JP4833663B2 - C2-fluoropyrrolo [2,1-c] [1,4] benzodiazepine dimer - Google Patents

Description

The present invention relates to novel 2-fluoro-pyrrolo [2,1-c] [1,4] benzodiazepine dimers that are effective as potential antitumor agents. The present invention also relates to a method for producing a novel 2-fluoro-pyrrolo [2,1-c] [1,4] benzodiazepine dimer that is effective as a potential antitumor agent. In particular, the present invention relates to a method for producing a novel bis-2-fluoropyrrolo [2,1-c] [1,4] benzodiazepine effective as an anticancer agent. More particularly, the present invention relates to 1,1 ′-{[(bisalkane-1, N-diyl)] dioxy} bis [(11aS) -2-fluoro-7-methoxy with varying aliphatic chains for compounds. Provided is a process for preparing -1,2,3,11a-tetrahydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5-one and also describes anticancer (antitumor) activity . The structural formula of the novel bis-2-fluoro-pyrrolo [2,1-c] [1,4] benzodiazepine is as follows, where n is 3, 4, 5, 6, 7, 8, 9, 10.

BACKGROUND OF THE INVENTION Pyrrolo [2,1-c] [1,4] benzodiazepine antitumor antibiotics are commonly known as anthramycin class compounds. In the past few years, there has been an increasing interest in the development of new pyrrolo [2,1-c] [1,4] benzodiazepines (PBD). These PBDs bind exclusively to exocyclic N2-guanine in a small groove of DNA via an acid-labile amine bond to an electrophilic imine at the N10-C11 position, a sequence-selective DNA-binding antitumor antibiotic (Kunimoto S .; Masuda T .; Kanbayashi N .; Hamada M .; Naganawa H .; Miyamoto M .; Takeuchi T .; Unezawa H., J. Antibiot. 1980, 33, 665; Kohn KW ; Speous CK, J. Mol. Biol. 1970, 51, 551; Hurley LH; Gairpla C .; Zmijewski M., Biochem. Biophys. Acta 1977, 475, 521; Kaplan DJ; Hurley LH, Biochemistry 1981, 20, 7572 ).

  All biologically active PBDs have an (S) configuration at the chiral C11a position, which provides a molecule with a clockwise twist and a B-type double-stranded DNA spanning 3 base pairs. Allows PBD to follow the curvature of small grooves. Recently, a PBD dimer has been developed comprising two C2-exo-methylene substituted DC-81s constrained together through the C-8 position via an inert propanedioxy linker (Gregson SJ; Howard PW; Hartely JA: Brooks NA; Adams LJ; Jenkins, Kelland LR; Thurston DE, J. Mol. Chem. 2001, 44, 737).

  Recently developed to combine two PBD units through the C-8 position to generate a bifunctional alkylating agent that can crosslink DNA (Thurston DE; Bose DS; Thomson AS; Howard PW; Leoni A .; Croker SJ; Jenkins TC; Neidle S .; Hurley LH, J. Org. Chem. 1996, 61, 8141-8147). Recently, non-crosslinked mixed imine-amide PBD dimers have been synthesized that have significant DNA binding ability and potent anti-tumor activity (Kamal A .; Laxman N .; Ramesh G .; Ramulu P. Srinivas, U.S. Pat.No. 636233; Kamal A .; Ramesh G .; Laxman N .; Ramulu P .; Srinivas O .; Neelima K .; Kondapi AK; Srinu VB; Nagarajaram HM, J. Mol. 45, 4679).

  PBD is currently of considerable interest because it recognizes a specific base sequence of double-stranded DNA and subsequently forms a covalent bond thereto. Naturally occurring pyrrolo [2,1-c] [1,4] benzodiazepines belong to a group of antitumor antibiotics derived from Streptomyces species, and members of these families are anthramycin and tomaymycin , Civiromycin, kikamycin, neotramycin A and B, and DC-81.

  However, the clinical efficacy of these antibiotics is hampered by several limitations, such as poor water solubility and the development of cardiotoxicity and drug resistance and metabolic inactivation.

Objects of the invention The main object of the present invention is to provide new bis-2-fluoropyrrolo [2,1-c] [1,4] benzodiazepines which are effective as antitumor agents.
Another object of the present invention is to provide a novel fluoropyrrolo [2,1-c] [1,4] benzodiazepine effective as an antitumor agent.

Accordingly , the present invention provides a compound of formula IX:

(Where n is 3 to 10)
A novel fluoropyrrolo [2,1-c] [1,4] benzodiazepine is provided.
In one embodiment of the invention, the compound of the formula IX is 1,1 ′-{[(propane-1,3-diyl) dioxy] bis [(11aS) -2-fluoro-7-methoxy-1,2, 3,11a-tetra-hydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5-one]}.
In another embodiment of the present invention, the compound of formula IX is 1,1 ′-{[(butane-1,4-diyl) dioxy] bis [(11aS) -2-fluoro-7-methoxy-1,2, 3,11a-tetra-hydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5-one]}.

  In another embodiment of the present invention, the compound of formula IX is 1,1 ′-{[(pentane-1,5-diyl) dioxy] bis [(11aS) -2-fluoro-7-methoxy-1,2,3 , 11a-tetra-hydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5-one]}.

The present invention also includes Formula IX comprising the following steps:

(Where n is 3 to 10)
A process for preparing bis-2-fluoropyrrolo [2,1-c] [1,4] benzodiazepines is provided:
(a) reacting methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-hydroxypyrrolidine-2-carboxylate dissolved in an organic solvent;
(b) Cool the solution and add dropwise a solution of diethylaminosulfur trifluoride (DAST) in an organic solvent;
(c) Isolating methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxylate with DIBAL-H formed in the presence of organic solvent And then cool down

(d) isolating the resulting methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde;
(e) protecting methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde with EtSH in the presence of an organic solvent;
(f) isolating (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal;
(g) (2S) -N- [4-Benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal is reacted with a debenzylating agent to obtain a compound of formula VI:

(2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal of
(h) (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl]-of formula VI at reflux temperature in an aprotic water-miscible organic solvent and in the presence of a mild inorganic base 4-Fluoropyrrolidine-2-carboxaldehyde diethylthioacetal is reacted with a dibromoalkane and the formula VII:

(Where n is 3 to 10)
1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal],
(i) reducing the compound of formula VII with SnCl ₂ .2H ₂ O to reflux temperature in the presence of an organic solvent and formula VIII:

(Where n is 3 to 10)
1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-amino-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal],
(j) reacting a compound of formula VIII with a deprotecting agent to give a bis-2-fluoropyrrolo [2,1-c] [1,4] of formula IX, where n is as defined above Benzodiazepine is produced.

In one embodiment of the invention, the organic solvent used in steps (a), (b) and (c) comprises CH ₂ Cl ₂ .
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 dropping in step (b) is carried out over 40 minutes.
In another embodiment of the present invention, step (c) is performed 15 hours after step (b).
In yet another embodiment of the invention, the cooling in step (c) is carried out at a temperature of -78 ° C for 45 minutes.

In another embodiment of the invention, step (e) is performed at room temperature in the presence of an organic solvent.
In yet another embodiment of the present invention, a mild selected from the group consisting of K ₂ CO ₃ , CsCO ₃ and BaCO _{3 in} an aprotic water miscible organic solvent selected from the group consisting of acetone, acetonitrile and DMF. (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal of formula VI with dibromoalkane in the presence of a simple inorganic base .

In another embodiment of the invention, step (h) is performed for 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 still other embodiments of the invention, the deprotecting agent comprises a combination of HgCl ₂ and HgO in CH ₃ CN / H ₂ O.

The present invention also includes Formula IX comprising the following steps:

(Where n is 3 to 10)
A method for producing a bis-2-fluoropyrrolo [2,1-c] [1,4] benzodiazepine is provided:
(a) Formula VI:

(2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal;
(b) (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl]-of formula VI at reflux temperature in an aprotic water-miscible organic solvent and in the presence of a mild inorganic base 4-Fluoropyrrolidine-2-carboxaldehyde diethylthioacetal is reacted with a dibromoalkane and the formula VII:

(Where n is 3 to 10)
1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal] is isolated;
(c) reducing the compound of formula VII with SnCl ₂ .2H ₂ O to reflux temperature in the presence of an organic solvent and formula VIII:

(式中nは3〜10である)

(Where n is 3 to 10)

1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-amino-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal] is isolated;
(d) reacting a compound of formula VIII with a deprotecting agent to give a bis-2-fluoropyrrolo [2,1-c] [1,4] of formula IX, where n is as defined above Benzodiazepine is produced.

In yet another embodiment of the present invention, a mild selected from the group consisting of K ₂ CO ₃ , CsCO ₃ and BaCO _{3 in} an aprotic water miscible organic solvent selected from the group consisting of acetone, acetonitrile and DMF. (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal of formula VI with dibromoalkane in the presence of a simple inorganic base .

In another embodiment of the invention, step (b) is performed for 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 still other embodiments of the invention, the deprotecting agent comprises a combination of HgCl ₂ and HgO in CH ₃ CN / H ₂ O.

Detailed Description of the Invention The present invention relates to (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2- of formula VI in an aprotic water-miscible organic solvent. Bis-2-fluoropyrrolo [2,1-c] [1,4] benzodiazepines of formula IX, wherein n is 3 to 10, comprising reacting carboxaldehyde diethylthioacetal with dibromoalkane A method of manufacturing the same is provided. The solvent is preferably selected from the group consisting of acetone, acetonitrile and DMF. The process is also carried out in the presence of a mild inorganic base such as K ₂ CO ₃ , CsCO ₃ and BaCO ₃ and up to reflux temperature for 48 hours.

The resulting 1,1 ′-{[(alkane-1, N-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoro of formula VII Pyrrolidine-2-carboxaldehyde diethylthioacetal] is isolated by conventional methods and the compound of formula VII (wherein n is 3-10) is converted to SnCl ₂ .2H ₂ O at reflux temperature in the presence of an organic solvent. Reduce with. The resulting 1,1 ′-{[(alkane-1, N-diyl) dioxy] bis [(2-amino-5-methoxy-1,4) of formula VIII (where n is 3 to 10) -Phenylene) carbonyl]} bis [4-fluoro-pyrrolidine-2-carboxaldehyde diethylthioacetal] is isolated by known methods. The compound of formula VIII is then reacted in a conventional manner with a known deprotecting agent to give a novel bis-2-fluoropyrrolo [2,1-c] of formula IX, where n is as defined above. [1,4] Benzodiazepines are produced.

In the alternative, this method may first comprise methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-hydroxypyrrolidine-dissolved in an organic solvent such as CH ₂ Cl _2. The 2-carboxylate is reacted and the solution is cooled to -78 ° C. To this cooled solution, a solution of an organic solvent such as diethylaminosulfur trifluoride (DAST) in CH ₂ Cl ₂ is added dropwise over 40 minutes. After 15 hours, methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxylate of formula II was isolated with DIBAL-H of formula III It is then cooled to −78 ° C. over 45 minutes in the presence of an organic solvent such as CH ₂ Cl ₂ .

  The resulting methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde is isolated by conventional methods and methylated in the presence of an organic solvent at room temperature. (2S) -N- [4-Benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde is protected with EtSH. The resulting (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal was then isolated by known methods and any Reaction with a benzyl agent produces (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 shown above.

The precursor, methyl (2S) -N- (4-benzyloxy-5-methoxy-2-nitrobenzoyl) -4-hydroxypyrrolidine-2-carboxylate (intermediate of DC-81) was prepared by literature methods (Thurston DE; Murthy VS; Langley DR; Jones GB, Synthesis 1990, 81).
Some representative compounds of formula IX of the present invention are described below:
1) 1,1 '-{[(propane-1,3-diyl) dioxy] bis [(11aS) -2-fluoro-7-methoxy-1,2,3,11a-tetra-hydro-5H-pyrrolo [ 2,1-c] [1,4] benzodiazepin-5-one]};
2) 1,1 '-{[(butane-1,4-diyl) dioxy] bis [(11aS) -2-fluoro-7-methoxy-1,2,3,11a-tetra-hydro-5H-pyrrolo [ 2,1-c] [1,4] benzodiazepin-5-one]};
3) 1,1 '-{[(pentane-1,5-diyl) dioxy] bis [(11aS) -2-fluoro-7-methoxy-1,2,3,11a-tetra-hydro-5H-pyrrolo [ 2,1-c] [1,4] benzodiazepin-5-one]}.

The reaction scheme is described below:

These novel analogs of pyrrolo [2,1-c] [1,4] benzodiazepines substituted at the C-2 position showed promising anticancer activity in various cell lines. The synthesized molecules have enormous biological significance with potential sequence selective DNA binding. This resulted in a new, similar design and synthesis comprising:
1. Fluoro substitution at the C-2 position of the DC-81 intermediate.
2. Ether bond between two fluoro DC-81 monomers at C-8 position.
3. Reflux the reaction mixture for 24-48 hours.
4. Synthesis of fluoro PBD antitumor antibiotic dimer imine.
5. Purification by column chromatography using various solvents such as ethyl acetate, hexane, dichloromethane and methanol.

Representative compounds of formula IX include:

  The following examples are illustrative of the invention and are therefore not to be construed as limiting the invention.

Example 1 .
(2S) -N- (4-hydroxy-5-methoxy-2-nitrobenzoyl) -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal VI (418 mg, of formula VI in dry acetone (40 ml) 1 mmol), 1,3-dibromopropane (101 mg, 0.5 mmol) and K ₂ CO ₃ (414 mg, 3 mmol) were refluxed for 48 hours. After the reaction was complete as indicated by TLC, EtOAc-hexane (7: 3), the reaction mixture was poured onto 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 pure 1,1 ′-{[(propane -1,3-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] VII was obtained. H ¹ NMR (CDCl ₃ , 200 MHz): δ1.2-1.39 (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, 4H), 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-fluoropyrrolidine-2-carboxaldehyde diethyl Thioacetal] VII (876 mg, 1.0 mmol) was dissolved in methanol (10 ml), and SnCl ₂ .2H ₂ O (1.124 g, 5.0 mmol) was added thereto and refluxed for 1.5 hours. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO ₃ solution and then extracted with ethyl acetate (3 × 20 ml). The combined organic phases were dried over Na ₂ SO ₄ and evaporated in vacuo to give crude 1,1 ′-{[(propane-1,3-diyl) dioxy] bis [(2-amino-5-methoxy- 1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] was obtained.

1,1 '-{[(propane-1,3-diyl) dioxy] bis [(2-amino-5-methoxy-1,4- in CH ₃ CN / H ₂ O (3: 1, 15 ml) Phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] VIII (846 mg, 1 mmol), HgCl ₂ (794 mg, 2.93 mmol) and HgO (686 mg, 3.18 mmol) Stir at room temperature for 12 hours until TLC (EtOAc) shows complete disappearance of the starting material. The organic layer was then evaporated in vacuo and the residue was diluted with EtOAc. To this was added saturated NaHCO ₃ solution slowly at room temperature and the mixture was filtered through celite and washed with ethyl acetate.

The filtrate was evaporated in vacuo to yield crude 1,1 ′-{[(propane-1,3-diyl)] dioxy} bis [(11aS) -2-fluoro-7-methoxy-1,2,3 of formula IXa. , 11a-tetrahydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5-one], which was further purified by column chromatography on silica gel, eluting first with ethyl acetate. Traces of mercury salts were removed and further eluted with CHCl ₃ -methanol (8.5: 1.5). H ¹ NMR (CDCl ₃ , 200 MHz): δ 2.15-2.45 (m, 6H), 3.7-3.9 (m, 6H), 4.01 (s, 6H), 4.22-4.3 (m, 4H), 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 .
(2S) -N- (4-Hydroxy-5-methoxy-2-nitrobenzoyl) -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal VI (418 mg, 1 mmol) in dry acetone (20 ml) A solution of 1,4-dibromobutane (107 mg, 0.5 mmol) and K ₂ CO ₃ (414 mg, 3 mmol) was refluxed for 48 hours. After the reaction was complete as indicated by TLC, EtOAc-hexane (7: 3), the reaction mixture was poured onto 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 and eluted with EtOAc-hexane (1: 1) to give pure 1,1 ′-{[(butane -1,4-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] VII was obtained. H ¹ NMR (CDCl ₃ , 200 MHz): δ1.29-1.4 (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, 4H), 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-fluoropyrrolidine-2-carboxaldehyde diethyl Thioacetal] VII (890 mg, 1.0 mmol) was dissolved in methanol (10 ml), and SnCl ₂ .2H ₂ O (1.124 g, 5.0 mmol) was added thereto and refluxed for 1.5 hours. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO ₃ solution and then extracted with ethyl acetate (3 × 20 ml). The combined organic phases were dried over Na ₂ SO ₄ and evaporated in vacuo to give crude 1,1 ′-{[(butane-1,4-diyl) dioxy] bis [(2-amino-5 of formula VIII -Methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] was obtained.

1,1 ′-{[(butane-1,4-diyl) dioxy] bis [(2-amino-5-methoxy-1) of formula VIII in CH ₃ CN / H ₂ O (3: 1, 15 ml) , 4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] (861 mg, 1 mmol), HgCl ₂ (794 mg, 2.93 mmol) and HgO (687 mg, 3.18 mmol) The solution was stirred at room temperature for 12 hours until TLC (EtOAc) showed complete disappearance of starting material. The organic layer was then evaporated in vacuo and the residue was diluted with EtOAc. To this was added saturated NaHCO ₃ solution slowly at room temperature and the mixture was filtered through celite and washed with ethyl acetate.

The filtrate was evaporated in vacuo to give crude 1,1 ′-{[(butane-1,4-diyl)] dioxy} bis [(11aS) -2-fluoro-7-methoxy-1,2,3,11a- Tetrahydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5-one] IXb was obtained, which was further purified by column chromatography on silica gel, eluting first with ethyl acetate Mercury salts were removed and further eluted with CHCl ₃ -methanol (9: 1). H ¹ NMR (CDCl ₃ , 200 MHz): δ1.94-2.09 (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 .
(2S) -N- (4-Hydroxy-5-methoxy-2-nitrobenzoyl) -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal VI (418 mg, 1 mmol) in dry acetone (20 ml) A solution of 1,5-dibromopentane (114 mg, 0.5 mmol) and K ₂ CO ₃ (414 mg, 3 mmol) was refluxed for 48 hours. After the reaction was complete as indicated by TLC, EtOAc-hexane (7: 3), the reaction mixture was poured onto 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 and eluted with EtOAc-hexane (1: 1) to give pure 1,1 ′-{[(pentane. -1,5-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] VII was obtained. H ¹ NMR (CDCl ₃ , 200 MHz): δ1.2-1.42 (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-1,5-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2- Carboxaldehyde diethylthioacetal] (905 mg, 1.0 mmol) was dissolved in methanol (10 ml), and SnCl ₂ .2H ₂ O (1.124 g, 5.0 mmol) was added thereto and refluxed for 1.5 hours. The reaction mixture was then carefully adjusted to pH 8 with saturated NaHCO ₃ solution and then extracted with ethyl acetate (3 × 20 ml). The combined organic phases were dried over Na ₂ SO ₄ and evaporated in vacuo to give crude 1,1 ′-{[(pentane-1,5-diyl) dioxy] bis [(2-amino-5 of formula VIII -Methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] was obtained.

1,1 ′-{[(pentane-1,5-diyl) dioxy] bis [(2-amino-5-methoxy-1) of formula VIII in CH ₃ CN / H ₂ O (3: 1, 15 ml) , 4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal] (875 mg, 1 mmol), HgCl ₂ (794 mg, 2.93 mmol) and HgO (687 mg, 3.18 mmol) The solution was stirred at room temperature for 12 hours until TLC (EtOAc) showed complete disappearance of starting material. The organic layer was then evaporated in vacuo and the residue was diluted with EtOAc. To this was added saturated NaHCO ₃ solution slowly at room temperature and the mixture was filtered through celite and washed with ethyl acetate.

The filtrate was evaporated in vacuo to give crude 1,1 ′-{[(pentane-1,5-diyl)] dioxy} bis [(11aS) -2-fluoro-7-methoxy-1,2,3,11a- Tetrahydro-5H-pyrrolo [2,1-c] [1,4] benzodiazepin-5-one] IXc was obtained, which was further purified by column chromatography on silica gel, eluting first with ethyl acetate Mercury salts were removed and further eluted with CHCl ₃ -methanol (9: 1). H ¹ NMR (CDCl ₃ , 200 MHz): δ1.58-1.81 (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 conducted at the National Cancer Institute (USA).

Cytotoxicity:
Compounds IXa and IXc in 60 human tumor cells derived from 9 cancer types (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer and breast cancer) Evaluated in vitro. For each compound, a dose response curve for each cell line was measured at 10-fold dilution with a minimum of 5 concentrations. Cell viability and proliferation were estimated using a 48 hour continuous drug exposure protocol and using a sulforhodamine B (SRB) protein assay. Concentrations that caused 50% cell growth inhibition (GI50), complete cell growth inhibition (TGI, 0% proliferation) and 50% cell death (LC50, -50% proliferation) compared to controls were calculated. The average graph midpoint values of log ₁₀ TGI and log ₁₀ LC50 and log ₁₀ GI50 for IXa and IXc are listed in Table 1. As evidenced by the average graph pattern, compound IXc exhibits an important distribution of activity and selectivity for various cell lines. The average graph midpoint of log ₁₀ TGI and log ₁₀ LC50 showed a similar pattern to the average graph midpoint of log ₁₀ GI50.

  The antitumor activity of two representative compounds is listed in Table 2. Compared with the data in Table 3, the importance of alkane spacers becomes clear. As the alkane spacer increased from 3 to 5, the cytotoxic activity was moderately enhanced. The 5-carbon spacer of compound IXc gives adequate fit in the small groove of double helix DNA and shows slightly higher activity in this series of compounds IXa and IXc.

Claims (31)

Formula IX:

A novel pyrrolo [2,1-c] [1,4] benzodiazepine, wherein n is 3-10. The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having The following structure:

The novel pyrrolobenzodiazepine according to claim 1 having Formula IX below:

(Where n is 3 to 10)
In the process for producing bis-2-fluoropyrrolo [2,1-c] [1,4] benzodiazepine,
(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 dropwise a solution of diethylaminosulfur trifluoride (DAST) in an organic solvent;
(c) Isolating methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxylate with DIBAL-H formed in the presence of organic solvent And cooling;
(d) isolating the resulting methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde;
(e) protecting methyl (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde with EtSH in the presence of an organic solvent;
(f) isolating (2S) -N- [4-benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal;
(g) (2S) -N- [4-Benzyloxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal is reacted with a debenzylating agent to obtain a compound of formula VI:

(2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal of
(h) (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] of formula VI in an aprotic water-miscible organic solvent and in the presence of a mild inorganic base up to reflux temperature. 4-Fluoropyrrolidine-2-carboxaldehyde diethylthioacetal is reacted with dibromoalkane and formula VII:

(Where n is 3 to 10)
1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal] is isolated;
(i) reducing the compound of formula VII with SnCl ₂ .2H ₂ O to reflux temperature in the presence of an organic solvent and formula VIII:

(Where n is 3 to 10)
1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-amino-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal] is isolated;
(j) reacting a compound of formula VIII with a deprotecting agent to give a bis-2-fluoropyrrolo [2,1-c] [1,4] of formula IX, where n is as defined above Produce benzodiazepines;
A method comprising that. Step (a), the organic solvent used comprises an CH ₂ Cl ₂ at (b) and (c), The method of claim 10.   The process according to claim 10, wherein in step (a) the solution is cooled to a temperature of -78 ° C.   11. The method according to claim 10, wherein the dropping in step (b) is carried out over 40 minutes.   11. The method according to claim 10, wherein step (c) is performed 15 hours after step (b).   The process according to claim 10, wherein the cooling in step (c) is carried out at a temperature of -78 ° C for 45 minutes.   The process according to claim 10, wherein step (e) is carried out in the presence of an organic solvent at room temperature. 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 K ₂ CO ₃ , CsCO ₃ and BaCO ₃ 11. The (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal of formula VI is reacted with a dibromoalkane. Method.   The method of claim 10, wherein step (h) is carried out for about 48 hours.   11. The process according to claim 10, wherein the reduction in step (i) is carried out in the presence of an organic solvent comprising methanol. Deprotecting agent comprises a combination of HgCl ₂ and HgO in CH ₃ CN / H ₂ O, The method of claim 10. Formula IX below:

(Where n is 3 to 10)
In the process for producing bis-2-fluoropyrrolo [2,1-c] [1,4] benzodiazepine,
(a) Formula VI:

(2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde-diethylthioacetal;
(b) (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl]-of formula VI at reflux temperature in an aprotic water-miscible organic solvent and in the presence of a mild inorganic base 4-Fluoropyrrolidine-2-carboxaldehyde diethylthioacetal is reacted with a dibromoalkane and the formula VII:

(Where n is 3 to 10)
1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-nitro-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal] is isolated;
(c) reducing the compound of formula VII with SnCl ₂ .2H ₂ O to reflux temperature in the presence of an organic solvent and formula VIII:

(Where n is 3 to 10)
1,1 '-{[(alkane-1, N-diyl) dioxy] bis [(2-amino-5-methoxy-1,4-phenylene) carbonyl]} bis [4-fluoropyrrolidine-2-carboxaldehyde Diethylthioacetal] is isolated;
(d) reacting a compound of formula VIII with a deprotecting agent to give a bis-2-fluoropyrrolo [2,1-c] [1,4] of formula IX, where n is as defined above Produce benzodiazepines;
A method comprising that. In the presence of a mild inorganic base selected from the group consisting of K ₂ CO ₃ , CsCO ₃ and BaCO _{3 in} an aprotic water-miscible organic solvent selected from the group consisting of acetone, acetonitrile and DMF 23. The method of claim 21, wherein (2S) -N- [4-hydroxy-5-methoxy-2-nitrobenzoyl] -4-fluoropyrrolidine-2-carboxaldehyde diethylthioacetal of VI is reacted with dibromoalkane.   24. The method of claim 21, wherein step (b) is carried out for about 48 hours.   The process according to claim 21, wherein the reduction in step (c) is carried out in the presence of an organic solvent comprising methanol. Deprotecting agent comprises a combination of HgCl ₂ and HgO in CH ₃ CN / H ₂ O, The method of claim 21.   A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula IX and a pharmaceutically acceptable additive. 27. A pharmaceutical composition according to claim 26 for the treatment of cancer. 28. A pharmaceutical composition according to claim 26 or 27 for administration to a mammal. 30. The pharmaceutical composition according to claim 28 , wherein the mammal is a human. 30. The cancer according to any one of claims 27 to 29, wherein the cancer is selected from the group consisting of leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer and breast cancer. Pharmaceutical composition . Leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, of formula IX for the manufacture of a pharmaceutical composition for the treatment of cancer selected from the group consisting of renal cancer, prostate cancer and breast cancer Use of compounds.