Title of Invention	C2-FLUORO SUBSTITUTED PIPERAZINE LINKED PYRROLO[2,1-C)[1,4]BENZODIAZEPINE DIMERS AND A PROCESS FOR THE PREPARATION THEREOF
Abstract	The present invention provides a compounds of general formula IXa-d, useful as potential antitumour agents and pharmaceutical composition comprising these compounds exhibits binding affinity with calf thymus (CT) DNA at a molar ratio of 1:5 in aqueous sodium phosphate buffer at pH of 7.00. The present invention further provides a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1 c][1,4]benzodiazepine of formula IX.

Title of Invention

C2-FLUORO SUBSTITUTED PIPERAZINE LINKED PYRROLO[2,1-C)[1,4]BENZODIAZEPINE DIMERS AND A PROCESS FOR THE PREPARATION THEREOF

Abstract

The present invention provides a compounds of general formula IXa-d, useful as potential antitumour agents and pharmaceutical composition comprising these compounds exhibits binding affinity with calf thymus (CT) DNA at a molar ratio of 1:5 in aqueous sodium phosphate buffer at pH of 7.00. The present invention further provides a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1 c][1,4]benzodiazepine of formula IX.

Full Text	FIELD OF THE INVENTION The present invention relates to C2-Fluoro substituted piperazine linked pyrrolo2,1-c][1,4]benzodiazepine dimers of formula IX (Formula removed) The present invention also relates to a process for the preparation of pyrrolo[2,1-c][1,4benzodiazepine. More particularly, it provides a process for the preparation of 1,1 '-{[(bisalkane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], and 1,1 '-{[(bisalkane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one, with aliphatic chain length variations (n = 3-10) for the compounds and it also describes the DNA-binding ability and anticancer (antitumour) activity. BACKGROUND OF THE INVENTION Pyrrolo[2,1-c][1,4]benzodiazepine antitumour antibiotics are commonly known as anthramycin class of compounds. In the last few years, a growing interest has been shown in the development of new pyrrolo[2,1-c][1 ^benzodiazepines (PBDs). These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within the minor groove of duplex DNA via an acid-labile aminal bond to the electrophilic imine at the N10-C11 position (Kunimoto, S.; Masuda, T.; Kanbayashi, N.; Hamada, M.; Naganawa, H.; Miyamoto, M.; Takeuchi, T.; Unezawa, H. J. Antibiot, 1980, 33, 665.; Kohn, K. W. and Speous, C. L. J. Mol. Biol., 1970, 51, 551.; Hurley, L. H.; Gairpla, C. and Zmijewski, M. Biochem. Biophys. Acta. 1977, 475, 521.; Kaplan, D. J. and Hurley, L. H. Biochemistry, 1981, 20, 7572). The molecules have a right-handed twist, which allows them to follow the curvature of the minor groove of B-form double-stranded DNA spanning three base pairs. A recent development has been the linking of two PBD units through their C-8 positions to give bisfunctional-alkylating agents capable of cross-linking DNA (Thurston, D. E.; Bose, D. S.; Thomson, A. S.; Howard, P. W.; Leoni, A.; Croker, S. J.; Jenkins, T. C; Neidle, S. and Hurley, L H. J. Org. Chem. 1996, 61, 8141). (Formula removed) Recently, PBD dimers have been developed that comprise of two C2-exo-methylene substituted DC-81 subunits tethered through their C-8 position via an inert propanedioxy linker (Gregson, S. J.; Howard, P. W.; Hartely, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T. C; Kelland, L. R. and Thurston, D. E. J. Med. Chem. 2001, 44, 737). A non-cross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent antitumour activity (Kamal, A.; Ramesh, G. Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu, V. B.; Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679). Recently, some new pyrrolobenzodiazepine (PBD) hybrids have been synthesized that have significant DNA binding ability and potent antitumour activity(Kamal, A.; Srinivas, O.; Ramulu, P.; Ramesh, G.; Kumar, P. P. Bioorg. Med. Chem. Lett. 2003, 13, 3577). Recently mono-fluoro and difluoro pyrrolo[2,1-c][1 ,4]benzodiazepines have been synthesized (O'Neil, A.;Thomoson.S.; Kalindjian, S. B.; Jenkins, T.C.; Tetrahedron Letf.2003, 44, 7809). Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines belong to a group of antitumour antibiotics derived from Streptomyces species. Recently, there is much impetus for the PBD systems as they can recognize and bind to specific sequence of DNA. Examples of naturally occurring PBDs include anthramycin, DC-81, tomaymycin, sibiromycin and neothramycin. However, the clinical efficacy for these antibiotics is hindered by several limitations, such as poor water solubility, cardio toxicity, development of drug resistance and metabolic inactivation. OBJECTIVES OF THE INVENTION The main object of the present invention is to provide C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines useful as antitumour agents. Yet another object of this invention is to provide pharmaceutical compositions comprising C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4] benzodiazepines useful as anti-cancer agents. Yet another object of this present invention is to provide C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines as potential DNA-binding agents. Yet another object of this invention is to provide a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4] benzodiazepines. SUMMARY OF THE INVENTION According, the present invention provides C2-Fluoro substituted piperazine linked pyrrolo[2,1 c][1,4]benzo- diazepine of formula IX. (Formula removed) In an embodiemnt of the present invnetion the compound C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine of general formula IX is represented by the group of following compounds: 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one](IXb); 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one](IXc); 1,1'-{[(bisProPane-1,N-diy\|)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd); 1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) and 1,1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5/-/-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf). In yet another embodiment the compound C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers, exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group of lung, colon, breast, ovarian, leukemia, Renal, Melanoma, Prostate and CNS cell lines In yet another embodiment the compound C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers (IXa-f) exhibits binding affinity with calf thymus (CT) DNA at a molar ratio of about 1:5 in aqueous sodium phosphate buffer at pH of 7.00. The preset invention further provides a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine formula IX (Formula removed) and the said process comprising the step of: (a) Preparing (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)4,fluorinated- pyrrolidine-2-carboxaldehydediethylthioacetal of formula IV by known method. (Formula removed) (b) reacting the compound of formula IV obtained in step (a) with dibromoalkane in a dry aprotic water miscible organic solvent, in the presence of mild inorganic base, under reflux, for a period up to 48 hours, purifying the resultant crude product by known method to obtain the compound of (2S)-N-[4-n-bromoalkoxy)-5-methoxy-2-nitrobenzoyl)- 4,fluorinated-pyrrolidine-2-carboxaldehydediethylthioacetal offormulaV. (Formula removed) (c) reacting the compound of formula V obtained in step (b) with piperazine of formula VI in a dry aprotic water miscible organic solvent, in the presence of mild inorganic base, under reflux, for a period of 45-48 hours, followed by pouring the resultant reaction mixture on to the water and extracting and purifying the resultant crude product by known method to obtain the compound 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis (11aS)-2-fluorineted-7-methoxy-2- nitrobenzoylpyrrolidin-2-carboxaldehydediethyl thioacetal] of formula VII (Formula removed) (d) reducing the compound of formula VII obtained in step (c) with SnCI2. 2H2O in an organic solvent, under reflux, for a period of 1-2 hours, at a pH of 8 in the presence of saturated alkalibicorbonate solution, followed by extraction with an organic solvent and drying the resultant organic phase over Na2SO4 and evaporating the solvent under vacuum to obtain the resultant compound. 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis(11 aS)-2-fluorineted-7-methoxy-2-amino benzoylpyrrolidin-2-carboxaldehyddi- ethylthioacetal] of formula VIII. (Formula removed) (e) reacting the compound of formula VIII with mercurous chloride and calcium carbonate in the presence of an aqueous organic solvent wherein organic solvent to water ratio is about 4:1, under stirring, at a temperature of 25-30°C, for a period of about 12 hours, followed by the evaporation of organic layer to obtain the crude residue and purifying the residue by known method to obtain the desired product of 1,1'-{[(bisalkane-1 ,Ndiyl)piperazine]dioxy}bis[(11 aS)-2-fluorinated-7-methoxy-1,2,3,11a-tetra- hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI. (Formula removed) wherein R=R1=F or F2; n= 3-10 In yet another embodiment the dibromoalkane used in step (a) is selected from the group consisting of 1,3-dibromopropane, 1,4-dibromobutane and 1,5- dibromopantane. In yet another embodiment the dry organic solvent used in step (b) and (c) is selected from acetone, acetonitrile and DMF. In yet another embodiment the mild inorganic base used in step (b) and (c) is selected from K2CO3, BaCO3 and CsCO3. In yet another embodiment the compound of formula VII used in step (c) is selected from the group consisting of 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2- nitrobenzoyl pyrrolidin-2-carboxaldehydediethylthioacetal] (Vila); 1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy- 2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllb); 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2- nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vile); 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy- 2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlld); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-2-difluoro-7-methoxy-2- nitrobenzoylpyrrolidin-2-carboxaldehyd diethylthioacetal] (Vile) and 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy- 2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllf). In yet another embodiment the compound of formula VIII obtained in step (d) is selected from the group consisting of 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Villa); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllb); 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllc); 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllld); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllle)and 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllf). In yet another embodiment the organic solvent used in step (d) are ethyl acetate, chloroform and methanol. In yet another embodiment the alkalibicorbonate used in step (d) is sodiumbicorbonate. In yet another embodiment the organic solvent used in step (d) is methanol. In yet another embodiment the organic solvent used in step (e) is acetonitrile. In yet another embodiment the cpmpound 1,1'—[[(bisalkane-1 ,N-diyl) piperazine]dioxy}bis[(11aS)-2-fluorinated-7-metoxy-1,2,3,11 a-tetrahyd ro-5H- pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI obtained in step (e) is represented by a group of following compounds: 1,1 '-{[(bispropane-1, N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy 1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa); 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXb); 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy 1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1 -c][ 1,4]benzodiazepine-5-one] (IXc); 1,1'-([(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5W-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd); 1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) And 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf). In still another embodiment A pharmaceutical composition useful as anti tumor agent comprising an effective amount of one or more analogues 1,1'-{[(bisalkane- 1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3, -11 a-tetrahyd ro-5H- pyrrolo[2,1-c][1,4]benzodiazepine-5-one] or 1,1'-{[(bisalkane-1 ,N-diyl) piperazine]bis(11aS)-2-2-difluoro-7-methoxy-1,2,3,11 a-tetrahyd ro-5H-pyrrolo [2,1-c][1,4]benzodiazepine-5-one optionally along with pharmaceutically acceptable additives. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepines of formula (IXa-f) of the drawing accompanying the specification where n is 3 to 10 which comprises: methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluori natedpyrrolidine-2-carbonate of formula I was reduced with DIBAL-H in presence of organic solvent like CH2CI2 cooled to -78 °C for a period of 45 min isolating methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2-carboxaldehyde II by conventional methods, protecting the above compound of formula II with EtSH in presence of organic solvent at room temperature isolating the methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidi- ne-2-carboxaldehydediethylthioacetal III by known methods, reacting the above said thio compound of formula III with known debenzylating agents in a conventional manner to give (2S)-N-[4-hydroxy-5- methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2-carboxaldehydediethylthioacetal of formula IV. Accordin-gly, the present process provides a process for preparation for C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines of formula of the drawing accompanying the specification where n is 3 to 10 which comprises: reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2- carboxaldehydediethylthioacetal of formula IV dibromoalkanes in an aprotic water miscible organic solvent like acetone, acetonitrile, THF, and DMF in presence of a mild inorganic bases like K2CO3, CSCO3, and BaCO3 upto refluxing temperature for a period upto 48 hours, isolating (2S)-N-[4-(n-bromoalkoxy)-5-methoxy-2-nitrobenzoyl]- 4-fluorinatedpyrrolidine-2-carboxaldehydediethylthio- acetal of formula V with piperazine of formula VI in presence of mild inorganic bases like K2CO3, CSCO3, and BaCO3 and in the presence of aprotic water miscible organic solvent up to refluxing for a period of 48 hours isolating 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis(11aS)-2-fluorineted-7-methoxy-2-nitrobenzoylpyrrolidin-2- carboxaldehyddiethylthioactal] VII where n is 3 to 10 by conventional method, reducing the above nitro compound of formula VII with SnCl2 .2H2O in presence of organic solvent upto a reflux temperature, isolating the 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis(11aS)-2-fluorineted-7-methoxy-2-aminobenzoylpyrrolidin-2- carboxaldehydediethylthioactal] of where n is 3 to 10 by know methods, reacting the above said amino compound of formula VIII with known deprotecting agent in a conventional manner to give novel 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis[(11 aS)-2-fluorinated-7-methoxy-1,2,3, 11 a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] of formula IX where in n are as stated above. The precursor, methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2-carbonate of formula I (intermediate of DC-81) was prepared by literature method (Dc Luca, L; Giacomelli, G.; Porcheddu, A.Org. Lett. 2001,3, 3041; Demange, L; Menez, A.; Dugave, C. Tetrahedron. Lett. 1998, 39, 1169; Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R. Bioorg. Med. Chem. Lett. 2004, 14,2669; ; Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.; Laxman, E.; Murthy, Y. L. N. Bioorg. Med. Chem. Lett. 2004, 14, 5699; Thurston, D.E.; Murthy, V. S.; Langley, D.R.; Jones, G.B. Synthesis, 1990, 81). Same representative compound of formula IX present invention are given below 1) 1,1 '-{[(bisProPane-1,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy 1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] 2) 1,1 '-{[(b'sbutane-l ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] 3) 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-metoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] 4) 1.1 '-{[(bispropane-1,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7- methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] 5) 1,1 '-{[(biSDutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] 6) 1,1 '-{[(Dispentane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]. These new analogues of pyrrolo[2,1-c][1,4]benzodiazepines dimers substituted at C2-position linked at C8 position through piperazine moiety have shown promising anticancer activity in various cell lines. The molecules synthesized are of immense biological significance with potential sequence selective DNA-binding property. This resulted in design and synthesis of new congeners as illustrated in Schemel and Scheme 2, which comprise: 1. Ether linkage at C-8 position fluoro substituted at C2-position of DC-81 intermediates with piperazine moiety 2. Refluxing the reaction mixture for 24-48 h. 3. Synthesis of C8-linked C2-fluorosubstituted PBD antitumour antibiotic dimer imines. 4. Purification by column chromatography using different solvents like ethyl acetate, hexane, dichloromethane, chloromethane, and methanol. The process of preparation of new non-cross linking C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine is disclosed and claimed in applicant's co-pending application no. (Formula removed) The following examples are given by way of illustration and therefore should not be construed to the present limit of the scope of invention. Example 1 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-corboxaldehydediethylthioacetal IV (418mg, 1mmol), 1,3-dibromopropane (0.365ml, 3mmol) and K2CO3 (825mg, 5mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-corboxaldehydediethylthioacetal of formula V (432mg, 82%). 1H NMR: (CDCI3, 200 MHz): 5 1.31-1.40 (m, 6H), 2.28-2.48 (m, 2H), 2.49-2.64 (m, 2H), 2.68-2.91 (m, 6H), 3.64 (m, 2H), 3.99 (s, 3H), 4.25 (t, 2H, J = 6.0), 4.56 (d, 1H, J = 6.7), 4.76 (m, 1H), 5.0-5.33 (m, 1H), 6.88 (s, 1H), 7.68 (s, 1H) LCMS: m/z 539.4 (M++Na). A solution of (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (539 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg, 10mmol) in dry acetone (20 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal] (441 mg, 82%). 1H NMR (CDCI3, 200 MHz): 5 1.29-1.36 (m, 12H), 2.02-2.11 (m, 4H), 2.46-2.69 (m, 12H), 2.73-2.89 (m, 8H), 3.39-3.62 (m, 4H), 3.93-3.94 (t, 4H), 4.17 (t, 4H), 4.52 (d, 2H, J = 6.79), 4.72 (q, 2H, J = 6.79), 5.07-5.29 (m, 2H), 6.84 (s, 2H), 7.65 (s, 2H) ESIMS: m/z 1003 (M+). The 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro7-methoxy-2- nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1004 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCI2.2H2O (2.25mg, 10 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 ml_). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy} bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethyl- thioacetal](803mg, 80%). A solution of the 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal].of formula VIII (944mg, 1 mmol), HgCl2 (1035mg, 5 mmol) and CaCO3 (500mg, 5 mmol)in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude. 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula IXa, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (613mg, 65%). 1H NMR (CDCI3, 200 MHz): 51.73-2.1 (m, 8H), 2.45-2.76 (m, 8H), 3.46-3.91 (m, 10H), 3.93 (s, 6H), 4.02-4.25 (m, 4H), 5.35-5.48 (dt, 2H), 6.85 (s, 2H), 7.49 (s, 2H), 7.86 (d, 2H, J = 3.66 Hz) ESIMS: m/z 695 (M++1). Example 2 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal IV (418 mg, 1 mmol), 1,4-dibromobutane (0.35ml 3 mmol) and K2CO3 (675mg, 5mmol) in dry acetone (30 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexan (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexan (1:1) gave the pure (2S)-/V-[4-(5-bromobutanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (355mg, 85%). 1H NMR: (CDCI3, 200 MHz): 6 1.26-1.43 (m, 6H), 2.01-2.46 (m, 4H), 2.49-2.67 (m, 2H), 2.70-2.95 (m, 6H), 3.58 (m, 2H), 3.99 (s, 3H), 4.25 (t, 2H, J = 6.0), 4.55 (d, 1H, J = 6.7), 4.73-4.79 (m, 1H), 5.0-5.33 (m, 1H), 6.89 (s, 1H), 7.69 (s, 1H) LCMS: m/z 553 (M+). A solution of (2S)-N-[4-(5-bromobutanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehyde diethyl thioacetal of formula V. (553 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (4014 mg, 10 mmol) in dry acetone (30 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bibutane-1,N-diyl) piperazine]dioxy}bis(11aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carbox-aldehyddiethylthioactal] (447 mg, 81%). 1H NMR (CDCI3, 200 MHz): 5 1.29-1.41 (m, 12H), 1.7-1.97 (m, 8H), 2.33-2.70 (m, 12H), 2.71-2.89 (m, 8H), 3.39-3.66 (m, 4H), 3.90-3.93 (t, 4H), 3.96 (s, 6H), 4.1 (t, 4H), 4.5 (d, 2H, J = 7.5), 4.2 (q, 2H, J = 6.79), 5.0-5.27 (m, 2H), 6.84 (s, 2H), 7.60 (s, 2H). The 1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1032 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCI2.2H20 (2025 mg, 10 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 ml_). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy} bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethyl thioactal] (825mg, 80%). A solution of the 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. of formula VIM (968 mg, 1 mmol), HgCI2 (1035 mg, 5 mmol) and CaCO3 (500 mg, 5 mmol) in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula IXb, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (580 mg, 60%). 1H NMR (CDCI3, 200 MHz): 5 1.26-1.94 (m, 8H), 2.11-2.47 (m, 4H), 2.59-2.75 (m, 8H), 3.48-3.86 (m, 10H), 3.93 (s, 6H), 4.0-4.25 (m, 4H), 5.27-5.55 (m, 2H), 6.82 (s, 2H), 7.49 (s, 2H), 7.85 (d, 2H, J = 4.4 Hz). Example 3 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal IV (418mg, 1 mmol), 1,5-dibromopantane (0.5 ml, 0.5 mmol) and K2CO3 (690 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(6-bromopentanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (355 mg, 85%). 1H NMR: (CDCb 200MHz): 5 1.26-1.41 (m, 6H), 1.74-2.0 (m, 4H), 2.35-2.47 (m, 2H), 2.49-2.65 (m, 2H), 2.70-2.92 (m, 6H), 3.58-3.66 (m, 2H), 3.98 (s, 3H), 4.25 (t, 2H, J = 6.0), 4.57 (d, 1H, J = 6.7), 4.75-4.85 (m, 1H), 5.0-5.34 (m,1H), 6.88 (s, 1H). 7.70 (s, 1H)LCMS:m/z567(M+). A solution of (2S)-N-[4-(6-bromopentanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (567 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10mmol) in dry acetone (40 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bipentane-1,N-diyl) piperazine]dioxy}bis(11aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carbox- aldehydediethylthioactal] (453 mg, 80%). 1H NMR (CDCI3, 200 MHz): 6 1.25-1.36 (m, 12H), 1.39-1.89 (m, 12H), 2.25-2.61 (m, 12H), 2.70-2.86 (m, 8H), 3.39-3.63 (m, 4H), 3.96 (s, 6H), 4.0 (t, 4H), 4.12 (t, 4H), 4.54 (d, 2H, J= 6.79), 4.75 (q, 2H, J= 6.0), 5.0-5.3 (m, 2H), 6.84 (s, 2H), 7.62 (s, 2H). The 1,1 '-{[(bipentane-1, N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1060 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCl2.2H2O (2.25 mg, 10 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispentane-1,/\/-diyl)piperazine] dioxyjbis (11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethyl- thioactal] (837 mg, 79%). A solution of the 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal].of formula VIII (1000 mg, 1 mmol), HgCI2 (1355 mg, 5 mmol) and CaCO3 (500 mg, 5 mmol)in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula IXc, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (560 mg, 56%). 1H NMR (CDCI3. 200 MHz): 6 1.44-2.09 (m, 12H), 2.30-2.51 (m, 4H), 2.52-3.0 (m, 8H), 3.47-3.87 (m, 10H), 3.93 (s, 6H), 4.0-4.1 (m, 4H), 5.27-5.58 (m, 2H), 6.9 (s, 2H), 7.49 (s, 2H), 7.9 (d, 2H, J = 4.6 Hz). Example 4 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine -2-carboxaldehydediethylthioacetal IV (436 mmol), 1,3-dibromopropane (0.3 ml, 3 mmol) and K2CO3 (690 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (370 mg, 85%). 1H NMR (CDCI3, 200 MHz): 5 1.36-1.42 (m, 6H), 2.39-2.47 (m, 2H), 2.62-2.95 (m, 6H), 3.48-3.58 (m, 2H), 3.64 (t, 2H, J = 6.0 Hz), 3.96 (s, 3H), 4.26 (t, 2H, J = 5.2 Hz), 4.82 (d, 1H), 4.89-4.96 (m, 1H), 6.77(s, 1H), 7.72 (s, 1H) LCMS: m/z 580 (M++23). A solution of (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoro pyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (557 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10mmol) in dry acetone (30 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bispropane-1,N-diyl) piperazine]dioxy}bis(11aS)-2,2-difluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal] (417 mg 75%). 1H NMR (CDCI3, 200 MHz): 6 1.25-1.39 (m, 12H), 2.0-2.14 (m, 4H), 2.58-2.66 (m, 8H), 2.69-2.88 (m, 12H), 3.45-3.79 (m, 8H), 3.94 (s, 6H), 4.1 (t, 4H), 4.78 (d, 2H), 4.85-4.96 (m, 2H), 6.7 (s, 2H), 7.6 (s, 1H) ESIMS: m/z 1039 (M+). The 1,1'-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2,2-difluoro7-methoxy -2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1039 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCl2.2H2O (2.25 mg, 10.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x20 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispropane-1,N-diyl)piperazine] dioxy} bis (11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydedieth yithioactal] (779 mg, 75%). A solution of the 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. Of formula VIII (976 mg, 1.0 mmol), HgCI2 (1355 mg, 5.0 mmol) and CaCO3 (686 mg, 5.0 mmol)in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula Xld, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCb-methanol (9:1) (745 mg, 55%). 1H NMR (CDCI3, 200 MHz): 6 1.45-1.92 (m, 4H), 1.99-2.20 (m, 4H), 2.48-2.99 (m, 8H), 3.1-3.88 (m, 10H), 3.96 (s, 6H), 3.98-4.24 (m, 4H), 6.80 (s, 2H), 7.49 (s, 2H), 7.82 (d, 2H, J = 3.8 Hz). Example 5 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4,4-difluoro pyrrolidine-2-corboxaldehyde diethylthioacetal IV (436, mg 1 mmol), 1,4-dibromobutane (0.35 ml, 3 mmol) and K2CO3 (690 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(5-bromobutanoxy)-5-methoxy-2-nitro benzoyl)-4,4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (353 mg.81%). 1H NMR (CDCI3, 200 MHz): 5 1.28-1.40(m, 6H), 2.0-2.5 (m, 4H), 2.58-2.79 (m, 6H), 3.51 (t, 2H), 3.75- (m, 2H), 3.96- (s, 3H), 4.10 (t, 2H), 4.79 (d, 1H), 4.85 (m, 1H), 6.74 (s, 1H) 7.6 (s, 1H) LCMS: m/z 594 (M++Na). A solution of (2S)-N-[4-(5-bromobutanoxy)-5-methoxy-2-nitrobenzoyl)-4,4-fluoro pyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (571 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10mmol) in dry acetone (40 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bisbutane-1,N-diyl) piperazine]dioxy}bis(11aS)-2,2-difluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehyddiethylthioactal] (485 mg, 85%). 1H NMR (CDCI3, 200 MHz): 5 1.33-1.48 (m, 12H), 1.66-1.98 (m, 8H), 2.40-2.50 (m, 8H), 2.63-2.94 (m, 12H), 3.42-3.83 (m, 8H), 3.92 (s, 6H), 4.11 (t, 4H), 4.77 (d, 2H), 4.83-4.94 (m, 2H), 6.72 (s, 2H), 7.62 (s, 2H) ESIMS: m/z 1067 (M+). The 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis(11aS)-2,2-difluoro7- methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1067 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCI2.2H2O (2.25 mg, 10.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x20 ml_). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1 '-{[(bisbutane-1 ,N-diyl) piperazine]dioxy} bis(11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehyde- diethylthioacetal] (810mg, 76%). A solution of the 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2- difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. Of formula VIII (1007 mg, 1 mmol), HgCI2 (1355 mg, 5.0 mmol) and CaCO3 (500 mg, 5 mmol) in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 1,1'-[[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7- methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula IXe, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (523 mg, 52%). 1H NMR (CDCI3, 200 MHz): 5 1.56-1.94 (m, 8H), 1.99-2.35 (m, 4H), 2.49-2.99 (m, 8H), 3.29-3.87 (m, 10H), 3.93 (s, 6H), 3.98-4.37 (m, 4H), 6.89 (s, 2H), 7.46 (s, 2H), 7.83 (d, 2H, J =3.67). Example 6 A solution of (2S)N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine -2-carboxaldehydediethylthioacetal IV (436 mg, 1 mmol), 1,5-dibromopentane (0.37ml 3 mmol) and K2CO3 (1380 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(6-bromopropoxy)-5-methoxy-2-nitro benzoyl)-4,4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (374 mg, 86%). 1H NMR (CDCI3,200 MHz): 5 1.06-1.36 (m, 6H), 1.40-2.1 (m, 4H), 2.58-2.88 (m, 6H), 3.52 (t, 2H), 3.70-3.97 (m, 2H), 3.97 (s, 3H), 4.15 (t, 2H), 4.80 (d, 1H), 4.91-5.02 (m, 1H), 6.75 (s, 1H), 7.6 (s, 1H) LCMS: m/z 608 (M++Na). A solution of (2S)-N-[4-(6-bromopentanoxy)-5-methoxy-2-nitrobenzoyl)-4,4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (585 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10 mmol) in dry acetone (40 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bispentane-1,N-diyl) piperazine]dioxy}bis(11aS)-2,2- difluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal] (462 mg, 79%). 1H NMR (CDCI3,200 MHz): 6 1.34-1.40 (m, 12H), 1.47-1796 (m, 12H), 2.36-2.49 (m, 4H), 2.50-2.66 (m, 8H), 2.68-2.90 (m, 8H), 3.37-3.80 (m, 8H), 3.94 (s, 6H), 4.08 (t, 4H), 4.77(d,2H), 4.85-4.91 (m, 2H), 6.72 (s, 2H), 7.63 (s, 2H) ESIMS: m/z 1095 (M+). The 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2,2-difluoro7-methoxy -2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1095 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCl2.2H2O (2.25 mg, 10.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy} bis(11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydedi- ethylthioactal] (744 mg, 68%). A solution of the 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-di-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. Of formula VIII (1035 mg, 1 mmol), HgCI2 (1355 mg, 5 mmol) and CaC03 (500 mg, 5.0 mmol) in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHC03 was added slowly at room temperature and the mixture is filtered through celite and washed with ethyl acetate. The filterate is evaporated in vacuum to get crude 1,1'-{[(bispentane-1,N-diyl)piperazine] dioxy} bis [(11aS)-2-2-difluoro-7-metoxy-1,2,3,11a-tetrahydro-5/7-pyrrolo[2,1-c][1,4]benzo- diazepine-5-one], of formula IXf, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (569 mg, 55%). 1H NMR (CDCI3,200 MHz): 6 1.54-2.03 (m, 12H), 2.22-2.47 (m, 4H), 2.50-2.73 (m, 8H), 3.35-3.85 (m, 10H), 3.94 (s, 6H), 3.97-4.26 (m, 4H), 6.71 (s, 2H), 7.47 (s, 2H), 7.79 (d, 2H J = 3.6) ESIMS: m/z 787 (M++H). Biological Activity: some of in vitro biological activity studies were carried out at the National Cancer Institute, Maryland, USA. Cytotoxicity: The compounds (IXa) 1,1'-{[(bispropane-1,N-diyl)piperazine] dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1 -c][1,4] benzodiazepine-5-one] and (IXd) 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis [(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1 c][1,4]benzo diazepine-5-one]. The above compounds were evaluated for in vitro anticancer activity against sixty human tumor cells derived from nine cancer types (leukemia, non small cell cancer, colon cancer CNS cancer, melanoma, ovarian cancer renal cancer, prostate cancer and breast cancer) as per NCI protocol. For each compound, dose response curves for each cell line were measured at a minimum of five concentrations at 10-fold dilution. A protocol of 48h continuous drug exposure was used, and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The concentration causing 50%cell growth inhibition (GI50), total cell growth inhibition (TGI, 0% growth) and 50% cell death (LC -50% growth) compared with the control was calculated. The mean graph midpoint values of log10TGI and log10 LC50 as well as log10 GI50 for IXa and IXd are listed in table 1. As demonstrated by mean graph pattern compounds IXa and IXd are exhibit an interesting profile of activity and selectivity for various cell lines. The mean graph midpoint of log10TGI and log10 LC50 showed similar pattern to the logio GI50 mean graph midpoints. Table 1 log10 GI50 log10TGI and log10LC50 mean graphs midpoint (MG_MID) of in vitro cytotoxicity data for the compounds IXa and IXd against human tumor cell lines. (Table removed) The in vitro anticancer activity for two representative compounds have given in table 2. Among them IXa exhibits a wide spectrum of activity against fifty nine cell lines in nine cell panels, with GI50 value of IG50 values against in leukemia cancer CCRF-CEM, SR, 9.79x10-7 uM and 8.82x10-7µM the cytotoxicity of IXa and IXd in selected cancer cell lines have been illustrated in Table 2. (Table removed) Thermal denaturation studies: The DNA binding affinity of the C2-Fluoro substituted piperazine linked pyrrolo[2,1c][1,4]benzodiazepine dimers were subjected to thermal denaturation studies using calf thymus (CT) DNA (Jones, G. B.; Davey, C. L; Jenkins, T. C; Kamal, A.; Kneale, G. G.; Neidle, S.; Webster, G. D.; Thurston, D. E. Anti-cancer Drug Des. 1990, 5, 249. McConnaughie, A. W.; Jenkins, T. C. J. Med. Chem. 1995, 38, 3488). The studies for these compounds (IXa-f) were carried out by DNA/ligand molar ratio is 1:5 the increase in the helix melting temperature (m) for each compound was examined at 0 h. The DNA biding activity for these novel C2-Fluoro substituted piperazine linked pyrrolo[2,1c][1,4] benzodiazepine dimers have been examined by thermal denaturation studies using calf thymus (CT) DNA melting studies shows that these compounds stabilize the Tm for CT-DNA at pH 7.0, incubated at 37 °C, were PBD/DNA molar ratio is 1:5 interestingly, in this assay all compounds of fluoro substituted dimer (IXa-f) elevates the melting temperature CT-DNA by margin of 11-38 ° C after incubation for at 37 °C Data for DSB-120 and SJG-136 are included in Table-3 for comparison. The synthetic DC-81 dimer (DSB-120) gives a Tm 10.2°C and SJC-136 gives a Tm of 25 °C under identical experimental condition. (Table removed) DNA alone at pH 7.00 c 0.01, Tm = 69.6°C+ 0.01 (mean value from 10 separate determinations), all Tm values are [0.1 ■ 0.2 °C. b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100 uM and ligand concentration = 20 uM in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01]; literature value of SJC-136 ADVANTAGES OF THE INVENTION 1. The present invention provides a C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers useful as antitumour agents. 2. It also provides a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers. We claim 1. C2-Fluoro substituted piperazine linked pyrrolo[2,1c][1,4]benzo- diazepine of formula IX. (Formula removed) 2. C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine as claimed in claim 1 is represented by the group of following compounds: 1,1 '-{[(Dispropane-I, N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa); 1,1 '-{[(bisbutane-1, N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXb); 1.1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXc); 1.1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) and 1,1 '-[[(bispentane-1 ,H-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf). 3. C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers as claimed in claim 1, exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group of lung, colon, breast, ovarian, leukemia, Renal, Melanoma, Prostate and CNS cell lines 4. A process as claimed in claim 3, wherein the C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers (IXa-f) exhibits binding affinity with calf thymus (CT) DNA at a molar ratio of about 1:5 in aqueous sodium phosphate buffer at pH of 7.00. 5. A process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine formula IX (Formula removed) and the said process comprising the step of: (a) Preparing (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)4,fluorinated- pyrrolidine-2-carboxaldehydediethylthioacetal of formula IV by known method. (Formula removed) (b) reacting the compound of formula IV obtained in step (a) with dibromoalkane in a dry aprotic water miscible organic solvent, in the presence of mild inorganic base, under reflux, for a period up to 48 hours, purifying the resultant crude product by known method to obtain the compound of (2S)-N-[4-n-bromoalkoxy)-5-methoxy-2-nitrobenzoyl)- 4,fluorinated-pyrrolidine-2-carboxaldehydediethylthioacetal offormulaV. (Formula removed) (c) reacting the compound of formula V obtained in step (b) with piperazine of formula VI in a dry aprotic water miscible organic solvent, in the presence of mild inorganic base, under reflux, for a period of 45-48 hours, followed by pouring the resultant reaction mixture on to the water and extracting and purifying the resultant crude product by known method to obtain the compound 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis (11aS)-2-fluorineted-7-methoxy-2- nitrobenzoylpyrrolidin-2-carboxaldehydediethyl thioacetal] of formula VII (Formula removed) (d) reducing the compound of formula VII obtained in step (c) with SnCI2. 2H2O in an organic solvent, under reflux, for a period of 1-2 hours, at a pH of 8 in the presence of saturated alkalibicorbonate solution, followed by extraction with an organic solvent and drying the resultant organic phase over Na2SO4 and evaporating the solvent under vacuum to obtain the resultant compound. 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis(11aS)-2-fluorineted-7-methoxy-2-amino benzoylpyrrolidin-2-carboxaldehyddi- ethylthioacetal] of formula VIII. (Formula removed) (e) reacting the compound of formula VIII with mercurous chloride and calcium carbonate in the presence of an aqueous organic solvent wherein organic solvent to water ratio is about 4:1, under stirring, at a temperature of 25-30°C, for a period of about 12 hours, followed by the evaporation of organic layer to obtain the crude residue and purifying the residue by known method to obtain the desired product of 1,1'-{[(bisalkane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fiuorinated-7-methoxy-1,2,3,11a-tetra- hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI. (Formula removed) wherein R=R1=F or F2; n= 3-10 6. A process as claimed in claim 5, wherein the dibromoalkane used in step (a) is selected from the group consisting of 1,3-dibromopropane, 1,4-dibromobutane and 1,5-dibromopantane. 7. A process as in claimed in claim 5, wherein the dry organic solvent used in step (b) and (c) is selected from acetone, acetonitrile and DMF. 8. A process as claimed in claim 5, wherein the mild inorganic base used in step (b) and (c) is selected from K2CO3, BaCO3 and CsCO3. 9. A process as claimed in claim 5 wherein the compound of formula VII used in step (c) is selected from the group consisting of 1,1'-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2- nitrobenzoyl pyrrolidin-2-carboxaldehydediethylthioacetal] (Vlla); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy- 2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllb); 1,1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2- nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlle); 1,1'-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy- 2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlld); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-2-difluoro-7-methoxy-2- nitrobenzoylpyrrolidin-2-carboxaldehyd diethylthioacetal] (Vlle) and 1,14[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllf). 10.A process as claimed in claim 5, wherein the compound of formula VIII obtained in step (d) is selected from the group consisting of 1,1'-{[(Dispropane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Villa); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllb); 1,1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllc); 1I1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllld); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllle)and 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllf). 11. A process as claimed in claim 5, wherein the organic solvent used in step (d) are ethyl acetate, chloroform and methanol. 12. A process as claimed in claim 5, wherein the alkalibicorbonate used in step (d) is sodiumbicorbonate. 13. A process as claimed in claim 5, wherein the organic solvent used in step (d) is methanol. 14. A process as claimed in claim 5, wherein the organic solvent used in step (e) is acetonitrile. 15. A process as claimed in claim 5, wherein the cpmpound 1,1'-{[(bisalkane-1,N- diyl) piperazine]dioxy}bis[(11 aS)-2-fluorinated-7-metoxy-1,2,3,11 a-tetrahydro- 5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI obtained in step (e) is represented by a group of following compounds: 1,1'-{[(bispropane-1 ,H-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy 1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa); 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXb); 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy 1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXc); 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd); 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) And 1-1'-{[(bisperitane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difIuoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf). 16. A pharmaceutical composition useful as anti tumor agent comprising an effective amount of one or more analogues 1,1'-{[(bisalkane-1,N- diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3, -11 a-tetrahydro-5H- pyrrolo[2,1-c][1,4]benzodiazepine-5-one] or 1,1 '-[[(bisalkane-1 ,N-diyl) piperazine]bis(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo [2,1-c][1,4]benzodiazepine-5-one optionally along with pharmaceutically acceptable additives. 17. C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers and preparation for the preparation thereof, substantially as herein described with reference to the examples accompanying the specification.

Full Text

FIELD OF THE INVENTION
The present invention relates to C2-Fluoro substituted piperazine linked pyrrolo2,1-c][1,4]benzodiazepine dimers of formula IX
(Formula removed)
The present invention also relates to a process for the preparation of pyrrolo[2,1-c][1,4benzodiazepine. More particularly, it provides a process for the preparation of 1,1 '-{[(bisalkane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], and 1,1 '-{[(bisalkane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one, with aliphatic chain length variations (n = 3-10) for the compounds and it also describes the DNA-binding ability and anticancer (antitumour) activity.
BACKGROUND OF THE INVENTION
Pyrrolo[2,1-c][1,4]benzodiazepine antitumour antibiotics are commonly known as anthramycin class of compounds. In the last few years, a growing interest has been shown in the development of new pyrrolo[2,1-c][1 ^benzodiazepines (PBDs). These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within the minor groove of duplex DNA via an acid-labile aminal bond to the electrophilic imine at the N10-C11 position (Kunimoto, S.; Masuda, T.; Kanbayashi, N.; Hamada, M.; Naganawa, H.; Miyamoto, M.; Takeuchi, T.; Unezawa, H. J. Antibiot, 1980, 33, 665.; Kohn, K. W. and Speous, C. L. J. Mol. Biol., 1970, 51, 551.; Hurley, L. H.; Gairpla, C. and Zmijewski, M. Biochem. Biophys. Acta. 1977, 475, 521.; Kaplan, D. J. and Hurley, L. H. Biochemistry, 1981, 20, 7572). The molecules have a right-handed twist, which allows them to follow the curvature of the minor groove of B-form double-stranded DNA spanning three base pairs. A recent development has
been the linking of two PBD units through their C-8 positions to give bisfunctional-alkylating agents capable of cross-linking DNA (Thurston, D. E.; Bose, D. S.; Thomson, A. S.; Howard, P. W.; Leoni, A.; Croker, S. J.; Jenkins, T. C; Neidle, S. and Hurley, L H. J. Org. Chem. 1996, 61, 8141).
(Formula removed)
Recently, PBD dimers have been developed that comprise of two C2-exo-methylene substituted DC-81 subunits tethered through their C-8 position via an inert propanedioxy linker (Gregson, S. J.; Howard, P. W.; Hartely, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T. C; Kelland, L. R. and Thurston, D. E. J. Med. Chem. 2001, 44, 737). A non-cross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent antitumour activity (Kamal, A.; Ramesh, G. Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu,
V. B.; Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679). Recently, some new pyrrolobenzodiazepine (PBD) hybrids have been synthesized that have significant DNA binding ability and potent antitumour activity(Kamal, A.; Srinivas, O.; Ramulu, P.; Ramesh, G.; Kumar, P. P. Bioorg. Med. Chem. Lett. 2003, 13, 3577). Recently mono-fluoro and difluoro pyrrolo[2,1-c][1 ,4]benzodiazepines have been synthesized (O'Neil, A.;Thomoson.S.; Kalindjian, S. B.; Jenkins, T.C.; Tetrahedron Letf.2003, 44, 7809).
Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines belong to a group of antitumour antibiotics derived from Streptomyces species. Recently, there is much impetus for the PBD systems as they can recognize and bind to specific sequence of DNA. Examples of naturally occurring PBDs include anthramycin, DC-81, tomaymycin, sibiromycin and neothramycin.
However, the clinical efficacy for these antibiotics is hindered by several limitations, such as poor water solubility, cardio toxicity, development of drug resistance and metabolic inactivation.
OBJECTIVES OF THE INVENTION
The main object of the present invention is to provide C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines useful as antitumour agents.
Yet another object of this invention is to provide pharmaceutical compositions comprising C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4] benzodiazepines useful as anti-cancer agents.
Yet another object of this present invention is to provide C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines as potential DNA-binding agents.
Yet another object of this invention is to provide a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4] benzodiazepines.
SUMMARY OF THE INVENTION
According, the present invention provides C2-Fluoro substituted piperazine linked pyrrolo[2,1 c][1,4]benzo- diazepine of formula IX.
(Formula removed)

In an embodiemnt of the present invnetion the compound C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine of general formula IX is represented by the group of following compounds:
1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one](IXb); 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one](IXc); 1,1'-{[(bisProPane-1,N-diy|)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd);
1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) and 1,1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5/-/-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf).
In yet another embodiment the compound C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers, exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group of lung, colon, breast, ovarian, leukemia, Renal, Melanoma, Prostate and CNS cell lines
In yet another embodiment the compound C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers (IXa-f) exhibits binding affinity with calf thymus (CT) DNA at a molar ratio of about 1:5 in aqueous sodium phosphate buffer at pH of 7.00.
The preset invention further provides a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine formula IX
(Formula removed)

and the said process comprising the step of:
(a) Preparing (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)4,fluorinated-
pyrrolidine-2-carboxaldehydediethylthioacetal of formula IV by known
method.
(Formula removed)

(b) reacting the compound of formula IV obtained in step (a) with
dibromoalkane in a dry aprotic water miscible organic solvent, in the
presence of mild inorganic base, under reflux, for a period up to 48
hours, purifying the resultant crude product by known method to obtain
the compound of (2S)-N-[4-n-bromoalkoxy)-5-methoxy-2-nitrobenzoyl)-
4,fluorinated-pyrrolidine-2-carboxaldehydediethylthioacetal offormulaV.
(Formula removed)

(c) reacting the compound of formula V obtained in step (b) with piperazine
of formula VI in a dry aprotic water miscible organic solvent, in the
presence of mild inorganic base, under reflux, for a period of 45-48
hours, followed by pouring the resultant reaction mixture on to the water
and extracting and purifying the resultant crude product by known
method to obtain the compound 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis (11aS)-2-fluorineted-7-methoxy-2-
nitrobenzoylpyrrolidin-2-carboxaldehydediethyl thioacetal] of formula VII
(Formula removed)

(d) reducing the compound of formula VII obtained in step (c) with SnCI2.
2H2O in an organic solvent, under reflux, for a period of 1-2 hours, at a
pH of 8 in the presence of saturated alkalibicorbonate solution, followed
by extraction with an organic solvent and drying the resultant organic
phase over Na2SO4 and evaporating the solvent under vacuum to
obtain the resultant compound. 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis(11 aS)-2-fluorineted-7-methoxy-2-amino
benzoylpyrrolidin-2-carboxaldehyddi- ethylthioacetal] of formula VIII.
(Formula removed)

(e) reacting the compound of formula VIII with mercurous chloride and
calcium carbonate in the presence of an aqueous organic solvent
wherein organic solvent to water ratio is about 4:1, under stirring, at a temperature of 25-30°C, for a period of about 12 hours, followed by the evaporation of organic layer to obtain the crude residue and purifying the residue by known method to obtain the desired product of 1,1'-{[(bisalkane-1 ,Ndiyl)piperazine]dioxy}bis[(11 aS)-2-fluorinated-7-methoxy-1,2,3,11a-tetra- hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI.
(Formula removed)

wherein R=R1=F or F2; n= 3-10 In yet another embodiment the dibromoalkane used in step (a) is selected
from the group consisting of 1,3-dibromopropane, 1,4-dibromobutane and 1,5-
dibromopantane.
In yet another embodiment the dry organic solvent used in step (b) and (c) is
selected from acetone, acetonitrile and DMF.
In yet another embodiment the mild inorganic base used in step (b) and (c)
is selected from K2CO3, BaCO3 and CsCO3.
In yet another embodiment the compound of formula VII used in step (c) is
selected from the group consisting of
1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-
nitrobenzoyl pyrrolidin-2-carboxaldehydediethylthioacetal] (Vila);
1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-
2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllb);
1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-
nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vile);
1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-
2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlld);
1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-2-difluoro-7-methoxy-2-
nitrobenzoylpyrrolidin-2-carboxaldehyd diethylthioacetal] (Vile) and
1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-
2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllf).
In yet another embodiment the compound of formula VIII obtained in step (d)
is selected from the group consisting of
1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Villa); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllb); 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllc); 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllld); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllle)and 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllf). In yet another embodiment the organic solvent used in step (d) are ethyl
acetate, chloroform and methanol.
In yet another embodiment the alkalibicorbonate used in step (d) is
sodiumbicorbonate.
In yet another embodiment the organic solvent used in step (d) is methanol. In yet another embodiment the organic solvent used in step (e) is acetonitrile. In yet another embodiment the cpmpound 1,1'—[[(bisalkane-1 ,N-diyl)
piperazine]dioxy}bis[(11aS)-2-fluorinated-7-metoxy-1,2,3,11 a-tetrahyd ro-5H-
pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI obtained in step (e) is
represented by a group of following compounds:
1,1 '-{[(bispropane-1, N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy 1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa); 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXb);
1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy 1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1 -c][ 1,4]benzodiazepine-5-one] (IXc); 1,1'-([(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5W-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd);
1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) And
1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf).
In still another embodiment A pharmaceutical composition useful as anti tumor
agent comprising an effective amount of one or more analogues 1,1'-{[(bisalkane-
1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3, -11 a-tetrahyd ro-5H-
pyrrolo[2,1-c][1,4]benzodiazepine-5-one] or 1,1'-{[(bisalkane-1 ,N-diyl)
piperazine]bis(11aS)-2-2-difluoro-7-methoxy-1,2,3,11 a-tetrahyd ro-5H-pyrrolo [2,1-c][1,4]benzodiazepine-5-one optionally along with pharmaceutically acceptable additives.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepines of formula (IXa-f) of the drawing accompanying the specification where n is 3 to 10 which comprises: methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluori natedpyrrolidine-2-carbonate of formula I was reduced with DIBAL-H in presence of organic solvent like CH2CI2 cooled to -78 °C for a period of 45 min isolating methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2-carboxaldehyde II by conventional methods, protecting the above compound of formula II with EtSH in presence of organic solvent at room temperature isolating the methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidi- ne-2-carboxaldehydediethylthioacetal III by known methods, reacting the above said thio compound of formula III with
known debenzylating agents in a conventional manner to give (2S)-N-[4-hydroxy-5-
methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2-carboxaldehydediethylthioacetal of
formula IV. Accordin-gly, the present process provides a process for preparation for
C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines of formula
of the drawing accompanying the specification where n is 3 to 10 which comprises:
reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2-
carboxaldehydediethylthioacetal of formula IV dibromoalkanes in an aprotic water
miscible organic solvent like acetone, acetonitrile, THF, and DMF in presence of a
mild inorganic bases like K2CO3, CSCO3, and BaCO3 upto refluxing temperature for a
period upto 48 hours, isolating (2S)-N-[4-(n-bromoalkoxy)-5-methoxy-2-nitrobenzoyl]-
4-fluorinatedpyrrolidine-2-carboxaldehydediethylthio- acetal of formula V with
piperazine of formula VI in presence of mild inorganic bases like K2CO3, CSCO3, and
BaCO3 and in the presence of aprotic water miscible organic solvent up to refluxing
for a period of 48 hours isolating 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis(11aS)-2-fluorineted-7-methoxy-2-nitrobenzoylpyrrolidin-2-
carboxaldehyddiethylthioactal] VII where n is 3 to 10 by conventional method,
reducing the above nitro compound of formula VII with SnCl2 .2H2O in presence of
organic solvent upto a reflux temperature, isolating the 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis(11aS)-2-fluorineted-7-methoxy-2-aminobenzoylpyrrolidin-2-
carboxaldehydediethylthioactal] of where n is 3 to 10 by know methods, reacting the
above said amino compound of formula VIII with known deprotecting agent in a
conventional manner to give novel 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis[(11 aS)-2-fluorinated-7-methoxy-1,2,3, 11 a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] of formula IX where in n are as stated above.
The precursor, methyl(2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluorinatedpyrrolidine-2-carbonate of formula I (intermediate of DC-81) was prepared by literature method (Dc Luca, L; Giacomelli, G.; Porcheddu, A.Org. Lett. 2001,3, 3041; Demange, L; Menez, A.; Dugave, C. Tetrahedron. Lett. 1998, 39, 1169; Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R. Bioorg. Med. Chem. Lett. 2004, 14,2669; ; Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.; Laxman, E.; Murthy, Y. L. N.
Bioorg. Med. Chem. Lett. 2004, 14, 5699; Thurston, D.E.; Murthy, V. S.; Langley,
D.R.; Jones, G.B. Synthesis, 1990, 81).
Same representative compound of formula IX present invention are given below
1) 1,1 '-{[(bisProPane-1,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]
2) 1,1 '-{[(b'sbutane-l ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]
3) 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-metoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]
4) 1.1 '-{[(bispropane-1,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-
methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]
5) 1,1 '-{[(biSDutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one]
6) 1,1 '-{[(Dispentane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one].
These new analogues of pyrrolo[2,1-c][1,4]benzodiazepines dimers substituted at C2-position linked at C8 position through piperazine moiety have shown promising anticancer activity in various cell lines. The molecules synthesized are of immense biological significance with potential sequence selective DNA-binding property. This resulted in design and synthesis of new congeners as illustrated in Schemel and Scheme 2, which comprise:
1. Ether linkage at C-8 position fluoro substituted at C2-position of DC-81 intermediates with piperazine moiety
2. Refluxing the reaction mixture for 24-48 h.
3. Synthesis of C8-linked C2-fluorosubstituted PBD antitumour antibiotic dimer imines.
4. Purification by column chromatography using different solvents like ethyl acetate, hexane, dichloromethane, chloromethane, and methanol.
The process of preparation of new non-cross linking C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine is disclosed and claimed in applicant's co-pending application no.
(Formula removed)

The following examples are given by way of illustration and therefore should not be construed to the present limit of the scope of invention.
Example 1
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-corboxaldehydediethylthioacetal IV (418mg, 1mmol), 1,3-dibromopropane (0.365ml, 3mmol) and K2CO3 (825mg, 5mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-corboxaldehydediethylthioacetal of formula V (432mg, 82%).
1H NMR: (CDCI3, 200 MHz): 5 1.31-1.40 (m, 6H), 2.28-2.48 (m, 2H), 2.49-2.64 (m, 2H), 2.68-2.91 (m, 6H), 3.64 (m, 2H), 3.99 (s, 3H), 4.25 (t, 2H, J = 6.0), 4.56 (d, 1H, J = 6.7), 4.76 (m, 1H), 5.0-5.33 (m, 1H), 6.88 (s, 1H), 7.68 (s, 1H) LCMS: m/z 539.4 (M++Na).
A solution of (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (539 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg, 10mmol) in dry acetone (20 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal] (441 mg, 82%).
1H NMR (CDCI3, 200 MHz): 5 1.29-1.36 (m, 12H), 2.02-2.11 (m, 4H), 2.46-2.69 (m, 12H), 2.73-2.89 (m, 8H), 3.39-3.62 (m, 4H), 3.93-3.94 (t, 4H), 4.17 (t, 4H), 4.52 (d, 2H, J = 6.79), 4.72 (q, 2H, J = 6.79), 5.07-5.29 (m, 2H), 6.84 (s, 2H), 7.65 (s, 2H) ESIMS: m/z 1003 (M+).
The 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro7-methoxy-2-
nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1004 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCI2.2H2O (2.25mg, 10 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 ml_). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy} bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethyl- thioacetal](803mg, 80%).
A solution of the 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal].of formula VIII (944mg, 1 mmol), HgCl2 (1035mg, 5 mmol) and CaCO3 (500mg, 5 mmol)in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude. 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula IXa, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (613mg, 65%).
1H NMR (CDCI3, 200 MHz): 51.73-2.1 (m, 8H), 2.45-2.76 (m, 8H), 3.46-3.91 (m, 10H), 3.93 (s, 6H), 4.02-4.25 (m, 4H), 5.35-5.48 (dt, 2H), 6.85 (s, 2H), 7.49 (s, 2H), 7.86 (d, 2H, J = 3.66 Hz) ESIMS: m/z 695 (M++1).
Example 2
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal IV (418 mg, 1 mmol), 1,4-dibromobutane (0.35ml 3 mmol) and K2CO3 (675mg, 5mmol) in dry acetone (30 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexan (6:4), the reaction
mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexan (1:1) gave the pure (2S)-/V-[4-(5-bromobutanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (355mg, 85%).
1H NMR: (CDCI3, 200 MHz): 6 1.26-1.43 (m, 6H), 2.01-2.46 (m, 4H), 2.49-2.67 (m, 2H), 2.70-2.95 (m, 6H), 3.58 (m, 2H), 3.99 (s, 3H), 4.25 (t, 2H, J = 6.0), 4.55 (d, 1H, J = 6.7), 4.73-4.79 (m, 1H), 5.0-5.33 (m, 1H), 6.89 (s, 1H), 7.69 (s, 1H) LCMS: m/z 553 (M+).
A solution of (2S)-N-[4-(5-bromobutanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehyde diethyl thioacetal of formula V. (553 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (4014 mg, 10 mmol) in dry acetone (30 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bibutane-1,N-diyl) piperazine]dioxy}bis(11aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carbox-aldehyddiethylthioactal] (447 mg, 81%). 1H NMR (CDCI3, 200 MHz): 5 1.29-1.41 (m, 12H), 1.7-1.97 (m, 8H), 2.33-2.70 (m, 12H), 2.71-2.89 (m, 8H), 3.39-3.66 (m, 4H), 3.90-3.93 (t, 4H), 3.96 (s, 6H), 4.1 (t, 4H), 4.5 (d, 2H, J = 7.5), 4.2 (q, 2H, J = 6.79), 5.0-5.27 (m, 2H), 6.84 (s, 2H), 7.60 (s, 2H).
The 1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1032 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCI2.2H20 (2025 mg, 10 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 ml_). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to
afford the crude. The 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy} bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethyl thioactal] (825mg, 80%).
A solution of the 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. of formula VIM (968 mg, 1 mmol), HgCI2 (1035 mg, 5 mmol) and CaCO3 (500 mg, 5 mmol) in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 1,1'-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula IXb, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (580 mg, 60%).
1H NMR (CDCI3, 200 MHz): 5 1.26-1.94 (m, 8H), 2.11-2.47 (m, 4H), 2.59-2.75 (m, 8H), 3.48-3.86 (m, 10H), 3.93 (s, 6H), 4.0-4.25 (m, 4H), 5.27-5.55 (m, 2H), 6.82 (s, 2H), 7.49 (s, 2H), 7.85 (d, 2H, J = 4.4 Hz).
Example 3
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal IV (418mg, 1 mmol), 1,5-dibromopantane (0.5 ml, 0.5 mmol) and K2CO3 (690 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(6-bromopentanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (355 mg, 85%).
1H NMR: (CDCb 200MHz): 5 1.26-1.41 (m, 6H), 1.74-2.0 (m, 4H), 2.35-2.47 (m, 2H), 2.49-2.65 (m, 2H), 2.70-2.92 (m, 6H), 3.58-3.66 (m, 2H), 3.98 (s, 3H), 4.25 (t, 2H, J = 6.0), 4.57 (d, 1H, J = 6.7), 4.75-4.85 (m, 1H), 5.0-5.34 (m,1H), 6.88 (s, 1H). 7.70 (s, 1H)LCMS:m/z567(M+).
A solution of (2S)-N-[4-(6-bromopentanoxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (567 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10mmol) in dry acetone (40 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bipentane-1,N-diyl) piperazine]dioxy}bis(11aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carbox- aldehydediethylthioactal] (453 mg, 80%). 1H NMR (CDCI3, 200 MHz): 6 1.25-1.36 (m, 12H), 1.39-1.89 (m, 12H), 2.25-2.61 (m, 12H), 2.70-2.86 (m, 8H), 3.39-3.63 (m, 4H), 3.96 (s, 6H), 4.0 (t, 4H), 4.12 (t, 4H), 4.54 (d, 2H, J= 6.79), 4.75 (q, 2H, J= 6.0), 5.0-5.3 (m, 2H), 6.84 (s, 2H), 7.62 (s, 2H).
The 1,1 '-{[(bipentane-1, N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1060 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCl2.2H2O (2.25 mg, 10 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispentane-1,/\/-diyl)piperazine] dioxyjbis (11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethyl- thioactal] (837 mg, 79%).
A solution of the 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal].of
formula VIII (1000 mg, 1 mmol), HgCI2 (1355 mg, 5 mmol) and CaCO3 (500 mg, 5 mmol)in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula IXc, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (560 mg, 56%).
1H NMR (CDCI3. 200 MHz): 6 1.44-2.09 (m, 12H), 2.30-2.51 (m, 4H), 2.52-3.0 (m, 8H), 3.47-3.87 (m, 10H), 3.93 (s, 6H), 4.0-4.1 (m, 4H), 5.27-5.58 (m, 2H), 6.9 (s, 2H), 7.49 (s, 2H), 7.9 (d, 2H, J = 4.6 Hz).
Example 4
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine -2-carboxaldehydediethylthioacetal IV (436 mmol), 1,3-dibromopropane (0.3 ml, 3 mmol) and K2CO3 (690 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (370 mg, 85%).
1H NMR (CDCI3, 200 MHz): 5 1.36-1.42 (m, 6H), 2.39-2.47 (m, 2H), 2.62-2.95 (m, 6H), 3.48-3.58 (m, 2H), 3.64 (t, 2H, J = 6.0 Hz), 3.96 (s, 3H), 4.26 (t, 2H, J = 5.2 Hz), 4.82 (d, 1H), 4.89-4.96 (m, 1H), 6.77(s, 1H), 7.72 (s, 1H) LCMS: m/z 580 (M++23).
A solution of (2S)-N-[4-(4-bromopropoxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoro pyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (557 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10mmol) in dry acetone (30 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bispropane-1,N-diyl) piperazine]dioxy}bis(11aS)-2,2-difluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal] (417 mg 75%).
1H NMR (CDCI3, 200 MHz): 6 1.25-1.39 (m, 12H), 2.0-2.14 (m, 4H), 2.58-2.66 (m, 8H), 2.69-2.88 (m, 12H), 3.45-3.79 (m, 8H), 3.94 (s, 6H), 4.1 (t, 4H), 4.78 (d, 2H), 4.85-4.96 (m, 2H), 6.7 (s, 2H), 7.6 (s, 1H) ESIMS: m/z 1039 (M+).
The 1,1'-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2,2-difluoro7-methoxy -2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1039 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCl2.2H2O (2.25 mg, 10.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x20 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispropane-1,N-diyl)piperazine] dioxy} bis (11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydedieth yithioactal] (779 mg, 75%).
A solution of the 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. Of formula VIII (976 mg, 1.0 mmol), HgCI2 (1355 mg, 5.0 mmol) and CaCO3 (686 mg, 5.0 mmol)in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHCO3 was added slowly at room temperature and the mixture is filtered through celite and washed with
ethylacetate. The filterate is evaporated in vacuum to get crude 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula Xld, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCb-methanol (9:1) (745 mg, 55%).
1H NMR (CDCI3, 200 MHz): 6 1.45-1.92 (m, 4H), 1.99-2.20 (m, 4H), 2.48-2.99 (m, 8H), 3.1-3.88 (m, 10H), 3.96 (s, 6H), 3.98-4.24 (m, 4H), 6.80 (s, 2H), 7.49 (s, 2H), 7.82 (d, 2H, J = 3.8 Hz).
Example 5
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4,4-difluoro pyrrolidine-2-corboxaldehyde diethylthioacetal IV (436, mg 1 mmol), 1,4-dibromobutane (0.35 ml, 3 mmol) and K2CO3 (690 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(5-bromobutanoxy)-5-methoxy-2-nitro benzoyl)-4,4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (353 mg.81%).
1H NMR (CDCI3, 200 MHz): 5 1.28-1.40(m, 6H), 2.0-2.5 (m, 4H), 2.58-2.79 (m, 6H), 3.51 (t, 2H), 3.75- (m, 2H), 3.96- (s, 3H), 4.10 (t, 2H), 4.79 (d, 1H), 4.85 (m, 1H), 6.74 (s, 1H) 7.6 (s, 1H) LCMS: m/z 594 (M++Na).
A solution of (2S)-N-[4-(5-bromobutanoxy)-5-methoxy-2-nitrobenzoyl)-4,4-fluoro pyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (571 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10mmol) in dry acetone (40 ml) was refluxed for 48h. After the completion of reaction as indicated by
TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bisbutane-1,N-diyl) piperazine]dioxy}bis(11aS)-2,2-difluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehyddiethylthioactal] (485 mg, 85%).
1H NMR (CDCI3, 200 MHz): 5 1.33-1.48 (m, 12H), 1.66-1.98 (m, 8H), 2.40-2.50 (m, 8H), 2.63-2.94 (m, 12H), 3.42-3.83 (m, 8H), 3.92 (s, 6H), 4.11 (t, 4H), 4.77 (d, 2H), 4.83-4.94 (m, 2H), 6.72 (s, 2H), 7.62 (s, 2H) ESIMS: m/z 1067 (M+).
The 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis(11aS)-2,2-difluoro7-
methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1067 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCI2.2H2O (2.25 mg, 10.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x20 ml_). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1 '-{[(bisbutane-1 ,N-diyl) piperazine]dioxy} bis(11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehyde- diethylthioacetal] (810mg, 76%).
A solution of the 1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-
difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. Of
formula VIII (1007 mg, 1 mmol), HgCI2 (1355 mg, 5.0 mmol) and CaCO3 (500 mg, 5
mmol) in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC
(EtOAc), indicates complete loss of starting material. Then organic layer is
evaporated in vacuum and the residue is diluted with EtOAc. To this saturated
NaHCO3 was added slowly at room temperature and the mixture is filtered through
celite and washed with ethylacetate. The filterate is evaporated in vacuum to get
crude 1,1'-[[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-
methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of
formula IXe, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (523 mg, 52%).
1H NMR (CDCI3, 200 MHz): 5 1.56-1.94 (m, 8H), 1.99-2.35 (m, 4H), 2.49-2.99 (m, 8H), 3.29-3.87 (m, 10H), 3.93 (s, 6H), 3.98-4.37 (m, 4H), 6.89 (s, 2H), 7.46 (s, 2H), 7.83 (d, 2H, J =3.67).
Example 6
A solution of (2S)N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine -2-carboxaldehydediethylthioacetal IV (436 mg, 1 mmol), 1,5-dibromopentane (0.37ml 3 mmol) and K2CO3 (1380 mg, 5 mmol) in dry acetone (40 ml) was refluxed for 48 h. After the completion of reaction as indicated by TLC, EtOAc-hexane (6:4), the reaction mixture was poured on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (1:1) gave the pure (2S)-N-[4-(6-bromopropoxy)-5-methoxy-2-nitro benzoyl)-4,4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V (374 mg, 86%).
1H NMR (CDCI3,200 MHz): 5 1.06-1.36 (m, 6H), 1.40-2.1 (m, 4H), 2.58-2.88 (m, 6H), 3.52 (t, 2H), 3.70-3.97 (m, 2H), 3.97 (s, 3H), 4.15 (t, 2H), 4.80 (d, 1H), 4.91-5.02 (m, 1H), 6.75 (s, 1H), 7.6 (s, 1H) LCMS: m/z 608 (M++Na).
A solution of (2S)-N-[4-(6-bromopentanoxy)-5-methoxy-2-nitrobenzoyl)-4,4-fluoropyrrolidine-2-carboxaldehydediethylthioacetal of formula V. (585 mg, 1mmol), piperazine (43mg, 0.5mmol) of the formula VI and K2CO3 (1380 mg,10 mmol) in dry acetone (40 ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc, the reaction mixture was on to the water and then extracted with ethylacetate. Evaporation of the organic layer gave the crude product, which was further purified by column chromatography on silica gel eluting with EtOAc-hexane (9:1) gave the pure 1,1'-{[(bispentane-1,N-diyl) piperazine]dioxy}bis(11aS)-2,2-
difluoro7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal] (462 mg, 79%).
1H NMR (CDCI3,200 MHz): 6 1.34-1.40 (m, 12H), 1.47-1796 (m, 12H), 2.36-2.49 (m, 4H), 2.50-2.66 (m, 8H), 2.68-2.90 (m, 8H), 3.37-3.80 (m, 8H), 3.94 (s, 6H), 4.08 (t, 4H), 4.77(d,2H), 4.85-4.91 (m, 2H), 6.72 (s, 2H), 7.63 (s, 2H) ESIMS: m/z 1095 (M+).
The 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2,2-difluoro7-methoxy -2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. VII (1095 mg, 1.0 mmol) was dissolved in methanol (20ml) and added SnCl2.2H2O (2.25 mg, 10.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3 solution and then extracted with ethyl acetate (3x30 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude. The 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy} bis(11aS)-2-2-difluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydedi- ethylthioactal] (744 mg, 68%).
A solution of the 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-di-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioactal]. Of formula VIII (1035 mg, 1 mmol), HgCI2 (1355 mg, 5 mmol) and CaC03 (500 mg, 5.0 mmol) in CH3CN/H2O (4:1,16 ml) was stirred at room temperature for 12 h. Until TLC (EtOAc), indicates complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this saturated NaHC03 was added slowly at room temperature and the mixture is filtered through celite and washed with ethyl acetate. The filterate is evaporated in vacuum to get crude 1,1'-{[(bispentane-1,N-diyl)piperazine] dioxy} bis [(11aS)-2-2-difluoro-7-metoxy-1,2,3,11a-tetrahydro-5/7-pyrrolo[2,1-c][1,4]benzo- diazepine-5-one], of formula IXf, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CHCI3-methanol (9:1) (569 mg, 55%).
1H NMR (CDCI3,200 MHz): 6 1.54-2.03 (m, 12H), 2.22-2.47 (m, 4H), 2.50-2.73 (m, 8H), 3.35-3.85 (m, 10H), 3.94 (s, 6H), 3.97-4.26 (m, 4H), 6.71 (s, 2H), 7.47 (s, 2H), 7.79 (d, 2H J = 3.6) ESIMS: m/z 787 (M++H).
Biological Activity: some of in vitro biological activity studies were carried out at the National Cancer Institute, Maryland, USA.
Cytotoxicity: The compounds (IXa) 1,1'-{[(bispropane-1,N-diyl)piperazine] dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1 -c][1,4] benzodiazepine-5-one] and (IXd) 1,1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis [(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11 a-tetrahydro-5H-pyrrolo[2,1 c][1,4]benzo diazepine-5-one].
The above compounds were evaluated for in vitro anticancer activity against sixty human tumor cells derived from nine cancer types (leukemia, non small cell cancer, colon cancer CNS cancer, melanoma, ovarian cancer renal cancer, prostate cancer and breast cancer) as per NCI protocol. For each compound, dose response curves for each cell line were measured at a minimum of five concentrations at 10-fold dilution. A protocol of 48h continuous drug exposure was used, and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The concentration causing 50%cell growth inhibition (GI50), total cell growth inhibition (TGI, 0% growth) and 50% cell death (LC -50% growth) compared with the control was calculated. The mean graph midpoint values of log10TGI and log10 LC50 as well as log10 GI50 for IXa and IXd are listed in table 1. As demonstrated by mean graph pattern compounds IXa and IXd are exhibit an interesting profile of activity and selectivity for various cell lines. The mean graph midpoint of log10TGI and log10 LC50 showed similar pattern to the logio GI50 mean graph midpoints.
Table 1 log10 GI50 log10TGI and log10LC50 mean graphs midpoint (MG_MID) of in vitro cytotoxicity data for the compounds IXa and IXd against human tumor cell lines.

(Table removed)
The in vitro anticancer activity for two representative compounds have given in table 2.
Among them IXa exhibits a wide spectrum of activity against fifty nine cell lines in nine cell panels, with GI50 value of IG50 values against in leukemia cancer CCRF-CEM, SR, 9.79x10-7 uM and 8.82x10-7µM the cytotoxicity of IXa and IXd in selected cancer cell lines have been illustrated in Table 2.
(Table removed)

Thermal denaturation studies:
The DNA binding affinity of the C2-Fluoro substituted piperazine linked pyrrolo[2,1c][1,4]benzodiazepine dimers were subjected to thermal denaturation studies using calf thymus (CT) DNA (Jones, G. B.; Davey, C. L; Jenkins, T. C; Kamal, A.; Kneale, G. G.; Neidle, S.; Webster, G. D.; Thurston, D. E. Anti-cancer Drug Des. 1990, 5, 249. McConnaughie, A. W.; Jenkins, T. C. J. Med. Chem. 1995, 38, 3488). The studies for these compounds (IXa-f) were carried out by DNA/ligand molar ratio is 1:5 the increase in the helix melting temperature (m) for each compound was examined at 0 h. The DNA biding activity for these novel C2-Fluoro substituted piperazine linked pyrrolo[2,1c][1,4] benzodiazepine dimers have been examined by thermal denaturation studies using calf thymus (CT) DNA melting studies shows that these compounds stabilize the Tm for CT-DNA at pH 7.0, incubated at 37 °C, were PBD/DNA molar ratio is 1:5 interestingly, in this assay all compounds of fluoro substituted dimer (IXa-f) elevates the melting temperature CT-DNA by margin of 11-38 ° C after incubation for at 37 °C Data for DSB-120 and SJG-136 are included in Table-3 for comparison. The synthetic DC-81 dimer (DSB-120) gives a Tm 10.2°C and SJC-136 gives a Tm of 25 °C under identical experimental condition.
(Table removed)

DNA alone at pH 7.00 c 0.01, Tm = 69.6°C+ 0.01 (mean value from 10 separate determinations), all Tm values are [0.1 ■ 0.2 °C. b For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100 uM and ligand concentration = 20 uM in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01]; literature value of SJC-136
ADVANTAGES OF THE INVENTION
1. The present invention provides a C2-Fluoro substituted piperazine linked
pyrrolo[2,1-c][1,4]benzodiazepine dimers useful as antitumour agents.
2. It also provides a process for the preparation of C2-Fluoro substituted
piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers.

We claim
1. C2-Fluoro substituted piperazine linked pyrrolo[2,1c][1,4]benzo- diazepine
of formula IX.
(Formula removed)

2. C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine as
claimed in claim 1 is represented by the group of following compounds:
1,1 '-{[(Dispropane-I, N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa); 1,1 '-{[(bisbutane-1, N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXb); 1.1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXc); 1.1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd);
1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) and 1,1 '-[[(bispentane-1 ,H-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf).
3. C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine
dimers as claimed in claim 1, exhibits an in vitro anticancer/antitumour activity
against human cancer cell lines selected from the group of lung, colon, breast,
ovarian, leukemia, Renal, Melanoma, Prostate and CNS cell lines
4. A process as claimed in claim 3, wherein the C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine dimers (IXa-f) exhibits binding affinity with calf thymus (CT) DNA at a molar ratio of about 1:5 in aqueous sodium phosphate buffer at pH of 7.00.
5. A process for the preparation of C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine formula IX
(Formula removed)
and the said process comprising the step of:
(a) Preparing (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)4,fluorinated-
pyrrolidine-2-carboxaldehydediethylthioacetal of formula IV by known
method.
(Formula removed)

(b) reacting the compound of formula IV obtained in step (a) with
dibromoalkane in a dry aprotic water miscible organic solvent, in the
presence of mild inorganic base, under reflux, for a period up to 48
hours, purifying the resultant crude product by known method to obtain
the compound of (2S)-N-[4-n-bromoalkoxy)-5-methoxy-2-nitrobenzoyl)-
4,fluorinated-pyrrolidine-2-carboxaldehydediethylthioacetal offormulaV.
(Formula removed)

(c) reacting the compound of formula V obtained in step (b) with piperazine
of formula VI in a dry aprotic water miscible organic solvent, in the
presence of mild inorganic base, under reflux, for a period of 45-48
hours, followed by pouring the resultant reaction mixture on to the water
and extracting and purifying the resultant crude product by known
method to obtain the compound 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis (11aS)-2-fluorineted-7-methoxy-2-
nitrobenzoylpyrrolidin-2-carboxaldehydediethyl thioacetal] of formula VII
(Formula removed)
(d) reducing the compound of formula VII obtained in step (c) with SnCI2.
2H2O in an organic solvent, under reflux, for a period of 1-2 hours, at a
pH of 8 in the presence of saturated alkalibicorbonate solution, followed
by extraction with an organic solvent and drying the resultant organic
phase over Na2SO4 and evaporating the solvent under vacuum to
obtain the resultant compound. 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis(11aS)-2-fluorineted-7-methoxy-2-amino
benzoylpyrrolidin-2-carboxaldehyddi- ethylthioacetal] of formula VIII.
(Formula removed)

(e) reacting the compound of formula VIII with mercurous chloride and
calcium carbonate in the presence of an aqueous organic solvent
wherein organic solvent to water ratio is about 4:1, under stirring, at a temperature of 25-30°C, for a period of about 12 hours, followed by the evaporation of organic layer to obtain the crude residue and purifying the residue by known method to obtain the desired product of 1,1'-{[(bisalkane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-fiuorinated-7-methoxy-1,2,3,11a-tetra- hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI.
(Formula removed)

wherein R=R1=F or F2; n= 3-10
6. A process as claimed in claim 5, wherein the dibromoalkane used in step (a) is
selected from the group consisting of 1,3-dibromopropane, 1,4-dibromobutane and 1,5-dibromopantane.
7. A process as in claimed in claim 5, wherein the dry organic solvent used in
step (b) and (c) is selected from acetone, acetonitrile and DMF.
8. A process as claimed in claim 5, wherein the mild inorganic base used in step
(b) and (c) is selected from K2CO3, BaCO3 and CsCO3.
9. A process as claimed in claim 5 wherein the compound of formula VII used
in step (c) is selected from the group consisting of
1,1'-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-
nitrobenzoyl pyrrolidin-2-carboxaldehydediethylthioacetal] (Vlla);
1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-
2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllb);
1,1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-
nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlle);
1,1'-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-
2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlld);
1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-2-difluoro-7-methoxy-2-
nitrobenzoylpyrrolidin-2-carboxaldehyd diethylthioacetal] (Vlle) and
1,14[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-2-difluoro-7-methoxy-2-nitrobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllf). 10.A process as claimed in claim 5, wherein the compound of formula VIII obtained in step (d) is selected from the group consisting of 1,1'-{[(Dispropane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Villa); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllb); 1,1 '-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllc); 1I1'-{[(bispropane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllld); 1,1 '-{[(bisbutane-1 ,N-diyl)piperazine]dioxy}bis(11 aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vllle)and 1,1'-{[(bispentane-1,N-diyl)piperazine]dioxy}bis(11aS)-2-fluoro-7-methoxy-2-aminobenzoylpyrrolidin-2-carboxaldehydediethylthioacetal] (Vlllf).
11. A process as claimed in claim 5, wherein the organic solvent used in step (d) are ethyl acetate, chloroform and methanol.
12. A process as claimed in claim 5, wherein the alkalibicorbonate used in step (d) is sodiumbicorbonate.
13. A process as claimed in claim 5, wherein the organic solvent used in step (d) is methanol.
14. A process as claimed in claim 5, wherein the organic solvent used in step (e) is acetonitrile.
15. A process as claimed in claim 5, wherein the cpmpound 1,1'-{[(bisalkane-1,N-
diyl) piperazine]dioxy}bis[(11 aS)-2-fluorinated-7-metoxy-1,2,3,11 a-tetrahydro-
5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one], of formula XI obtained in step (e)
is represented by a group of following compounds:
1,1'-{[(bispropane-1 ,H-diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy 1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXa);
1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXb); 1,1'-{[(bispentane-1 ,N-diyl)piperazine]dioxy}bis[(11aS)-2-fluoro-7-methoxy 1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXc); 1,1 '-{[(bispropane-1 ,N-diyl)piperazine]dioxy}bis[(11 aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXd);
1,1'-{[(bisbutane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXe) And
1-1'-{[(bisperitane-1,N-diyl)piperazine]dioxy}bis[(11aS)-2-2-difIuoro-7-methoxy-1,2,3,11a-tetrahydro-5N-pyrrolo[2,1-c][1,4]benzodiazepine-5-one] (IXf).
16. A pharmaceutical composition useful as anti tumor agent comprising an
effective amount of one or more analogues 1,1'-{[(bisalkane-1,N-
diyl)piperazine]dioxy}bis[(11 aS)-2-fluoro-7-methoxy-1,2,3, -11 a-tetrahydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepine-5-one] or 1,1 '-[[(bisalkane-1 ,N-diyl)
piperazine]bis(11aS)-2-2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepine-5-one optionally along with pharmaceutically
acceptable additives.
17. C2-Fluoro substituted piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine
dimers and preparation for the preparation thereof, substantially as herein
described with reference to the examples accompanying the specification.

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http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=0O/YKny95fnlPNkyUt/3lg==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

278139

Indian Patent Application Number

608/DEL/2008

PG Journal Number

52/2016

Publication Date

16-Dec-2016

Grant Date

14-Dec-2016

Date of Filing

11-Mar-2008

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	AHMED KAMAL	INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY, HYDERABAD, INDIA-500007
2	RAJENDER	INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY, HYDERABAD, INDIA-500007
3	METUKU KASHIREDDY	INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY, HYDERABAD,INDIA-500007
4	GORRE BALAKISHAN	INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY, HYDERABAD,INDIA-500007

PCT International Classification Number

A61K31/5517 ; A61P35/00 ; C07D487/04

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA