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WO2005063758A1 - C2-FLUORO PYRROLO[2,1-c][1,4]BENZODIAZEPINE DIMERS - Google Patents

C2-FLUORO PYRROLO[2,1-c][1,4]BENZODIAZEPINE DIMERS Download PDF

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WO2005063758A1
WO2005063758A1 PCT/IN2003/000448 IN0300448W WO2005063758A1 WO 2005063758 A1 WO2005063758 A1 WO 2005063758A1 IN 0300448 W IN0300448 W IN 0300448W WO 2005063758 A1 WO2005063758 A1 WO 2005063758A1
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methoxy
bis
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fluoro
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PCT/IN2003/000448
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Ahmed Kamal
Peram Surakattula Murali Mohan Reddy
Depatla Rajasekhar Reddy
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Council Of Scientific And Industrial Research
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Priority to JP2005512747A priority Critical patent/JP4833663B2/en
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Priority to GB0614749A priority patent/GB2425309B/en
Priority to AU2003300705A priority patent/AU2003300705A1/en
Publication of WO2005063758A1 publication Critical patent/WO2005063758A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to novel 2-fluoro-pyrrolo[2,l- c][l,4]benzodiazepine dimers useful as potential antitumour agents.
  • the present invention also relates to a process for the preparation of novel 2-fluoro-pyrrolo[2,l- c][l,4]benzodiazepine dimers useful as potential antitumour agents.
  • the present invention particularly relates to a process for the preparation of new bis-2-fluoro- pyrrolo[2,l-c][l,4] benzodiazepines useful as anticancer agents.
  • Pyrrolo[2,l-c][l,4]benzodiazepine antitumour antibiotics are commonly known as anthramycin class of compounds.
  • PBDs pyrrolo[2,l-c][l,4]benzodiazepines
  • PBD dimers have been developed that comprise two C2-exo- methylene- substituted DC-81 subunits tethered through their C-8 position via an inert propanedioxy linker.
  • Naturally occurring pyrrolo[2,l-c][l,4]benzodiazepines belong to a group of antitumour antibiotics derived from Streptomyces species with family members including anthramycin, tomaymycin, sibiromycin, chicamycin, neothramycins A and B, and DC-81.
  • the main object of the invention is to provide new bis-2-fluoro. pyrrolo[2,l- c][l,4]benzodiazepines useful as antitumour agents.
  • Another object of the invention is to provide a process for the preparation of novel fiuoro pyrrolo[2,l-c][l,4]benzodiazepines useful as antitumour agents.
  • Summary of the invention Accordingly the present invention provides fiuoro pyrrolo[2,l- c][l,4]benzodiazepine dimers of formula IX where n is 3 to 10.
  • the compound of formula IX is 1,1'- ⁇ [(propane- 1,3 -diyl)dioxy]bis[(l 1 aS)-2-fluoro-7-methoxy- 1 ,2,3, 11 a-tetra-hydro-5H- pyrrolo[2, l-c][l,4]benzodiazepin-5-one] ⁇ .
  • the compound of formula IX is 1,1'- ⁇ [(butane-l,4-diyl)dioxy]bis[(l laS)-2-fluoro-7-methoxy- 1,2,3, 1 la-tetra-hydro-5H- pyrrolo[2,l-c][l,4]benzodiazepin-5-one] ⁇ .
  • the compound of formula IX is 1,1'- ⁇ [(pentane- 1 , 5-diyl)dioxy]bis[( 11 aS)-2-fluoro-7-methoxy- 1,2,3,11 a-tetra-hydro-5H- pyrrolo[2, 1 -c][ 1 ,4]benzodiazepin-5-one] ⁇ .
  • the present invention also provides a process for the preparation of bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX
  • Formula IX where n is 3 to 10 which comprises: (a) reacting methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- hydroxypyrrolidine-2-carboxylate dissolved in an organic solvent, (b) cooling the solution and adding a solution of diethylaminosulfurtrifluoride (DAST) in an organic solvent drop wise; (c) isolating the methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxylate with DIBAL-H formed in the presence of an organic solvent and cooling;
  • DAST diethylaminosulfurtrifluoride
  • step (j) reacting the compound of formula VIII with a deprotecting agent to obtain bis 2- fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX wherein n is as stated above.
  • the organic solvent used in steps (a), (b) aiid (c) comprises CH2CI2.
  • step (a) the solution is cooled to a temperature of -78°C.
  • step (c) is carried out after 15 hours of step (b).
  • step (c) is done to a temperature of -78°C and for a period of 45 minutes.
  • step (e) is carried out in presence of an organic solvent and at room temperature.
  • step (h) is carried out for a period of about 48 hours.
  • step (i) is carried out in the presence of an organic solvent comprising methanol.
  • the deprotecting agent comprises a combination of HgCl 2 and HgO in CH 3 CN/H 2 O.
  • the present invention also provides a process for the preparation of bis 2-fluoro pyrrolo[2, l-c][l,4]benzodiazepines of formula IX Formula IX where n is 3 to 10, which comprises:
  • step (b) is carried out for a period of about 48 hours.
  • the reduction in step (c) is carried out in the presence of an organic solvent comprising methanol.
  • the deprotecting agent comprises a combination of HgCl 2 and HgO in CH 3 CN/H 2 O..
  • the present process provides a process for the preparation of bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX as given above where n is 3 to 10 which comprises reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoro-2- carboxaldehyde diethylthioacetal of formula VI with a dibromoalkane in an aprotic water miscible organic solvents.
  • the solvent is preferably chosen from acetone, acetonitrile, and DMF.
  • the reaction is also carried out in the presence of a mild inorganic bases such as K 2 CO 3 , CsCO 3 and BaCO 3 and up to refluxing temperature for a period of 48 hours.
  • a mild inorganic bases such as K 2 CO 3 , CsCO 3 and BaCO 3
  • the 1,1' - ⁇ [(alkane-l,N-diyl)dioxy ⁇ bis[(2-nitro-5-methoxy-l,4- phenylene) carbonyl] bis [4-fluoropyrrolidin-2-carboxaldehyde diethylthioacetal] of formula VII formed where n is 3-10 is then isolated by conventional methods and reduced with SnCl 2 .2H 2 O in presence of organic solvent up to a reflux temperature.
  • the process comprises first reacting methyl (2S)-N-[4- benzyloxy-5-methoxy-2-nitrobenzoyl]-4-hydroxypyrrolidine-2-carboxylate dissolved in an organic solvent such as CH 2 C1 2 and cooling the solution to -78°C. To this cooled solution, a solution of diethylaminosulfurtrifluoride (DAST) in an organic solvent such as CH 2 C1 2 is added drop wise over a period of 40 min.
  • DAST diethylaminosulfurtrifluoride
  • Cytotoxicity Compounds IXa and IXc were evaluated for in vitro against sixty human tumour cells derived from nine cancer types (leukemia, non-small-cell lung, colon, CNS, melanoma, ovarian, prostate, and breast cancer). For each compound, dose response curves against each cell line were measured at a minimum of five concentrations at 10 fold dilutions. A protocol of 48 h continuous drug exposure was used and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The concentration causing 50 % cell growth inhibition (GI50), total cell growth inhibition (TGI, 0% growth) and 50% cell death (LC50, -50% growth) compared with the control was calculated.
  • GI50 % cell growth inhibition
  • TGI total cell growth inhibition
  • LC50 50% cell death
  • log 10 GI50 log 10 TGI and log 10 LC50 mean graphs midpoints (MG_MID) of in vitro Cytotoxicity data for the compounds IXa and IXc against human tumour cell lines.
  • Compound Log ⁇ o GI50 LogioTGI Log ⁇ 0 LC50 UXa A2 ⁇ 75 ⁇ 431 IXc -7.14 -6,27 -4.87

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Abstract

The present invention relates to novel 2-fluoro-pyrrolo[2,1-c][1,4]benzodiazepine dimers of formula (IX) where n is 3 to 10 useful as antitumour agents and to a process for the preparation thereof.

Description

C2-FLUORO PYRROLO[2,l-c][l,4]BENZODIAZEPINE DIMERS Field of the invention The present invention relates to novel 2-fluoro-pyrrolo[2,l- c][l,4]benzodiazepine dimers useful as potential antitumour agents. The present invention also relates to a process for the preparation of novel 2-fluoro-pyrrolo[2,l- c][l,4]benzodiazepine dimers useful as potential antitumour agents. The present invention particularly relates to a process for the preparation of new bis-2-fluoro- pyrrolo[2,l-c][l,4] benzodiazepines useful as anticancer agents. More particularly, it provides a process for the preparation of l,l'-{[(bisalkane-l,N-diyl)] dioxy) bis [(1 laS) - 2 - fluoro - 7 - methoxy - 1, 2, 3, 1 la - tetrahydro - 5H -pyrrolo [2, ϊ-c] [1, 4] benzodiazepin-5-one, with aliphatic chain length variations for the compounds and also describes the anticancer (antitumour) activity. The structural formula of novel bis- 2-fluoro-pyrrolo[2,l-c][l,4]benzodiazepine is as follows, wherein n= 3,4,5,6,7,8,9,10.
Figure imgf000002_0001
Background of the invention Pyrrolo[2,l-c][l,4]benzodiazepine antitumour antibiotics are commonly known as anthramycin class of compounds. In the last few years, a growing interest has been shown in the development of new pyrrolo[2,l-c][l,4]benzodiazepines (PBDs). These PBDs are a family of sequence selective DNA-binding antitumour antibiotics that bind exclusively to the exocyclic N2-guanine in the minor groove of DNA via an acid-labile aminal bond to the electrophilic imine at the N10-C11 position. (Kunimoto, S.; Masuda, T.; Kanbayashi, N.; Hamada, M.; Naganawa, H.; Miyamoto, M.; Takeuchi, T.; Unezawa, H. J. Antibiot., 1980, 33, 665.; Kohn, K. W.; Speous, C. L. J Mol. Biol, 1970, 51, 551.; Hurley, L. H. Gairpla, C; Zmijewski, M. Biochem. Biophys. Ada., 1977, 475, 521.; Kaplan, D. 1; Hurley, L. H. Biochemistry, 1981, 20, 7572.) All biologically active PBDs possess the (S) configuration at the chiral Cl la position which provides the molecule with 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. Recently, PBD dimers have been developed that comprise two C2-exo- methylene- substituted DC-81 subunits tethered through their C-8 position via an inert propanedioxy linker. (Gregson, S. J.; Howard, P. W.; Hartely, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, Kelland, L. R.; Thurston, D. E. J. Med. Chem., 2001, 44, 737.). A recent development has been the linking of two PBD units through their C-8 positions to give bisfunctional alkylating agents capable of cross-linking DNA (Thurston, D. E.; Bose, D. S.; Thomson, A. S.; Howard, P. W.; Leoni, A.; Croker, S. J.; Jenkins, T. C; Neidle, S.; Hurley, L. H. J. Org. Chem., 1996, 61, 8141-8147). Recently, noncross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent anti- tumour activitiy (Kamal, A.; Laxman, N.; Ramesh, G.; Ramulu, P; Srinivas, US Patent 636233; Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu, V. B.; Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679.). PBDs are of considerable current interest due to their ability to recognize and subsequently form covalent bonds to specific base sequences of double-stranded DNA. Naturally occurring pyrrolo[2,l-c][l,4]benzodiazepines belong to a group of antitumour antibiotics derived from Streptomyces species with family members including anthramycin, tomaymycin, sibiromycin, chicamycin, neothramycins A and B, and DC-81.
Figure imgf000003_0001
anthramycin C2-exo-met ylene-sύbstituted DC-81
Figure imgf000003_0002
DC-81 dimeis (n=3-5); DSB-120 (n=3)
Figure imgf000003_0003
However, the clinical efficacy for these antibiotics is hindered by several limitations, such as poor water solubility and cardiotoxicity and development of drug resistance and metabolic inactivation. Object of the invention The main object of the invention is to provide new bis-2-fluoro. pyrrolo[2,l- c][l,4]benzodiazepines useful as antitumour agents. Another object of the invention is to provide a process for the preparation of novel fiuoro pyrrolo[2,l-c][l,4]benzodiazepines useful as antitumour agents. Summary of the invention Accordingly the present invention provides fiuoro pyrrolo[2,l- c][l,4]benzodiazepine dimers of formula IX where n is 3 to 10.
Figure imgf000004_0001
Formula EX In one embodiment of the invention, the compound of formula IX is 1,1'- { [(propane- 1,3 -diyl)dioxy]bis[(l 1 aS)-2-fluoro-7-methoxy- 1 ,2,3, 11 a-tetra-hydro-5H- pyrrolo[2, l-c][l,4]benzodiazepin-5-one]} . In another embodiment of the invention, the compound of formula IX is 1,1'- {[(butane-l,4-diyl)dioxy]bis[(l laS)-2-fluoro-7-methoxy- 1,2,3, 1 la-tetra-hydro-5H- pyrrolo[2,l-c][l,4]benzodiazepin-5-one]}. In another embodiment of the invention, the compound of formula IX is 1,1'- { [(pentane- 1 , 5-diyl)dioxy]bis[( 11 aS)-2-fluoro-7-methoxy- 1,2,3,11 a-tetra-hydro-5H- pyrrolo[2, 1 -c][ 1 ,4]benzodiazepin-5-one] } . The present invention also provides a process for the preparation of bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX
Figure imgf000004_0002
Formula IX where n is 3 to 10, which comprises: (a) reacting methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- hydroxypyrrolidine-2-carboxylate dissolved in an organic solvent, (b) cooling the solution and adding a solution of diethylaminosulfurtrifluoride (DAST) in an organic solvent drop wise; (c) isolating the methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxylate with DIBAL-H formed in the presence of an organic solvent and cooling;
(d) isolating methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxaldehyde formed;
(e) protecting methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxaldehyde with EtSH in presence of an organic solvent;
(f) isolating (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidine-2- carboxaldehyde diethylthioacetal; (g) reacting the (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidine- 2-carboxaldehyde diethylthioacetal with a debenzylating agent to obtain (2S)-N-[4- hydroxy - 5 - methoxy - 2 - nitrobenzoyl] - 4 - fluoropyrrolidine - 2 - carboxaldehyde - diethylthioacetal of formula NI,
Figure imgf000005_0001
Formula VI
(h) reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoro-2-carboxaldehyde diethylthioacetal of formula VI with a dibromoalkane in an aprotic water miscible organic solvent and in the presence of a mild inorganic base up to refluxing temperature and isolating l,l'-{[(alkane-l,N-diyl)dioxy}bis[(2-nitro-5-methoxy- 1,4-phenylene) carbonyl] bis [4-fluoropyrrolidin-2-carboxaldehyde diethylthioacetal] of formula VII where n is 3-10
(EtS)2H θ2 γ O 2C) A>y 02 CH(SEt)2 o , o Formula VII
(i) reducing the compound of formula VTI with SnCl2 .2H2O in presence of organic solvent up to a reflux temperature and isolating l,l'-{[(alkane-l,N- diyl)dioxy}bis[(2-amino-5-methoxy- 1 ,4-phenylene)carbonyl]]bis [4-fluoro- pyrrolidin-2-carboxaldehyde diethylthioacetal]] of formula VIII where n is 3-10
Figure imgf000006_0001
Formula VIK
(j) reacting the compound of formula VIII with a deprotecting agent to obtain bis 2- fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX wherein n is as stated above. In one embodiment of the invention, the organic solvent used in steps (a), (b) aiid (c) comprises CH2CI2. In another embodiment of the invention, in step (a) the solution is cooled to a temperature of -78°C. In another embodiment of the invention, the drop wise addition in step (b) is carried out for a period of 40 min. In another embodiment of the invention, step (c) is carried out after 15 hours of step (b). In yet another embodiment of the invention, the cooling in step (c) is done to a temperature of -78°C and for a period of 45 minutes. In another embodiment of the invention, step (e) is carried out in presence of an organic solvent and at room temperature. In yet another embodiment of the invention, the (2S)-N-[4-hydroxy-5-methoxy-
2-nitrobenzoyl]-4-fluoro-2-carboxaldehyde diethylthioacetal of formula VI is reacted with a dibromoalkane in an aprotic water miscible organic solvent selected from the group consisting of acetone, acetonitrile and DMF and in the presence of a mild inorganic base selected from the group consisting of K2CO3, CsCO3 and BaCO3. In another embodiment of the invention, step (h) is carried out for a period of about 48 hours. In another embodiment of the invention, the reduction in step (i) is carried out in the presence of an organic solvent comprising methanol. In yet another embodiment of the invention, the deprotecting agent comprises a combination of HgCl2 and HgO in CH3CN/H2O. The present invention also provides a process for the preparation of bis 2-fluoro pyrrolo[2, l-c][l,4]benzodiazepines of formula IX
Figure imgf000007_0001
Formula IX where n is 3 to 10, which comprises:
(a) (2S)-N-[4-hydroxy - 5 - methoxy - 2 - nitrobenzoyl] - 4 - fluoropyrrolidine - 2 carboxaldehyde - diethylthioacetal of formula VI,
Figure imgf000007_0002
Formula VI
(b) reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoro-2-carboxaldehyde diethylthioacetal of formula VI with a dibromoalkane in an aprotic water miscible organic solvent and in the presence of a mild inorganic base up to refluxing temperature and isolating l,l'-{[(alkane-l,N-diyl)dioxy}bis[(2-nitro-5-methoxy- 1,4-phenylene) carbonyl] bis [4-fluoropyrrolidin-2-carboxaldehyde diethylthioacetal] of formula VII where n is 3-10
Figure imgf000007_0003
Formula VII
(c) reducing the compound of formula VII with SnCl2 .2H2O in presence of organic solvent up to a reflux temperature and isolating l,l'-{[(alkane-l,N- diyl)dioxy}bis[(2-amino-5-methoxy-l,4-phenylene)carbonyl]]bis [4-fluoro- pyrrolidin-2-carboxaldehyde diethylthioacetal]] of formula VIII where n is 3-10
Figure imgf000007_0004
Formula VIEl
(d) reacting the compound of formula VIII with a deprotecting agent to obtain bis 2- fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX wherein n is as stated above. In yet another embodiment of the invention, the (2S)-N-[4-hydroxy-5-methoxy- 2-nitrobenzoyl]-4-fluoro-2-carboxaldehyde diethylthioacetal of formula VI is reacted with a dibromoalkane in an aprotic water miscible organic solvent selected from the group consisting of acetone, acetonitrile and DMF and in the presence of a mild inorganic base selected from the group consisting of K2CO3, CsCO3 and BaCO3. In another embodiment of the invention, step (b) is carried out for a period of about 48 hours. In another embodiment of the invention, the reduction in step (c) is carried out in the presence of an organic solvent comprising methanol. In yet another embodiment of the invention, the deprotecting agent comprises a combination of HgCl2 and HgO in CH3CN/H2O.. Detailed description of the invention The present process provides a process for the preparation of bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX as given above where n is 3 to 10 which comprises reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoro-2- carboxaldehyde diethylthioacetal of formula VI with a dibromoalkane in an aprotic water miscible organic solvents. The solvent is preferably chosen from acetone, acetonitrile, and DMF. The reaction is also carried out in the presence of a mild inorganic bases such as K2CO3, CsCO3 and BaCO3 and up to refluxing temperature for a period of 48 hours. The 1,1' -{[(alkane-l,N-diyl)dioxy}bis[(2-nitro-5-methoxy-l,4- phenylene) carbonyl] bis [4-fluoropyrrolidin-2-carboxaldehyde diethylthioacetal] of formula VII formed where n is 3-10 is then isolated by conventional methods and reduced with SnCl2 .2H2O in presence of organic solvent up to a reflux temperature. The 1 , 1 ' - { [(alkane- 1 ,N-diyl)dioxy }bis[(2-amino-5-methoxy- 1 ,4- phenylene)carbonyl]]bis [4-fluoro-pyrrolidin-2-carboxaldehyde diethylthioacetal]] of formula VIII formed where n is 3-10 is then isolated by known methods. The compound of formula VIII is then reacted with a known deprotecting agent in a conventional manner to obtain the novel bis 2-fluoro pyrrolo[2,l- c][l,4]benzodiazepines of formula IX wherein n are as stated above. In the alternate, the process comprises first reacting methyl (2S)-N-[4- benzyloxy-5-methoxy-2-nitrobenzoyl]-4-hydroxypyrrolidine-2-carboxylate dissolved in an organic solvent such as CH2C12 and cooling the solution to -78°C. To this cooled solution, a solution of diethylaminosulfurtrifluoride (DAST) in an organic solvent such as CH2C12 is added drop wise over a period of 40 min. After 15 hours methyl (2S)-N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidme-2-carboxylate of formula II with DLBAL-H formula III is isolated and then cooled in the presence of organic solvent such as CH2C12 -78°C for a period of 45 min. The methyl (2S)-N-[4-benzyloxy- 5-methoxy-2-nitrobenzoyl]-4-fluoropyrroUdine-2-carboxaldehyde formed is isolated by conventional methods and protected with EtSH in the presence of organic solvent at room temperature. The (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxaldehyde diethylthioacetal obtained is then isolated by known methods and reacted with any conventional debenzylating agent to give (2S)-N- [4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoropyrrolidine-2-carboxaldehyde- diethylthioacetal of formula VI. The compound of formula VI is then converted to the compound of formula IX in the manner indicated above. The precursor, methyl (2S)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)-4- hydroxypyrrolidine-2-carboxylate (intermediates of DC-81) was prepared by literature methods (Thurston, D.E.; Murthy, V. S.; Langley, D. R; Jones, G. B. Synthesis, 1990, 81.)
Some representative compounds of formula IX of present invention are given below:
1) l,l'-{ [(propane- l,3-diyl)dioxy]bis[(l laS)-2-fluoro-7-methoxy-l,2,3,l la-tetra- hydro-5H-pyrrolo[2, 1 -c] [ 1 ,4]benzodiazepin-5 -one] }
2) 1 , 1 ' - { [(butane- 1 ,4-diyl)dioxy]bis[(l 1 aS)-2-fluoro-7-methoxy- 1 ,2,3 , 11 a-tetra- hydro-5H-pyrrolo[2, l-c][l,4]benzodiazepin-5-one]}
3) 1 , 1 '- { [(pentane- 1 ,5-diyl)dioxy]bis[(l 1 aS)-2-fluoro-7-methoxy- 1 ,2,3, 11 a-tetra- hydro-5H-pyrrolo [2, 1 -c] [ 1 ,4]benzodiazepin-5 -one] } The reaction scheme is given below:
Figure imgf000010_0001
Br— (CH2) -Br K2C03/acetone
Figure imgf000010_0002
HgCI2/CaC03
Figure imgf000010_0003
These new analogues of pyrrolo[2,l-c][l,4]benzodiazepine dimers substituted at C-2 position have shown promising anticancer activity in various cell lines. The molecules synthesized are of immense biological significance with potential sequence selective DNA-binding property. This resulted in design and synthesis of new congeners, which comprise: The fiuoro substitution at C-2 position of DC-81 intermediates. The ether linkage between two fiuoro. DC-81 monomers at C-8 position. Refluxing the reaction mixture for 24-48 h. Synthesis of fiuoro PBD antitumour antibiotic dimer imines. Purification by column chromatography using different solvents like ethylacetate, hexane, dichloromethane and methanol. Representative compounds of Formula IX include
Figure imgf000011_0001
Formula IXa
Figure imgf000011_0002
Formula IXb
Figure imgf000011_0003
Formula f c
Figure imgf000011_0004
Formula IXd
Figure imgf000011_0005
Formula IXe
Figure imgf000011_0006
Formula IXf.
Figure imgf000011_0007
Formula IXg
Figure imgf000011_0008
Formula IXh The following examples are given by way of illustration and therefore should not be construed to the present limit of the scope of invention. Example 1 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoro pyrrolidine 2-carboxaldehyde diethylthioacetal VI (418 mg, 1 mmol), 1, 3- dibromopropane (101 mg, 0.5 mmol) and K2CO3 (414 mg, 3 mmol) in dry acetone (40 ml) 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) to give the pure l,l'-{[(Propane-l,3 diyl)dioxy]bis[2-nitro-5- methoxy- 1 ,4-phenylene) carbonyl] }bis[4-fluoro pyrrolidine-2-carboxaldehyde diethylthioacetal] VII. H1 NMR (CDC13, 200 MHz): δ 1.2-1.39 (m, 12H), 2.4-2.68 (m, 6H), 2.7-2.9 (m, 8H), 3.41-3.62 (m, 4H), 3.99 (s, 6H), 4.29-4.4 (m, 4 H), 4.52 (d, J = 3.9 Hz, 2H), 4.69-4.79 (m, 2H), 5.05 (t, 1H), 6.85 (s, 2H), 7.63 (s, 2H). FAB MASS: 877 (M+H) 1 , 1 ' - { [(Propane- 1 ,3 diyl)dioxy]bis[2-nitro-5-methoxy- 1 ,4-phenylene) carbonyl] }bis[4-fluoro pyrrolidine-2-carboxaldehyde diethyl thioacetal] VII (876 mg, 1.0 mmol) was dissolved in methanol (10 mL) and to this was added SnCl2.2H2O (1.124 g, 5.0 mmol) and 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 Na2SO and evaporated under vacuum to afford the crude l,l'-{[(Propane-l,3 diyl)dioxy]bis[2- amino-5-methoxy-l,4-phenylene) carbonyl] }bis[4-fluoro pyrrolidine-2-carboxaldehyde diethyl thioacetal]. A solution of the 1,1 '-{[(Propane- 1,3 diyl)dioxy]bis[2-amino-5-methoxy-l,4- pheny- lene) carbonyl] }bis[4-fluoro pyrrolidine-2-carboxaldehyde diethylthioacetal] VIII (846 mg, 1 mmol), HgCl2 (794 mg, 2.93 mmol) and HgO (686 mg, 3.18 mmol) in CH3CN/H2O (3: 1, 15 ml) was stirred at room temperature for 12h until TLC (EtOAc), indicated complete loss of starting material. Then organic layer is evaporated in vacuum and the residue is diluted with EtOAc. To this, saturated NaHCO3 solution was added slowly at room temperature and the mixture was filtered through celite and washed with ethylacetate. The filtrate was evaporated in vacuum to get crude 1,1'- {[(propane-l,3-diyl)]dioxy}bis[(l laS)-2-fluoro-7-methoxy-l,2,3, 1 la-tetrahydro-5H- pyrrolo[2,l-c][l,4] benzodiazepin-5-one] of formula IXa, which was further purified by column chromatography on silica gel eluting first with ethyl acetate to remove traces of mercuric salts and further eluted with CΗCl3-methanol (8.5:1.5). H1 NMR (CDC13, 200 MHz): δ 2.15-2.45 (m, 6H), 3.7-3.9 (m, 6H), 4.01(s, 6H), 4.22-4.3 (m, 4 H), 5.05 (t, 1H), 5.20 (t, 1H), 6.80 (s, 2H), 7.42 (s, 2H), 7.80 (d, 2H, J= 4.2 Hz). FAB MASS: 569 (M+H) Example 2 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoro pyrrolidine 2-carboxaldehyde diethylthioacetal VI (418 mg, 1 mmol), 1,4- dibromobutane (107 mg, 0.5 mmol) and K2CO3 (414 mg, 3 mmol) in dry acetone (20 ml) 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 ethyl acetate. 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) to give the purel,l'-{[Butane-l,4-diyl)dioxy]bis(2-nitro-5- methoxy- 1 ,4-phenylene) carbonyl] } bis[4-fluoro pyrroilidine-2-carboxaldehyde diethylthioacetal] VII. H1 NMR (CDC13, 200 MHz): δ 1.29-1.4 (m, 12H), 2.1-2.2 (m, 4H), 2.49-2.61 (m, 4H), 2.7-2.9 (m, 8H), 3.4 - 3.7 (m, 4H), 3.92 (s, 6H), 4.27 (t, 4 H), 4.58 (d, 2H), 4.70-4.85 (m, 2H), 5.08 (t, 1H), 5.29 (t, 1H), 6.82 (s, 2H), 7.65 (s, 2H). FAB MASS: 891 (M+H) 1 , 1 ' - { [Butane- 1 ,4-diyl)dioxy]bis(2-nitro-5-methoxy- 1 ,4-phenylene) carbonyl]} bis[4-fluoro pyrrolidine-2-carboxaldehyde diethylthioacetal] of formula VII (890 mg, 1.0 mmol) was dissolved in 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 Na2SO and evaporated under vacuum to afford the crude of purel,r-{[Butane-l,4-diyl)dioxy]bis(2-amino-5-methoxy-l,4- phenylene) carbonyl] }bis[4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal of formula VIII. A solution of l,l'-{[Butane-l,4-diyl)dioxy]bis(2-amino-5-methoxy-l,4- phenylene) carbonyl] }bis[4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal of formula VIII (861 mg, 1 mmol), HgCl2 (794 mg, 2.93 mmol) and HgO (687 mg, 3.18 mmol) in CH3CN/H_O (3:1, 15 ml) was stirred at room temperature for 12h until TLC (EtOAc), indicated complete loss of starting material. Then organic layer was evaporated in vacuum and the residue is diluted with EtOAc. To this, saturated NaHCO3 solution was added slowly at room temperature and the mixture is filtered through celite and washed with ethyl acetate. The filtrate was evaporated in vacuum to get crude l,l'-{[(butane-l,4-diyl)]dioxy}bis[(l laS)-2-fluoro-7-methoxy-l,2,3,l la- tetrahydro-5H-pyrrolo[2, l-c][l,4] benzodiazepin-5-one IXb, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with CHCl3-methanol (9:1). H1 NMR (CDC13, 200 MHz): δ 1.94-2.09 (m, 4H), 2.1-2.5 (m, 4H), 3.5 - 3.82 (m, 6H), 3.98 (s, 6H), 4.1-4.37 (m, 4H), 5.29 (t, 1H), 5.5 (t, 1H), 6.80 (s, 2H), 7.45 (s, 2H), 7.80 (d, 2H, J = 4.3 Hz). FAB MASS: 583 (M+H) Example 3 A solution of (2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)-4-fluoro pyrrolidine 2-carboxaldehyde diethylthioacetal VI (418 mg, 1 mmol), 1,5- dibromopentane (114 mg, 0.5 mmol) and K2CO3 (414 mg, 3 mmol) in dry acetone (20 ml) 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) to give the purel,l'-{[Pentane-l,5-diyl)dioxy]bis(2-nitro-5- methoxy-l,4-phenylene) carbonyl] }bis[4-fluoro pyrroilidine-2-carboxaldehyde diethyl thioacetal VII. H1 NMR (CDC13, 200 MHz): δ 1.2-1.42 (m, 12H), 1.65-2.1 (m, 6H), 2.4 - 2.61 (m, 4H), 2.7-2.91 (m, 8H), 3.29-3.67 (m, 4H), 3.99 (s, 6H), 4.09-4.25 (m, 4H), 4.52-4.68 (m, 2H), 4.82 (d, 2H), 5.10 (t, 1H), 5.32 (t, 1H), 6.89 (s, 2H), 7.69 (s, 2H). FAB MASS: 905 (M+H) 1,1 '-{[Pentane-l,5-diyl)dioxy]bis(2-nitro-5-methoxy-l,4-phenylene) carbonyl]} bis[4-fluoro pyrroilidine-2-carboxaldehyde diethylthioacetal]. of formula VII (905 mg, 1.0 mmol) was dissolved in methanol (10 ml) and to it was added SnCl2.2H2θ (1.124 g, 5.0 mmol) and 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 Na SO4 and evaporated under vacuum to afford the crude -{[Pentane-l,5-diyl)dioxy]bis(2-amino- 5-methoxy-l,4-phenylene)carbonyl]}bis[4-fluoropyrrolidine-2-carboxaldehyde diethyl thioacetal. of formula VIII. A solution of l,l'-{[Pentane-l,5-diyl)dioxy]bis(2-amino-5-methoxy-l,4- phenylene) carbonyl] }bis[4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal of formula VIII (875 mg, 1 mmol), HgCl2 (794 mg, 2.93 mmol) and HgO (687 mg, 3.18 mmol) in CH.3CN/H2 (3: 1, 15 ml) was stirred at room temperature for 12h until TLC (EtOAc) indicated complete loss of starting material. Then organic layer was evaporated in vacuum and the residue was diluted with EtOAc. To this, saturated NaHCO3 solution was added slowly at room temperature and the mixture was filtered through celite and washed with ethylacetate. The filtrate was evaporated in vacuum to get crude 1,1 '-{[(pentane-l,5-diyl)]dioxy}bis[(l laS)-2-fluoro-7-methoxy-l,2,3, 11a- tetrahydro-5H-pyrrolo[2,l-c][l,4] benzodiazepin-5-one IXc, which was further purified by column chromatography on silica gel eluting first with ethylacetate to remove traces of mercuric salts and further eluted with CHCl3-methanol (9:1). H1 NMR (CDC13, 200 MHz): δ 1.58-1.81 (m, 4H), 1.90-2.01 (m, 2H), 2.38 - 2.50 (m, 4H), 3.08-3.24 (m, 4H), 4.01-4.20 (m, 4H), 4.92 (s, 6H), 5.21 (t, 1H), 5.5 (t, 1H), 6.81 (s, 2H), 7.49 (s, 2H), 7.83 (d, 2H, J= 4.4 Hz). FAB MASS: 597 (M+H)
Biological Activity: In vitro biological activity studies were carried out at National Cancer Institute (USA).
Cytotoxicity: Compounds IXa and IXc were evaluated for in vitro against sixty human tumour cells derived from nine cancer types (leukemia, non-small-cell lung, colon, CNS, melanoma, ovarian, prostate, and breast cancer). For each compound, dose response curves against each cell line were measured at a minimum of five concentrations at 10 fold dilutions. A protocol of 48 h continuous drug exposure was used and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The concentration causing 50 % cell growth inhibition (GI50), total cell growth inhibition (TGI, 0% growth) and 50% cell death (LC50, -50% growth) compared with the control was calculated. The mean graph midpoint values of logioTGI and log10LC50 as well as log10 GI50 for IXa and IXc are listed in Table 1. As demonstrated by mean graph pattern, compound IXc exhibits an interesting profile of activity and selectivity for various cell lines. The mean graph mid point of log10 TGI and log10 LC50 showed similar pattern to the log10 GI50 mean graph mid points. Table 1. log10 GI50 log10 TGI and log10LC50 mean graphs midpoints (MG_MID) of in vitro Cytotoxicity data for the compounds IXa and IXc against human tumour cell lines. Compound Logιo GI50 LogioTGI Logι0LC50 UXa A2Ϊ 75 ^431 IXc -7.14 -6,27 -4.87
Table 2. In vitro one dose primary anticancer assaya bisfluorinated PBDs of formula IXa, and LXc PBD Growth percentages
(Lung) (Breast) (CNS) NCI-H460 MCF7 SF-268
IXa 0 0 0
LXc 0 0 0 aOne dose of IXa and IXc at 10"4 molar concentration The anticancer activity for two representative compounds has been given in
Table 2. The comparison of the data of Table 3 reveals the importance of the alkane spacer. As the alkane spacer increased from 3-5 the cytotoxic activity has moderately enhanced. The 5 carbon spacer of compound IXc confers a suitable fit in the minor groove of double helix DNA and shows slightly higher activity in this series of compounds IXa and IXc.
Table 3. Log GI50 (inhibitory concentration) Values for Compounds IXa, and IXc cancer Compound Compound IXa EXc leukemia 5.668 7.794 non-small-cell 5.258 7.318 lung colon 5.285 7.064 CNS 5.543 7.625 melanoma 5.490 7.301 ovarian 5.310 6.620 renal 5.315 7.492 prostate 5.180 7.430 breast 5.490 7.234 Each cancer type represents the average of six to eight different cancer cell lines. 2005/063758
Figure imgf000017_0001
MEAN GRAPH TABLE 4
Figure imgf000018_0001

Claims

We Claim:
1. A novel pyrrolo[2, 1 -c] [1 ,4]benzodiazepine of formula IX where n is 3 to 10.
Figure imgf000019_0001
2. A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000019_0002
A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000019_0003
4. A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000019_0004
5. A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000019_0005
6. A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000019_0006
7. A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000020_0001
A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000020_0002
9. A novel pyrrolobenzodiazepine as claimed in claim 1 of the structure
Figure imgf000020_0003
10. A process for the preparation of bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX
Figure imgf000020_0004
Formula IX where n is 3 to 10, which comprises: (a) reacting methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- hydroxypyrrolidine-2-carboxylate dissolved in an organic solvent, (b) cooling the solution and adding a solution of diethylaminosulfurtrifluoride (DAST) in an organic solvent drop wise; (c) isolating the methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxylate with DIBAL-H formed in the presence of an organic solvent and cooling; (d) isolating methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxaldehyde formed; (e) protecting methyl (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxaldehyde with EtSH in presence of an organic solvent; (f) isolating (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxaldehyde diethylthioacetal; (g) reacting the (2S)-N-[4-benzyloxy-5-methoxy-2-nitrobenzoyl]-4- fluoropyrrolidine-2-carboxaldehyde diethylthioacetal with a debenzylating agent to obtain (2S)-N- [4-hydroxy - 5 - methoxy - 2 - nitrobenzoyl] - 4 - fluoropyrrolidine - 2 - carboxaldehyde - diethylthioacetal of formula VI,
Figure imgf000021_0001
Formula VI
(h) reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoro-2- carboxaldehyde diethylthioacetal of formula VI with a dibromoalkane in an aprotic water miscible organic solvent and in the presence of a mild inorganic base up to refluxing temperature and isolating 1, l'-{ [(alkane- 1,N- diyl)dioxy}bis[(2-nitro-5-methoxy-l,4-phenylene) carbonyl] bis [4- fluoropyrrolidin-2-carboxaldehyde diethylthioacetal] of formula VII where n is 3-10
Figure imgf000021_0002
Formula VII
(i) reducing the compound of formula VII with SnCl2 .2H2O in presence of organic solvent up to a reflux temperature and isolating l,l'-{[(alkane-l,N- diyl)dioxy}bis[(2-amino-5-methoxy-l,4-phenylene)carbonyl]]bis [4-fluoro- pyrrolidin-2-carboxaldehyde diethylthioacetal]] of formula VIII where n is 3-10
Figure imgf000021_0003
Formula VIII
(j) reacting the compound of formula VIII with a deprotecting agent to obtain bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX wherein n is as stated above.
11. A process as claimed in claim 10 wherein the organic solvent used in steps (a), (b) and (c) comprises CH2C12.
12. A process as claimed in claim 10 wherein in step (a) the solution is cooled to a temperature of -78°C.
13. A process as claimed in claim 10 wherein the drop wise addition in step (b) is carried out for a period of 40 min.
14. A process as claimed in claim 10 wherein step (c) is carried out after 15 hours of step (b).
15. A process as claimed in claim 10 wherein the cooling in step (c) is done to a temperature of -78°C and for a period of 45 minutes.
16. A process as claimed in claim 10 wherein step (e) is carried out in presence of an organic solvent and at room temperature.
17. A process as claimed in claim 10 wherein the the (2S)-N-[4-hydroxy-5-methoxy- 2-nitrobenzoyl]-4-fluoro-2-carboxaldehyde diethylthioacetal of formula VI is reacted with a dibromoalkane in an aprotic water miscible organic solvent selected from the group consisting of acetone, acetonitrile and DMF and in the presence of a mild inorganic base selected from the group consisting of K2CO3, CsCO3 and BaCO3.
18. A process as claimed in claim 10 wherein step (h) is carried out for a period of about 48 hours.
19. A process as claimed in claim 10 wherein the reduction in step (i) is carried out in the presence of an organic solvent comprising methanol.
20. A process as claimed in claim 10 wherein the deprotecting agent comprises a combination of HgCl2 and HgO in CH3CN/H2O.
21. A process for the preparation of bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX
Figure imgf000022_0001
Formula IX where n is 3 to 10, which comprises: (a) (2S)-N- [4-hydroxy - 5 - methoxy - 2 - nitrobenzoyl] - 4 - fluoropyrrolidine - 2 - carboxaldehyde - diethylthioacetal of formula VI,
Figure imgf000023_0001
Formula VI (b) reacting (2S)-N-[4-hydroxy-5-methoxy-2-nitrobenzoyl]-4-fluoro-2- carboxaldehyde diethylthioacetal of formula VI with a dibromoalkane in an aprotic water miscible organic solvent and in the presence of a mild inorganic base. up to refluxing temperature and isolating 1,1 '-{[(alkane- 1,N- diyl)dioxy}bis[(2-nitro-5-methoxy-l,4-phenylene) carbonyl] bis [4- fluoropyrrolidin-2-carboxaldehyde diethylthioacetal] of formula VII where n is 3-10
Figure imgf000023_0002
Formula VII (c) reducing the compound of formula VII with SnCl2 .2H2O in presence of organic solvent up to a reflux temperature and isolating l,l'-{[(alkane-l,N- diyl)dioxy}bis[(2-amino-5-methoxy-l,4-phenylene)carbonyl]]bis [4-fluoro- pyrrolidin-2-carboxaldehyde diethylthioacetal]] of formula VIII where n is 3-10
Figure imgf000023_0003
Formula VIII (d) reacting the compound of formula VIII with a deprotecting agent to obtain bis 2-fluoro pyrrolo[2,l-c][l,4]benzodiazepines of formula IX wherein n is as stated above.
22. A process as claimed in claim 21 wherein the (2S)-N-[4-hydroxy-5-methoxy-2- nitrobenzoyl]-4-fluoro-2-carboxaldehyde diethylthioacetal of formula VI is reacted with a dibromoalkane in an aprotic water miscible organic solvent selected from the group consisting of acetone, acetonitrile and DMF and in the presence of a mild inorganic base selected from the group consisting of K2CO3, CsCO3 and BaCO3.
23. A process as claimed in claim 21 wherein step (b) is carried out for a period of about 48 hours.
24. A process as claimed in claim 21 wherein the reduction in step (c) is carried out in the presence of an organic solvent comprising methanol.
25. A process as claimed in claim 21 wherein the deprotecting agent comprises a combination of HgCl2 and HgO in CH3CN/H2O.
26. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula IX and pharmaceutically acceptable additives.
27. Method for the treatment of cancer in a patient suffering from the same, said method comprising administering to the patient a pharmaceutically effective amount of a compound of formula IX.
28. A method as claimed in claim 27 wherein the patient is a mammal.
29. A method as claimed in claim 27 wherein the mammal is a human being.
30. A method as claimed in claim 27 wherein the cancer is selected from the group consisting of leukemia, non-small cell, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast.
31. Use of a compound of formula IX for the treatment of cancer selected from the group consisting of leukemia, non-small cell, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast in a subject suffering from the same.
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