Chem. Res. Toxicol. 1988,1, 258-268
258
Articles Pyrrole[ 1,4]benrodiazepine Antitumor Antibiotics: Relationship of DNA Alkylation and Sequence Specificity to the Biological Activity of Natural and Synthetic Compounds Laurence H. Hurley,* Teri Reck, David E. T h u r s t o n , + and David R. Langley Drug Dynamics Institute and Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712
Kenneth G. Holden, Robert P. Hertzberg, J o h n R. E. Hoover, F. Faucette, Shau-Ming Mong, and Randall K. Johnson
Gregory Gallagher, Jr., Leo
Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406 Received June 2, 1988
The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease I11 stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease I11 which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry a t C - l l a . These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LDm), and for antitumor activity (ILS,) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.
Introduction Anthramycin and tomaymycin (Table I) are two of the best known examples of the pyrrolo[1,4]benzodiazepine [P(1,4)BI1group of antibiotics, which are potent antitumor compounds produced by various Actinomycetes (1-6). The biological potency of the P(1,4)Bs is generally attributed to their ability to bond covalently to DNA (7-9). While anthramycin, sibiromycin, and neothramycins A and B have been used experimentally in the treatment of human cancer (2,lO-13),their real potential as clinical candidates is reported to be compromised by either cardiotoxicity ( 10, 14,15),tissue necrosis at the site of injection (15),or, in the case of the neothramycins, lack of efficacy (13).
* Address correspondence to this author. t Present address: School of Pharmacy and Biomedical Science,
Portsmouth Polytechnic, King Henry 1st St., Portsmouth, Hants PO1 2DZ, England.
Mechanism of action studies have led to considerable insight into the three-dimensional structures of the P(1,4)B-DNA adducts (416-20), and some of the biochemical (21) and biological consequences (22,23) of DNA modification have been described. The ultimate DNA-reactive species is proposed to be the N-10-C-11 imine form of the P(1,4)Bwhich undergoes nucleophilic attack from N-2 of guanine to form an aminal linkage (24) (see Figure 1). Since the two enantiomeric C-11 carbinolamines react with DNA via the N - 1 0 4 1 1 imine and are in equilibrium in Abbreviations: bp, base pair; BAP, bacterial alkaline phosphatase; CPK, Corey, Pauling, Koltun; D W , dithiothreitol;EDTA, ethylenediaminetetraacetic acid; ex0 111, exonuclease III; ip, intraperitoneal, W E , methidiumpropyl-EDTA; P(1,4)B, pyrrolo[l,4]benzodiazepine; PNK, polynucleotide kinase; Pu, purine: Py, pyrimidine; SAR, structure-activity relationship;TAE, 40 mM Tris-acetate, pH 8.0, and 50 mM EDTA TBE, 50 mM Tris-borate and 10 m M EDTA, pH 8.3; TEMED, A’,”,N’,N’-tetramethylethylenediamine; Tris, tris(hydroxymethy1)aminomethane.
0893-228~/88/2701-0258$01.50/0 0 1988 American Chemical Society
Pyrrolo[l,,Ubenzodiazepine Antitumor Antibiotics
Chem. Res. Toxicol., Vol. 1, No. 5, 1988 259
Table I. In Vitro and in Vivo Biological Activity of Natural and Synthetic P(1,4)Bs comDounds IC,, pMa LD,, malkab anthramycin
0.4
1.1
118 f 49
tomaymycin
0.02
2.2
87 f 6
V
0.02
2.2
76
VI
1.0
VI1
k E q H
P3W I L L ,
12
VI11
1.0
IX, llas
0.06
x, llaR
8.0
*5
12
40
100
NDd
71
NDd
35
40 NDd
>100
aConcentration that inhibits proliferation of B16 melanoma cells by 50% on continuous exposure. *Single ip dose which kills 50% of female B6D2F1 mice. Increase in lifespan produced in mice bearing ip P388 leukemia at the maximally tolerated dose given ip on days 1 and 5. dNot determined.
to examine the effect of structural modification of various portions of the P(1,4)B nucleus and its substituents. These same compounds were also subjected to a DNA alkylation assay that measures bonding reactivity as well as sequence selectivity. In addition to demonstrating a relationship between DNA bonding and biological activity, information concerning the relationship between DNA sequence specificity and antitumor efficacy was obtained. Figure 1. Proposed mechanism for alkylation of DNA by the adduct. P(1,4)Bs to form the P(1,4)B-(N-a-guanine)-DNA
solution, they are biologically equivalent. The resultant P(1,4)B-DNA adduct is snugly cradled in the minor groove of DNA and covers a 2-3 bp region (8) for which a sequence specificity has been demonstrated (25). CPK models of the (N-Zguanine)-DNA adducts have been used to rationalize the published SAR for a variety of natural and synthetic P(1,4)B analogues (26). Versatile synthetic routes leading to the carbinolamine-containing P(1,4)B nucleus have been recently published from these laboratories and others (27-35), which now allows ready access to a variety of P(1,4)B analogues. In this article we describe the results of in vitro and in vivo evaluation of the biological activity of two naturally occurring compounds and a number of synthetic analogues (Table I) designed
Materials and Methods Electrophoretic materials [acrylamide, bis(acrylamide), TEMED, urea, and ammonium persulfate] were obtained from Bio-Rad. Restriction endonucleases and exo I11 were from New England Biolabs. T4 polynucleotide kinase (T4-PNK) was from BRL. BAP was obtained both from Sigma and BRL, and [y32P]ATPwas from ICN. X-ray film, intensifying screens, and developing chemicals were from Kodak. Ml3mpl RF DNA was isolated from Escherichia coli JMlOl by the CsClz ultracentrifugation method described by Messing (36). Anthramycin and tomaymycin were obtained from Hoffmann LaRoche and Fujisawa Cos., respectively. Compounds 1-111 (Table I) were synthesized by the reductive cyclization procedure previously described (34). Compound V was prepared as previously described from tomaymycin (24). (1lRS,1 laRS)-8-Chloro-ll-hydroxy-1,2,3,10,11,1 la-hexahydro-5W-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (IV). 4Chloro-2-nitrobenzoic acid (12 g, 60 mmol) and S0C12 (6.0 mL,
260 Chem. Res. Toxicol., Vol. I, No. 5, 1988 82 mmol) in dry benzene (100 mL) was heated under reflux for 3 h, followed by evaporation of the solvent to afford 4-chloro-2nitrobenzoyl chloride as a light brown oil. After stripping from benzene three times to remove excess SOC12,the oil was dissolved in dry THF (65 mL) and added dropwise to a stirred solution of proline (8.22 g, 71 mmol) and triethylamine (13 mL) in water (65 mL) maintained at 0 "C in an ice bath. After warming to room temperature and stirring for a further 1.25 h, the T H F was evaporated in vacuo, followed by the addition of water (200 mL). The solution was acidified with 5% HC1, decanted from the resulting gum, and then replaced with fresh water. The gum crystallized upon scratching, and the decanted solution afforded a precipitate upon standing at 0 "C. The combined solid was washed with water and then ether to afford (2R)-N-(4-chloro-2nitrobenzoy1)proline (14.90 g, 84%): 90-MHz 'H NMR (DMSO-dB)6 1.60-2.55 (4 H, m), 3.09-3.75 (2 H, m), 4.30-4.55 (1H, m), 7.50 (1H, d, J = 9.2 Hz), 7.93 (1H, d, J = 9.2 Hz and fine coupling of 1.6 Hz), 8.38 (1H, d, J = 1.6 Hz). A suspension of the (2R)-N-(4-chloro-2-nitrobenzoyl)proline prepared above (5.0 g, 1.7 mmol), oxalyl chloride (9.6 mL, 110 mmol), and DMF (4 drops) in dry benzene (50 mL) was stirred a t room temperature for 30 min, followed by evaporation of the benzene in vacuo. The residue was dissolved in dry acetone (50 mL),triphenylphosphine added (8.65 g, 33 mmol), and the solution cooled in an ice bath. (Ph3P)&d3H4(10.3 g, 23 mmol) was then added in portions and the mixture stirred a t low temperature for a further 15 min, followed by removal of the copper salt by fitration. The filtrate was reduced to a low volume and additional copper salt filtered off. The filtrate was then evaporated in vacuo to afford a pale yellow oil, which was dissolved in a small volume of ethyl acetate and purified by flash chromatography on silica gel, eluting with ethyl acetate/toluene (l:l), to afford (2%')N-(4-chloro-2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde (1.9 g, 40%) as a pale yellow oil: 90-MHz 'H NMR (CDCl,) 6 1.50-2.42 (4 H, m), 3.25 (2 H, t, J = 6.4 Hz), 4.70 (1H, t, J = 6.9 Hz), 7.50 (1H, d, J = 8.6 Hz), 7.71 (1H, d, J = 8.6 Hz with fine coupling of 1.7 Hz),8.22 (1 H, d, J = 1.7 Hz), 9.75 (1 H, s). The nitro aldehyde prepared above (1.9 g, 6.7 mmol) was dissolved in 2,2-dimethoxypropane (10 mL) and methanol (10 mL). Methanesulfonic acid (0.46 mL) was added with stirring under nitrogen and stirring continued for a further 2 h when the TLC (EtOAc/toluene, 1:l) indicated a complete loss of starting material. The solvent was removed by evaporation in vacuo and the residue stripped once from toluene, followed by purification by flash chromatography on silica gel (EtOAc/toluene, 1:l).The sample was then triturated with petroleum ether and the precipitate collected to yield (2RS)-N-(4-chloro-2-nitrobenzoyl)pyrrolidine2-carboxaldehyde dimethyl acetal (1.5 g, 68%): 90-MHz 'H NMR (CDC13) 6 1.59-2.50 (4 H, m), 3.00-3.33 (2 H, m), 3.57 (6 H, s), 4.30-4.65 (1 H, m), 4.94 (1 H, d , J = 1.2 Hz), 7.40 (1H, d, J = 7.6 Hz), 7.73 (1H, d, J = 7.6 Hz with fine coupling 1.2 Hz), 8.21 (1 H, d, J = 1.2 Hz). A mixture of the nitro acetal prepared as described above (1.5 g, 4.6 mmol) and 10% Pd/C (80 mg) was suspended in dry methanol (100 mL) and hydrogenated with shaking at atmospheric pressure for 7-8 h, after which time TLC (silica gel, EtOAc/ toluene, 1:l) indicated a complete loss of starting material. After removal of the catalyst by filtration, the solvent was evaporated to a low volume. The residue was taken up in EtOAc/toluene (1:l) and purified by flash chromatography using the same solvent system. The fluorescent product of highest R value was (2RS)-N-(2-amino-4-chlorobenzoyl)pyrrolidine-2-carboxaldehyde dimethyl acetal (0.4 g, 30%): 90-MHz 'H NMR (CDC13) 6 1.46-2.30 (4 H, m), 3.30-3.70 (2 H, m), 3.50 (6 H, s), 4.59-4.77 (1 H, m), 6.65 (d, J = 8.0 Hz), 7.02-7.32 (2 H, m). Some dechlorinated products of lower R, values were also obtained. The amino acetal prepared above (0.22 g, 0.74 mmol) was dissolved in 50% CH3CN/H20 (5 mL) and trifluoroacetic acid (0.05 mL) added with stirring at room temperature. Reaction was complete after 3 h as judged by HPLC (reversed phase; CH3CN/H20,7:3). Further stirring overnight gave a precipitate of (11RS,llaRS)-8-chloro-ll-hydroxy-1,2,3,1O,ll,lla-hexahydro-5H-pyrrolo[2,1-c] [1,4]benzodiazepin-5-one (IV) and the corresponding imine (60 mg, 32%): 90-MHz 'H NMR (CDCl,/DMSO-d,J 6 1.69-2.32 (m), 3.30-3.91 (m), 4.80 (d, J = 8.8 Hz), 5.04 (s), 6.56-7.41 (m), 7.70 (d, J = 2.3 Hz), 7.80 (d, J
Hurley et al. = 3.7 Hz), 8.20 (d, J = 1.6 Hz).
(1laRS)-1,2,3,10,11,1 la-Hexahydro-7,8,1l-trimethoxy-5Hpyrrolo[2,1-c][ 1,4]benzodiazepin-5-one (VI). Trifluoroacetic anhydride (6.2 mL, 44 mmol) was added to a stirred suspension of 2-amino-4,5-dimethoxybenzoic acid (3.94 g, 20 mmol) in ethyl acetate (50 mL). After 1 h a t room temperature, the resulting solution was evaporated to dryness and stripped twice from toluene to give 6,7-dimethoxy-2-(trifluoromethyl)-4H-1,3-benzoxazin-4-one (5.29 g, 96%): 'H NMR (90 MHz, CDC13) 6 4.04 (3 H, s), 4.05 (3 H, s), 7.17 (1H, s), 7.57 (1 H, s). Without further purification, the benzoxazinone (2.75 g, 10 mmol) was added in portions over 15 min to a stirred solution of (S)-2-pyrrolidinemethanol (1.01 g, 10 mmol) in acetonitrile (20 mL). After being stirred for 1 h, the reaction mixture was evaporated to a residue of N - [ 4,5-dimethoxy-2-(trifluoroacetamido)benzoyl] - (S)-2pyrrolidinemethanol (3.76 g), which was used in the next step without purification. To a solution of crude product from the previous reaction (3.76 g, 10 mmol) in CHZClz(150 mL) and DMSO (15 mL) were added sodium acetate (2.05 g, 25 mmol) and pyridinium chlorochromate (10.75 g, 50 mmol). After being stirred overnight, the reaction mixture was chromatographed on silica (110 g) eluted with CHzCl2. Crystallization from CH30H/EtOAc gave 7,8-dimethoxy1,2,3,10,11,1la-hexahydro-ll-hydroxy-lO-(trifluoroacetyl)-5Hpyrrolo[2,1-c][l,4]benzodiazepin-5-one (2.33 g, 60% overall), mp 196-198 "C. Anal. (C16H17F3N205) C, H, N. 'H NMR (90 MHz, CDC1,) 6 1.8-2.2 (4 H, m), 3.18 (3 H, s), 3.2-3.6 (3 H, m), 3.81 (3 H, s), 3.85 (3 H, s), 6.75-6.95 (2 H, m). A suspension of the above N-(trifluoroacety1)benzodiazepinone (0.75 g, 1 mmol) and Bio-Rad AG3-X4A resin (200-400 mesh, free base form, 2.5 g) in CH30H was stirred a t room temperature for 4 days. The resin was removed by filtration and the filtrate evaporated to a residue (1.92 g) which was recrystallized from EtOAc/cyclohexane to give (llR,llaRS)-1,2,3,10,ll,lla-hexahydro-7,8,1l-trimethoxy-5H-pyrrolo[2,l-c] [1,4]benzodiazepin-5one (VI) (125 mg, 21%), mp 127-129 "C (dec). Anal. (C1J+,-,NZO4) C, H, N. 'H NMR (90 MHz, CDC13) 6 1.7-2.4 (4 H, m) 3.32 (3 H, s), 3.4-4.2 (3 H, m), 4.51 (1H, d, J = 6.3 Hz), 5.74 (1 H, d, J = 6.3 Hz), 6.21 (1H, s), 7.53 (1 H, s). 7,8-Dimethoxy-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (VII). The synthesis was carried out in a manner similar to that [ 1,4]benzodiazepinreported (37) for 8-methyl-5H-pyrrolo[2,1-c] 5-one. A solution of 2-amino-4,5-dimethoxybenzoic acid (4.93 g, 25 mmol) in 150 mL of dry toluene containing 25 mL of thionyl chloride was heated at 100 "C for 2 h. The toluene was evaporated at reduced pressure, and the solid residue was dried over sodium hydroxide in a vacuum desiccator. A solution of the crude sulfonamide anhydride in 200 mL of anhydrous ether was treated with pyrrole-2-carboxaldehyde sodium salt (2.92 g, 25 mmol; prepared from pyrrole-2-carboxaldehyde and sodium hydride). The resulting light yellow solid (8.15 g) was removed by filtration, dried in vacuo over PzO5, and stored at -20 "C. A portion of this material (1g) was distributed between 25 mL each of ethyl acetate and 5% aqueous NaZCO3. After adding more ethyl acetate to dissolve the solid that had precipitated, the phases were separated and the aqueous phase was extracted with 25 mL of ethyl acetate. The combined organic phases were washed twice with water, decolorized with charcoal, dried, and evaporated a t reduced pressure to give 0.25 g (31%) [1,4]benzodiazepin-5-one (VII), of 7,8-dimethoxy-5H-pyrrolo[2,1-c] which proved to be a powerful olfactory and skin irritant. IR, 'H NMR, and I3C NMR spectra were consistent with the assigned structure. (1laRS)-7,8-Dimethoxy-ll-(methylthio)-3,10,11,1 la-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (VIII). A solution of 2-amino-4,5-dimethoxybenzoic acid (1.97 g, 11mmol) in 5 mL of dry DMF was treated with a solution of 1.78 g (11 mmol) of N,N'-carbonyldiimidazole in 10 mL of dry DMF dropwise over 1 h with stirring at 50 "C. The reaction was stirred at 50 "C for 1 h and at room temperature for 3 days. The solids from the reaction mixture were collected by filtration, washed with a small volume of cold DMF followed by Et20, and dried in vacuo to give 1.50 g (67%) of 6,7-dimethoxy-2H-3,1-benzoxazine-2,4(1H)-dione as white crystals, mp 270-271 "C [lit. (38) 274-275 "C]: IR (Nujol) 1715, 1760 cm-'; 'H NMR (90 MHz, DMSO-d,/CDCl,) 6 6.63 (1 H, s), 7.22 (1 H, s), 3.83 (3 H, s), 3.90
Chem. Res. Toricol., Vol. 1, No. 5, 1988 261
Pyrrolo[l,4] benzodiazepine Antitumor Antibiotics (3 H, s), 6.63 (1 H, s), 7.22 (1H, s). A mixture of 1.20 g (5.4 mmol) of the above benzoxazinedione and 0.61 g (5.4 mmol) of (S)-3,4-dehydroproline in 13 mL of dry DMSO was heated at 100 "C for 3l/, h. The reaction was cooled, poured into 130 mL of ice-HzO, allowed to stand at 0 "C for 1 h, filtered, washed with THF followed by EhO, and dried to give 0.85 g (57%) of 7,8-dimethoxy-3,10,1l,lla-tetrahydro-5Hpyrrole[ 2,l-c] [ 1,4]benzodiazepine-5,ll-dione. Recrystallization from 40 mL of hot acetonitrile afforded 728 mg of flocculent white needles, mp 245-250 "C: IR (Nujol) 1635,1685 cm-'; 'H NMR (90 MHz, DMS0-d6/CDCl3) 6 3.83 (6 H, s), 4.22-4.47 (2 H, m), 4.70-4.93 (1H, m), 5.80-6.27 (2 H, m) 6.71 (1H, s), 7.32 (1H,
(2 H, m), 6.32 (1 H, s), 7.29 (1 H, 8 ) . (1laS)- a n d (llaR)-8-(Benzyloxy)-7-methoxy-1,2,3,1la-
tetrahydro-5H-pyrrolo[2,1-cI[ 1,4]benzodiazepin-5-ones(IX and X). The natural llaS isomer (IX) was synthesized by using
the convergent diethyl dithioacetal technique of Langley and Thurston (35)and will be reported elsewhere. The unnatural llaR isomer (X) was synthesized by using the related linear diethyl dithioacetal route (35),but starting with D-prOline. Analytical data for the l l a R isomer and related intermediates are given below. (2R )-N-[4-(Benzyloxy)-5-methoxy-2-nitrobenzoyl]proline (89.4% yield, TLC/ethyl acetate): IR (Nujol) 3680-3100 (COOH), 2920,2880,2825,1725 and 1625 (C=O), 1575 (C=C), 1510 and 5). A mixture of 728 mg (2.6 mmol) of 7,8-dimethoxy1370 (NO,), 1450,1280,1275,1060,720 cm-l; lH NMR (CDC13) 3,l0,ll,lla-tetrahydro-5H-pyrrolo[2,1-c] [ 1,4]benzodiazepine1.75-2.54 (4 H, m), 3.00-3.38 (2 H, m), 3.88 and 3.98 (3 H, 8 , minor 5,11-dione, 446 mg (5.3 mmol) of powdered NaHC03, and 706 mg and major rotamen of OCH3, 1:5.7), 4.68-4.99 (1H, m), 5.14 and (3.2 mmol) of P2S6in 14 mL of acetonitrile was refluxed for 15 5.20 (2 H, s, minor and major rotamers of OCH2Ph, 1:4.8), 5.89 min; TLC indicated complete disappearance of starting material. (1 H, e), 6.86 (1H, s), 7.40 (5 H, s), 7.77 (1H, s); MS m / e (re1 The reaction was allowed to cool to room temperature over 30 intensity) 400 (M+, l),352 (ll), 322 (lo), 286 (9), 226 (12), 217 min, poured into 250 mL of H20, extracted with CH2Cl,, washed (3), 202 (6), 160 (4), 136 (a), 121 (6), 105 (7), 91 (100);mp 166-167 once with HzO, and evaporated in vacuo. The residue was slurried "c (yellow needles from ethyl acetate/hexane); [ct]=D +25.1" (c in EhO, filtered, and dried to give 0.62 g (81%) of crude product. 0.1552, CHC13). Anal. Calcd for CmH&I2O7:400.1270. Fouiid: This was crystallized from 60 mL of hot EtOH to give 418 mg 400.1279. of 7,8-dimethoxy-3,10,1l,lla-tetrahydro-5-oxo-5H-pyrrolo[2,1- M e t h y l (2R ) - N -[4-(benzyloxy)-5-met h o x y - t - n i t r o c][1,4]benzodiazepine-11-thioneas flocculent pale yellow needles, benzoyl]pyrrolidine-2-carboxylate(88% yield, TLC/ethyl mp 250-253 "C: IR (Nujol) 1635 cm-'; 'H NMR (90 MHz, acetate): IR (Nujol) 2915, 2850, 1760 and 1645 (C=O), 1590 DMSO-d6/CDC13)6 3.85 (6 H, s), 4.1-4.4 (2 H, m) 4.92-5.2 (1H, (C=C), 1513 and 1380 (NO,), 1452,1426,1338,1288,1227,1199, m), 5.83-6.23 (2 H, m), 6.93 (1H, s), 7.33 (1 H, s). 1175, 1071, 1020, 869, 840, 765, 737 cm-'; 'H NMR (CDC13) 6 A mixture of 2.0 g (6.9 mmol) of 7,8-dimethoxy-3,10,1l,lla- 1.70-2.59 (4 H, m), 3.01-3.43 (2 H, m), 3.52 and 3.80 (3 H, s, minor tetrahydr0-5-0~0-5H-py~rolo[2,1-c] [1,4]benzodiazepine-ll-thione, and major rotamers of C02CH3,1:2.7), 3.91 and 3.98 (3 H, s, minor 1.37 mL of methyl iodide, and 1.2 g of anhydrous K&O3 in 54 and major rotamers of OCH3, 1:2.3), 4.64-4.87 (1H, m), 5.19 (2 mL of DMF and 54 mL of THF was stirred at room temperature H, s), 6.81 and 6.88 (1H, s, minor and major rotamen of H6,1:2.3), for 4 h. The T H F was removed in vacuo, and the residual syrup 7.23-7.58 (5 H, m), 7.77 (1H, s); MS m / e (re1 intensity) 414 (M+, was poured into 60 mL of saturated NaCl solution. The prell),355 (32), 286 (loo),198 (5), 196 (51, 190 (9), 128 (13), 121 (42), cipitate was taken up in ethyl acetate and separated from the 91 (93), 65 (25), mp 134-135 "C (yellow needles from ethyl aceaqueous phase which was also extracted with ethyl acetate. The tate/hexane); [aIz6D+20.3" (c 0.3440, CHC13). Anal. Calcd for combined organic extracts were treated with charcoal, concenCzlHzzN207:414.1427. Found 414.1418. trated in vacuo, and evaporated once from carbon tetrachloride. (2R ) - N - [4-( Benzyloxy)-li-met hoxy-2-nitrobenzoyl]The residual syrup was crystallized from 75 mL of EtOH/H,O pyrrolidine-2-carboxaldehyde(92.2% yield, TLC/ethyl aceby seeding and refrigeration to give 1.6 g (76%) of 7,a-dimethtate): IR (Nujol) 2910,2848,1734 and 1640 (C=O), 1578 (C=C), [ 1,4]benzooxy-3,lla-dihydro-1 l-(methylthio)-5H-pyrrolo[2,1-c] 1518 and 1379 (NO,), 1453,1430,1331,1280,1219,1064,762 cm-'; diazepin-&one as a yellow powder, mp 85 "C: IR (CHC13 film) 'H NMR (CDC13) 6 1.60-2.44 (4 H, m), 3.04-3.43 (2 H, m), 3.96 1630 cm-'; 'H NMR (90 MHz, CDC13) 6 2.45 (3 H, s), 3.97 (6 H, and 4.00 (3 H, s, minor and major rotamers of OCH3, 1:3.5), s), 4.3-4.52 (2 H, m), 4.67-4.83 (1H, m), 5.88-6.31 (2 H, m), 6.75 4.58-4.91 (1H, m), 5.21 (2 H, s), 6.89 (1H, s), 7.25-7.60 (5 H, m), (1H, s), 7.47 (1 H, s). 7.80 (1H, s), 9.39 and 9.84 (1H, dd, J = 2 Hz, minor and major A mixture of 144 mg (0.45 mmol) of 7,8-dimethoxy-3,lla-dirotamers of CHO, 1:3.1); mp 160.5-162 "C (yellow plates from hydro-ll-(methylthio)-5H-pyrrolo[2,l-c] [ 1,4]benzodiazepin-5-one, ethyl acetate/hexane/diethyl ether); [ c t I z 6 ~+16.8" (c 0.2672, 30 mL of THF, 3 mL of saturated aqueous KH2P04,and aluCHC13). minum amalgam prepared from 184 mg of aluminum foil ac(2R ) - N - 4-(Benzyloxy)-5-methoxy-2-nitrobenzoyl][ cording to the procedure of Keck et al. (39)was stirred efficiently pyrrolidine-2-carboxaldehydediethyl dithioacetal (85.7 % a t 0 "C for 18 h; HPLC indicated 65% conversion. The reaction yield, TLC/ethyl acetate): IR (neat) 2960, 2920, 2864, 1632 was diluted with an additional 10 mL of THF, 1 mL of saturated (C=O), 1573 (C=C), 1510 and 1330 (NO& 1445,1418,1370,1270, aqueous KH2P04,and Al/Hg prepared from 61 mg of foil. After 1218,1179,1058,988,863,792,750,691cm-'; 'H NMR (CDC13) a total of 35 h conversion ceased at about 75%. The reaction 1.30 (6 H, t, J = 8 Hz), 1.54-2.41 (4 H, m), 2.51-3.00 (4 H, m), mixture was diluted with 25 mL each of water and acetone and 3.06-3.41 (2 H, m), 3.94 (3 H, s), 4.50-4.84 (1H, m), 4.88 (1 H, filtered while still cold. The inorganic salts were washed with d, J = 4 Hz), 5.19 (3 H, s), 6.84 (1H, e), 7.18-7.55 (5 H, m), 7.75 wet acetone, and the combined fiitrates were concentrated in vacuo (1H, s); MS m / e (re1 intensity) 490 (M+, 2), 355 (19), 286 (80), at
