanism of action of KPU-02 (and CA4) is primarily due to inhibition of MT polymerization, rather than suppression of microtubule dynamics.

Table 24. MT dynamic instability at the mitotic block.
sd
CLC
Control sd NPI-2358 sd CA4 sd
Ml (» an rates i/min) irowth 9.03 4.66 11.89 6.23 10.59 4.75 11.31 8.46
hortenina 32.86 18.06 31.87 15.12 40.21 18.90 29.17 17.13
M (n an duration n) irowth 0.41 0.27 0.31 0.15 0.27 0.12 0.26 0.14
ihortertrB 0.20 0.07 0.20 0.07 0.21 0.09 0.23 0.29
.ttenuation 0.58 0.38 0.73 0.36 0.45 0.32 0.56 0.43
% !me spent Srowth 41.83 29.66 35.31 33.07
hortenina 18.57 20.53 25.19 25.96
( kttenualten 39.60 49.81 39.50 40.97
Fti >q. of(ml»-1) ^atastrophe 1.15 1.27 1.52 1.57
1 lescue 3.05 2.79 1.90 2.95
D' n. (urn/rain) 9.87 10.07 13.87 11.31
MMl s/cetls 16/30 nutes 40.66 11/33 43.69 13/39 39.62 8/26 35.29

Table 25. jiMT dynamic instability at the mitotic block.

Mi tan rates (pm/min) Jrowth Control . 9.03 . sd 4.66 NPI-2358 10.63 sd 6.78 CM 11.21 sd 5.64
Shortening 32.85 18.06 34.06 1Z84 29.88 18.87
M tan duration (mln) Jrowth ' 0.41 0.27 0.30 0.18 0.33 0.18
ihorteninn 0.20 0.07 0.22 0.08 0.22 0.08
attenuation 0.58 0.38 0.61 0.41 0.55 0.44
% time spent Growth 41.83 33.02 43.64
Shortening 18.57 20.92 20.13
^ttenuatton 39.60 46.06 36.23
Fi jo., of (rrtn-1 ) ;atastroorie 1.15 1.22 1.09
?escue 3.05 2.60 2.79
D namidty'fpm/mln) 9.87 10.64 10.91
MM s/cells lutes 30/16 40.66 25/11 33.13 29/9 39.14
[OSS3] The examples described above are set forth solely to assist in the understanding of the invention. Thus, those skilled in the art will appreciate that the disclosed Methods and compounds encompass and may otherwise provide further derivatives of dehydrcjiphenylahistins.
IBB

[0554] One skilled in the art would readily appreciate that the present invention is well adapted to obtain, for example, the ends and advantages mentioned, as well as others inherent. The methods and procedures described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention.
[0555] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
[0556] As noted above, all patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are hereby incorporated by reference herein to the extent; allowable by law, such that each individual patent and publication may be treated as specifically and individually indicated to be incorporated by reference.
[0557] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically
disclosed herein. The terms and expressions which have been employed are used as terms of
i description and not of limitation, and there is no intention that in the use of such! terms and
expressions indicates the exclusion of equivalents of the features shown and defscribed or portions thereof. It is recognized that various modifications are possible within thfs scope of the invention. Thus, it should be understood that although the present inventioi| has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in tjjie art, and that such modifications and variations are considered to be falling within the of the invention.



WE CLAIM:
1. A diketopiperazine having the structure of Formula (I):
(Formula Removed)
wherein
R1, R1' and R1'is selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C1C6 alkyl, unsaturated C2-C6 alkenyl, C5-C12 cycloalkyl, C2-C6 cycloalkenyl, C1-C6 alkoxy, amino, nitro, hydroxy, cyano, C1-C6 alkylthio;
R4 is a C1-6 alkyl group;
R6 is a hydrogen atom;
R1, R1' and R1" are either covalently bound to one another or are not covalently bound to one another;
R2, R3, and R5 are each a hydrogen atom;
X1 and X2 are an oxygen atom;
Y is a nitrogen atom;
n is an integer equal to zero or one;
Z, for each separate n, if non-zero, and Z1, Z2, Z3 and Z4 are each separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom; and
the dashed bonds may be either single or double bonds;
and wherein said compound induces vasculature collapse.
2. The diketopiperazine as claimed in claim 1, wherein R4 is a C1-C4 alkyl group.
3. The diketopiperazine as claimed in claim 1, wherein
R1, R1', and R1" are each separately selected from the group consisting of a hydrogen atom, C1-4 alkyl, cyclohexyl, trifluoromethyl, ethenyl, thiomethyl, trifluoromethoxy, methoxy, ethoxy, hydroxy, nitro, cyano, amino, and a halogen;
R4 is a tert-butyl group; and
Z, for each separate n, if non-zero, and Z1, Z2, Z3 and Z4 are each separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom.
4. The diketopiperazine as claimed in claim 1, wherein n is one and Z, for each separate n, if non-zero, and Z1, Z2, Z3 and Z4 are each separately selected from a carbon atom.
5. The diketopiperazine as claimed in claim 4, wherein n is one and Z, for each separate n, if non-zero, and Z1, Z2, Z3 and Z4 are each separately selected from a carbon atom to form a six-membered aromatic ring.
6. The diketopiperazine as claimed in claim 1, wherein R1, R1', and R1" are the same substituent.
7. The diketopiperazine as claimed in claim 6, wherein R1, R1, and R1" are a hydrogen atom.
8. The diketopiperazine as claimed in claim 1, wherein two of R1, R1', and R1" are the same substituent.
9. The diketopiperazine as claimed in claim 1, wherein the C1-C6 alkyl is a methyl or t-butyl substituent.
10. The diketopiperazine as claimed in claim 1, wherein the halogen atom is chlorine.
11. The diketopiperazine as claimed in claim 1, wherein Z, for each separate n, if non-zero, and Z1, Z2, Z3 and Z4 collectively form a pyridine ring, a thiophene ring, or a furanyl ring.
12. The diketopiperazine as claimed in claim 1, wherein at least one of R1, R1',
and R1" is a methoxy substituent.
13. The diketopiperazine as claimed in claim 1, wherein n is equal to zero.
14. The diketopiperazine as claimed in claim 1, wherein n is equal to one.
15. The diketopiperazine as claimed in claim 1, wherein the compound is
selected from the group consisting of KPU-2, KPU-6, KPU-8, KPU-9, KPU-10, KPU-11,
KPU-12, KPU-13, KPU-14, KPU-15, KPU-16, KPU-17, KPU-18, KPU-19, KPU-20,
KPU-21, KPU-22, KPU-23, KPU-24, KPU-25, KPU-26, KPU-27, KPU-28, KPU-29,
KPU-30, KPU-31, KPU-32, KPU-33, KPU-34, KPU-35, KPU-36, KPU-37, KPU-38,
KPU-39, KPU-40, KPU-41, KPU-42, KPU-43, KPU-44, KPU-45, KPU-46, KPU-47,
KPU-48, KPU-49, KPU-50, KPU-51, KPU-52, KPU-53, KPU-54, KPU-55, KPU-56,
KPU-57, KPU-58, KPU-59, KPU-60, KPU-61, KPU-62, KPU-63, KPU-64, KPU-65,
KPU-66, KPU-67, KPU-68, KPU-69, KPU-77, KPU-79, KPU-80, KPU-81, KPU-82,
KPU-83, KPU-84, KPU-85, KPU-86, KPU-87, KPU-88, KPU-89, KPU-90, KPU-91,
KPU-92, KPU-93, KPU-94, KPU-95, KPU-96, and KPU-97.
16. The diketopiperazine as claimed in claim 1, wherein the compound is KPU-
2.
17. A pharmaceutical composition comprising a pharmaceutically effective amount of diketopiperazine as claimed in claim 1 together with a pharmaceutically acceptable carrier therefore.

Documents:

4720-DELNP-2006-Abstract-(01-05-2012).pdf

4720-delnp-2006-abstract.pdf

4720-delnp-2006-assignment.pdf

4720-DELNP-2006-Claims-(01-05-2012).pdf

4720-delnp-2006-claims.pdf

4720-DELNP-2006-Correspondence Others-(01-05-2012).pdf

4720-DELNP-2006-Correspondence Others-(27-12-2011).pdf

4720-delnp-2006-correspondence-others.pdf

4720-delnp-2006-description (complete).pdf

4720-delnp-2006-drawings.pdf

4720-DELNP-2006-Form-1-(01-05-2012).pdf

4720-delnp-2006-form-1.pdf

4720-DELNP-2006-Form-2-(01-05-2012).pdf

4720-delnp-2006-form-2.pdf

4720-delnp-2006-form-26.pdf

4720-DELNP-2006-Form-3-(27-12-2011).pdf

4720-delnp-2006-form-3.pdf

4720-DELNP-2006-Form-5-(01-05-2012).pdf

4720-delnp-2006-form-5.pdf

4720-DELNP-2006-GPA-(01-05-2012).pdf

4720-delnp-2006-pct-101.pdf

4720-delnp-2006-pct-210.pdf

4720-DELNP-2006-Petition-137-(01-05-2012).pdf

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Patent Number 253042
Indian Patent Application Number 4720/DELNP/2006
PG Journal Number 25/2012
Publication Date 22-Jun-2012
Grant Date 19-Jun-2012
Date of Filing 17-Aug-2006
Name of Patentee NEREUS PHARMACEUTICALS, INC.
Applicant Address 10480 WATERIDGE CIRCLE, SAN DIEGO, CA 92121 (US)
Inventors:
# Inventor's Name Inventor's Address
1 PALLADINO, MICHAEL, A. 3357 FORTUNA RANCH ROAD, OLIVENHAIN, CA 92024 (US).
2 LLOYD, GEORGE, KENNETH 13932 DODDER COURT, POWAY, CA 92064 (US).
3 NICHOLSON, BENJAMIN 3936 GOLDFINCH STREET, SAN DIEGO, CA 92103 (US),
4 HAYASHI, YOSHIO 3-9-18-301 HESO, RITTO, SHIGA 520-3031 (JP),
PCT International Classification Number C07D 403/06
PCT International Application Number PCT/US2005/003636
PCT International Filing date 2005-02-04
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/542,073 2004-02-04 U.S.A.
2 60/624,262 2004-11-01 U.S.A.