Title of Invention	"A PROCESS FOR THE PREPARATION OF NEW PYRROLO[2,1-C][1,4] BENZODIAZEPINES AS POTENTIAL ANTITUMOUR AGENTS"
Abstract	A process for preparation of new pyrrolo [2.1-c] [1,4] benzodiazepines useful as potential antitumour agents by reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzyl]pyrrolidine 2-carbaxy carbaldehyde diethyl thioacetal with dibromo alkane in an aprotic water miscible organic solvents in presence of a mild inorganic base up to refluxing temperature for a period up to 48 hours, isolating (2S)-N-[4-(3- bromo alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal with dilactam in presence of an inorganic mild base in presence of aprotic water miscible organic solvent up to refluxing temperature for a period up to 48 hours, isolating 8-{[2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2.3,5,l 0,11, 1 la-hydro-lH-pyrrolo[2,l,-c][ 1,4] benzodiazepine-5,ll-done reducing the above nitro with SnCl2O in presence of organic solvent up to a reflux temperature. Isolating the 8- j [2S)-N-5-methoxy-2-aminobenzoyl]prrolidin-2-carboxy carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5,1-,11, lla-hydro-lH-pyrrolo[2,l,-c][l,4] benzodiazepine-5,11-donc with known deprotecting agent in a conventional manner to give novel pyrrolo[2.1-c][ 1,4\| benzodiazepines.

Title of Invention

"A PROCESS FOR THE PREPARATION OF NEW PYRROLO[2,1-C][1,4] BENZODIAZEPINES AS POTENTIAL ANTITUMOUR AGENTS"

Abstract

A process for preparation of new pyrrolo [2.1-c] [1,4] benzodiazepines useful as potential antitumour agents by reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzyl]pyrrolidine 2-carbaxy carbaldehyde diethyl thioacetal with dibromo alkane in an aprotic water miscible organic solvents in presence of a mild inorganic base up to refluxing temperature for a period up to 48 hours, isolating (2S)-N-[4-(3- bromo alkoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal with dilactam in presence of an inorganic mild base in presence of aprotic water miscible organic solvent up to refluxing temperature for a period up to 48 hours, isolating 8-{[2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2.3,5,l 0,11, 1 la-hydro-lH-pyrrolo[2,l,-c][ 1,4] benzodiazepine-5,ll-done reducing the above nitro with SnCl2O in presence of organic solvent up to a reflux temperature. Isolating the 8- j [2S)-N-5-methoxy-2-aminobenzoyl]prrolidin-2-carboxy carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5,1-,11, lla-hydro-lH-pyrrolo[2,l,-c][l,4] benzodiazepine-5,11-donc with known deprotecting agent in a conventional manner to give novel pyrrolo[2.1-c][ 1,4| benzodiazepines.

Full Text	The present invention relates to a process for preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines useful as potential antitumour agents. More particularly, it provides a process for the preparation of 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2. 3, 4,lla-tetrahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-8-yloxyalkyloxy}-(l laR)-2,3,5, 10,11.1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-dione. with aliphatic chain length variations for the compounds and their 2-hydroxy, 2-acetyloxy derivatives. In the last few years, a growing interest has been shown in the development of new pyrrolo[2,l-c][l,4]benzodiazepines (PBDs). These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within in the minor groove of duplex DNA via an acid-labile aminal bond to the electrophilic imine at the N10-C11 position. (Ref. Kunimoto, S. Masuda, T. Kanbayashi, N. Hamada. M. Naganawa, H. Miyamoto, M. Takeuchi, T. and Unezawa, H. J. Antibiot., 1980, 33, 665.; Kohn, K.W. and Speous, C.L. J. Mol. Bioi, 1970, 57, 551.; Hurley, L.H. Gairpla, C. and Zmijewski, M. Biochem. Biophys. Acta., 1977, 475, 521.; Kaplan, D.J. and Hurley, L.H. Biochmestry, 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 (Ref: 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, 8141-8147). Naturally occurring pyrrolo[2,l-c][l,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 recognise and bind to specific sequence of DNA. Examples of naturally occurring PBD's include anthramycin, tomaymycin. sibiromycin and neothramycin. (Formula Removed) In the last decade a number of PBD's have been designed and synthesized to explore them as DNA-sequence selective agents particularly C-8 linked PBD dimers. (Ref: Bose, D. S. Thomson, A. S. Ching, J. A. Hartley, J. A. Berardini, M. D. Jenkins, T. C. Neidle, S. Hurley, L. H. Thurston, D. E. J. Am. Chem. Soc. 1992, 114, 4939). The main object of present invention is to provide a new class of C-8 linked PBD dimers. wherein one PBD has an imine functionality while the other has an amide group. It has been envisaged that such a mixed dimer could offer more insight not only for the covalent binding but also the role played by non-covalent interactions with DNA-bases. Pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-diones are a class of compounds that bind to DNA by non-covalent interactions such as hydrophobic, Vander walls interactions and hydrogen bonding between ring substituents and DNA, are also responsible for the influence on the sequence selectivity. Further, some dilactams such as (7-methoxy-2-methylcarbonyloxy-5,1 l-dioxo-(2S)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]- benzodiazepine-5,ll-dione-8-yl acetate has been reported to possess significant in vivo antitumour activity in the P388 rat model. (Ref: Kaneko, T. Wong, H. Doyle. T. W. Rose, W. C. Bradner. W. T. J. Med. Chem. 1985, 25, 388). The main object of the present invention is to provide a process for the preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI useful as antitumour agents. A process for preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI of the drawing accompanying the specification wherein R is H, OH, OAc and RI is H, and n is 3 to 5 which comprises: reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzyl]-pyrrolidin-[2-carbaxy- carbaldehyde diethyl thioacetal of formula I where in RI is H with dibromoalkanes in an aprotic water miscible organic solvents in presence of a mild inorganic base upto refluxing temperature for a period upto 48 hours, isolating (2S)-N-[4-(3-bromoalkoxy)-5-methoxy-2- nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II where in RI is H and reacting the above compound of formula II with dilactam of formula III wherein R is H. OH, OAc in presence of an inorganic mild base in presence of aprotic water miscible organic solvents upto refluxing temperature for a period upto 48 hours isolating 8-{[(2S)-N- 5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-alkoxy-7-methoxy- 2,3,5,10,1 l,lla-hydro-lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,ll-dione of formula IV where in R is H, OH, OAc and RI is H and n is 3-5 by conventional methods, reducing the above nitro compounds of formula IV with SnCl2 .2H2O in presence of organic solvent upto a reflux temperature, isolating the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2- carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5,10,11, 11 a-hydro-1 H-pyrrolo[2,1 ,- c][l,4]benzodiazepine-5,l 1-dione of formula V, where in R is H, OH, OAc and R1 is H and (Scheme Removed) n is 3-5 by known methods, reacting the above said amino compound of formula V with known deprotecting agents in a conventional manner to give novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI wherein R. RI and n as stated above. The present invention provides a process for the preparation of pyrrolo[2,l-c][l,4]benzodiazepines of formula VI of the drawing accompanying this specification where R is H, OH, OAc and RI is H and n is 3 to 5, which comprises: reacting t (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaxy carbaldehyde diethyl thioacetal of formula I where in RI is H with dibromoalkanes in the presence of organic solvents such as acetone, THF and DMF. In an embodiment of the inventon the inorganic bases used may be such as K^COa, BaCOs and NaiC03 upto refluxing temperatures for a period in the range of 24 to 48 hours, affording the (2S)-N-[4-(3-bromopropoxy)-5-methoxy- 2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetol, (2S)-N-[4-(4- bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetol, (2S)-N-[4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II, where in RI is H. In another embodiment of the invention, reacting compounds of the formula II with debenzylated dilactam of formula III where in R is H, OH, OAc (ref: Kaneko. E.; Wang, H.; Doyle, T. W. The J. Antibiotics, 1984, 3, 300) and organic solvents used may be aprotic water miscible solvents such as acetone. THF, DMF. In yet another embodiment of the invention the inorganic bases used may such as K2CO3, BaCO3 and Na2CO3, upto refluxing temperature for a period in the range of 24 to 48 hours, affording the 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2- carbaldehyde diethylthioacetal]-propoxy-7-methoxy-2,3,5,10,11,11 a-hydro-1 H-pyrrolo[2,1 ,- c] [ 1,4] benzo-diazepine-5,11 -dione, 8-{ [(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2- carbaldehyde diethylthioacetal]-butoxy-7-methoxy-2.3,5.10,l 1,1 la-hydro- lH-pyrrolo[2,l, - c][1.4]benzo-diazepine-5,l 1-dione, 8-{[(2S)-N-5-methoxy-2-nitrobenzoyi]pyrrolidin-2- carbaldehyde diethylthioacetal]-pentyloxy-7-methoxy-2.3,5,10,11,11 a-hydro-1 H-pyrrolo [2,l,-c][l,4] benzodiazepine-5,11-dione of compounds of the formula IV where in R is H, OH. OAc and RI is H. Reacting above said nitro thio acetal compounds of the formula IV with the reducing agent SnCl2. 2H2O in the presence of organic solvents such as MeOH, DMF amd 1,4-dioxane and their mixture upto reflux temperature, affording the 8-{[(2S)-N-5- methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-propoxy-7-methoxy- 2,3,5,10,11, 1 la-hydro-lH-pyrrolo-[2,l,-c][l,4]benzodiazepine-5,l 1-dione. 8-{[(2S)-N-5- methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-butoxy-7-methoxy- 2,3.5,10, 11,11 a-hydro-lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,l 1-dione, 8-{[(2S)-N-5- methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-penryloxy-7- methoxy-2,3,5,10, 11,11 a-hydro-lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,l 1-dione of formula V, where in R is H, OH, OAc and RI is H by conventional methods, reacting the amino compounds of the formula V with known deprotecting agents such as HgCl2/HgO, HgCl2/CaCO3 in presence of organic solvents such as acetonitrile, MeOH in a conventional manner recovering 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2, 3, 4, 11 a-tetrahydro-1H- pyrrolo[2,l-c][l,4] benzodiazepine-8-yloxypropoxy}-(l laR)-2,3,5,10,l 1,1 la-hexahyro-lH- pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, 8-methoxy-7-{4-[7-methoxy-5-oxo-(l laS)- 2, 3, 4, 11 a-tetrahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-8-yloxybutoxy}-(l laR)-2,3,5,10, 11,1 la-hexahyro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, 8-methoxy-7-{5-[7- methoxy-5-oxo-(llaS)- 2, 3, 4, lla-tetrahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-8- yloxypentyloxy}-(l laR)-2,3,5,10,l 1,1 la-hexahyro-lH-pyrrolo[2,l-c][l,4]benzodiazepine- 5.11-dione of the formula VI, from the above reaction mixture by known methods wherein R, RI and n are stated above. The precursor, (2S)-N-(4-hydroxy-2-methoxy-2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula I (intermediates of DC-81) prepared by literature methods (Ref: Thurston, D.E.; Murthy, V. S.; Langley, D. R.; Jones, G. B. Synthesis, 1990,81) « • Some representative compounds of formula VI present invention are given below 1) 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2, 3, 4, lla-tetrahydro-lH-pyrrolo[2,l- c][l,4]benzodiazepine-8-yloxypropoxy}-(l laR)-2,3,5,10,l 1,11 a-hexahyro-1H- pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5,11 -dione 2) 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2, 3, 4, lla-tetrahydro-lH-pyrrolo-[2,l- c][l,4]benzodiazepine-8-yloxybutoxy}-(l laR)-2,3,5,10,11,11 a-hexahyro-1H- pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5,11 -dione 3) 8-methoxy-7-{3-[7-methoxy-5-oxo-(l laS)-1,2,3,1 la-tetrahydro-lH-pyrrolo-[2,l- c][l,4]-benzodiazepine-8-yloxypentyloxy}-(l laR)-2,3,5,10,11,11 a-hexahyro-1H- pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5,11 -dione 4) 2-hydroxy-8-methoxy-7-3-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-1H- pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]propoxy-(2R,l laS)-2,3,5,10,11,1 la- hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine -5,11-dione 5) 2-hydroxy-8-methoxy-7-4-[7-methoxy-5-oxo-(l Ia5)-2,3,5,l 1 a-tetrahydro-1H- pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]butoxy-(2R,l IaS)-2,3,5,10,l 1,1 la- hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine -5,11-dione 6) 2-hydroxy-8-methoxy-7-5-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-1H- pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]pentyloxy-(2R,l laS)-2,3,5,10,11,1 la- hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine -5.11-dione 7) 8-methoxy-7-3[7-methoxy-5-oxo-(l laS)-2,3.5,l la-tetrahydro-lH-pyrrolo[2,l- c][l,4]benzodiaze-pine-8-yloxy)propoxy)-5,l \-dioxo-(2R,l laR)-2,3,5,10,l 1.1 la- hexahydro-1H-pyrrolo[2,1 -c][1,4]benzodiazepine-2-yl acetate f 8) 8-methoxy-7-4-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-lH-pyrrolo[2,l- c][l,4]benzodiaze-pine-8-yloxy)butoxy)-5,l l-dioxo-(2R,l laR)-2,3,5,10,l 1,1 la- hexahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-2-yl acetate 9) 8-methoxy-7-(5-(7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-lH-pyrrolo[2,l- c][l,4]benzodiazepine-8-yloxy)pentyloxy)-5,l 1-dioxo-(2R, 11 aR)-2,3,5,10,11,1 la- hexahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-2-yl acetate 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 Scheme-1, which comprise: 1. The ether linkage at C-8 position of DC-81 intermediates with dilactams. 2. Refluxing the reaction mixture for 24-48 h. 3. Synthesis of C-8 linked PBD antitumour antibiotic imines. 4. Purification by column chromatography using different solvents like ethylacetate, hexane, dichloromethane and methanol. The following examples are given by way of illustrations and therefore should not be construpted to the present limit of the scope of invention. Example 1 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1.3-dibromopropane (505mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the • • completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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 EtOAchexane (1:1) gave the pure (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II. 1'H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 2H), 2.6-2.9 (m, 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, IH), 4.85 (d, IH), 6.82 (s, lH),7.75(s, IH). A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (520mg, 1 mmol), 8-hydroxy-7-methoxy-(1 laS)-2.3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione (262mg, 1 mmol) of the formula III and K2CO3(414mg, 3 mmol) in dry acetone (20ml) 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 (8:2) gave the pure 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-propoxy-7-methoxy- 2.3.5.10,11,1 la-hexahydro-lH-pyrrolo[2.1-c][l,4]benzo-diazepine-5.11 -dione. of comp¬ounds of formula IV. 1H NMR (CDC13)δ 1.2-1.5 (m, 6H), 1.7-2.25 (m,8H), 2.23-2.5 (m, 2H). 2.6-2.9 (m, 4H), 3.2-3.3 (m. 4H), 3.6-3.8 (m, 2H), 3.8-4.0 (s, 6H), 4.01 (m. 1H), 4.2-4.35 (m. 2H). 4.65 (m, 1H), 4.85 (d, 1H), 6.5 (s, 1H), 6.8 (s, 1H), 7.38 (s, 1H), 7.7 (s, 1H), 8.21 (s, 1H, NH exchangeable); FAB MS 703 (M+H)+. • The 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-propoxy-7-methoxy-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione of formula IV (702mg, 1.0 mmol) was dissolved in dichloro-methane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-propoxy-7-methoxy-2,3,5,10,11,1 la-hexahydro-1H-pyrrolo[2,l-c] [1,4] benzodiazepine-5,11-dione of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal} -propoxy-7-methoxy-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c]-[ 1,4]benzo-diazepine-5,11-dione of formula V (672mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 8-methoxy-7-{3-[7-methoxy- 5-oxo-( 1 laS)-2,3,l la-tetrahydro-lH-pyrrolo[2,l-c] [l,4]-benzodiazepine-8-yloxypropoxy}- (11 aR)-2.3,5,10,11,11 a-hexahyro-1 H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5.11 -dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with methanohCHCl3 (2:8). 1HNMR (DMSO-d6+CDC13) δ1.89-2.5 (m, 10H), 3.4-4.05 (m, 13H), 4.1-4.4 (m, 3H), 6.6 (s, t 1H), 6.82 (s, 1H), 7.4 (s, 1H), 7.5 (s, 1H), 7.65 (d, 1H), 9.9 (s, 1H, NH exchangeable); FAB MS: 549 (M+H)+. Example 2 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,4-dibromobutane (540mg, 2.5 mmol) and K2C03 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s, 1H), 7.75 (s, 1H). A solution of (2S)-N-[4-(4-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (534mg, 1 mmol), 8-hydroxy-7-methoxy-(1 laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrol[2,l-c][l,4]benzodiazepine-5,l 1-dione (262mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 (8:2) gave the pure 8-{[(2S)-N-5-methoxy-2- nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thio-acetal}-butoxy-7-methoxy- 2,3,5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiaze-pine-5,l 1-dione of formula IV. 1H NMR (CDC13) δ 1.2-1.5 (m, 6H), 1.7-2.25 (m,8H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H), 3.2-3.3 (m, 4H), 3.6-3.8 (m, 2H), 3.8-4.0 (s, 6H), 4.01 (m, 1H), 4.2-4.35 (m, 2H), 4.65 (m, 1H), 4.85 (d, 1H), 6.5 (s, 1H), 6.8 (s, 1H), 7.38 (s, 1H), 7.7 (s, 1H), 8.21 (s, 1H, NH exchangeable); The 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-butoxy-7-methoxy-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzo-diazepine-5,l 1-dione (716mg, 1.0 mmol) of the formula IV was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H20 (1.124 g, 5.0 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 Na2S04 and evaporated under vacuum to afford the crude 8-{[(2S)-N-5-methoxy-2-aminobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal}-butoxy-7-methoxy-2,3,5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-5,l 1-dione of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal} -butoxy-7-methoxy-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c] [ 1,4]benzodi-azepine-5,11-dione of formula V. (686mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 8-methoxy-7-{3-[7-methoxy-5-oxo-(l laS)-2, 3, 4, 1 la-tetrahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-8-yloxy- « f butoxy} -(11 aR)-2,3,5,10,11,11 a-hexahyro-1 H-pyrrolo- [2,1 -c] [ 1,4]benzodiazepine-5,11 -dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with methanol:CHCl3 (2:8). 1HNMR (DMSO-d6+CDCl3) δ1.89-2.5 (m, 12H), 3.4-4.05 (m, 13H), 4.1-4.4 (m, 3H), 6.6 (s, 1H), 6.82 (s, 1H), 7.4 (s, 1H), 7.5 (s, 1H), 7.65 (d, 1H), 9.9 (s, 1H, NH exchangeable). Example 3 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,5-dibromopentane (575mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m. 6H), 2.6-2.9 (m, 4H), 3.15-3.33 (m. 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s. lH),7.75(s, 1H). A solution of (2S)-N-[4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. (548mg, 1 mmol), 8-hydroxy-7- methoxy-(l laS)-2,3.5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11- dione (262mg, 1 mmol) III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 (8:2) gave the pure 8-{[(2S)-N-5-methoxy-2-nitrobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal}-pentyloxy-7-methoxy-2.3,5.10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione of formula IV. 'H NMR (CDC13) 5 1.2-1.5 (m, 6H), 1.7-2.25 (m,8H), 2.23-2.5 (m, 6H), 2.6-2.9 (m, 4H), 3.2-3.3 (m, 4H), 3.6-3.8 (m, 2H), 3.8-4.0 (s, 6H), 4.01 (m, 1H), 4.2-4.35 (m, 2H), 4.65 (m, 1H), 4.85 (d. 1H), 6.5 (s, 1H), 6.8 (s, 1H), 7.38 (s. 1H), 7.7 (s, 1H), 8.21 (s, 1H, NH exchangeable) The 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-pentyloxy-7-methoxy-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c] [ 1,4]ben-zodiazepine-5,11-dione of formula IV (730mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCOs 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 8-{[(2S)-N-5-methoxy-2- aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-pentyloxy-7-methoxy-2.3.5.10.11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]ben-zodiazepine-5,l1-dione of formula V. A solution of 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal} -pentyloxy- 7-methoxy-2.3,5,10,11,11 a-hexahydro-1 H-pyrrolo [2,1 -c] [ 1,4] -ben-zodiazepine-5,11-dione formula V. (700mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1. 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)-2, 3, 4, lla-tetrahydro-5H-pyrrolo[2,l-c][l,4]-benzodiazepine-8-yloxypentyloxy}-(l laR)-2,3,5,10,11,1 la-hexahyro-lH-pyrrolo[2,l-c] [l,4]benzodiazepine-5,11-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with methanol:CHCl3 (2:8). 1HNMR (DMSO-d6+CDCl3) δ 1.89-2.5 (m, 14H), 3.4-4.05 (m, 13H), 4.1-4.4 (m, 3H), 6.6 (s, 1H), 6.82 (s, 1H), 7.4 (s, 1H), 7.5 (s, 1H), 7.65 (d, 1H), 9.9 (s, 1H, NH exchangeable). Example 4 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,3-dibromopropane (505mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m. 6H), 1.7-2.2 (m. 4H), 2.23-2.5 (m, 2H), 2.6-2.9 (m, 4H), 3.15-3.33 (m. 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m. 1H), 4.85 (d, 1H), 6.82 (s,lH), 7.75 (s,lH). A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (520mg, 1 mmol), 2,8-dihydroxy-7-methoxy-(2R..1 laR)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione (278mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 EtOAcrhexane (9:1) gave the pure 8-{[(2S)-N-5-methoxy-2-nitrobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal }-propoxy-2-hydroxy-7-methoxy(2R. 11aR)-2,3,5,10,ll,lla-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-dione. of compounds of formula IV. 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-propoxy-2-hydroxy-7-methoxy (2R, 1 laR)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,11-dione, of compounds of formula IV (718mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H20 (1.124 g, 5.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3solution and then extracted with ethyl acetate (3x20 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude8- ([(2S)-N-5-methoxy-2-arninobenzoyl]pyrrolidine 2-carbaldehyde diethyl thio- acetal}- propoxy-2-hydroxy-7-methoxy(2R, 1 laR)-2.3.5.10.11,1 la-hexahydro-lH-pyrrolo[2,l- c'][l,4]benzodiazepine-5.11-dione. of compounds of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-propoxy-2-hydroxy-7-methoxy(2R. 1 laR)-2,3,5,10,l 1,1 la-hexahydro-lH- pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, of compounds of formula V (688mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 2-hydroxy-8-methoxy-7-3-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-1H-pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]propoxy-(2R,1 laS)-2,3,5,10,11,11 a-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8). Example 5 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,4-dibromobutane (540mg, 2.5 mmol) and K.2C03 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m. 4H), 2.23-2.5 (m, 4H). 2.6-2.9 (m. 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s, lH),7.75(s, 1H). • « A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (534mg, 1 mmol), 2,8-dihydroxy-7-methoxy-(2R.I laR)-2,3.5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11-dione (278mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-{[(2S)-N-5-methoxy-2-nitrobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal }-butoxy-2-hy droxy- 7-methoxy(2R. 1 laR)-2.3.5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c]-[l,4]benzodiazepine-5.11-dione. of compounds of formula IV. A solution of 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-butoxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3,5,10,11,1 la-hexahydro-lH- pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, of compounds of formula IV (732mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine-2-carbaldehyde diethyl thio-acetal}-butoxy-2-hydroxy-7-methoxy(2R. 1 laR)-2.3.5,10.11.1 la-hexahydro-lH-pyrrolo[2,l-c][1.4]benzodiazepine-5,l 1-dione, of compounds of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}butoxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3,5,10.11,1 la-hexa-hydro-lH-pyrrolo- [2,l-c][l,4]benzodiazepine-5,l 1-dione, of compounds of formula V. (702 mg, 1 mmol), HgCh (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 2-hydroxy-8-methoxy-7-4-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-lH-pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]butyloxy-2R,1 laS)-2,3,5,10,l 1,1 la-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8). Example 6 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,5-dibromopentane (575mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 6H). 2.6-2.9 (m, 4H), 3.15-3.33 (m. 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m. 1H), 4.85 (d, 1H), 6.82 (s. 1H), 7.75 (s, 1H). A solution of (2S)-N-[4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (548mg, 1 mmol), 2,8-dihydroxy-7-methoxy-(2R, laR)-2,3,5,10,11,11 a-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11-dione (278mg, 1 mmol) formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-butoxy-2-hydroxy-7-methoxy(2R. 11 aR)-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c]-[ 1,4]benzodiazepine-5.11 -dione. of compounds of formula IV. A solution of 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }pentyloxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3,5,10,l 1,11 a-hexahydro-1H- pyrrolo[2,l-c][l,4]benzodiazepine-5,11-dione, of compounds of formula IV (746mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2-2H2O (1.124 g, 5.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCOs solution and then extracted with ethyl acetate (3x20 mL). The combined organic phase was dried over Na2SO4and evaporated under vacuum to afford the crude 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}pentyloxy-2-hydroxy-7-methoxy(2R. 1 laR)-2.3,5.10.11,11 a-hexahydro-1H- pyrrolo[2.1-c][l,4]benzodiazepine-5.11-dione. of compounds of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}pentyloxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3.5,10,l 1.11 a-hexa-hydro-1H- * • pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-dione, of compounds of formula V. (716 mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The fllterate is evaporated in vacuum to get crude 2-hydroxy-8-methoxy-7-5-[7-methoxy-5-oxo-(l laS)-2,3,5,1 la-tetrahydro-1H-pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]pentyloxy-(2R, IaS)2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzo-diazepine -5,11-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with methanohCHCl3 (2:8). Example 7 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,3-dibromopropane (505mg, 2.5 mmol) and K2C03 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ1.3-1.4 (m. 6H), 1.7-2.2 (m. 4H). 2.23-2.5 (m. 2H), 2.6-2.9 (m. 4H). 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t. 2H), 4.62-4.78 (m. IH), 4.85 (d, IH), 6.82 (s. IH), 7.75 (s, IH). • * A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (520mg, 1 mmoi), 8-hydroxy-7-methoxy-5,ll-dioxo-(2R,l laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-2-yl acetate (320mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-(3-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)propoxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2.3,5,10.11,1 la-hexahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-2-yl acetate of compounds of formula IV.8-(3-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l-pyrrolylcarbonyi)-2-methoxy-5-nitrophenoxy)propoxy)-7-methoxy-5,11 -dioxo-(2R, 11 aS)-2,3,5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV (760mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 Na2SO4and evaporated under vacuum to afford the crude 8-(3-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l- pyrrolylcarbonyl)-2-methoxy-5-aminophenoxy)propoxy)-7-methoxy-5.11 -dioxo-(2R, 11 aS)- 2.3,5,10.11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate. of compounds of formula V. A solution of the 8-(3-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-lH-l-pyrrolylcarbonyl)- 2-methoxy-5-aminophenoxy)propoxy)-7-methoxy-5,l l-dioxo-(2/?,l laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V (730mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H20 (3:1. 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 7-methoxy-8-(4-(7-methoxy-5-oxo-(llaR)-2,3,5,lla-tetrahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-8-yloxy)butoxy)-5,11 -dioxo-(2R, 11 aS)-2.3.5.10.11,lla-hexa-hydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of formula VI. which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8). Example 8 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,4-dibromobutane (540mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), § 6.82 (s, 1H), 7.75 (s, 1H). A solution of (2S)-N-[4-(4-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (534mg, 1 mmol), 8-hydroxy-7-methoxy-5,ll-dioxo-(2£,l laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-2-yl acetate (320mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-(4-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)butoxy)-7-methoxy-5,l l-dioxo-(2R,l IaS)-2,3,5,10,l 1,1 la-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV. 8-(4-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)butoxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2,3,5,10,l 1,11 a-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV (774mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 8-(4-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2-methoxy-5-arninophenoxy)butoxy)-7-methoxy-5.1 l-dioxo-(2R,l laS)-2,3,5.10,11.1 la- hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V. • A solution of the 8-(4-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-aminophenoxy)butoxy)-7-methoxy-5,l l-dioxo-(2R,l IaS)2,3,5,10.1 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V (744mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 7-methoxy-8-(4-(7-methoxy-5-oxo-( 11 aR)-2,3,5,11 a-tetrahydro-1 H-pyrrolo[2,1 -c] [ 1,4]-benzodiazipine-8-yloxy)butoxy)-5,l l-dioxo-(2R.l laS)-2,3,5,10,l 1,11 a-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8). Example 9 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,5-dibromopentane (575mg, 2.5 mmol) and K2CO3(414mg, 3 mrnol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC. EtOAc:hexane (7:3), 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-bromopentyloxy)-5-methoxy-2- nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II. • § 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s, 1H), 7.75 (s, 1H). A solution of 5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal 1 of formula II (548mg, 1 mmol), 8-hydroxy-7-methoxy-5,1 l-dioxo-(2R,l laS)-2,3,5,10,11,1 1 a-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiaze-pine-2-yl acetate (320mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2-methoxy-5-nitrophen-oxy)pentyloxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2,3,5,10,l 1,11 a-hexahydro-1H-pyrrolo-[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV. 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2.S)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)pentyloxy)-7-methoxy-5.1 l-dioxo-(2R,l Ia5)-2,3,5.10,l 1,11 a-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV (788mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2- methoxy-5-aminophenoxy)pentyloxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2,3,5,10,l 1,1 la- hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V. A solution of the 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-l/H-l-pyrrolylcarbonyl)-2-methoxy-5-aminophenoxy)pentyloxy)-7-methoxy-5,l 1 -dioxo-(2R, 11aS)-2,3,5,10,\1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V (758mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 7-methoxy-8-(4-(7-methoxy-5-oxo-(llaR)-2,3,5,lla-tetrahydro-1 H-pyrrolo[2,1 -c] [ 1,4]benzodiaze-pine-8-yloxy)butoxy)-5,11 -dioxo-(2R, 11 aS)-2,3.5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8). Biological Activity: In vitro anticancer activity studies were carried out at National Cancer Institute (USA) and the thermal denaturation of DNA were performed at School of Life Sciences, University of Hyderabad. Table 1 Thermal denaturation with calf thymus DNAa, at a [PBD]:[DNA] molar ratio of 1:5b and in vitro one dose primary anticancer assayc in the NCI-H460, MCF 7 and SF-268 for VI, wherein R is H, RI is H, OH, OAc and n is 3 to 5. (Table Removed) aFor CT-DNA at pH 7.00±0.01, ΔTm = 66.5°C±0.01 (mean value from 60 separate determinations), all ΔTm values ± 0.1-0.2°C. bFor a 1:5 molar ratio of [ligand]:[DNA], where CT-DNA concentration = 100 µM in aqueous buffer [10 mM sodium phosphate +1 M EDTA, pH 7.00+0.01. C0ne dose of VI (n is 3 to 5) at 10-4 molar concentration. Interestingly, the data presented in Table 1 shows that as the size of the linker spacer increases from 3 to 5 the DNA stabilization is also enhances. In this assay for a 1 :5 molar ratio of (PBD):DNA), one of this mixed imine-amide PBD dimer VI, where n is 5, elevates the helix melting temperature of CT DNA remarkably to 17.0°C after incubation for 18h at 37°C. In similar conditions, the dimer having two imino functionalities i.e., DSB-120 provides a ΔTm of 15.4°C. On the other hand, the naturally occurring DC-81 having only one imino group exhibits a ΔTm of 0.7°C. This demonstrates that the compound VI, where n is 5 containing a single imino functionality shows a very significant DNA binding affinity. To the best of our knowledge, this is the first synthetic non-cross-linking molecule which has exhibited a remarkable DNA binding effect that is similar to the naturally occurring sibiromycin (ΔTm= 16.3°C at 18h). This data indicates that non-covalent interactions play an important role for the enhancement of DNA binding affinity. The preliminary anti-cancer assay carried out in the three human cell lines: lung (NCI-H460), breast (MCF7) and CNS (SF-268) exhibit significant anticancer activity for these compounds as illustrated in Table 1. We Claim: A process for preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI of the drawing accompanying the specification wherein R is H, OH, OAc and R1 is H, and n is 3 to 5 which comprises: reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzyl]-pyrrolidin -2-carbaxy carbaldehyde diethyl thioacetal of formula I where in R1 is H with dibromoalkane in an aprotic water miscible organic solvents in presence of a mild inorganic base upto refluxing temperature for a period upto 48 hours, isolating (2S)-N-[4-(3-bromoalkoxy)-5-methoxy-2- nitrobenzoyl]pyrrolidine-2-carboxy 1 carbaldehydediethyl thoacetal of formula II where in R1 is H, reacting the above compound of formula II with dilactam of formula III wherein R is H, OH, OAc in presence of an inorganic mild base in presence of aprotic water miscible organic solvent upto refluxing temperature for a period upto 48 hours, isolating 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5, 1 0, 1 1 , 1 la-hydro- lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,l 1-dione of formula IV where in R is H, OH, OAc and R1 is H and n is 3-5 by conventional methods, reducing the above nitro compounds of formula IV with SnCl2 .2H2O in presence of organic solvent upto a reflux temperature, isolating the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-carboxy carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5,10,l 1, 1 la-hydro-lH-pyrrolo[2,l,-c][l,4] benzodiazepine-5,1 1-dione of formula V, wherein R is H, OH, OAc, and RI is H and n is 3-5 by known methods, reacting the above said amino compounds of formula V with known deprotecting agent in a conventional manner to give novel pyrrolo[2,l-c][l,4] benzodiazepines of formula VI wherein R, R1 and n as stated above. 2. A process as claimed in claim 1, wherein nitrodiethyl thioacetal pyrrolo[2,l- c][l,4]benzodiazepines used is such as C-8 linked to dilactam without or with substitutions in the proline ring such as H or OH. 3. A process as claimed in claimsl-2, wherein dibromoalkane used is having 3 carbon to 5 carbons. 4. A process as claimed in claim 1-3, wherein the mild base used is selected from BaCO3, Na2C03and K2CO3. 5. A process as claimed in claim 1-3, wherein water miscible aprotic solvent used is selected from acetone or N,N-dimethyl formamide, THF. 6. A Process as claimed in claims 1-5, wherein the deprotecting agent used is selected from HgCl2/HgO, HgCl2/CaC03. 7. A process as claimed in claims 1-6, wherein the reducing agent used is SnCl2.H2O 8. A process for the preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines substantially as herein described with reference to the examples and drawing accompanying this specification.

Full Text

The present invention relates to a process for preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines useful as potential antitumour agents. More particularly, it provides a process for the preparation of 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2. 3, 4,lla-tetrahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-8-yloxyalkyloxy}-(l laR)-2,3,5, 10,11.1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-dione. with aliphatic chain length
variations for the compounds and their 2-hydroxy, 2-acetyloxy derivatives.
In the last few years, a growing interest has been shown in the development of new pyrrolo[2,l-c][l,4]benzodiazepines (PBDs). These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within in the minor groove of duplex DNA via an acid-labile aminal bond to the electrophilic imine at the N10-C11 position. (Ref. Kunimoto, S. Masuda, T. Kanbayashi, N. Hamada. M. Naganawa, H. Miyamoto, M. Takeuchi, T. and Unezawa, H. J. Antibiot., 1980, 33, 665.; Kohn, K.W. and Speous, C.L. J. Mol. Bioi, 1970, 57, 551.; Hurley, L.H. Gairpla, C. and Zmijewski, M. Biochem. Biophys. Acta., 1977, 475, 521.; Kaplan, D.J. and Hurley, L.H. Biochmestry, 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 (Ref: 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, 8141-8147).
Naturally occurring pyrrolo[2,l-c][l,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 recognise and bind to specific sequence of DNA. Examples

of naturally occurring PBD's include anthramycin, tomaymycin. sibiromycin and neothramycin.

(Formula Removed)
In the last decade a number of PBD's have been designed and synthesized to explore them as DNA-sequence selective agents particularly C-8 linked PBD dimers. (Ref: Bose, D. S. Thomson, A. S. Ching, J. A. Hartley, J. A. Berardini, M. D. Jenkins, T. C. Neidle, S. Hurley, L. H. Thurston, D. E. J. Am. Chem. Soc. 1992, 114, 4939). The main object of present invention is to provide a new class of C-8 linked PBD dimers. wherein one PBD has an imine functionality while the other has an amide group. It has been envisaged that such a mixed dimer could offer more insight not only for the covalent binding but also the role played by non-covalent interactions with DNA-bases.
Pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-diones are a class of compounds that bind to DNA by non-covalent interactions such as hydrophobic, Vander walls interactions and hydrogen bonding between ring substituents and DNA, are also responsible for the influence on the sequence selectivity. Further, some dilactams such as (7-methoxy-2-methylcarbonyloxy-5,1 l-dioxo-(2S)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]-

benzodiazepine-5,ll-dione-8-yl acetate has been reported to possess significant in vivo antitumour activity in the P388 rat model. (Ref: Kaneko, T. Wong, H. Doyle. T. W. Rose, W. C. Bradner. W. T. J. Med. Chem. 1985, 25, 388).
The main object of the present invention is to provide a process for the preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI useful as antitumour agents. A
process for preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI of the
drawing accompanying the specification wherein R is H, OH, OAc and RI is H, and n is 3 to
5 which comprises: reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzyl]-pyrrolidin-[2-carbaxy-
carbaldehyde diethyl thioacetal of formula I where in RI is H with dibromoalkanes in an
aprotic water miscible organic solvents in presence of a mild inorganic base upto refluxing
temperature for a period upto 48 hours, isolating (2S)-N-[4-(3-bromoalkoxy)-5-methoxy-2-
nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II where in
RI is H and reacting the above compound of formula II with dilactam of formula III wherein
R is H. OH, OAc in presence of an inorganic mild base in presence of aprotic water miscible
organic solvents upto refluxing temperature for a period upto 48 hours isolating 8-{[(2S)-N-
5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-
2,3,5,10,1 l,lla-hydro-lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,ll-dione of formula IV
where in R is H, OH, OAc and RI is H and n is 3-5 by conventional methods, reducing the
above nitro compounds of formula IV with SnCl2 .2H2O in presence of organic solvent upto a
reflux temperature, isolating the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-
carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5,10,11, 11 a-hydro-1 H-pyrrolo[2,1 ,-
c][l,4]benzodiazepine-5,l 1-dione of formula V, where in R is H, OH, OAc and R1 is H and
(Scheme Removed)
n is 3-5 by known methods, reacting the above said amino compound of formula V with known deprotecting agents in a conventional manner to give novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI wherein R. RI and n as stated above.
The present invention provides a process for the preparation of pyrrolo[2,l-c][l,4]benzodiazepines of formula VI of the drawing accompanying this
specification where R is H, OH, OAc and RI is H and n is 3 to 5, which comprises: reacting
t
(2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaxy carbaldehyde diethyl
thioacetal of formula I where in RI is H with dibromoalkanes in the presence of organic
solvents such as acetone, THF and DMF. In an embodiment of the inventon the inorganic
bases used may be such as K^COa, BaCOs and NaiC03 upto refluxing temperatures for a
period in the range of 24 to 48 hours, affording the (2S)-N-[4-(3-bromopropoxy)-5-methoxy-
2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetol, (2S)-N-[4-(4-
bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl
thioacetol, (2S)-N-[4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy
carbaldehyde diethyl thioacetal of formula II, where in RI is H. In another embodiment of the
invention, reacting compounds of the formula II with debenzylated dilactam of formula III
where in R is H, OH, OAc (ref: Kaneko. E.; Wang, H.; Doyle, T. W. The J. Antibiotics,
1984, 3, 300) and organic solvents used may be aprotic water miscible solvents such as
acetone. THF, DMF. In yet another embodiment of the invention the inorganic bases used
may such as K2CO3, BaCO3 and Na2CO3, upto refluxing temperature for a period in the
range of 24 to 48 hours, affording the 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-
carbaldehyde diethylthioacetal]-propoxy-7-methoxy-2,3,5,10,11,11 a-hydro-1 H-pyrrolo[2,1 ,-
c] [ 1,4] benzo-diazepine-5,11 -dione, 8-{ [(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-

carbaldehyde diethylthioacetal]-butoxy-7-methoxy-2.3,5.10,l 1,1 la-hydro- lH-pyrrolo[2,l, -
c][1.4]benzo-diazepine-5,l 1-dione, 8-{[(2S)-N-5-methoxy-2-nitrobenzoyi]pyrrolidin-2-
carbaldehyde diethylthioacetal]-pentyloxy-7-methoxy-2.3,5,10,11,11 a-hydro-1 H-pyrrolo [2,l,-c][l,4] benzodiazepine-5,11-dione of compounds of the formula IV where in R is H, OH. OAc and RI is H. Reacting above said nitro thio acetal compounds of the formula IV
with the reducing agent SnCl2. 2H2O in the presence of organic solvents such as MeOH,
DMF amd 1,4-dioxane and their mixture upto reflux temperature, affording the 8-{[(2S)-N-5-
methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-propoxy-7-methoxy-
2,3,5,10,11, 1 la-hydro-lH-pyrrolo-[2,l,-c][l,4]benzodiazepine-5,l 1-dione. 8-{[(2S)-N-5-
methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-butoxy-7-methoxy-
2,3.5,10, 11,11 a-hydro-lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,l 1-dione, 8-{[(2S)-N-5-
methoxy-2-aminobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-penryloxy-7-
methoxy-2,3,5,10, 11,11 a-hydro-lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,l 1-dione of
formula V, where in R is H, OH, OAc and RI is H by conventional methods, reacting the
amino compounds of the formula V with known deprotecting agents such as HgCl2/HgO,
HgCl2/CaCO3 in presence of organic solvents such as acetonitrile, MeOH in a conventional
manner recovering 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2, 3, 4, 11 a-tetrahydro-1H-
pyrrolo[2,l-c][l,4] benzodiazepine-8-yloxypropoxy}-(l laR)-2,3,5,10,l 1,1 la-hexahyro-lH-
pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, 8-methoxy-7-{4-[7-methoxy-5-oxo-(l laS)- 2,
3, 4, 11 a-tetrahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-8-yloxybutoxy}-(l laR)-2,3,5,10,
11,1 la-hexahyro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, 8-methoxy-7-{5-[7-
methoxy-5-oxo-(llaS)- 2, 3, 4, lla-tetrahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-8-
yloxypentyloxy}-(l laR)-2,3,5,10,l 1,1 la-hexahyro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-

5.11-dione of the formula VI, from the above reaction mixture by known methods wherein R, RI and n are stated above.
The precursor, (2S)-N-(4-hydroxy-2-methoxy-2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula I (intermediates of DC-81) prepared by literature methods (Ref: Thurston, D.E.; Murthy, V. S.; Langley, D. R.; Jones, G. B.
Synthesis, 1990,81)
«
•
Some representative compounds of formula VI present invention are given below
1) 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2, 3, 4, lla-tetrahydro-lH-pyrrolo[2,l-
c][l,4]benzodiazepine-8-yloxypropoxy}-(l laR)-2,3,5,10,l 1,11 a-hexahyro-1H-
pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5,11 -dione
2) 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)- 2, 3, 4, lla-tetrahydro-lH-pyrrolo-[2,l-
c][l,4]benzodiazepine-8-yloxybutoxy}-(l laR)-2,3,5,10,11,11 a-hexahyro-1H-
pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5,11 -dione
3) 8-methoxy-7-{3-[7-methoxy-5-oxo-(l laS)-1,2,3,1 la-tetrahydro-lH-pyrrolo-[2,l-
c][l,4]-benzodiazepine-8-yloxypentyloxy}-(l laR)-2,3,5,10,11,11 a-hexahyro-1H-
pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5,11 -dione
4) 2-hydroxy-8-methoxy-7-3-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-1H-
pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]propoxy-(2R,l laS)-2,3,5,10,11,1 la-
hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine -5,11-dione
5) 2-hydroxy-8-methoxy-7-4-[7-methoxy-5-oxo-(l Ia5)-2,3,5,l 1 a-tetrahydro-1H-
pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]butoxy-(2R,l IaS)-2,3,5,10,l 1,1 la-
hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine -5,11-dione

6) 2-hydroxy-8-methoxy-7-5-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-1H-
pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]pentyloxy-(2R,l laS)-2,3,5,10,11,1 la-
hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine -5.11-dione
7) 8-methoxy-7-3[7-methoxy-5-oxo-(l laS)-2,3.5,l la-tetrahydro-lH-pyrrolo[2,l-
c][l,4]benzodiaze-pine-8-yloxy)propoxy)-5,l \-dioxo-(2R,l laR)-2,3,5,10,l 1.1 la-
hexahydro-1H-pyrrolo[2,1 -c][1,4]benzodiazepine-2-yl acetate
f
8) 8-methoxy-7-4-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-lH-pyrrolo[2,l-
c][l,4]benzodiaze-pine-8-yloxy)butoxy)-5,l l-dioxo-(2R,l laR)-2,3,5,10,l 1,1 la-
hexahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-2-yl acetate
9) 8-methoxy-7-(5-(7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-lH-pyrrolo[2,l-
c][l,4]benzodiazepine-8-yloxy)pentyloxy)-5,l 1-dioxo-(2R, 11 aR)-2,3,5,10,11,1 la-
hexahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-2-yl acetate
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 Scheme-1, which comprise:
1. The ether linkage at C-8 position of DC-81 intermediates with dilactams.
2. Refluxing the reaction mixture for 24-48 h.
3. Synthesis of C-8 linked PBD antitumour antibiotic imines.
4. Purification by column chromatography using different solvents like ethylacetate,
hexane, dichloromethane and methanol.

The following examples are given by way of illustrations and therefore should not be construpted to the present limit of the scope of invention.
Example 1
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1.3-dibromopropane (505mg, 2.5 mmol)
and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the
•
• completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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 EtOAchexane (1:1) gave the pure (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II. 1'H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 2H), 2.6-2.9 (m, 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, IH), 4.85 (d, IH), 6.82 (s, lH),7.75(s, IH).
A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (520mg, 1 mmol), 8-hydroxy-7-methoxy-(1 laS)-2.3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione (262mg, 1 mmol) of the formula III and K2CO3(414mg, 3 mmol) in dry acetone (20ml) 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 (8:2) gave the pure 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-propoxy-7-methoxy-

2.3.5.10,11,1 la-hexahydro-lH-pyrrolo[2.1-c][l,4]benzo-diazepine-5.11 -dione. of comp¬ounds of formula IV.
1H NMR (CDC13)δ 1.2-1.5 (m, 6H), 1.7-2.25 (m,8H), 2.23-2.5 (m, 2H). 2.6-2.9 (m, 4H), 3.2-3.3 (m. 4H), 3.6-3.8 (m, 2H), 3.8-4.0 (s, 6H), 4.01 (m. 1H), 4.2-4.35 (m. 2H). 4.65 (m, 1H), 4.85 (d, 1H), 6.5 (s, 1H), 6.8 (s, 1H), 7.38 (s, 1H), 7.7 (s, 1H), 8.21 (s, 1H, NH
exchangeable); FAB MS 703 (M+H)+.
•
The 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-propoxy-7-methoxy-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione of formula IV (702mg, 1.0 mmol) was dissolved in dichloro-methane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-propoxy-7-methoxy-2,3,5,10,11,1 la-hexahydro-1H-pyrrolo[2,l-c] [1,4] benzodiazepine-5,11-dione of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal} -propoxy-7-methoxy-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c]-[ 1,4]benzo-diazepine-5,11-dione of formula V (672mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 8-methoxy-7-{3-[7-methoxy-

5-oxo-( 1 laS)-2,3,l la-tetrahydro-lH-pyrrolo[2,l-c] [l,4]-benzodiazepine-8-yloxypropoxy}-
(11 aR)-2.3,5,10,11,11 a-hexahyro-1 H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-5.11 -dione of
formula VI, which was further purified by column chromatography on silica gel eluting first
with ethylacetate to remove traces of mercuric salts and further eluted with methanohCHCl3
(2:8).
1HNMR (DMSO-d6+CDC13) δ1.89-2.5 (m, 10H), 3.4-4.05 (m, 13H), 4.1-4.4 (m, 3H), 6.6 (s,
t 1H), 6.82 (s, 1H), 7.4 (s, 1H), 7.5 (s, 1H), 7.65 (d, 1H), 9.9 (s, 1H, NH exchangeable); FAB MS: 549 (M+H)+.
Example 2
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,4-dibromobutane (540mg, 2.5 mmol) and K2C03 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s, 1H), 7.75 (s, 1H).
A solution of (2S)-N-[4-(4-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (534mg, 1 mmol), 8-hydroxy-7-methoxy-(1 laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrol[2,l-c][l,4]benzodiazepine-5,l 1-dione (262mg,

1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 (8:2) gave the pure 8-{[(2S)-N-5-methoxy-2-
nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thio-acetal}-butoxy-7-methoxy-
2,3,5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiaze-pine-5,l 1-dione of formula IV. 1H NMR (CDC13) δ 1.2-1.5 (m, 6H), 1.7-2.25 (m,8H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H), 3.2-3.3 (m, 4H), 3.6-3.8 (m, 2H), 3.8-4.0 (s, 6H), 4.01 (m, 1H), 4.2-4.35 (m, 2H), 4.65 (m, 1H), 4.85 (d, 1H), 6.5 (s, 1H), 6.8 (s, 1H), 7.38 (s, 1H), 7.7 (s, 1H), 8.21 (s, 1H, NH exchangeable);
The 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-butoxy-7-methoxy-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzo-diazepine-5,l 1-dione (716mg, 1.0 mmol) of the formula IV was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H20 (1.124 g, 5.0 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 Na2S04 and evaporated under vacuum to afford the crude 8-{[(2S)-N-5-methoxy-2-aminobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal}-butoxy-7-methoxy-2,3,5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-5,l 1-dione of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal} -butoxy-7-methoxy-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c] [ 1,4]benzodi-azepine-5,11-dione of formula V. (686mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO

(687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 8-methoxy-7-{3-[7-methoxy-5-oxo-(l laS)-2, 3, 4, 1 la-tetrahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-8-yloxy-
«
f
butoxy} -(11 aR)-2,3,5,10,11,11 a-hexahyro-1 H-pyrrolo- [2,1 -c] [ 1,4]benzodiazepine-5,11 -dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with methanol:CHCl3 (2:8).
1HNMR (DMSO-d6+CDCl3) δ1.89-2.5 (m, 12H), 3.4-4.05 (m, 13H), 4.1-4.4 (m, 3H), 6.6 (s, 1H), 6.82 (s, 1H), 7.4 (s, 1H), 7.5 (s, 1H), 7.65 (d, 1H), 9.9 (s, 1H, NH exchangeable).
Example 3
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,5-dibromopentane (575mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II.

1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m. 6H), 2.6-2.9 (m, 4H), 3.15-3.33 (m. 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s. lH),7.75(s, 1H).
A solution of (2S)-N-[4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. (548mg, 1 mmol), 8-hydroxy-7-
methoxy-(l laS)-2,3.5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11-
dione (262mg, 1 mmol) III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 (8:2) gave the pure 8-{[(2S)-N-5-methoxy-2-nitrobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal}-pentyloxy-7-methoxy-2.3,5.10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione of formula IV. 'H NMR (CDC13) 5 1.2-1.5 (m, 6H), 1.7-2.25 (m,8H), 2.23-2.5 (m, 6H), 2.6-2.9 (m, 4H), 3.2-3.3 (m, 4H), 3.6-3.8 (m, 2H), 3.8-4.0 (s, 6H), 4.01 (m, 1H), 4.2-4.35 (m, 2H), 4.65 (m, 1H), 4.85 (d. 1H), 6.5 (s, 1H), 6.8 (s, 1H), 7.38 (s. 1H), 7.7 (s, 1H), 8.21 (s, 1H, NH exchangeable) The 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal}-pentyloxy-7-methoxy-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c] [ 1,4]ben-zodiazepine-5,11-dione of formula IV (730mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCOs 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 8-{[(2S)-N-5-methoxy-2-

aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-pentyloxy-7-methoxy-2.3.5.10.11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]ben-zodiazepine-5,l1-dione of formula V. A solution of 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal} -pentyloxy- 7-methoxy-2.3,5,10,11,11 a-hexahydro-1 H-pyrrolo [2,1 -c] [ 1,4] -ben-zodiazepine-5,11-dione formula V. (700mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO
(687mg, 3.18 mmol) in CH3CN/H2O (3:1. 15ml) was stirred at room temperature for 12h
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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 8-methoxy-7-{3-[7-methoxy-5-oxo-(llaS)-2, 3, 4, lla-tetrahydro-5H-pyrrolo[2,l-c][l,4]-benzodiazepine-8-yloxypentyloxy}-(l laR)-2,3,5,10,11,1 la-hexahyro-lH-pyrrolo[2,l-c] [l,4]benzodiazepine-5,11-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with methanol:CHCl3 (2:8).
1HNMR (DMSO-d6+CDCl3) δ 1.89-2.5 (m, 14H), 3.4-4.05 (m, 13H), 4.1-4.4 (m, 3H), 6.6 (s, 1H), 6.82 (s, 1H), 7.4 (s, 1H), 7.5 (s, 1H), 7.65 (d, 1H), 9.9 (s, 1H, NH exchangeable).
Example 4
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,3-dibromopropane (505mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m. 6H), 1.7-2.2 (m. 4H), 2.23-2.5 (m, 2H), 2.6-2.9 (m, 4H), 3.15-3.33 (m. 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m. 1H), 4.85 (d, 1H),
6.82 (s,lH), 7.75 (s,lH).
A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (520mg, 1 mmol), 2,8-dihydroxy-7-methoxy-(2R..1 laR)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione (278mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 EtOAcrhexane (9:1) gave the pure 8-{[(2S)-N-5-methoxy-2-nitrobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal }-propoxy-2-hydroxy-7-methoxy(2R. 11aR)-2,3,5,10,ll,lla-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-dione. of compounds of formula IV.
8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-propoxy-2-hydroxy-7-methoxy (2R, 1 laR)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5,11-dione, of compounds of formula IV (718mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H20 (1.124 g, 5.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO3solution and then extracted with ethyl acetate (3x20 mL). The combined

organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude8-
([(2S)-N-5-methoxy-2-arninobenzoyl]pyrrolidine 2-carbaldehyde diethyl thio- acetal}-
propoxy-2-hydroxy-7-methoxy(2R, 1 laR)-2.3.5.10.11,1 la-hexahydro-lH-pyrrolo[2,l-
c'][l,4]benzodiazepine-5.11-dione. of compounds of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl
thioacetal}-propoxy-2-hydroxy-7-methoxy(2R. 1 laR)-2,3,5,10,l 1,1 la-hexahydro-lH-
pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, of compounds of formula V (688mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 2-hydroxy-8-methoxy-7-3-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-1H-pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]propoxy-(2R,1 laS)-2,3,5,10,11,11 a-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8).
Example 5
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,4-dibromobutane (540mg, 2.5 mmol) and K.2C03 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m. 4H), 2.23-2.5 (m, 4H). 2.6-2.9 (m. 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s, lH),7.75(s, 1H).
• «
A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (534mg, 1 mmol), 2,8-dihydroxy-7-methoxy-(2R.I laR)-2,3.5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11-dione (278mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-{[(2S)-N-5-methoxy-2-nitrobenzoyljpyrrolidine 2-carbaldehyde diethyl thioacetal }-butoxy-2-hy droxy- 7-methoxy(2R. 1 laR)-2.3.5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c]-[l,4]benzodiazepine-5.11-dione. of compounds of formula IV.
A solution of 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl
thioacetal }-butoxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3,5,10,11,1 la-hexahydro-lH-
pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione, of compounds of formula IV (732mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine-2-carbaldehyde diethyl thio-acetal}-butoxy-2-hydroxy-7-methoxy(2R. 1 laR)-2.3.5,10.11.1 la-hexahydro-lH-pyrrolo[2,l-c][1.4]benzodiazepine-5,l 1-dione, of compounds of formula V. A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl
thioacetal}butoxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3,5,10.11,1 la-hexa-hydro-lH-pyrrolo-

[2,l-c][l,4]benzodiazepine-5,l 1-dione, of compounds of formula V. (702 mg, 1 mmol), HgCh (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 2-hydroxy-8-methoxy-7-4-[7-methoxy-5-oxo-(l laS)-2,3,5,l la-tetrahydro-lH-pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]butyloxy-2R,1 laS)-2,3,5,10,l 1,1 la-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-5,l 1-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8).
Example 6
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,5-dibromopentane (575mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 6H). 2.6-2.9 (m, 4H), 3.15-3.33 (m. 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m. 1H), 4.85 (d, 1H),
6.82 (s. 1H), 7.75 (s, 1H).
A solution of (2S)-N-[4-(5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (548mg, 1 mmol), 2,8-dihydroxy-7-methoxy-(2R, laR)-2,3,5,10,11,11 a-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-5.11-dione (278mg, 1 mmol) formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl thioacetal }-butoxy-2-hydroxy-7-methoxy(2R. 11 aR)-2,3,5,10,11,11 a-hexahydro-1 H-pyrrolo[2,1 -c]-[ 1,4]benzodiazepine-5.11 -dione. of compounds of formula IV.
A solution of 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidine 2-carbaldehyde diethyl
thioacetal }pentyloxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3,5,10,l 1,11 a-hexahydro-1H-
pyrrolo[2,l-c][l,4]benzodiazepine-5,11-dione, of compounds of formula IV (746mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2-2H2O (1.124 g, 5.0 mmol) was refluxed for 1.5 h. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCOs solution and then extracted with ethyl acetate (3x20 mL).

The combined organic phase was dried over Na2SO4and evaporated under vacuum to afford
the crude 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl
thioacetal}pentyloxy-2-hydroxy-7-methoxy(2R. 1 laR)-2.3,5.10.11,11 a-hexahydro-1H-
pyrrolo[2.1-c][l,4]benzodiazepine-5.11-dione. of compounds of formula V.
A solution of the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidine 2-carbaldehyde diethyl
thioacetal}pentyloxy-2-hydroxy-7-methoxy(2R, 1 laR)-2,3.5,10,l 1.11 a-hexa-hydro-1H-
* •
pyrrolo[2,l-c][l,4]benzodiazepine-5,ll-dione, of compounds of formula V. (716 mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The fllterate is evaporated in vacuum to get crude 2-hydroxy-8-methoxy-7-5-[7-methoxy-5-oxo-(l laS)-2,3,5,1 la-tetrahydro-1H-pyrrolo-[2,l-c][l,4]benzodiazepine-8-yloxy]pentyloxy-(2R, IaS)2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzo-diazepine -5,11-dione of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with methanohCHCl3 (2:8).
Example 7
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,3-dibromopropane (505mg, 2.5 mmol) and K2C03 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II. 1H NMR: (CDC13) δ1.3-1.4 (m. 6H), 1.7-2.2 (m. 4H). 2.23-2.5 (m. 2H), 2.6-2.9 (m. 4H). 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t. 2H), 4.62-4.78 (m. IH), 4.85 (d, IH), 6.82 (s. IH), 7.75 (s, IH).
•
*
A solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (520mg, 1 mmoi), 8-hydroxy-7-methoxy-5,ll-dioxo-(2R,l laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-2-yl acetate (320mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-(3-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)propoxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2.3,5,10.11,1 la-hexahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-2-yl acetate
of compounds of formula IV.8-(3-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l-pyrrolylcarbonyi)-2-methoxy-5-nitrophenoxy)propoxy)-7-methoxy-5,11 -dioxo-(2R, 11 aS)-2,3,5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV (760mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 Na2SO4and evaporated
under vacuum to afford the crude 8-(3-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l-
pyrrolylcarbonyl)-2-methoxy-5-aminophenoxy)propoxy)-7-methoxy-5.11 -dioxo-(2R, 11 aS)-
2.3,5,10.11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate. of
compounds of formula V.
A solution of the 8-(3-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-lH-l-pyrrolylcarbonyl)-
2-methoxy-5-aminophenoxy)propoxy)-7-methoxy-5,l l-dioxo-(2/?,l laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V (730mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H20 (3:1. 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 7-methoxy-8-(4-(7-methoxy-5-oxo-(llaR)-2,3,5,lla-tetrahydro-1H-pyrrolo[2,1 -c] [ 1,4]benzodiazepine-8-yloxy)butoxy)-5,11 -dioxo-(2R, 11 aS)-2.3.5.10.11,lla-hexa-hydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of formula VI. which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8).
Example 8
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,4-dibromobutane (540mg, 2.5 mmol) and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC, EtOAc:hexane (7:3), 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-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II. 1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H),
3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H),
§
6.82 (s, 1H), 7.75 (s, 1H).
A solution of (2S)-N-[4-(4-bromobutoxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde diethyl thioacetal of formula II (534mg, 1 mmol), 8-hydroxy-7-methoxy-5,ll-dioxo-(2£,l laS)-2,3,5,10,l 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiazepine-2-yl acetate (320mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-(4-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)butoxy)-7-methoxy-5,l l-dioxo-(2R,l IaS)-2,3,5,10,l 1,1 la-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV.
8-(4-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)butoxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2,3,5,10,l 1,11 a-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV (774mg, 1.0 mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O

(1.124 g, 5.0 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 8-(4-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2-methoxy-5-arninophenoxy)butoxy)-7-methoxy-5.1 l-dioxo-(2R,l laS)-2,3,5.10,11.1 la-
hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V.
•
A solution of the 8-(4-(4-(2-di(ethylsulfanyl)methyl-(25)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-aminophenoxy)butoxy)-7-methoxy-5,l l-dioxo-(2R,l IaS)2,3,5,10.1 1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V (744mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude
7-methoxy-8-(4-(7-methoxy-5-oxo-( 11 aR)-2,3,5,11 a-tetrahydro-1 H-pyrrolo[2,1 -c] [ 1,4]-benzodiazipine-8-yloxy)butoxy)-5,l l-dioxo-(2R.l laS)-2,3,5,10,l 1,11 a-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8).
Example 9
A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carbaldehyde diethyl thioacetal of formula I (400mg, 1 mmol), 1,5-dibromopentane (575mg, 2.5 mmol)

and K2CO3(414mg, 3 mrnol) in dry acetone (20ml) was refluxed for 48h. After the completion of reaction as indicated by TLC. EtOAc:hexane (7:3), 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-bromopentyloxy)-5-methoxy-2-
nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal of formula II.
• §
1H NMR: (CDC13) δ 1.3-1.4 (m, 6H), 1.7-2.2 (m, 4H), 2.23-2.5 (m, 4H), 2.6-2.9 (m, 4H), 3.15-3.33 (m, 2H), 3.6 (t, 2H), 3.95 (s, 3H); 4.25 (t, 2H), 4.62-4.78 (m, 1H), 4.85 (d, 1H), 6.82 (s, 1H), 7.75 (s, 1H).
A solution of 5-bromopentyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxy carbaldehyde dieth-yl thioacetal 1 of formula II (548mg, 1 mmol), 8-hydroxy-7-methoxy-5,1 l-dioxo-(2R,l laS)-2,3,5,10,11,1 1 a-hexahydro-lH-pyrrolo[2,l-c][l,4]-benzodiaze-pine-2-yl acetate (320mg, 1 mmol) of the formula III and K2CO3 (414mg, 3 mmol) in dry acetone (20ml) 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 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2-methoxy-5-nitrophen-oxy)pentyloxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2,3,5,10,l 1,11 a-hexahydro-1H-pyrrolo-[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV. 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2.S)-tetrahydro-lH-l-pyrrolylcarbonyl)-2-methoxy-5-nitrophenoxy)pentyloxy)-7-methoxy-5.1 l-dioxo-(2R,l Ia5)-2,3,5.10,l 1,11 a-hexahydro-1H-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of compounds of formula IV (788mg, 1.0

mmol) was dissolved in dichloromethane (5 mL), methanol (10 mL) and added SnCl2.2H2O (1.124 g, 5.0 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 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-1 H-1 -pyrrolylcarbonyl)-2-
methoxy-5-aminophenoxy)pentyloxy)-7-methoxy-5,l l-dioxo-(2R,l laS)-2,3,5,10,l 1,1 la-
hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V. A solution of the 8-(5-(4-(2-di(ethylsulfanyl)methyl-(2S)-tetrahydro-l/H-l-pyrrolylcarbonyl)-2-methoxy-5-aminophenoxy)pentyloxy)-7-methoxy-5,l 1 -dioxo-(2R, 11aS)-2,3,5,10,\1,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate, of compounds of formula V (758mg, 1 mmol), HgCl2 (794mg, 2.93 mmol) and HgO (687mg, 3.18 mmol) in CH3CN/H2O (3:1, 15ml) was stirred at room temperature for 12h 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 thorough celite and washed with ethylacetate. The filterate is evaporated in vacuum to get crude 7-methoxy-8-(4-(7-methoxy-5-oxo-(llaR)-2,3,5,lla-tetrahydro-1 H-pyrrolo[2,1 -c] [ 1,4]benzodiaze-pine-8-yloxy)butoxy)-5,11 -dioxo-(2R, 11 aS)-2,3.5,10,11,1 la-hexahydro-lH-pyrrolo[2,l-c][l,4]benzodiazepine-2-yl acetate of formula VI, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with MeOH:CHCl3 (2:8).

Biological Activity: In vitro anticancer activity studies were carried out at National Cancer Institute (USA) and the thermal denaturation of DNA were performed at School of Life Sciences, University of Hyderabad.
Table 1 Thermal denaturation with calf thymus DNAa, at a [PBD]:[DNA] molar ratio of 1:5b and in vitro one dose primary anticancer assayc in the NCI-H460, MCF 7 and SF-268 for VI, wherein R is H, RI is H, OH, OAc and n is 3 to 5.

(Table Removed)
aFor CT-DNA at pH 7.00±0.01, ΔTm = 66.5°C±0.01 (mean value from 60 separate determinations), all ΔTm values ± 0.1-0.2°C. bFor a 1:5 molar ratio of [ligand]:[DNA], where CT-DNA concentration = 100 µM in aqueous buffer [10 mM sodium phosphate +1
M EDTA, pH 7.00+0.01. C0ne dose of VI (n is 3 to 5) at 10-4 molar concentration.
Interestingly, the data presented in Table 1 shows that as the size of the linker spacer
increases from 3 to 5 the DNA stabilization is also enhances. In this assay for a 1 :5 molar ratio of (PBD):DNA), one of this mixed imine-amide PBD dimer VI, where n is 5, elevates the helix melting temperature of CT DNA remarkably to 17.0°C after incubation for 18h at 37°C. In similar conditions, the dimer having two imino functionalities i.e., DSB-120 provides a ΔTm of 15.4°C. On the other hand, the naturally occurring DC-81 having only one imino group exhibits a ΔTm of 0.7°C. This demonstrates that the compound VI, where n is 5 containing a single imino functionality shows a very significant DNA binding affinity. To the best of our knowledge, this is the first synthetic non-cross-linking molecule which has exhibited a remarkable DNA binding effect that is similar to the naturally occurring sibiromycin (ΔTm= 16.3°C at 18h). This data indicates

that non-covalent interactions play an important role for the enhancement of DNA binding affinity. The preliminary anti-cancer assay carried out in the three human cell lines: lung (NCI-H460), breast (MCF7) and CNS (SF-268) exhibit significant anticancer activity for these compounds as illustrated in Table 1.

We Claim:
A process for preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines of formula VI of the
drawing accompanying the specification wherein R is H, OH, OAc and R1 is H, and n is 3 to
5 which comprises: reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzyl]-pyrrolidin -2-carbaxy carbaldehyde diethyl thioacetal of formula I where in R1 is H with dibromoalkane in an aprotic water miscible organic solvents in presence of a mild inorganic base upto refluxing
temperature for a period upto 48 hours, isolating (2S)-N-[4-(3-bromoalkoxy)-5-methoxy-2-

nitrobenzoyl]pyrrolidine-2-carboxy 1 carbaldehydediethyl thoacetal of formula II where in R1 is H, reacting the
above compound of formula II with dilactam of formula III wherein R is H, OH, OAc in presence of an inorganic mild base in presence of aprotic water miscible organic solvent upto refluxing temperature for a period upto 48 hours, isolating 8-{[(2S)-N-5-methoxy-2-nitrobenzoyl]pyrrolidin-2-carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5, 1 0, 1 1 , 1 la-hydro- lH-pyrrolo[2,l,-c][l,4]benzodiazepine-5,l 1-dione of formula IV where in R is H, OH, OAc and R1 is H and n is 3-5 by conventional methods, reducing the above nitro compounds of formula IV with SnCl2 .2H2O in presence of organic solvent upto a reflux temperature, isolating the 8-{[(2S)-N-5-methoxy-2-aminobenzoyl]pyrrolidin-2-carboxy carbaldehyde diethylthioacetal]-alkoxy-7-methoxy-2,3,5,10,l 1, 1 la-hydro-lH-pyrrolo[2,l,-c][l,4] benzodiazepine-5,1 1-dione of formula V, wherein R is H, OH, OAc, and RI is H and n is 3-5 by known methods, reacting the above said amino compounds of formula V with known deprotecting agent in a conventional manner to give novel pyrrolo[2,l-c][l,4] benzodiazepines of formula VI wherein R, R1 and n as stated above.

2. A process as claimed in claim 1, wherein nitrodiethyl thioacetal pyrrolo[2,l-
c][l,4]benzodiazepines used is such as C-8 linked to dilactam without or with substitutions in
the proline ring such as H or OH.
3. A process as claimed in claimsl-2, wherein dibromoalkane used is having 3 carbon to 5
carbons.
4. A process as claimed in claim 1-3, wherein the mild base used is selected from BaCO3,
Na2C03and K2CO3.
5. A process as claimed in claim 1-3, wherein water miscible aprotic solvent used is selected
from acetone or N,N-dimethyl formamide, THF.
6. A Process as claimed in claims 1-5, wherein the deprotecting agent used is selected from
HgCl2/HgO, HgCl2/CaC03.
7. A process as claimed in claims 1-6, wherein the reducing agent used is
SnCl2.H2O
8. A process for the preparation of novel pyrrolo[2,l-c][l,4]benzodiazepines substantially as
herein described with reference to the examples and drawing accompanying this
specification.

Documents:

116-del-2001-abstract.pdf

116-del-2001-claims.pdf

116-del-2001-correspondence-others.pdf

116-del-2001-correspondence-po.pdf

116-del-2001-description (complete).pdf

116-del-2001-drawings.pdf

116-del-2001-form-1.pdf

116-del-2001-form-2.pdf

116-DEL-2001-Form-3.pdf

116-del-2001-form-4.pdf

116-del-2001-petition-138.pdf

« Previous Patent

Next Patent »

Patent Number

230908

Indian Patent Application Number

116/DEL/2001

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

28-Feb-2009

Date of Filing

31-Jan-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG NEW DELHI-110001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	AHMED KAMAL	FROM INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY,HYDERABAD-500 007,INDIA
2	NALLAN LAXMAN	FROM INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY,HYDERABAD-500 007,INDIA
3	GUJJAR RAMESH	FROM INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY,HYDERABAD-500 007,INDIA
4	PODDUTOORI RAMULU	FROM INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY,HYDERABAD-500 007,INDIA
5	OLEPU SRINIVAS	FROM INDIAN INSTITUTE OF CHEMICAL TECHNOLOGY,HYDERABAD-500 007,INDIA

PCT International Classification Number

A61K 31/33

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA