EP2262809A1 - Benzophenone-piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids as potential anticancer agents and process for the preparation thereof - Google Patents

Benzophenone-piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids as potential anticancer agents and process for the preparation thereof

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Publication number
EP2262809A1
EP2262809A1 EP08873530A EP08873530A EP2262809A1 EP 2262809 A1 EP2262809 A1 EP 2262809A1 EP 08873530 A EP08873530 A EP 08873530A EP 08873530 A EP08873530 A EP 08873530A EP 2262809 A1 EP2262809 A1 EP 2262809A1
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European Patent Office
Prior art keywords
benzodiazepine
pyrrolo
benzophenone
methoxy
chloro
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EP08873530A
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German (de)
French (fr)
Inventor
Ahmed Kamal
Bandari Rajendra Prasad
Adla Malla Reddy
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Council of Scientific and Industrial Research CSIR
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Council of Scientific and Industrial Research CSIR
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    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to benzophenone-piperazine linked pyrrolo[2,1- c][1 , ⁇ benzodiazepine hybrids and a process for the preparation there of. More particularly It relates to 7-Methoxy-8-[n- ⁇ /1 -[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazino-acetamide] alkyloxy ⁇ -(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4] benzodiazepine-5-one with aliphatic chain length variations useful as anticancer (antitumour) agent.
  • the structural formula of these benzophenone-piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines hybrids is given below.
  • Pyrrolo[2,1-c][1 ,4]benzodiazepine antitumour antibiotics are commonly known as anthramycin class of compounds.
  • PBDs pyrrolo [2, 1-c][1 ⁇ benzodiazepines
  • antibiotics react covalently with DNA to form an N2-guanine adduct that lies within the minor groove of duplex DNA via an acid-labile aminal bond to the electrophilic imine at the N10-C11 position
  • PBD dimers have been developed that comprise of two C2-exo-methylene substituted DC-81 subunits tethered through their C-8 position via an inert propanedioxy linker (Gregson, S. J.; Howard, P. W.; Hartely, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T. C; Kelland, L R. and Thurston, D. E. J. Med. Chem. 2001, 44, 737).
  • a non-cross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent antitumour activity (Kamal, A.; Ramesh, G.
  • Naturally occurring pyrrolo[2,1-c][1 , ⁇ benzodiazepines belong to a group of antitumour antibiotics derived from Streptomyces species. Recently, there is much impetus for the PBD systems as they can recognize and bind to specific sequence of DNA. Examples of naturally occurring PBDs include anthramycin, DC-81 , tomaymycin, sibiromycin and neothramycin.
  • the main objective of the present invention is to provide novel benzophenone - piperazine linked pyrrolo[2,1-c][1 , ⁇ benzodiazepine hybrids, useful as antitumour agents.
  • Yet another object of this invention is to provide a process for the preparation of novel benzophenone-piperazine linked pyrrolo[2,1-c][1 , ⁇ benzodiazepine hybrids.
  • the present invention provides a novel benzophenone-piperazine linked pyrrolo[2,1-c][1 , ⁇ benzodiazepine hybrid of general formula 5
  • n 3, 4, 5, 6, 8
  • novel benzophenone-piperazine linked pyrrolo[2,1-c][1 , ⁇ benzodiazepine hybrid according to claim 1 is represented by the group of the following compounds:
  • novel benzophenone-piperazine linked pyrrolo [2,1- c][1 ,4]benzodiazepine hybrid exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group consisting of lung (Hop-62), cervix (SiHa), breast (MCF7, Zr-75-1), colon (Colo205), prostate (DU 145, PC3) and oral (DWD, HT1080) cell lines.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid used for in vitro activity against Colo205 for IC50 is in the range of 13 to 80 ⁇ m, at an exposure period of at least 48 hrs.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against DU145 for IC50 is in the range of 12 to 80 ⁇ m, at an exposure period of at least 48 hrs.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against DWD for IC50 is in the range of 8 to 80 ⁇ m, at an exposure period of at least 48 hrs. In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 , ⁇ benzodiazepine hybrids used for in vitro activity against HoP62 for IC50 is in the range of 11 to 48 ⁇ m, at an exposure period of at least 48 hrs.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1,4]benzodiazepine hybrids used for in vitro activity against HT1080 for IC50 is in the range of 14 to 36 ⁇ m, at an exposure period of at least 48 hrs.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against MCF7 for IC50 is in the range of 22 to about 80 ⁇ m, at an exposure period of at least 48 hrs.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against PC3 for IC50 is in the range of 28 to about 80 ⁇ m, at an exposure period of at least 48 hrs.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1,4]benzodiazepine hybrids used for in vitro activity against SiHa for IC50 is in the range of 36 to about 80 ⁇ m, at an exposure period of at least 48 hrs.
  • the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against Zr-75-1 for IC50 is in the range of 29 to about 80 ⁇ m, at an exposure period of at least 48 hrs.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising benzophenone linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid, its derivatives, analogues, salts or mixture thereof optionally with pharmaceutically acceptable carriers, adjuvants and additives.
  • benzophenone-piperazine linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid used is represented by a general formula 5,
  • n 3, 4, 5, 6, 8 and the said process comprising the steps of: a) reacting (2S)-N-[(n-bromoalkyloxy)-3-methoxy-2-nitrobenzoyl)]pyrrolidine-2- carboxaldehyde diethylthioacetal of formula 1
  • n 3, 4, 5, 6, 8 4a-e c) reacting the above said amino compound of formula 4 obtained in step (b) with a deprotecting agent of the kind of mercuric chloride by known method to obtain the desired compound of formula 5
  • the mild inorganic base used in steps (a) is potassium carbonate.
  • aprotic organic solvent used in step (a) is acetone and acetonitrile
  • organic solvent used in step (c) is acetonitrile and acetone
  • the alcohol used in step (b) is selected from methanol and ethanol.
  • the compounds of formula 5a-e obtained are represented by a group of the following compounds:
  • the benzophenone-piperazine linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid of formula 5a-e exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group consisting of lung, cervix, breast, colon, prostate and oral cell lines.
  • Reagents and conditions (i) K 2 CO 3 , acetone,18 h, refllux, 90-92%; (ii) SnCI 2 .2H 2 O, MeOH, 2 h, reflux, 8! 87%; (iii) HgCI 2 -CaCO 3 , CH 3 CN-H 2 O (4:1), 12 h, rt, 68-71 %.
  • This compound was prepared according to the method described for the compound 3 a by employing 2S- ⁇ /-[4-(4-bromobutoxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2- carboxaldehyde (1b) diethylthioacetal (535 mg, 1.0 mmol) was added anhydrous potassium carbonate (690 mg, 5.0 mmol) and ⁇ /1-4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide 2 (392 mg, 1.0 mmol) to obtain the pure product 3b (710 mg, 84% yield).
  • This compound was prepared according to the method described for the compound 4a by reducing 2S- ⁇ /- ⁇ 4-[4- ⁇ /1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] butyl)oxy-5-methoxy-2-nitrobenzoyl ⁇ pyrrolidine-2-carboxaldehyde diethylthioacetal 3b (846 mg, 1.0 mmol) using SnCI 2 .2H 2 O (1.12 g, 5.0 mmol). The amino compound 4b obtained was (795 mg, 97% yield).
  • This compound was prepared according to the method described for the compound 5a employing 2S- ⁇ /-4- ⁇ /1 -[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]butyl) oxy-5-methoxy-2-aminobenzoyl ⁇ pyrrolidine-2-carboxaldehyde diethylthioacetal 4b (816 mg, 1.0 mmol) and HgCI 2 (582 mg, 2.26 mmol), CaCO 3 (230 mg, 2.46 mmol) in acetonitrile-water (4: 1) to obtain the pure product 5b (330 mg, 58% yield).).
  • This compound was prepared according to the method described for the compound 3a by employing 2S- ⁇ /-[4-(5-bromopenyloxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2- carboxaldehyde diethylthioacetal (1c) (549 mg, 1.0 mmol) was added anhydrous potassium carbonate (690 mg, 5.0 mmol) and ⁇ /1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide 2 (392 mg, 1.0 mmol) to obtain the pure product 3c (740 mg, 87% yield).
  • This compound was prepared according to the method described for the compound 5a employing 2S- ⁇ /-[5- ⁇ /1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] pentyl)oxy-5-methoxy-2-aminobenzoyl ⁇ pyrrolidine-2-carboxaldehyde diethylthioacetal 4c (830 mg, 1.0 mmol) and HgCI 2 (590 mg, 2.26 mmol), CaCO 3 (244 mg, 2.46 mmol) in acetonitrile-water (4:1) to obtain the pure product 5c (330 mg, 58% yield).
  • This compound was prepared according to the method described for the compound 3a by employing 2S- ⁇ /-[4-(6-bromohexyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2- carboxaldehyde diethylthioacetal 1d (563 mg, 1.0 mmol) was added anhydrous potassium carbonate (690 mg, 5.0 mmol) and ⁇ /1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide 2 (392 mg,1.0 mmol) to obtain the pure product (3d) (775 mg, 88% yield).
  • This compound was prepared according to the method described for the compound 4a by reducing 2S- ⁇ // ' 4-/6- ⁇ /1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] hexyl)oxy-5-methoxy-2-nitrobenzoyl ⁇ pyrrolidine-2-carboxaldehyde diethyl thioacetal 3d (874 mg, 1.0 mmol) using SnCI 2 .2H 2 O (1.12 g, 5.0 mol). The amino compound 4d obtained was (842 mg, 97% yield).
  • the C8-linked benzophenone-piperazine-PBD hybrids have been tested against sixty human tumour cell lines derived from nine cancer types (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) as per NCI protocol.
  • dose response curves for individual cell lines have been measured at a minimum of five concentrations at 10 fold dilutions.
  • a protocol of 48 h continuous drug exposure has been used, and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth.
  • the concentration for 50% cell growth inhibition (Gl 50 ), total cell growth inhibition (TGI, 0% growth) and 50% cell death (LC 50 , 50% growth) compared with the control has been calculated (Figure-2).
  • Compounds 5b and 5d have been evaluated for their in vitro cytotoxicity in eleven cell lines from nine human cancer cell lines of colon (Colo205), lung (Hop-62), cervix (SiHa), prostate (DU145, PC3), oral (DWD, HT1080), and breast (MCF7, Zr-75-1) origin. The results are expressed as percent of cell growth determined relative to that of untreated control cells (Table 1). The representative compounds 5b and 5d have shown significant cytotoxicity against some cancer cell lines.
  • Each cancer type represents the average of six to eight different cancer cell lines.
  • 5b-d exhibits a wide spectrum of activity against fifty six cell lines in nine cell panels, with Gl 50 value of ⁇ 20 nM.
  • the growth of HOP-62, NCI-H23 cell lines were affected by compound 5b with Gl 50 values as 20.1 , 24.2 and 27.1 nM respectively.
  • the Gl 50 values of compound 5d against colon cancer HCC-2988, HCT-116 and KM12 cell lines are 21.3, 21.8 and 23.7 nM respectively.
  • the Gl 50 values for compound 5b against CNS SF-295, SF-539, SNB-19 and SNB-75 cell lines are in a range of 14.1-33.5 nM.
  • Compound 5b exhibits activity against fifty-six cell lines in nine cancer cell panels with Gl 50 values of ⁇ 10 ⁇ M.
  • Compound 5d exhibits activity against fifty-seven cell lines in nine cancer cell panels, Gl 50 values of ⁇ 10 ⁇ M.
  • In vitro cytotoxicity of compounds 5b and 5d in selected cancer cell lines has been illustrated in Table 2.
  • the average Gl 50 values for each cancer panel of compounds 5b and 5d have been illustrated in Table 3.
  • the mean graph mid point values of Iog 10 TGI and log 10 LC 50 as well as log 10 Gl 50 for 5b and 5d are listed in Table-1. As demonstrated by mean graph pattern, compounds 5b and 5d exhibit an interesting profile of activity and selectivity for various cell lines.
  • the mean graph mid points of log-io TGI and log 10 LCg 0 have shown similar pattern to the logTM Gl 50 mean graph mid points.
  • Table-3. log 10 Gl 50 , logioTGI and log 10 LC 50 mean graphs midpoints(MG_MID) of in vitro cytotoxicity data for the compounds 5b and 5d against human tumour cell lines.
  • Thermal denaturation studies Compounds have been subjected to thermal denaturation studies with duplex-form calf thymus DNA (CT-DNA) using a modification of a reported procedure.
  • Working solutions in aqueous buffer (10 mM NaH 2 PO 4 ZNa 2 HPO 4 , 1 mM Na 2 EDTA, pH 7.00 + 0.01) containing CT-DNA (100 nm in phosphate) and the PBD (20nm) have been prepared by addition of concentrated PBD solutions in DMSO to obtain a fixed [PBD]/[DNA] molar ratio of 1:5.
  • the DNA-PBD solutions have been incubated at 37 0 C for 0 and 18 h prior to analysis.
  • the present invention provides novel pyrrolo[2,1-c][1 , ⁇ benzodiazepine hybrids useful as antitumour agents. ;
  • novel C8-linked benzophenone-piperazine-PBD hybrids that have been synthesized exhibited significant DNA-binding ability and showed cytotoxic activity against sixty human tumour cell lines. Some of these hybrids exhibited promising DNA-binding among these hybrids the compound 5c show high DNA-binding ability (9.6 0 C).

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Abstract

The present invention provides benzophenone-piperazine linked pyrrolo[2,1- c][1, 4]benzodiazepine hybrids of general formula 5a-e, useful as potential antitumour agents against human cancer cell lines. The present invention further provides a process for the preparation of pyrrolo[2,1-c][1, 4]benzodiazepine hybrids of general formula (5a-e).

Description

"BENZOPHENONE-PIPERAZINE LINKED PYRROLO[2,1-C][1,4]BENZODIAZEPINE . HYBRIDS AS POTENTIAL ANTICANCER AGENTS AND PROCESS FOR THE
PREPARATION THEREOF"
FIELD OF THE INVENTION
The present invention relates to benzophenone-piperazine linked pyrrolo[2,1- c][1 , ^benzodiazepine hybrids and a process for the preparation there of. More particularly It relates to 7-Methoxy-8-[n-{Λ/1 -[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazino-acetamide] alkyloxy}-(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4] benzodiazepine-5-one with aliphatic chain length variations useful as anticancer (antitumour) agent. The structural formula of these benzophenone-piperazine linked pyrrolo[2,1-c][1,4]benzodiazepines hybrids is given below.
Formula 5a-e, n = 3, 4, 5, 6 or 8
BACKGROUND OF THE INVENTION
Pyrrolo[2,1-c][1 ,4]benzodiazepine antitumour antibiotics are commonly known as anthramycin class of compounds. In the last few years, a growing interest has been shown in the development of new pyrrolo [2, 1-c][1 ^benzodiazepines (PBDs). These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within the minor groove of duplex DNA via an acid-labile aminal bond to the electrophilic imine at the N10-C11 position (Kunimoto, S.; Masuda, T.; Kanbayashi, N.; Hamada, M.; Naganawa, H.; Miyamoto, M.; Takeuchi, T.; Unezawa, H. J. Antibiot, 1980, 33, 665.; Kohn, K. W. and Speous, C. L. J. MoI. Biol., 1970, 51, 551.; Hurley, L. H.; Gairpla, C. and Zmijewski, M. Biochem. Biophys. Acta., 1977, 475, 521; Kaplan, D. J. and Hurley, L. H. Biochemistry, 1981 , 20, 7572). The molecules have a right-handed twist, which allows them to follow the curvature of the minor groove of B-form double-stranded DNA spanning three base pairs. A recent development has been the linking of two PBD units through their C-8 positions to give bisfunctional- alkylating agents capable of cross-linking DNA (Thurston, D. E.; Bose, D. S.; Thomson, A. S.; Howard, P. W.; Leoni, A.; Croker, S. J.; Jenkins, T. C; Neidle, S. and Hurley, L H. J. Org. Chem. 1996, 61, 8141).
anthramycin
DC-81 dimers (n = 3-5); DSB-120 (n = 3) imine-amide PBD dimers; n = 3-5
Recently, PBD dimers have been developed that comprise of two C2-exo-methylene substituted DC-81 subunits tethered through their C-8 position via an inert propanedioxy linker (Gregson, S. J.; Howard, P. W.; Hartely, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T. C; Kelland, L R. and Thurston, D. E. J. Med. Chem. 2001, 44, 737). A non-cross-linking mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent antitumour activity (Kamal, A.; Ramesh, G. Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.; Kondapi, A. K.; Srinu, V. B.; Nagarajaram, H. M. J. Med. Chem. 2002, 45, 4679). Recently, some new pyrrolobenzodiazepine (PBD) hybrids have been synthesized that have significant DNA binding ability and potent antitumour activity.( Kamal, A.; Srinivas, O.; Ramulu, P.; Ramesh, G.; Kumar, P. P. Bioorg. Med. Chem. Lett. 2003, 13, 3577).
Naturally occurring pyrrolo[2,1-c][1 , ^benzodiazepines belong to a group of antitumour antibiotics derived from Streptomyces species. Recently, there is much impetus for the PBD systems as they can recognize and bind to specific sequence of DNA. Examples of naturally occurring PBDs include anthramycin, DC-81 , tomaymycin, sibiromycin and neothramycin.
However, the clinical efficacy for these antibiotics is hindered by several limitations, such as poor water solubility, cardio toxicity, development of drug resistance and metabolic inactivation.
OBJECTIVES OF THE INVENTION
The main objective of the present invention is to provide novel benzophenone - piperazine linked pyrrolo[2,1-c][1 , ^benzodiazepine hybrids, useful as antitumour agents.
Yet another object of this invention is to provide a process for the preparation of novel benzophenone-piperazine linked pyrrolo[2,1-c][1 , ^benzodiazepine hybrids.
SUMMARY OF THE INVENTION
Accordingly the present invention provides a novel benzophenone-piperazine linked pyrrolo[2,1-c][1 , ^benzodiazepine hybrid of general formula 5
Formula 5a-e
Where in, n = 3, 4, 5, 6, 8
In an embodiment of the present invention the novel benzophenone-piperazine linked pyrrolo[2,1-c][1 , ^benzodiazepine hybrid according to claim 1 is represented by the group of the following compounds:
7-Methoxy-8-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]propyloxy}- (11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one (5a); 7-Methoxy-8-[4-{A/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]butyloxy}- (11 aS)-1 ,2, 3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]ben2odiazepine-5-one(5b); 7-Methoxy-8-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] pentyloxy}- (11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1-c][1 ,4]benzodiazepine-5-one(5c); 7-Methoxy-8-[6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]hexyloxy}- (11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one (5d); 7-Methoxy-8-[8-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]octyloxy}- (11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1-c][1 ,4]benzodiazepine-5-one (5e);
In yet another embodiment the structural formula of the representative compounds of benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid are:
Formula 5a-e n = 3, 4, 5, 6, 8
In yet another embodiment the novel benzophenone-piperazine linked pyrrolo [2,1- c][1 ,4]benzodiazepine hybrid exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group consisting of lung (Hop-62), cervix (SiHa), breast (MCF7, Zr-75-1), colon (Colo205), prostate (DU 145, PC3) and oral (DWD, HT1080) cell lines.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid used for in vitro activity against Colo205 for IC50 is in the range of 13 to 80μm, at an exposure period of at least 48 hrs.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against DU145 for IC50 is in the range of 12 to 80μm, at an exposure period of at least 48 hrs.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against DWD for IC50 is in the range of 8 to 80μm, at an exposure period of at least 48 hrs. In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 , ^benzodiazepine hybrids used for in vitro activity against HoP62 for IC50 is in the range of 11 to 48 μm, at an exposure period of at least 48 hrs.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1,4]benzodiazepine hybrids used for in vitro activity against HT1080 for IC50 is in the range of 14 to 36μm, at an exposure period of at least 48 hrs.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against MCF7 for IC50 is in the range of 22 to about 80μm, at an exposure period of at least 48 hrs.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against PC3 for IC50 is in the range of 28 to about 80μm, at an exposure period of at least 48 hrs.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1,4]benzodiazepine hybrids used for in vitro activity against SiHa for IC50 is in the range of 36 to about 80μm, at an exposure period of at least 48 hrs.
In yet another embodiment the concentration of benzophenone linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids used for in vitro activity against Zr-75-1 for IC50 is in the range of 29 to about 80μm, at an exposure period of at least 48 hrs.
The present invention further provides a pharmaceutical composition comprising benzophenone linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid, its derivatives, analogues, salts or mixture thereof optionally with pharmaceutically acceptable carriers, adjuvants and additives.
In yet another embodiment the benzophenone-piperazine linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid used is represented by a general formula 5,
Formula 5 wherein n = 3, 4, 5, 6, or 8. The present invention further provides a process for the preparation of benzophenone-piperazine linked pyrrolo[2,1-c][1, ^benzodiazepine hybrid of formula 5
wherein n = 3, 4, 5, 6, 8 and the said process comprising the steps of: a) reacting (2S)-N-[(n-bromoalkyloxy)-3-methoxy-2-nitrobenzoyl)]pyrrolidine-2- carboxaldehyde diethylthioacetal of formula 1
with Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide of below formula 2
in an aprotic water miscible organic solvent, in the presence of anhydrous mild inorganic base, under refluxing temperature in an oil bath, for a period of about 24 to 48 hrs, followed by the removal of inorganic base by filtration and evaporating the organic solvent to obtain the resultant crude product and purifying it by column chromatography to obtain the desired product of 2S-A/-[4-{n-(N1-[4-chloro-2-(2- chlorobenzoyl)phenyl]-2-piperazinoacetamide]alkyl)oxy-5-methoxy-2-nitrobenzoyl }pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula 3a-e, n = 3, 4, 5, 6, 8 3a-e
b) reducing (2SJ-Λ/-[4-{n-(N-1-[4-chloro-2-(2-chlorobenzoyl) phenyl]-2-pipera zinoacetamide]alkyl)oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxa-ldehyde diethyl-thioacetal of formula 3a-e with SnCI2.2H2O, in an alcohol, under reflux, followed by the evaporation of alcohol and adjusting the pH of the resultant product layer to about 8 to 9 by using a base like Sodium bicarbonate and potassium bicarbonate, followed by extraction with ethyl acetate and washing the combined organic phase with brine solution and evaporating the solvent to obtain the desired product of 2S-Λ/-[4-{n-(Λ/-/-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamid-e]alkyl)oxy-5-methoxy-2-amino benzoyl}pyrrolidine-2- carboxa-ldehyde diethylthioacetal of formula 4a-e,
n = 3, 4, 5, 6, 8 4a-e c) reacting the above said amino compound of formula 4 obtained in step (b) with a deprotecting agent of the kind of mercuric chloride by known method to obtain the desired compound of formula 5
In yet anther embodiment the mild inorganic base used in steps (a) is potassium carbonate.
In yet anther embodiment the aprotic organic solvent used in step (a) is acetone and acetonitrile
In yet anther embodiment the organic solvent used in step (c) is acetonitrile and acetone
In yet anther embodiment the alcohol used in step (b) is selected from methanol and ethanol. In yet anther embodiment the compounds of formula 5a-e obtained are represented by a group of the following compounds:
7-Methoxy-8-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]propyloxy}-
(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5a); 7-Methoxy-8-[4-{Λ/1 -[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] butyloxy}-
(11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one(5b);
7-Methoxy-8-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]pentyloxy}-
(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5c);
7-Methoxy-8-[6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]hexyloxy}- (11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one (5d);
7-Methoxy-8-[8-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]octyloxy}-
(11 aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one (5e);
In still anther embodiment the benzophenone-piperazine linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid of formula 5a-e exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group consisting of lung, cervix, breast, colon, prostate and oral cell lines.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a process for preparation of pyrrolo[2,1- c][1 ,4]benzodiazepine hybrids of formula 5a-e of the drawing accompanying the specification where n = 3-6, and 8 which comprises reacting {Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide of formula 2 with (2S)-Λ/-[(n-bromoalkyloxy)-3-methoxy-2- nitrobenzoyl)]pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula 1 in presence of CH3COCH3/K2CO3 for a period of 48 h with isolating (2S)-{Λ/1-[n-{4-chloro-2-(2- chlorobenzoyl)phenyl]-2-piperazinoacetamide)alkyl]oxy-5-methoxy-2-nitrobenzoyl} pyrrolidine -2-carboxaldehyde diethylthioacetal of formula 3a-e by conventional methods. Reducing the above nitro compound of formula 3a-e with SnCI2.2H2O in presence of organic solvent with reflux temperature, resulting with the formation of (2S)-Λ/-1-[4-chloro-2-(2- chlorobenzoyl)phenyl]-2-piperazinoacetamide]alkyloxy]}-5-methoxy-2-aminobenzoyl} pyrrolidine-2-carboxaldeh yde diethylthioacetal 4a-e. Reacting the above said amino compound of formula 4a-e with known deprotecting agents in a conventional manner to give novel pyrrolo[2,1-c][1 ,4]benzodiazepine hybrids of formula 5a-e where 'n' is as stated above. The precursors, {Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide of formula 2 (Kentaro Hirai, Teruyuki Ishiba, Hirohiko Sugimoto, Toshio Fujishita, Yuji sukinoki, and Katsumi Hirose; J. Med. Chem. 1981, 24, 20-27). and (2SJ-Λ/-(4-hydroxy-5-methoxy-2- nitrobenzoyl)pyrrolidine-2-carboxaldehyde diethylthioacetal of formula 1 (Thurston, D. E.;
Morris, S. J.; Hartley, J. A. Chem. Commun. 1996, 563-565) have been prepared by literature methods.
Some representative compounds of formula 5a-e for the present inventions are given below
7-Methoxy-8-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]propyloxy}-
(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5a); 7-Methoxy-8-[4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]butyloxy}-
(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5b);
7-Methoxy-8-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]pentyloxy}-
(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5c);
7-Methoxy-8-[6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]hexyloxy}- (11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one (5d);
7-Methoxy-8-[8-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]octyloxy}-
(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one (5e);
These new analogues of pyrrolo[2,1-c][1 ,4]benzodiazepine hybrids linked at C-8 position have shown promising DNA binding activity and efficient anticancer activity in various cell lines. The molecules synthesized are of immense biological significance with potential sequence selective DNA-binding property. This resulted in design and synthesis of new congeners as illustrated in Scheme-1 , which comprise:
1. The ether linkage at C-8 position of DC-81 intermediates with {Λ/1-[4-chloro-2-(2- chlorobenzoyl)phenyl]-2-piperazinoacetamide moiety.
2. Refluxing the reaction mixtures for 48 h.
3. Synthesis of C-8 linked PBD antitumour antibiotic hybrid imines.
4. Purification by column chromatography using different solvents like ethyl acetate, hexane, dichloromethane and methanol.
Reagents and conditions: (i) K2CO3, acetone,18 h, refllux, 90-92%; (ii) SnCI2.2H2O, MeOH, 2 h, reflux, 8! 87%; (iii) HgCI2-CaCO3, CH3CN-H2O (4:1), 12 h, rt, 68-71 %.
Scheme: 1 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
2S-Λ/-[4-(3-{Λ/1-[4-chloro-2-(2-chIorobenzoyl)phenyl]-2-piperazinoacetamide]propyl)oxy-5- methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal(3a)
To a solution of 2S-Λ/-[4-(3-bromopropoxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2- carboxaldehyde diethylthioacetal (1a) (521 mg, 1.0 mmol) in dry acetone (20 ml_) was added anhydrous potassium carbonate (690 mg, 5.0 mmol) and Λ/1-[4-chloro-2-(2- chlorobenzoyl)phenyl]-2-piperazinoacetamide (2) (392 mg, 1.0 mmol). The reaction mixture was refluxed in an oil bath for 24 h and the reaction was monitored by TLC using ethyl acetate-hexane (6:4) as a solvent system. The potassium carbonate was then removed by suction filtration and the solvent was evaporated under vacuum to afford the crude product. This was further purified by column chromatography using ethyl acetate: hexane (6:4) as a solvent system to obtain the pure product (3a) (670 mg, 80% yield).
1H NMR (CDCI3): .51.22-1.39 (m, 6H), 1.69-2.32 (m, 8H), 2.51-2.56 (m, 4H), 2.66-2.86 (m, 8H), 3.19-3.26 (m, 4H), 3.92 (s, 3H), 4.06-4.13 (t, 2H, J=6.79 Hz), 4.63-4.70(m, 1 H), 4.81- 4.83 (d, 1 H, J = 3.77 Hz) 6.75 (s, 1 H), 7.23-7.60(m, 7H), 8.87-8.90 (d, 1 H, J = 9.06 Hz), 12.30(s, 1 H); FABMS: 832 (M+H)+.
2S-Λ/-/"4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]propyl)oxy-5- methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehydediethyl thioacetal (4a)
2S-Λ/-[4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] propyl)oxy- 5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal of formula 3a (832 mg, 1.0 mmol) was dissolved in methanol (10 ml_), SnCI2.2H2O (1.12 g, 5.0 mmol) was added and refluxed until the TLC indicated the completion of the reaction. The methanol was then evaporated in vacuum and the aqueous layer was then adjusted to pH 8 with 10% NaHCO3 solution and extracted with ethyl acetate (60 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to afford the crude amino diethylthioacetal 4a (790 mg, 97% yield), which was directly used in the next step.
7-Methoxy-8-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]propyloxy}- (11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one (5a) A solution of2S-Λ/-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyI)phenyl]-2-piperazinoacetamide ]propyl)oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal of formula 4a (802 mg, 1.0 mmol), HgCI2 (576 mg, 2.26 mmol) and CaCO3 (225 mg, 2.46 mmol) in acetonitrile-water (4:1) was stirred slowly at room temperature overnight until complete consumption of starting material as indicated by the TLC. The clear organic supernatant liquid was extracted with ethyl acetate and washed with saturated 5% NaHCO3 (20 ml_), brine (20 ml_) and the combined organic phase was dried over Na2SO4. The organic layer was evaporated in vacuum to afford a white solid, which was first eluted on a column chromatography with ethyl acetate to remove mercuric salts, and then with ethyl acetate to obtain the pure product 5a (330 mg, 58% yield).
1H NMR (CDCI3): 51.24-1.54 (m, 4H), 1.66-1.94 (m, 4H), 2.00-2.38 (m, 4H), 2.94-3.05 (m, 2H), 3.29-3.41 (m, 6H), 3.56-4.23 (m, 4H), 6.80 (s, 1 H), 7.25-7.70 (m, 8H), 8.87-8.89 (d, 1H1 J = 4.28Hz), 12.44 (s, 1 H); FABMS: 678 (M+H)+. Example-2
2S-Λ/-[4-(4-{Λ/'/-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]butyl)oxy-5- methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehydediethyl thioacetal (3b)
This compound was prepared according to the method described for the compound 3a by employing 2S-Λ/-[4-(4-bromobutoxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2- carboxaldehyde (1b) diethylthioacetal (535 mg, 1.0 mmol) was added anhydrous potassium carbonate (690 mg, 5.0 mmol) and Λ/1-4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide 2 (392 mg, 1.0 mmol) to obtain the pure product 3b (710 mg, 84% yield). 1H NMR (CDCI3): δ 1.22-1.39 (m, 8H), 1.70-2.32 (m, 8H), 2.52-2.57 (m, 4H1), 2.68-2.85 (m, 8H, 3.19-3.27 (m, 4H), 3.92(s, 3H), 4.06-4.12 (t, 2H, J=6.55 Hz), 4.64-4.70(m, 1 H), 4.82-4.83 (d, 1 H, J= 3.77Hz) 6.75 (s, 1 H), 7.22-7.60(m, 7H), 8.87-8.90 (d, 1 H, J = 9.06Hz), 12.30(s, 1 H); FABMS: 846 (M+H+).
2S-Λ/-{4-[4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]butyl)oxy-5- methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (4b)
This compound was prepared according to the method described for the compound 4a by reducing 2S-Λ/-{4-[4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] butyl)oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal 3b (846 mg, 1.0 mmol) using SnCI2.2H2O (1.12 g, 5.0 mmol). The amino compound 4b obtained was (795 mg, 97% yield).
7-Methoxy-8-[4-{Λ/1-[4-chloro-2-(2-chIorobenzoyl)phenyl]-2-piperazino acetamide]butyloxy}- (11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one (5b)
This compound was prepared according to the method described for the compound 5a employing 2S-Λ/-4-{Λ/1 -[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]butyl) oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal 4b (816 mg, 1.0 mmol) and HgCI2 (582 mg, 2.26 mmol), CaCO3 (230 mg, 2.46 mmol) in acetonitrile-water (4: 1) to obtain the pure product 5b (330 mg, 58% yield).).
1H NMR (CDCI3): 51.23-1.43 (m, 4H), 1.53-2.18 (m, 6H), 2.27-2.38 (m, 2H), 2.96-3.11 (m, 4H), 3.31-3.43 (m, 4H), 3.56-3.94 (m, 4H), 4.00-4.12(m, 2H), 6.78 (s, 1 H) 7.25-7.75 (m, 8H), 8.86-8.90 (d, 1 H, J = 4.36 Hz), 12.45(s, 1 H); FABMS: 692 (M+H)+. Example-3
2S-Λ/-[4-{5-(Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]pentyl)oxy-5- methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldedehydediethyl thioacetal (3c)
This compound was prepared according to the method described for the compound 3a by employing 2S-Λ/-[4-(5-bromopenyloxy)-5-methoxy-2-nitrobenzoyl] pyrrolidine-2- carboxaldehyde diethylthioacetal (1c) (549 mg, 1.0 mmol) was added anhydrous potassium carbonate (690 mg, 5.0 mmol) and Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide 2 (392 mg, 1.0 mmol) to obtain the pure product 3c (740 mg, 87% yield). 1H NMR (CDCI3): 51.20-1.39 (m, 8H), 1.72-2.33 (m, 10H), 2.50-2.56 (m, 8H) 2.60-2.85 (m, 4H), 3.19-3.26 (m, 4H), 3.91 (s, 3H), 4.05-4.13 (m, 2H), 4.62-4.69 (m, 1 H), 4.80-4.83(d, 1 H, J = 3.77Hz), 6.74 (s, 1 H), 7.22-7.60(m, 7H), 8.86-8. 90 (d, 1 H, J = 9.06Hz), 12.30(s, 1 H); FABMS: 860 (M+H) +.
2S-Λ/-{4-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]pentyl)oxy-5- methoxy-2-aminobenzoyl}pyrroli.dine-2-carboxaldehyde diethylthioacetal (4c) This compound was prepared according to the method described for the compound
4a by reducing A solution of 2S-Λ/-{4-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamideJpentyOoxy-δ-methoxy^-aminobenzoylJpyrrolidine^-carboxaldehyde diethyl thioacetal 3c (860 mg, 1.0 mmol) using SnCI2.2H2O (1.12 g, 5.0 mmol). The amino compound 4c obtained was (810 mg, 97% yield). 7-Methoxy-8-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]pentyloxy}- (HaSJ-I^^.Ha-tetrahydro-SH-pyrrolo^.i-clCI ^] benzodiazepine -5-one (5c)
This compound was prepared according to the method described for the compound 5a employing 2S-Λ/-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] pentyl)oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal 4c (830 mg, 1.0 mmol) and HgCI2 (590 mg, 2.26 mmol), CaCO3 (244 mg, 2.46 mmol) in acetonitrile-water (4:1) to obtain the pure product 5c (330 mg, 58% yield). 1H NMR (CDCI3): 5 1.22-1.39 (m, 4H), 1.72-2.33 (m, 8H), 2.60-2.85 (m, 4H), 2.66-2.86 (m, 3H), 3.19-3.26 (m, 4H), 3.92 (s, 3H), 4.06-4.13 (t, 2H, J=6.79 Hz), 6.75 (s, 1 H), 7.23-7.60(m, 8H), and 8.87-8. 90 (d, 1 H, J = 4.41 Hz), 12.30(s, 1 H); FABMS: 706 (M+H)+.
Example-4 2S-Λ/-[4-{6-(Λ/7-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazino acetamide] hexyl)oxy-5- methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal (3d)
This compound was prepared according to the method described for the compound 3a by employing 2S-Λ/-[4-(6-bromohexyloxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2- carboxaldehyde diethylthioacetal 1d (563 mg, 1.0 mmol) was added anhydrous potassium carbonate (690 mg, 5.0 mmol) and Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide 2 (392 mg,1.0 mmol) to obtain the pure product (3d) (775 mg, 88% yield).
1H NMR (CDCI3): 51.21-1.48 (m, 8H), 1.69-1.86 (m, 10H), 1.98-2.15(m, 4H), 2.23-2.39 (m, 4H), 2.59-2.98 (m, 6H), 3.18-3.30 (m, 4H), 3.93 (s, 3H) 4.02-4.12 (m, 2H), 4.63-4.69(m, 1 H), 4.81-4.83 (d, 1 H1 J = 3.77 Hz) 6.78 (s, 1 H), 7.25-7.61 (m, 7H), 8.86-8. 90 (d, 1 H, J = 9.06 Hz), 12.33(s, 1 H); FABMS: 874 [M+H]+.
2S-Λ//'4-/'6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]hexyl)oxy-5- methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (4d)
This compound was prepared according to the method described for the compound 4a by reducing 2S-Λ//'4-/6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] hexyl)oxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal 3d (874 mg, 1.0 mmol) using SnCI2.2H2O (1.12 g, 5.0 mol). The amino compound 4d obtained was (842 mg, 97% yield).
7-Methoxy-8-[6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] hexyloxy}-(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -][1 ,4]benzodiazepine-5-one (5d) This compound was prepared according to the method described for the compound
5a employing 2S-Λ/-[6-{Λ/1 -[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] hexyl)oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-corboxaldehyde diethylthioacetal 4d (844 mg, 1.0 mmol) and HgCI2 (597 mg, 2.26 mmol), CaCO3 (250 mg, 2.46 mmol) in acetonitrile- water (4:1) to obtain the pure product 5d (485 mg, 58% yield).
1H NMR (CDCI3): .51.22-1.39 (m, 6H), 1.69-2.32 (m, 8H), 2.51-2.56 (t, 4H, J=6.64 Hz), 2.66- 2.86 (m, 3H), 3.19-3.26 (m, 4H), 3.92 (s, 3H), 4.06-4.13 (t, 2H, J=6.79 Hz), 6.75 (s, 1H), 7.23-7.60(m, 8H), 8.87-8.90 (d, 1H, J = 5.06Hz), 12.30(s, 1H); MS(FAB): 720 (M+H)+.
Biological Activity: some of in vitro biological activity studies were carried out at the National Cancer Institute, Marryland, USA. Cytotoxicity:
The compounds 7-Methoxy-8-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-pipera zinoacetamide]propyloxy}-(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine -5-one(5a);
7-Methoxy-8-[4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]butyloxy }- (11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1-c][1 ,4]benzodiazepine-5-one(5b); 7-Methoxy-8-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]pentyloxy}- (11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5c); 7-Methoxy-8-[6-{Λ/1-[4-chloro-2-(2-chiorobenzoyl)phenyl]-2-piperazinoacetamide
]hexyloxy}-(11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one (5d); 7-Methoxy-8-[8-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide]octyloxy}- (11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one (5e);
BIOLOGICAL ACTIVITY OF C8-LINKED BENZOPHENONE-PIPERAZINE-PBD HYBRIDS In vitro Cytotoxicity
The C8-linked benzophenone-piperazine-PBD hybrids have been tested against sixty human tumour cell lines derived from nine cancer types (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) as per NCI protocol. For each compound, dose response curves for individual cell lines have been measured at a minimum of five concentrations at 10 fold dilutions. A protocol of 48 h continuous drug exposure has been used, and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The concentration for 50% cell growth inhibition (Gl50), total cell growth inhibition (TGI, 0% growth) and 50% cell death (LC50, 50% growth) compared with the control has been calculated (Figure-2).
Compounds 5b and 5d have been evaluated for their in vitro cytotoxicity in eleven cell lines from nine human cancer cell lines of colon (Colo205), lung (Hop-62), cervix (SiHa), prostate (DU145, PC3), oral (DWD, HT1080), and breast (MCF7, Zr-75-1) origin. The results are expressed as percent of cell growth determined relative to that of untreated control cells (Table 1). The representative compounds 5b and 5d have shown significant cytotoxicity against some cancer cell lines.
Table-1. Log Gl50 (concentration in mol/L causing 50% growth inhibition) values for benzophenone-piperazine-PBD hybrids
Compound 5b Compound 5d
CANCER CELL LINES
Leukemia -6.02 -6.39
Non-small-cell-lung -6.86 -6.74
Colon -5.87 -5.41
CNS -5.11 -5.43
Melanoma -4.34 -5.31
Ovarian -5.40 -5.18
Renal -5.39 -5.18
Prostate -5.50 -5.82
Breast -6.14 -6.64
Each cancer type represents the average of six to eight different cancer cell lines. Among them 5b-d exhibits a wide spectrum of activity against fifty six cell lines in nine cell panels, with Gl50 value of <20 nM. In the non-small cell lung cancer panel, the growth of HOP-62, NCI-H23 cell lines were affected by compound 5b with Gl50 values as 20.1 , 24.2 and 27.1 nM respectively. The Gl50 values of compound 5d against colon cancer HCC-2988, HCT-116 and KM12 cell lines are 21.3, 21.8 and 23.7 nM respectively. The Gl50 values for compound 5b against CNS SF-295, SF-539, SNB-19 and SNB-75 cell lines are in a range of 14.1-33.5 nM. Four cancer cell lines (OVCAR-4, OVCAR-5, OVCAR-8 and SK-OV-3) in the ovarian cancer cell panel were affected by compound 5d with Gl50 values of 21.6, 23.7, 29.6 and 69.8 nM respectively. In this study compound
Compound 5b exhibits activity against fifty-six cell lines in nine cancer cell panels with Gl50 values of < 10 μM. Compound 5d exhibits activity against fifty-seven cell lines in nine cancer cell panels, Gl50 values of < 10 μM. In vitro cytotoxicity of compounds 5b and 5d in selected cancer cell lines has been illustrated in Table 2. The average Gl50 values for each cancer panel of compounds 5b and 5d have been illustrated in Table 3.
Table 2. In vitro cytotoxicity of compounds 5b and 5d in selected cancer cell lines
The mean graph mid point values of Iog10 TGI and log10 LC50 as well as log10 Gl50 for 5b and 5d are listed in Table-1. As demonstrated by mean graph pattern, compounds 5b and 5d exhibit an interesting profile of activity and selectivity for various cell lines. The mean graph mid points of log-io TGI and log10 LCg0 have shown similar pattern to the log™ Gl50 mean graph mid points. Table-3. log10 Gl50, logioTGI and log10 LC50 mean graphs midpoints(MG_MID) of in vitro cytotoxicity data for the compounds 5b and 5d against human tumour cell lines.
Log10GI50 Log10TGI Log10 LC50
Compound
5b ^Tδϊ ^94 =449
5d -5.49 -5.97 -4.53
DNA-BINDING ABILITY OF NOVEL C8-LINKED BENZOPHENONE-PIPERAZINE-PBD HYBRIDS
Thermal denaturation studies Compounds have been subjected to thermal denaturation studies with duplex-form calf thymus DNA (CT-DNA) using a modification of a reported procedure. Working solutions in aqueous buffer (10 mM NaH2PO4ZNa2HPO4, 1 mM Na2EDTA, pH 7.00 + 0.01) containing CT-DNA (100 nm in phosphate) and the PBD (20nm) have been prepared by addition of concentrated PBD solutions in DMSO to obtain a fixed [PBD]/[DNA] molar ratio of 1:5. The DNA-PBD solutions have been incubated at 37 0C for 0 and 18 h prior to analysis. Samples have been monitored at 260 nm using a Beckman DU-800 spectrophotometer fitted with high performance temperature controller, and heated at 1 0C min"1 in the 40-110 0C range. DNA helix→coil transition temperatures (7" m) have been obtained from the maxima in the d(/A260)/dr derivative plots. Drug-induced alterations in DNA melting behavior are given by: Δ7m=7m(DNA+PBD) -Tm(DNA alone), where the Tm value for the PBD-free CT-DNA is 68.5 + 0.01. The fixed [PBD]/[DNA] ratio used has not resulted in binding saturation of the host DNA duplex for any compound examined.
The DNA-binding for these novel C8-linked benzophenone-piperazine-PBD hybrids has been examined by thermal denaturation studies using calf thymus (CT) DNA. Melting studies show that these compounds stabilize the thermal helix → coil or melting stabilization (ATm) for the CT-DNA duplex at pH 7.0, incubated at 37 0C, where PBD/DNA molar ratio is 1 :5. Interestingly, in this assay one of the benzophenone-piperazine-PBD hybrids (5c) elevates the helix melting temperature of CT-DNA by a margin of 9.6 0C after incubation for 18 h at 37 0C. Data for 5a-e and DC-81 are included in Table 4 for comparison.
Table-4.Thermal denaturation data for benzophenone-piperazine-PBD hybrids with calf thymus (CT) DNA [PBD]:[DNA] Δrm(°C)a after incubation at 37 0C for
PBD hybrids molar ratiob O h Ϊ8~h
__ __
5a 1 :5
5b 1:5 8.5 9.5
5c 1:5 8.9 9.6
5d 1 :5 8.2 9.5
1 :5 8.3 9.0
5e 1 :5 0.3 0.7 DCC--8811
a For CT-DNA alone at pH 7.00 ± 0.01 , Tm = 68.5 0C + 0.01 (mean value from 10 separate determinations), all ΔTm values are + 0.1 - 0.2 0C. b For a 1 :5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100 μM and ligand concentration = 20 μM in aqueous sodium phosphate buffer [10 mM sodium phosphate + 1 mM EDTA, pH 7.00 ± 0.01]. c The Δ7" m for PBD hybrids 5a, 5b, 5c, 5d and 5e at a [PBD]:[DNA] molar ratio of 1 :5 increased to a value of 9.3 0C , 9.5 0C, 9.6 0C, 9.5 and 9.0 0C after 18 h incubation respectively.
ADVANTAGES
The present invention provides novel pyrrolo[2,1-c][1 , ^benzodiazepine hybrids useful as antitumour agents. ;
The novel C8-linked benzophenone-piperazine-PBD hybrids that have been synthesized exhibited significant DNA-binding ability and showed cytotoxic activity against sixty human tumour cell lines. Some of these hybrids exhibited promising DNA-binding among these hybrids the compound 5c show high DNA-binding ability (9.6 0C).

Claims

We claim:-
1. Novel benzophenone-piperazine linked pyrrolo[2,1-c][1.^benzodiazepine hybrid of general formula 5
Formula 5a-e wherein, n = 3, 4, 5, 6 or 8
2. Benzophenone-piperazine linked pyrrolo[2,1-c][1 , ^benzodiazepine hybrid according to claim 1 is represented by the group of the following compounds:
7-Methoxy-8-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] propyloxy}-(11 aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one(5a); 7-Methoxy-8-[4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] butyloxy }-(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5b); 7-Methoxy-8-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] pentyloxy}-(11 aS)-1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5c); 7-Methoxy-8-[6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] hexyloxy}-(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one (5d); 7-Methoxy-8-[8-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide ]octyloxy}-(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one (5e);
3. Benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid according to claim 1 , wherein the structural formula of the representative compound are:
4. Benzophenone-piperazine linked pyrrolo[2,1-c][1 , ^benzodiazepine hybrid according to claim 1 , exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group consisting of lung (Hop-62), cervix (SiHa), breast (MCF7, Zr-75- 1), colon (Colo205), prostate (DU145, PC3) and oral (DWD, HT1080) cell lines.
5. Benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid according to claim 1 , wherein the concentration of the compound used for in vitro activity against Colo205 for IC50 is in the range of 13 to 80μm, at an exposure period of at least 48 hrs.
6. Benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrids according to claim 1 , wherein the concentration of the compound used for in vitro activity against DU145 for IC50 is in the range of 12 to 80μm, at an exposure period of at least 48 hrs.
7. Benzophenone-piperazine linked pyrrolo[2,1-c][1, ^benzodiazepine hybrids according to claim 1 , wherein the concentration of the compound used for in vitro activity against DWD for IC50 is in the range of 8 to 80μm, at an exposure period of at least 48 hrs.
8. Benzophenone-piperazine linked pyrrolo[2,1-c][1 , ^benzodiazepine hybrid according to claim 1 , wherein the concentration of the compound used for in vitro activity against
HoP62 for IC50 is in the range of 11 to 40 μm, at an exposure period of at least 48 hrs.
9. Benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid according to claim 1 , wherein the concentration of the compound used for in vitro activity against HT1080 for IC50 is in the range of 14 to 30μm, at an exposure period of at least 48 hrs.
10. Benzophenone-piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid according to claim 1 , wherein the concentration of the compound used for in vitro activity against MCF7 for IC50 is in the range of 22 to about 80μm, at an exposure period of at least 48 hrs.
11. Benzophenone-piperazine linked pyrrolo[2,1-c][1 ^benzodiazepine hybrid according to claim 1 , wherein the concentration of the compound used for in vitro activity against PC3 for IC50 is in the range of 28 to about 80μm, at an exposure period of at least 48 hrs.
12. Benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid according to claim 1 , wherein the concentration' of the compound used for in vitro activity against SiHa for IC50 is in the range of 36 to about 80μm, at an exposure period of at least 48 hrs.
13. Benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid according to claim 1 , wherein the concentration of the compound used for in vitro activity against Zr-
75-1 for IC50 is in the range of 29 to about 80μm, at an exposure period of at least 48 hrs.
14.A pharmaceutical composition comprising benzophenone-piperazine linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid, its derivatives, analogues, salts or mixture thereof optionally with pharmaceutically acceptable carriers, adjuvants and additives.
15.A pharmaceutical composition according to claim 14, wherein the benzophenone- piperazine linked pyrrolo[2,1-c][1 ,4]benzodiazepine hybrid used is represented by a general formula 5a-e,
Formula 5a-e wherein n = 3, 4, 5, 6, 8
16. A process for the preparation of benzophenone linked pyrrolo[2,1-c][1 ,4] benzodiazepine hybrid of formula 5a-e,
wherein n = 3, 4, 5, 6, 8 and the said process comprising the steps of: a) reacting (2S)-Λ/-[4-(n-bromoalkyl)oxy-5-methoxy-2-nitrobenzoyl] pyrrolidi ne-2- carboxaldehyde diethylthioacetal of formula 1
with piperazinobenzophenone derivative selected from the compound of formula 2
in the presence of K2CO3, in an organic solvent, under refluxing temperature to obtain the resultant nitro compound of 2S-Λ/-[4-{n-(Λ/7-[4-chloro-2-(2- chlorobenzoyOphenyO^-pipera∑inoacetamidelalkyOoxy-δ-methoxy^-nitrobenzoyl} pyrrolidine-2-carboxaldehyde diethylthioacetal of formula 3a-e. n = 3, 4, 5, 6 or 8 3a-e b) reducing(2Sj-Λ/-[4-{n-(Λ/7-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-pipera-zinoacet amide]alkyl)oxy-5-methoxy-2-nitrobenzoyl}pyrrolicline-2-carbox-aldehycle diethyl thioacetal of formula 3a-e with SnCI2.2H2O, in an alcohol, under reflux, followed by the evaporation of alcohol and adjusting the pH of the resultant product layer to about 8 by using a base, followed by extraction with ethyl acetate and washing the combined organic phase with brine solution and evaporating the solvent to obtain the desired 2S-Λ/-[4-{n-(Λ/7-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2- piperazinoacetamide] alkyl)oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carbox aldehyde diethylthioacetal of formula 4a-e,
n = 3, 4, 5, 6 or 8 4a-e c) reacting (2SJ-Λ/-[4-{n-(Λ/7-[4-chloro-2-(2-chlorobenzoyl) phenyl]-2-piperazine acetamide]alkyl)oxy-5-methoxy-2-aminobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal of formula 4 with mercuric chloride, in a mixture of water and organic solvent, in the presence of mild inorganic, under stirring, at a temperature of about 20-300C, for a period of 8-12 hrs, followed by the extraction of yellow organic supernatant and washing with sodium bi carbonate and brine, respectively, and evaporating the organic layer, under reduced pressure and further purified by column chromatography to obtain the desired product of benzophenone-piperazine linked pyrrolo[2,1-c][1 ,4] benzodiazepine hybrid of formula 5a-e.
17. A process according to claim 16, wherein the mild inorganic base used in step (a) is potassium carbonate.
18. A process according to claim 16, wherein the aprotic organic solvent used in step (a) is acetone and acetonitrile.
19. A process according to claim 16, wherein the organic solvent used in step (c) is acetonitrile and acetone.
20. A process according to claim 16, wherein the alcohol used in step (b) is selected from methanol and ethanol.
21. A process according to claim 16, wherein the compounds of formula 5 obtained are represented by a group of the following compounds: 7-Methoxy-8-[3-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide] propyloxy}-(11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one(5a);
7-Methoxy-8-[4-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide ]butyloxy }-(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5b); 7-Methoxy-8-[5-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide ]pentyloxy}-(11 aS)-1 ,2,3, 11 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepine-5-one(5c); 7-Methoxy-8-[6-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide
]hexyloxy}-(11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one (5d); 7-Methoxy-8-[8-{Λ/1-[4-chloro-2-(2-chlorobenzoyl)phenyl]-2-piperazinoacetamide ]octyloxy}-(11aS)-1 ,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepine-5-one (5e);
22. A process according to claim 16, wherein benzophenone-piperazine linked pyrrolo[2,1- c][1 ,4]benzodiazepine hybrid of formula 5a-e exhibits an in vitro anticancer/antitumour activity against human cancer cell lines selected from the group consisting of lung, cervix, breast, colon, prostate and oral cell lines.
EP08873530A 2008-03-26 2008-10-31 Benzophenone-piperazine linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids as potential anticancer agents and process for the preparation thereof Withdrawn EP2262809A1 (en)

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