NITROANILINE DERIVATIVES AND THEIR USE AS ANTITUMOR AGENTS

Abstract: The invention provides nitroaniline derivatives represented by general formula (I) where the nitro group is substituted at any one of the available benzene positions 2-6; where R and A separately represent the groups NO2, CN, COOR<1>, CONR<1>R<2>, CSNR<1>R<2> or SO2NR<1>R<2> and A is substituted at any one of the available benzene positions 2-6; where B represents N(CH2CH2halogen)2 or N(CH2CH2OSO2R<3>)2 substituted at any one of the available benzene positions; and where R<1>, R<2> and R<3> separately represent H, or lower alkyl optionally substituted with hydroxyl, ether, carboxy or amino functions, including cyclic structures, or R<1> and R<2> together with the nitrogen form a heterocyclic structure, and pharmaceutical preparations containing them. These compounds have activity as hypoxia-selective cytotoxins, reductively-activated prodrugs for cytotoxins, hypoxic cell radiosensitisers, and anticancer agents.

Description: NITROANILINE DERIVATIVES AND THEIR USE AS ANTI-TUMOUR AGENTS Alkylating agents are an important class of anticancer drugs, which express their cytotoxic and antitumour effects by forming adducts with cellular DNA (Garcia et al., Biochem. Pharmacol., 1988, 37, 3189). The present invention relates to novel nitroaniline-based alkylating agents having activity as hypoxia-selective cytotoxins, reductively-activated prodrugs for cytotoxins, hypoxic cell radiosensitisers, and anticancer agents, to methods of preparing the novel compounds, and to the use of these compounds as antitumour agents. In one aspect, the present invention relates to the class of nitroaniline derivatives represented by the general formula (I); where the nitro group is substituted at any one of the available benzene positions 2-6; where R and A separately represent the groups NO : , CN, COOR 1 , CONR x R : , CSNT^R 2 or SC j NR^ 2 and A is substituted at one of the available benzene positions 2-6; where B represents N(CH 2 CH,haIogen) : or N(CH : CH : OSO ; R 3 ) 2 substituted at any one of the available benzene positions; and where R 1 , R " and R J separately represent H, or lower alkyl optionally substituted with hydroxyl, ethe carboxyl or amino functions, including cyclic structures, or R l and R 2 together wit the nitrogen form a heterocyclic structure. Where R 1 , R 2 and/or R 3 represent lower alkyl, the group may contain, from 1 to carbon atoms. Where R 1 , R 2 and/or R J represent groups containing tertiary amines, the N-oxide of those tertiary amine moieties are also included. The compounds of formula (I) have cytotoxic and antitumour activity, and are useful as hypoxia-selective cytotoxiiis, reductively-activated prodrugs for hypoxic cell radiosensitisers, and anticancer agents. Some of the compounds of formula (I) form pharmaceutically-acceptable addition salts with both organic and inorganic acids, and these addition salts also form part of the present invention. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic. salicylic, malic, fumaric. succinic. ascorbic, maleic. methanesulfonic, isethionic and the like. Some of the compounds of formula (I) form pharmaceutically-acceptable addition salts with both organic and inorgamc bases, and these addition salts also form part of the present invention. Examples of suitable bases for salt formation are sodium and potassium carbonate, sodium and potassium hydroxide- ammonia, triethylamine, triethanolamine and the like. The compounds of formula (I) and the acid or base addition salts and the N-oxides thereof may be prepared by the processes outlined in the following Schemes 1 to 7. Specific but non-limitative examples of the processes outlined in Schemes 1 to 7 are given hereinafter in Examples A to H respectively Scheme 1 Y = OH, NH 2 X = OS0 2 Me, halogen. R = CONH, Scheme 2 X = OS0 2 Me. halogen Y* = Cl R = COOR 1 , CONR -R 2 Scheme 5 X = Cl R = CN or CSNH . Scheme 4 X = OS0 2 Me. halogen R = SO.NH- XI Scheme 5 Z = Cl, NH 2 , NR α R 2 R = CONH 2) CONR ! R 2 Scheme 6 X = OH, Cl Z = OEt, OH. Cl R = CONR-R 2 Scheme 7 x = OH, ci Z = OMe, OH, Cl R = CONR^R 2 The invention also relates to the preparation of cσπpounds of th general foππula I, the acid or base addition salts and the N-oxide thereof, by a process as outlined in any one of Schemes 1-7 above or b an obvious chemical equivalent thereof. The invention includes also those forms of the preparation processes according to which a ∞rrapound obtainable as intermediate at any stage of the process is used as starting material and the reiraining steps are carried out, or a -Starting material is used in the form of a derivative or salt or, especially, is formed under the reaction conditions. ϊhe following Table 1 sets out physicochemical data for 24 compounds within the general formula (I) , representative of it, and preparable by the processes of the invention. The following Examples A to H illustrate the preparation of compou representative of the general formula (I), and specifically the compounds liste Table 1. Example A : Preparation of 5-[N,N-bϊs(2-methanesulfonoxyethyl)aιnino] -2 -dinitrobenzamide (4) (la: X = OSO 2 e; R = CONH j ) and analogues by t method outlined in Scheme 1. Treatment of 5-chloro-2,4-dinitrobenzoic acid (II: Y = OH) [Goldstein, Stamm, R.Helv. ChirruActa., 1952,55, 1330-1332J with SOCl 2 followed by ammon gave5 chloro-2,4-dirLitrobenzamide (II: Y = NH 2 ), mp 210 °C (dec) [Goldstein, Stamm, R. Helv. Own. Ada., 1952, 35, 1330-1332 report mp 212 °C] . Η NM (CD 3 COCD 3 ) δ 8.68 (s, 1 H, H-3), 8.02 (s, 1 H, H-6), 7.50 (br, 2 H, CONH 2 ). Th amide was heated with diethanolamine in dioxan to give 5-[N,N-bis( -hydroxyethyl)]ami_Qθ-2,4-dinitrobeιιzamide (HI) (459e yield), mp (EtOA 176-178°C Η NMR (CD 3 SOCD 3 ) δ 8.47 (s, 1 H, ArH3), 8.07, 7.73 (2 br, 2 CONH 2 ), 7.35 (s, 1 H, ArH6), 4.80 (m, 2 H, OH), 3.83-3.20 (m, 8 H, CH 2 N, CH,0 Anal. (C u H 14 N 4 0 7 ) C,H,N. Methanesulfonyi chloride (5.17 ml, 0.067 mol) was added dropwise to a solution o the above amide diol (10.00 g, 0.032 mol) in dry pyridine (150 m ) at 0 °C. Afte a further 10 minutes at 0 °C, the solution was stirred at 20 °C for 30 minutes, an then volatiles were removed under reduced pressure at below 40 °C. The residu was partitioned between EtOAc and water, and the organic residue wa chromatographed on silica gel. Elution with EtOAc gave foreruns, followed by 5 -[N,N-bis(2-methanesulfonoxyethyl)amino]-2,4-dinitrobenzamide (4) (la: X = OS0 2 Me; R = CONH 2 ) (11.05 g, 74%), mp (sealed tube : ex EtOAc/petroleum ether) 60 °C. Η NMR(CD 3 COCD 3 ) δ 8.54 (s, 1 H, H-3), 7.65 (s, 1 H, H-6), 4.49 (t, J = 5.3 Hz, 4 H, CH 2 OS0 2 CH 3 ), 3.89 (t, J = 5.3 Hz, 4 H, CH 2 N), 3.09 (s, 6 H, S0 2 CH 3 ), 2.90 (br s, NH 2 ). 13 C NMR δ 167.01 (CONH 2 ), 148.56 (C-5), 140.45, 138.44 (C-2,4), 138.57 (C-l), 124.92, 123.06 (C-3.6), 67.65 ( H 2 OS0 2 CH 3 ), 52.02 (CH 2 N), 37.23 (S0 2 CH 3 ). Anal. (C 13 H 18 N 4 O n S 2 ) CH,N,S. The above dimesylate (4) (15.0 g, 31.9 mol) was dissolved in dry DMF (150 m )_ The solution was treated with solid LiCl (20 g), warmed to 100° C with vigorous stirring for lh, and then concentrated to dryness. The residue was partitioned between EtOAc and water, and the organic portion was washed well with 3N HC1, worked up to give a waxy solid, and chromatographed on silica. Elution with EtOAc/petroleum ether (1:1) gave foreruns, while EtOAc/petroleum ether (3:2) gave 5-[N,N-bis(2-chloroethyl)amino]-2,4-dir_itrobenzaπ_ide (1) (la, X = Cl, R = CONH 2 ) (7.51 g, 67%)which crystallised from EtOAc/petroleum ether as small orange needles, m.p. 109-111° C, Η NMR (CD 3 COCD 3 )δ 8.52 (s, 1H, H-3), 7.58 (s, 1H, H-6), 7.10 (br, 2H, CONH 2 ), 3.89-3.81 (m, 8H, NCH,CH 2 C1). 13 C NMR δ 167.10 (CONH 2 ), 148.31 (C-5), 140.08 (C-2), 138.84 (C-l), 138.20 (C-4), 124.94, 122.24 (C-3,6), 54.25 (CH 2 N), 42.04 (CH,C1) M /z 354,352,350 (M + , 2%), 303.301 (67,24%),222(25),185(26),171(29),100(74),65(25),63(56),56(57),36(100). FoundM÷ 354.0124; 352.0123; 350.0189. Anal. (C π H ι; Cl 2 N 4 0 5 ) C, H, N, I. A solution of the above dimesylate (4) (1.00 g, 2.12 mmol) in dry DMF (50 mL) was treated with solid NaBr (10.0 g), warmed to 120 C C with vigorous stirring for 15 min, and then concentrated to. dryness under reduced pressure. The residue was partitioned between EtOAc and water, and the organic portion was washed well with 3N HC1 and worked up. Chromatography of the residue on silica and elution with EtOAc/petroleum ether (1:1) gave 5-[N,N-bis(2-bromoethyl)amino]-2.4- -dinitrobenzamide (2) (la: X = Br, R = CONH,) (0.73 g, 78% yield), mp (EtOAc/petroleum ether) 126-128 °C. l H NMR (CD 3 COCD 3 ) δ 8.53 (s, 1 H, H-3), 7.58 (s, 1 H, H-6), 3.89 (t, / = 6.6 Hz, 4 H, CH,N), 3.73 (t, J = 6.6 Hz, 4 H τ CH 2 Br), 2.93 (br, 2 H, CONH 2 ). 13 C NMR δ 167.20 (CONH 2 ), 147.89 (C-5), 140.06, 138.26 (C-2,4), 138.75 (C-l), 124.95, 122.30 (C-3,6), 54.11 (CHUN), 29.S9 (CH 2 Br). Anal. (C π H 12 Br 2 N 4 0 5 ) C,H,N. A similar reaction using Nal gave 5-[N,N-bis(2-iodoethyl)amino]-2.4- -dinitrobenzamide (3) (la: X = I, R = CONH-,) in 92% yield, mp (EtOAc/petroleum ether) 170-172 °C. l H NMR (CD 3 COCD 3 ) δ 8.52 (s, 1 H. H-3), 7.55 (s, 1 H, H-6), 3.84 (t, = 7.2 Hz. 4 H, CH,N), 3.50 (t, / = 7.2 Hz. 4 H. CH 2 Br), 2.88 (br, 2 H, CONH,). 1 1 3 J C NMR δ 167.09 (CONH,), 147.17 (C 140.08, 138.33 (C-2,4), 138.74 (C-l), 124.94, 122.31 (C-3,6), 54.89 (CH 2 N), 1 (CH,I). Anal. (C 11 H 12 I 2 N 4 0 5 ) C,H,N,I. - Example B : Methyl 5-[N,N-bis(2-chloroethyl)amϊno]-2,4-dϊnitrobe_ιzoate (5) ( X = Cl) and analogues by the method outlined in Scheme 2. Methyl 5-chloro-2,4-dinitrobenzoate (IV) (1.24 g, 4.76 mmol) was treated w diethanolamine (1.00 g, 9.52 mmol) in dioxane (50 mL) at 50 °C for 3 h, and t residue after workup was chromatographed on silica gel. Elution with EtOAc " ga methyl 5-[N,N-bis(2-hydroxyethyl)amino]-2,4-dinitrobenzoate (V) (1.52 g, 97%), (EtOAc/petroleum ether) 104-106 °C. Η NMR (CDC1 3 ) δ 8.48 (s, 1 H, H-3), 7. (s, 1 H, H-6), 3.95 (s, 3 H, OCH 3 ), 3.81 (t, / = 5.0 Hz, 4 H, CH 2 OH), 3.60 (t, / 5.0 Hz, 4 H, CH,N), 2.20 (br, 2 H, OH). 13 C NMR δ 165.S9 (COOMe), 147. (C-5), 138.67, 136.21 (C-2,4), 133.30 (C-l), 124.22, 121.65 (C-3,6), 59.34 (CH 2 OH 54.59 (CH 2 N), 53.74 (OCH 3 ). Anal. C,H,N. A solution of this diol (1.50 g, 4.56 mmol) and Et 3 N (1.33 mL, 9.57 mmol) in dr CH,C1, (60 mL) was treated with MsCl (0.73 mL, 9.34 mmol) at 0 °C, and poure into saturated aqueous NaHC0 3 after 15 min. The crude dimesylate (VI X = OS0 2 Me) from workup was treated directly with excess LiCl in DMF a 120 °C for 30 min. The mixture was poured into brine, extracted with EtOAc worked up, and chromatographed on silica gel. Elution with EtOAc/petroleu ether gave methyl 5-[N,N-bis(2-chIoroethyI)amino]-2,4-dinitrobenzoate (5) (VI: = Cl; Y 1 = OMe) (1.34 g, 80%), mp (EtOAc/petroleum ether) 136.5-138 °C. Η NMR (CDCL j ) δ 8.54 (s, 1 H, H-3), 7.34 (s, 1 H, H-6), 3.97 (s, 3 H, OCH 3 ), 3.68 (br s, 8 H, NCH 2 CH 2 C1). 13 C NMR δ 165.30 (COOCH-), 147.22 (C5). 139.81, 137.82 (C-2,4), 133.39 (C-l), 124.33, 121.77 (C-3,6), 53.86 (OCH 3 ), 53.78 (CH,N), 40.69 (CH.CI). Anal. (C 12 H 13 C1 2 N 3 0 6 ) C,H,N,C1. Hydrolysis of (5) with 4N KOH in p-dioxane at 20 °C for 15 min gave a quantitativ yield of 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzoic acid (6) (lb: X = Cl R = COOH), mp (EtOAc/petroleum ether) 125-127 °C. Η NMR (CD 3 COCD 3 ) δ 8.56 (s, 1H, H-3), 7.72 (s, 1 H, H-6), 3.89-3.86 (br, 8 H, NCH 2 CH,C1), 3.78 (br, 5. COOH). 13 C NMR δ 166.02 (COOH), 148.52 (C-5), 140.25, 138.09 (C-2,4), 134.72 (C-l), 124.87, 122.58 (C-3,6), 54.12 (CH,N), 42.12 (CH,C1). Anal. (C 11 H 11 Cl,N 3 0 6 .y 2 EtOAc) C,H,N,C1. Treatment of (6) with excess SOCl 2 containing a drop of DMF under reflux for 20 min, followed by concentration to dryness and azeotroping with benzene, gave crude 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzoyl chloride (VI: X = Cl; Y 1 = Cl). This was dissolved in Me,CO (20 mL). cooled to 0°C, treated with excess 40% aqueous methvlamine. The product was purified by chromatography on silica gel, elution with EtOAc/petroleum ether (1:1) giving N-methyl [N,N-bis(2- -chloroethyl)amino]-2,4-dinitrobenzamide (S) (lb: X = Cl, R = CONHMe), mp (EtOAc/petroleum ether) 180.5 °C. Η NMR (CD 3 COCD 3 ) δ 8.53 (s, 1 H, ArH3), 7.75 (br, 1 H, CONH), 7.56 (s, 1 H, H-6), 3.88-3.80 (m, 8 H, NC/_,CH,C1), 2.90, 2.89 (2 s, 3 H, CONHMe). 13 C NMR δ 165.89 (CONH), 148.43 (C-5), 140.05, 138.26 (C-2,4), 138.94 (C-l), 125.05, 122.45 (C-3,6), 54.20 (CH,N), 42.10 (CH,C1), 26.69 (CONHCH 3 ). Anal. (C 12 H 14 C1 2 N 4 0 5 ) C.H,N,C1. Similar treatment of the acid chloride (VI: X = Cl; Y 1 = Cl) with ammonia or suitable amines gave : - 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (4) (lb: X = Cl, R = CONH 2 ) (67% yield), mp (EtOAc/petroleum ether) 109-111 °C. Η NMR (CD 3 COCD 3 ) δ 8.52 (s, 1 H, H-3), 7.58 (s, 1 H, H-6), 7.10 (br, 2 H, CONH 2 ), 3.89- 3.81 (m, 8 H, NCH,CH,C1). 13 C NMR δ 167.10 (CONH,), 148.31 (C-5), 140.08 (C-2), 138.84 (C-l), 138.20 (C-4), 124.94, 122.24 (C-3,6), 54.25 (CH,N), 42.04 (CH,C1). M /z 354, 352, 350 (M + ', 2%), 303, 301 (67, 24%), 222 (25), 185 (26), 171(29), 100(74), 65(25), 63(56), 56(57), 36(100). Found M τ' 354.0124; 352.0123; 350.0189. C n H 12 Cl,N 4 0 5 requires 354.0126; 352.0155; 350,018 Anal. ( C- uH^ (-UNA) C,H,N,C1. N,N-dimethyl [N,N-bis(2-cUoroethyl)amino]-2,4-dinitrobeι_za__ide (9) (l X = Cl, R = CONMe,) (65% yield), mp (CHCl 3 /petroleum ether) 130.5 ° Η NMR (CDC1 3 ) δ 8.61 (s, 1 H, H-3), 7.07 (s, 1 H, H-6), 3.62 (s, 8 NCH 2 CH 2 Cl), 3.07, 2.78 (2x 3 H, CON(CH 3 ) 2 )). 13 CNMR δ 166.12 148.33 (C-5), 138.88, 135.75 (C-2,4), 137.82 (C-l), 124.92, 120.00 (C-3,6), 53.5 (CH,N), 40.98 (CH,C1), 38.06, 34.85 (CON( __H 3 ) 2 )). Anal. (C 13 H 15 C1 2 N 4 0 5 ) C,H, - N-morpholino P -,N-bis(2-cUoroethyl)a_nino]-2,4-diiiitrobenzamide (10) (l X = Cl, R = CON(CH,CH,),0), mp (CHCl 3 /hexane) 140-142 °C. Η NM (CDC1 3 ) δ 8.66 (s, 1 H, ArH-3), 7.55 (s, 1 H, ArH-6), 3.68 (br, 10H, NCH EUC and CH 2 0), 3.60 & 3.34 (2 x m, 2 x 2 H, CH,NCO). 13 C NMR δ 165.22 (CON) 149.34 (C-5), 139.88, 137.00 (C-2,4), 138.26 (C-l), 125.59, 121.17 (C-3,6), 66.92 66.79 (CH 2 0), 54.22 (CH 2 N, 47.88, 42.80 (CONHCH 2 ), 42.26 (CH 2 C1) Anal. (C 15 H 18 C1,N 4 0 6 ) C,H,N. - N-[2-(dime±ylamino)ethyl] [N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (11) (lb: X = Cl, R = CONHCH,CH,NMe,), mp (hydrochloride salt from MeOH/EtOAc) 85-90 °C. Η NMR (D,0) δ 8.76 (s, 1H, ArH-3), 7.59 (s, 1H, ArH-6), 3.86-3.80 (m, 10H, NCH 2 CH 2 C1 and CH 2 NH + Me 2 ), 3.53 t, J = 6.1 Hz, 2H, CONHCH j ), 3.06 (s, 6H, NMe 2 ). 13 C NMR δ 170.77 (CONH), 166.12, 1451.39 (C-5), 140.53, 137.51 (C-2,4), 138.18 (C-l), 128.25, 123.39 (C-3,6), 58.46 (CH,N + HMe 2 ), 55.49 (CH,N), 45.67 (N + HMe,_), 43.97 (CH,C1), 38.54 (CONHOE ). Anal. (C^H^C NA-HCl) C,H,N. A solution of the free base of (11) (0.50 g, 1.18 mmol) in CH,C1, (15 mL) was treated with a solution of 2-benzenesulfonyl-3--phenyloxaziridine [Davis, F.; Stringer, CD. J. Org. Cherr , 1982, 47, 1775-1777]. (0.32 g, 1.24 mmol) in CH 2 C1 2 (3 mL) was added dropwise to. After lOmin, petroleum ether (10 mL) was added, the solution was cooled at - 30 °C overnight, and the resulting solid was dried under high vacuum to give N-(N 1 ,N 1 -dimethylaminoethyl) [N,N-bis(2-chloroethyl)a ino]- -2,4-dinitrobenzamide N α -oxide (14) ((lb: X = Cl, R = C0NHCH,CH,N(O)Me,) as a hygroscopic foam. Treatment with MeOH/HCl gave the hydrochloride salt (0.36g, 64%), mp (MeOH/'Pr 2 0) 165-169 C C. Η NMR (D,0/(CD 3 ),SO) δ 8.82 (s, 1 H, H-3), 7.46 (s, 1 H, H-6), 3.98, 3.93 (2xt, = 5.1 Hz, 2x2 H, CH 2 CH 2 ), 3.82 (t, / = 4.7 Hz, 4 H, CH,C1), 3.77 (t, / = 4.7 Hz, 4 H, CH,N), 3.57 (s, 6 H, N(CH 3 ) 2 ). 13 C NMR (D,0/(CD 3 ),SO) δ 170.71 (CONH), 151.63 (C-5), 140.68, 137.31 (C-3,6), 69.20 (CH 2 NOMe*χ), 59.03 (NOMe,), 55.40 (CH,N), 44.06 (CH,C1), 36.45 (CONH CH,). Anal. (C 15 H 21 C1,N 5 0 6 .HC1) C,H,N,C1. - N-[2-(morpholino)ethyl] [N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzaπ_idε (12) (lb: X = Cl, R = CONHCH,CH,N(CH : CH,) ; 0) as a gum. The hydrochloride salt crystallized from MeOH/ ! Pr 2 0, mp 119-120 °C. Η NMR (D,0) δ 8.81 (s, 1 H, H-3), 7.59 (s, 1 H, H-6), 4.07 (br, NH and HCl), 3.91 (t, J = 6.0 Hz, 4 H, N + CH,CH 2 0), 3.86-3.81 (m, 10 H, NCH,CH,C1 and CH,CH,N + ), 3.56 (t, J = 6.0 Hz, 6 H, N + CH,CH,0 and CONHCH,). 13 C NMR δ 171.17 (CONH), 151.69 (C-5), 140.61, 137.28 (C-2,4), 138.12 (C-l), 128.53, 123.48 (C-3,6), 66.30 (N + CH 2 C H 20), 58.07 (CH,N + ), 55.48 (CH,N), 54.65 (N + CH,CH,0), 44.09 (CH,C1), 36.84 (CONHCH,). Anal. (C 17 H 23 C1,N 5 0 5 .HC1. , _H,0) C,H,N,C1. - N-(2-hydroxyethyl) 5-[_^,N-bis(2-clιloroethyl)aιrLino]-2,4-dimtrobeι_zaιi-ide (16) (lb: X = Cl, R = CONHCH,CH,OH) (1.24 g, 85%), mp (EtOAc/petroleum ether) 157- 158 °C. X H NMR ((CD 3 ),SO) δ 8.73 t, / = 5.5 Hz, 1 H, CONH), 8.53 (s, 1 H, H-3), 7.45 (s, 1 H, H-6), 4.76 (t, / = 5.5 Hz, 1 H. OH), 3.82 (t, / = 6.0 Hz. 4 H, CH 2 C1), 3.68 (t, / = 6.0 Hz, 4 H, CH 2 N), 3.54 (dt, / = 6.1, 5.5 Hz, 2 H, CH,OH), 3.31 (dt, J = 6.1, 5.5 Hz, 2 H, CONHCH,). 13 C NMR ((CD 3 )SO) δ 164.44 (CONΗ), 147.11 (C-5), 137.87. 136.27 (C-2,4), 137.32 (C-l), 124.24, 121.01 (C-3,6), 59.32 (CΗ,OΗ), 52.44 (CH,N), 41.93 (CONHCH,), 41.65 (CH,C1). Anal. (C 13 H 16 C1 2 N,0 6 ) C,H,N,C1. -N-(2,3-dihydroxypropyl)5-[N,N-bis(2-chloroethyl)arr_ino]-2,4-dinitrobenzamide(17) (lb: X = Cl, R = CONHCH,CH(OH)CH,OH) (0.96 g, 59%) as a hygroscopic yellow foam. Η NMR ((CD 3 ) 2 SO) δ 8 JO (t, / = 5.7 Hz, 1 H, CONH), 8.53 ( H, H-3), 7.44 (s, 1 H, H-6), 4.83 (d, / = 4.9 Hz, 1 H, CHOH) 4.56 (t, J = 5.7 1 Η, CΗ,OH), 3.83 (t, / = 5.9 Hz, 4 H, CH 2 C1), 3.68 (t, / = 5.9 Hz, 4 H, CH 2 3.50-3.06 (m, 5 H, CONH CH 2 CHOHCH 2 OH). 13 C NMR ((CD 3 ),SO) δ 164. (CONH), 147.12 (C-5), 37.94, 136.40 (C-2,4), 137.40 (C-l), 124.29, 121.18 (C-3, 70.08 (CHOH), 63.77 (CH,OH), 52.55 (CH,N), 42.79 (CONHCH 2 ), 41.72. (CH, Anal. (C 14 H 1S CI 2 N 4 0 7 ) C,H,N,C1. crude N-(2-methoxycarbonylethyl) 5-[N,N-bis(2-chIoroethyl)amino]-2, dinitrobenzamide [from treatment with β-alanine methyl ester hydrochloride] (l X = Cl, R = CONHCH,CH,COOMe) as a yellow oil. This was immediate dissolved in THF (25 mL) and treated with aqueous KOH (10 mL of 2N) at 20 ° for 3h. The solution was then acidified with cone. HCl, extracted with EtOAc, a worked up as usual. Chromatography on silica, eluti g with EtOAc, gave N-( carboxyethyl) 5-[N,N-bis(2-cMoroethyl)amino]-2,4-dmitrobenzamide (15) (lb: X Cl, R = CONHCH 2 CH 2 COOH) (0.72 g, 68%), mp (EtOAc/petroleum ether) 15 153°C. l ΕL NMR ((CD 3 ),SO) δ 12.27 (br, 1 H, COOH) 8.79 (t, / = 5.6 Hz, 1 CONH), 8.54 (s, 1 H, H-3), 7.42 (s, 1 H, H-6) 3.83 (t, / = 6.0 Hz, 2 H, CH,C1), 3.6 (t, / = 6.0 Hz, 4 H, CH,N), 3.44 (dxt, / = 6.9, 2 H, 5.6 Hz, CONHCH^, 2.53 ( / = 6.9 Ηz, 2 Η, CH 2 , COOΗ). 13 C NMR ((CD 3 ),SO) δ 172.74 (COOΗ), 164.5 (CONΗ), 147.21 (C-5), 137.94, 136.13 (C-2,4), 137.29 (C-l), 124.37, 120.93 (C-3,6) 52.58 (CΗ,N), 41.72 (CH,C1), 35.24, 33.19 (CONHCH, H,). Anal. (C 14 H 16 C1,N 4 0 7 C,H,N,C1. (7) (Ic: X = Cl, R = CN) and 5-[N^-bis(2-chloroethyl)amino] -2,4- -dinitrothϊobenzamide (13) (Ic: X = Cl, R = CSNH j ) by the method outlined in Scheme 3. A solution of 5-[N,N-bis(2-chloroethyl)an_ino]-2,4-dinitrobenzamide (1) (VII) (0.20 g, 0.57 mmol) in SOC (5 mL) was heated under reflux in N, for 84 h. Excess SOC1, was removed under reduced pressure, and the residue was chromatographed on silica gel. Elution with EtOAc/petroleum ether (3:7) gave 5-[N,N-bis(2- chloroethyl)aιr_ino]-2,4-diιιitrobenzonitrile (7) (Ic: X = Cl, R = CN) (0.16 g, 87%), mp (CHC1 3 ) 127 °C. 1H NMR (CD 3 COCD 3 ) δ 8.79 (s, 1 H, H-3), 8.19 (s, 1 H, H-6), 3.93 (s, 8 H, NCH,CH 2 C1). 13 C NMR δ 148.55 (C-5), 140.98, 139.07 (C-2,4), 129.50, 126.10 (C-3,6), 115.05 (C-l), 112.84 (CN), 54.18 (CΗ 2 N), 42.20 (CH i). Anal. (C n H 10 Cl 2 N 4 O 4 ) C,H,N. A solution of (1) (VII) (0.50 g, 1.42 mmol) in p-dioxane (20 mL) was treated with P,S 5 (0.63 g, 2.84 mmol) and NaHC0 3 (0.24 g, 2.S4 mmol) was heated under reflux with stirring for 3 h. The residue after workup was chromatographed on silica gel, and elution with EtOAc/petroleum ether (1:4) gave 5-[N,N-bis(2- -chloroethyl)amino]-2,4-dinitrothiobenzamide (13) Ic: X = Cl, R = CSNH,) (0.48 g, 92%), mp (EtOAc/petroleum ether) 166-167 °C. Η NMR (CD 3 SOCD 3 ) δ 9.50, 9.34 (2xbr, 2 H, CSNH,), 8.50 (s, 1 H, H-3), 7.45 (s, 1 H H-6), 3.87, 3.82 (2xm. 8 H, NCH,CH,C1). 13 C NMR δ 199.46 (CS), 148.08 (C-5), 143.87 (C-l), 139.51. 136.99 (C-2,4), 125.23, 121.36 (C-3,6), 54.23 (CΗ,N), 42.02 (CH,Ci). Anal. (C n H 12 Cl,N 4 0 4 S) C,H,N,S. Example D : Preparation of 5-[N,N-bis(2-chIoroethyl)amino]-2, dϊnϊtrobenzenesulfonamide (18) (Id : X = Cl, R = SO-NH^) by the method outline in Scheme 4. " , A solution of 5-chloro-2,4-dinitrobenzenesulfonamide (VHI) [Herbert, R.B. an Hollman, R.G. Tetraliedron, 1965, 21, 663-675] (1.55 g, 5.5 mmol) an diethanolamine (1.16 g, 11 mmol) in dioxan (100 mL) was held at 60 °C for 1.5 h then adsorbed directly onto silica gel. Excess solvent was evaporated under reduce pressure, and the residue was chromatographed on silica gel, eluting wit EtOAc/MeOH (19:1), to gv/ 5-[N,N-bis(2-hydroxyethyl)ammo]-2,4 -dimtrobenzenesulfonamide (IX) (1.91 g, 99%), mp (EtOAc/petroleum ether) 138 139 °C. Η NMR ((CD 3 ),SO) δ 8.53 (s, 1 H, H-3), 7.97 (s, 1 H, H-6), 7,93 (br s, 2 H, SO,NH,), 4.88 (t, / = 5.1 Hz, 2 H, OH), 3.65 (dt, / = 5.2. 5.1 Hz, 4 H, CH.OH), 3.49 (t, J = 5.1 Hz, 4 H, NCH,). 13 C NMR δ 146.84 (C-5), 140.53 (C-l), 136.84, 134.39 (C-2,4), 125.70 (C-3), 54.27 (NCH,). Anal. (C 10 H 14 N 4 O 8 S) C,H,N. A stirred solution of the above diol (IX) (1.90 g, 5.43 mmol) and Et 3 N (1.90 mL, 14 mmol) in dry THF (60 mL) was treated dropwise at 0 °C with methanesulfonyl chloride (0.89 mL, 11 mmol). After a further 15 min, the solution was diluted with EtOAc, washed well with water, and worked up to give the crude dimesylate (Id: X = OSO,Me, R = SO,NH,). This was immediately dissolved in DMF (50 mL) containing LiCl (20 g), and the mixture stirred at 130 °C for 15 min before solvent was removed under reduced pressure. The residue was paπitioned between EtOAc and water, and the organic layer was worked up and chromatographed on silica gel. Elution with EtOAc gave 5-[N,N-bis(2-chloroethyl)aminσ]-2,4- -dinitrobenzenesulfαnamide (18) (Id : X = Cl, R = SO,NH,) (1.47 g, 64%), mp 153-154 °C (EtOAc/petroleum ether). l H NMR ((CD 3 ) 2 CO) δ 8.54. (S, 1H. H-3), 7.99 (br, 2H, S0 2 NH 2 ), 7.91 (S, 1H, R-6), 3.84 (t, / = 5.6 Hz, 4H, CH,C1), 3.67 (t, / = 5.6 Hz, 4H, CH 2 N). 13 C NMR ((CD 3 ) 2 CO) δ 146.13 (C-5), 140.64 (C-l), 138.86, 136.93 (C-2,4), 124.95, 121.77 (C-3,6), 52.47 (CH 2 N), 41.62 (CH 2 C1). Anal. (C 10 Hι 2 Cl 2 N 4 O 6 S) C,H,N,S. Example E : Preparation of 2-[N,N-bis(2-chloroethyl)amino]-3,5-dinitrobenzamid (19) (Ie: R = CONH^ and N-(N,N-dimethyIammoethyI) 2-[N,N-bis(2 -chloroethyl) amino] -3,5-dinitrobenzamide (20) (Ie: R = CONHCH,CH 2 NMe 2 ) b the method outlined in Scheme 5. Reaction of commercially-available 2-chloro-3,5-dinitrobenzoic acid (X) with SOC1 gave 2-chloro-3,5-dinitrobenzoyl chloride (XI: Z = Cl), which was quenched wit ammonia to give 2-chloro-3,5-dinitrobenzamide (XI: Z = NH,). A solution of thi amide (1.00 g, 4.07 mmol) and Et 3 N (1.42 g, 10 mmol) in /. -dioxane (30 mL) wa treated with N,N-bis(2-chloroethyl)amine hydrochloride (1.45 g, 8.14 mmol) at 50 ° for 18 h. The mixture was then poured into water and extracted with EtOAc to give an oil, which was chromatographed on silica gel. Elution with EtOAc/petroleum ether (1:1) gave 2-[N,N-bis(2-chloroethyl)amino]-3,5- dinitrobenzamide (19) (Ie: R = CONH 2 ) (1.15 g, 80%), mp (CHCl 3 /petroleum ether) 123-124 °C. J H NMR (CD 3 SOCD 3 ) δ 8.70 (d, / = 2.7 Hz, 1 H, H-4), 8.49 (d, / = 2.7 Hz, 1 H, H-6), 7.68, 7.35 (2xbr, 2H, CONH,), 3.80, 3.61 (2xt, / = 6.9 Hz, 8 H, NCH,CH,C1). 13 C NMR δ 167.94 (CONΗ,), 147.27 (C-2), 147.20. 142.84 (C-3,5), 137.70 (C-l), 128.40, 123.24 (C-4,6), 55.84 (CΗ,N), 42.2S (CH,C1). Anal. (C n H ι: ,Cl,N 4 0 5 ) C,H,N,C1. Similar treatment of the acid chloride (XI: Z = Cl) with N.N- -dimethylethylenediamine in water at 0 °C gave N-(N,N-dimethylaminoethyl 2-chloro-3,5-dinitrobenzamide (XI: Z = NHCH 2 CH,NMe,) as a viscous oil (5.6 g, 59%. The hydrochloride salt crystallised from MeOH/isopropyl ether, mp 220-222 °C. Η NMR (D,0) δ 8.99 (d, J = 2.3 Hz, 1 H, ArH4), 8.74 (d, J = 2.3 Hz. 1 H, ArH6), 3.89 (t, J = 6.3 Hz, 2 H, CH,N + Me,), 3.51 (t, J = 6.3 Hz, 2 H, CONHCH 2 ), 3.04 (s, 6 H, NMe,). 13 C NMR 169.32 (CONH), 150.SS (C-2), 148.70, 141.02 (C-3,5), 133.23 (C-l), 129.45, 125.27 (C-4,6), 58.64 (CH,N τ Me 2 ), 45.88 (N + Me 2 ), 37.94 (CONHCH,). Anal. (C n H 13 ClN 4 0 5 .HCl) C,H,N,C1. Reaction of this amide (XI: Z = NHCH 2 CH,NMe,) with bis(2-chloroethyl)amin hydrochloride and Et 3 N as above, followed by chromatography on silica gel an elution with EtOAc/MeOH (9:1) gave N-(N,N-dimethylaminoethyl) 2-[N,N-bis(2 chloroethyl)amino]-3,5-dinitrobenzamide (20) (Ie: R = CONHCH j CH j NMe,), m 5 (EtOAc/petroleum ether) 115-118 °C. Η NMR (CDC1 3 ) δ 8.63 (s, 2 H, ArH4) 7.41 (br, 1 H, CONH), 3.74 (t, / = 6.3 Hz, 4 H, CH 2 C1), 3.59 (t, / = 5.8 Hz, 2 H CONHCH,), 3.55 (t, / = 6.3 Hz, 4 H, NCH 2 CH,C1), 2.56 (t, / = 5.8 Hz, 2 H, CH,NMe 2 ), 2.27 (s, 6 H, NMe,). 13 C NMR 164.70 (CO), 145.93 (C-2), 144.86, 141.94 (C-3,5), 135.68 (C-l), 129.63 (C-4), 123.16 (C-6), 57.51 (CONHCH,), 54.82 10 (NCH,CH 2 C1), 45.12 (NMe,), 41.39 (NOLCH l), 37.67 (CONHCH,). Anal. C,H,N,C1. (The hydrochloride salt was too hygroscopic to handle). Example F : Preparation of 3-[N,N-bis(2-chloroethyI)amino]-2,6-dinitrobenzamide 15 (21) (If: R = CONH j ) and N-(N,N-dϊmethyIamiπoethyl) 3-[N,N-bis- (2-chloroethyl)amino]-2,6-dinitrobenzamide (22) (If: R = CONHCH H j NMe^ by the method outlined in Scheme 6. A solution of 3-chlorobenzoic acid (60 g, 0.38 mol) in c.H,S0, (600 ml) was treated 20 portionwise with fuming nitric acid (d 1.42) (150 ml), and the solution was warmed gradually with stirring to 140 °C in an open flask, and held at this temperature for 6h. After cooling overnight, ice-water was added cautiously, and after chilling to 5 °C for 3h the precipitated product was removed by filtration and washed well with water. Crystallisation from aqueous EtOH gave 5-chloro-2,4-dinitrobenzoic acid (II: - 25 Y = OH) (62 g, 66%) mp 180-183 °C [Goldstein, H.; Stamm, R. Helv. Chim. Acta., 1952, 35, 1330-1332 report mp 182-183 °Q_ The original filtrate and washings were combined and allowed to stand for several hours, depositing crystals of pure 3- -chloro-2,6-dinitrobenzoic acid (XH) (8.4 g, 9 %), mp (H,0) 162-163.5 °C. Η NMR (CD 3 S0CD 3 ) δ 10.50 (br, 1 H, COOH), 8.82 (d, J = 8.8 Hz, 1 H, H-5), 8.62 (d, 30 / = 8.8 Hz, 1 H, H-4). 13 C NMR δ 162.03 (COOH), 146.34 (C-2), 145.57 (C-6), 133.69 (C-5), 130.01 (C-l), 128.26 (C-4), 125.19 (C-3). Anal. ( HjClN ) C,H,N,CI. The 3-chloro-2,6-dinitrobenzoic acid (XII) was converted to the ethyl ester, which was reacted as above with diethanolamine to give crude ethyl 3-[N,N-bis- (2-hydroxyethyl)amino]-2,6-dinitrobenzoate (XHI: X = OH, Z =OEt). Reaction of this with MsCl/LiCl gave ethyl 3-[N,N-bis(2-chloroethyl)amino]-2,6- -dinitrobenzoate (XIII: X = Cl, Z = OEt), mp (EtOAc/petroleum ether) 84-87 °C. Η NMR (CD 3 COCD 3 ) δ 8.32 (d, J = 9.4 Hz, 1 H, H-5), 7.79 (d, = 9.4 Hz, 1 H, H-4) 4.39 (q, / = 7.1 Hz, 2 H, OCH,CH 3 ), 3.85-3.77 (m, 8 H, NCH,CH,C1), 1.34 (t, / = ' 7.1 Hz, 3 H, OCH 2 CH 3 ). 13 C NMR δ 163.24 (COOEt), 148.30 (C-3), 141.04, 138.80 (C-2,6), 128.84, 124.65 (C-4,5), 127.39 (C-l), 63.94 (OCH,CH 3 ), 54.39 (NCH 2 ), 42.00 (CH,C1) 13.89 (OCH,CH 3 ). Anal. (C 13 H 15 C1,N 3 0 6 ) C,H,N,CL An X-ray crystallographic detei ination was carried out on this compound to confirm the structure. Hydrolysis of this ester (XIII: X = Cl, Z = OEt) in aqueous KOH//7-dioxaπe (30 mL) at 20 °C for 18 h gave 3-[N,N-bis(2-chloroethyl)amino]-2,6-dinitrobenzoic acid (XIII: X = Cl, Z = OH), which was converted to the corresponding acid chloride (Xπi: X = Cl, Z = Cl). A solution of this in Me,CO at 0 °C was treated with excess aqueous ammonia, and the product was chromatographed on silica gel, EtOAc/petroleum ether (1:1) eluting 3-[N,N-bis(2-chloroethyl)amino]-2,6- dinitrobenzamide (21) (If: R = CONH 2 ) (0.93g), mp (EtOAc/petroleum ether) 140- 141.5°C. Η NMR (CD 3 COCD 3 ) δ 8.26 (d, = 9.3 Hz, 1 H, H-5), 7.71 (d, J = 9.3 Hz, 1 H, H-4), 7.63, 7.23 (2xbr, 2 H, CONH 2 ), 3.75 (s, 8 H, NCH 2 CH 2 C1). 13 C NMR δ 163.99 (CONΗ 2 ), 147.38 (C-3), 142.92, 139.76 (C-2,6), 130.48 (C-l), 128.22, 124.23 (C-4,5), 54.74 (NCH 2 ), 42.00 (CH,C1). Anal. (C n H 12 Cl 2 N 4 0 5 ) C,H,N,C1. Similar treatment of the acid chloride (XIII: X = Cl, Z = Cl) with N,N- -dimethylethylenediamine gave N-(N,N-dimethylaminoethyl) 3-[N,N-bis(2- -chloroethyl)amino]-2,6-dinitrobenzamide (22) (If: R = CONHCH,CH,NMe 2 ). Hie hydrochloride salt crystallised from MeOH/isopropyl ether, mp 180-182 °C. 1H NMR (D 2 0) δ 8.37 (d, J = 9.6 Hz, 1 H, ArH5), 7.63 (d, J = 9.6 Hz, 1 H, ArH4), 3.80-3.71 (m, 10 H, NCH 2 CH,C1 and CH,NMe,), 3.44 (t, J = 6.4 Hz, 2 H, CONHCH j ), 3.00 (s, 6 H, NMe,). 13 C NMR δ 168.75 (CONH), 150.91 (C-3 141.55, 138,72 (C-2,6), 131.61 (C-4), 130.45 (C-l), 125.85 (C-5), 58.31 (CH,N + Me, 55.74 (NCH^H l), 45.97 (N + Me 2 ), 44.16 (NCH j CH.CI), 37.91 (CONHCH, Anal. C,H,N,CL Example G : Preparation of 4-[N,N-bis(2-chloroe_hyl)aminb]-3,5-dinitrobenzaπιid (23) (Ig: X = Cl, R = CONH^ and N-(N,N-dimethyIaminoethyI) 4-[N,N-bis(2 chloroethyl) amino] -3,5-dinitrobenzamide (24) (Ig: X = Cl, R = CONHCH^H^TVfe j ) by the method outlined in Scheme 7. A solution of methyl 4-chloro-3,5-dinitrobenzoate (XIV) (10.0 g, 0.036 mol) and diethanolamine (7.66 g, 0.073 mol) in /. -dioxane (60 mL) was stirred at 50 °C for 5h. Volatiles were removed under reduced pressure, and the residue was adsorbed directly onto silica gel and chromatographed. Elution with EtOAc/petroleum ether (1:10) gave foreruns, and subsequent elution with EtOAc/petroleum ether (3:2) gave methyl 4-[N,N-bis(2-hydroxyethyl)amino]-3,5-dir_itrobenzoate (XV: X = OH, Z = OMe) (9.61 g, 80%), mp (CHCl 3 /petroleum ether) 101-103° C. Η NMR (CDCI 3 ) δ 8.52 (s, 2 H, ArH-2,6), 3.97 (s, 3 H, COOCH 3 ), 3.76 (dxt, / = 6.5, 4.7 Hz, 4 H, CH,OH), 3.26 (t, J = 4.7 Hz, 4 H, NCH,), 2.76 (t, / = 6.5 Hz, 2 H, OH). D C NMR δ 163.14 (COOCH 3 ), 145.50 (C-4), 142.54 (C-3,5), 131.19 (C-2,6), 123.59 (C-l), 59.16 (CH,OH)l, 54.58 (CH,N), 53.14 (OCH 3 ). Anal. (C^H^ A) C,H,N. MsCl (3.93 mL, 0.049 mol) was added dropwise at 0 °C to a solution of the above diol (XV: X = OH, Z = OMe) (7.30 g, 0.022 mol) and Et 3 N (7.72 mL, 0.055 mol) in CH 2 C1, (100 mL). After 15 min, the solution was washed several times with water and worked up to give the crude dimesylate, which was treated with LiCl in DMF at 120 °C for 5 min. Workup and chromatography on silica gel (elution with EtOAc/petroleum ether (1:4)) gave methyl 4-[N,N-bis(2-chloroethyl)_mino]-3,5- dinitrobenzoate (XV: X = Cl, Z = OMe) (6.21 g, 77%), mp (CHCl 3 /petroleum ether) 93-95 °C_ NMR (CDC1 3 ) δ 8.54 (s, 2 H, H-2,6), 3.99 (s, 3 H, COOCH 3 ) 5 3.63 (t. / = 6.9 Hz, 4 H, CH,C1), 3.43 (t, J = 6.9 Hz, 4 H, CH,N). 13 C NMR δ 162.90 (COOCH 3 ), 147.83 (C-4), 140.53 (C-3,5), 129.85 (C-2,6), 126.70 (C-l), 55.47 (CH 2 N), 53.23 (OCH 3 ), 41.18 (CH,C1). Anal. (C 12 H 13 C1 2 N 3 0 6 ) C,H,N,C1. Hydrolysis of this ester (XV: X = Cl, Z = OMe) with aqueous KOH/p -dioxane at 20 °C for 3 h gave a quantitative yield of 4-[N,N-bis(2-chloroethyl)amino]-3,5- -dinitrobenzoic acid (XV: X = Cl, Z = OH), mp EtOAc/petroleum ether) 164-166 °C. X NMR ((CD 3 ) 2 SO) δ 8.61 (s, 2 H, H -2,6), 3.78 (t, / = 6.9 Hz, 4 H, CH 2 C1), 3.52 (t, / = 6.9 Hz, 4 H, CH,N) (COOH not visible). 13 C NMR δ 164.12 (COOH), 149.12 (C-4), 141.40 (C-3,5), 130.70 (C-2,6), 128.19 (C-l), 56.14 (CH,N), 42.46 (CH,C1). Anal ( -πHn __UN 3 0 6 ) C,H,N,C1. The above acid (XV: X = Cl, Z = OH) was converted to the crude acid chloride (XV: X = Cl, Z = Cl), which was dissolved in dry Et,0 (100 mL), cooled to 5 °C, and treated dropwise with aqueous NH 3 (20 mL of a 3N solution, 60 mmol). After 10 min the solution was worked up, and the residue was chromatographed on silica. Elution with EtOAc/petroleum ether (3:7) gave foreruns, while EtOAc/petroleum ether (7:3) gave 4-[N,N-bis(2-chloroethyl)amino]-3,5-dinitrobenzaπ_ide (23) (Ig: X = Cl, R = CONH 2 ) (80% overall yield), mp (EtOAc/petroleum ether) 113-116 °C. Η NMR (CD 3 COCD 3 ) δ 8.61 (s, 2 H, H-2,6), 7.98, 7.24 (2xbr, 2 H, CONH 2 ), 3.75 (t, / = 7.0 Hz, 4 H, CH,C1), 3.50 (t, 4 H. CH,N). 13 C NMR δ 164.91 (CONH,), 149.32 (C-4), 140.07 (C-3,5), 132.18 (C-l), 128.91 (C-2,6), 56.37 (CH,N), 42.55 (CH,C1). Anal. (C n H 12 Cl 2 N 4 0 5 ) C,H,N,C1. Similar treatment of the crude acid chloride (XV: X = Cl, Z = Cl) with N,N- dimethylethylenediamine gave N-(N,N-dimethylaminoethyl) 4-[N,N-bis(2- -chloroethyl)amino] -3,5-dinitrobenzamid e (24) (Ig: X = Cl, R = CONHCH 2 CH,NMe 2 ). The hydrochloride salt crystallised from MeOH/isopropyl ether, mp 145-148 °C. Η NMR (CD 3 SOCD 3 ) δ 10.65 (br, 1 H, HCl), 9.49 (t, J = 5.4 Hz, 1 H, CONH), 8.75 (s, 2 H, ArH2,6), 3.71 (t, / = 6.9 Hz, 4 H, NCH 2 CH,C1), 3.44-3.28 (m, 8 H, NCH 2 CH,C1 and CH,CH,N + Me,), 2.83 (s, 6 H, N + Me 2 ). 13 C NMR δ 162.63 (CO), 147.43 (C-3,5), 138.75 (C-4), 130.22 (C-l), 128.31 (C-2,6), 55.44 (CH 2 N + Me 2 ), 54.75 (NCH 2 CH,C1), 42.22 (N τ Me,), 42.1 (NCH 2 CH 2 C1), 34.75 (CONHCH,). Anal. (Q J H 21 CI 2 N 5 O s _Hα) C,H,N,C1. The following Table 2 gives biological data for selected examples of the compounds listed in Table 1. The abbreviations used in Table 2 are : No. The number given the corresponding compound in Table 1. IC j o Growth inhibition studies were performed as described in detail elsewhere (W.R. Wilson, R.F. Anderson and W.A Denny. J. Med. Chem., 1989, 32, 23; G.J. Finlay, B.C. Baguley and W.R. Wilson Anal. Biochem., 1984, 139, 172.), using 200 viable AA8 or 300 viable UV4 cells plus 5000 lethally-irradiated AA.8 feeder cells per well in 96-well tissue culture dishes. AA8 and UV4 cells were maintained in logarithmic-phase growth in 25 cm 3 tissue culture flasks with subculture twice weekly by trypsinization. The growth medium was antibiotic-free α -MEM with 10% v/v heat-inactivated (56°C, 40 min) fetal calf serum. Doubling times were approximately 14 h for AAS and 15h for UV4 cells. Cultures were tested for mycoplasma contamination frequently, using a cytochemical staining method (IJR. Chen, Exp. Cell Res., 1977, 104, 255. Drugs were added 24 hours after initiating cultures in 96-well dishes, and cells were incubated under aerobic or hypoxic conditions for 18 hours before washing with fresh medium. The IC S0 was determined as the drug concentration needed to reduce the cell mass (protein content, measured after 72-78h by staining with methylene blue and measuring absorbance in a microplate photometer) to 50% of the mean value for 8 control cultures on the same 96-well plate. HF^ Ratio of IC 50 values for a compound after exposure as detailed above against AA8 and UV4 cell lines (HF = IC 50 (AA8)/IC 50 (UV4)). CT j o The product (in μM-hr) of the drug concentration times the exposure time to reduce cell survival to 10% of control values in the clonoeenic assav. air/N 2 Ratio of CT 10 values after exposure of UV4 cells as detailed abo under either aerobic (air) or hypoxic (N 2 ) conditions (= aerob CT 10 /hypoxic CT 10 ). HF R - 2 Ratio of IC 50 values for a compound after exposure as detailed abov against the UV4 cell line in the presence and absence of the E. co nitroreductase enzvme NR-2. Table 2: Biological activity of selected compounds of Table I The following Examples 1 to 3 show that compound 1 (referred to therein SN23862) is reductively activated by bacterial nitroreductase enzymes. The alkylating agent 5-(a_dridin-l-yl)-2,4-dinitrobenzamide (hereinafter designate CB 1954) has been known, almost for 20 years, as an interesting experimenta compound of unique selectivity. Although CB 1954 is structurally quite closel related to numerous other known alkylating agents which have a relatively broa range of activity, CB 1954 exhibits considerable activity against the Walker tumou cells in vivo or jn vitro but was thought to be virtually inactive against othe tumours. It was recently discovered that the selectivity of CB 1954 arose from the fact that it was not an anti-tumour agent per.se but was a prodrug for an anti-tumour agent generated from CB 1954 by a nitroreductase enzyme found in the Walker cell. This nitroreductase from the Walker cell was subsequently shown to be an enzyme known from other sources which was an NAD(P)H dehydrogenase (quinone) classified as EC.1.6.99.2, see Robertson et al, J. Biol Chem. 261, 15794-15799 (1986). In the course of the previous investigations with CB 1954, it was found that the Walker cell enzyme EC.1.6.99.2 had the ability to reduce the 4-nitro group of CB 1954 to the corresponding hydroxylamine and that it was the resulting 5-(aziridin-l-yI)-2-nitro-4-hydroxylamino-benzamide that was the active anti-tumour agent. PCT Application PCT/GB92/01947 of Cancer Research Campaign Technology Limited describes new nitroreductases, obtainable from bacterial sources, for example from Escheήchia coli and from Bacillus amyloliquifaciens, that are of interest in that not only are they capable of converting CB 1954 into an active anti-tumour agent, but also, unlike EC.1.6.99.2. capable of converting CB 1954 analogues which are also prodrugs into active anti-tumour agents. As is more particularly described in PCT Application PCT/GB92/01947 the _se prodrugs represents a clinically very valuable concept in cancer therapy sinc particularly where the prodrug is to be converted to an anti-tumour agent ι_ndeτ th influence of an enzyme that is linkable to a monoclonal antibody that will bind t a tumour associated antigen, the combination of such a prodrug with such a enzyme monoclonal/antibody conjugate represents a very powerful clinical agen Example 1. ' The effect of SN23862 on the survival of V79 cells in the presence of the E. coli nitroreductase. All treatments were for 2 hours at 37°C and the cells were then plated out for their resulting colony forming ability. The nitroreductase concentration was 2μg/m 1 and NADH was used as a co-factor. The initial cell density was 2xl0 5 /mL. TREATMENT %SURVIVAL % DRUG REDUCTION CONTROL 100 + 500/ M NADH 100 + 50/<M SN23862 100 < 1.0 + NADH + SN23862 100 < 1.0 + Nitroreductase 94 + NR + 50/tM SN23862 99 < 1.Q + NR + SN23862 + 500/.M NADH 0.007 95 Example 2. The effect of SN23862 on the survival of V79 cells in the presence of the B. amyloliquifaάens nitroreductase. All treatments were for 2 hours at 37°C and the cells were then plated out for their resulting colony forming ability. The nitroreductase concentration was 7.45/.g/ml and NADH was used as a co-factor. The initial cell density was 2x lO s /mL. TREATMENT %SURVIVAL DRUG REDUCTION CONTROL 100 . 500/- M NADH 120 + 50/.M SN23862 110 < 1.0 + NADH + SN23862 1 15 < 1.0 + Nitroreductase (BA) 96.8 + BA .- 50/.M SN23862 110 < 1.0 + BA + SN23862 + 500/.M NADU 6.0 > 95 Example 3. The effect of SN23862 on the survival of V79 or Mawi cells in the presence of the E. coli nitroreductase: A5B7 F(ab) 2 conjugate. All treatments were for 2 hours at 37°C and the cells were then plated out for their resulting colony forming ability. The conjugate concentration was 81/tg/ml (equivalent to 5.2/<g/ml nitroreductase) and NADH was used as a co-factor. The initial cell density was 2xl0 5 /mL. No attempt was made to remove unbound conjugate. TREATMENT % DRUG REDUCTION CONTROL + 500 ι M NADH + 50/ M SN23862 -. NADU + SN23862 + Conjugate (CG) + CG + 50/_M SN23862 + CG + SN23862 + 500/* M NADU 0.014 0.003 The accompanying Figure 1 shows radiosensitising propeπies of compound 1. The radiosensitising efficiency of compound 1 has been assessed in stirred suspensions of AA8 cells under hypoxic conditions using gamma irradiation 5- (cobalt 60) at a dose rate of 2.2 Gy/min at 37 °C. Drug and cells were mixed under hypoxic conditions 30 min before irradiation. Radiosensitisation of hypoxic cells was observed (Figure 1). The concentration for an enhancement ratio (ratio of radiation doses for 10% survival with and without drug) of 1.6 (C 1 6 ) was approximately lOOμM, and this extent of sensitisation was observed in the absence 10 of drug toxiciry (Figure 1). Compound 1 is therefore a hypoxic cell radiosensitiser as well as a hypoxia-selective cytotoxin. It is clear from the data of Table 2, Examples 1 to 3 and Figure 1 that the examples of the nitroaniline derivatives of general formula (I) listed in Table 2 15 include compounds which are active as cytotoxic agents, and which have the additional capability of being reductively activated by both mammalian tumour cells and bacterial nitroreductase enzymes, and are therefore suitable as prodrugs. The compounds are selectively toxic to hypoxic rumour cells, and therefore useful as anticancer drugs. The compounds are capable of sensitising hypoxic cells jn vitro 20. to ionising radiation, and thus are suitable as hypoxic cell radiosensitisers. The present invention therefore also provides pharmaceutical compositions, having antitumour and radiosensitising activity, and comprising at least one compound represented by the general formula (I), and one or more pharmaceutically- 5 acceptable carriers or diluents. The present invention further provides the use of at least one compound represented by the general formula (I) for the manufacture of pharmaceutical compositions for the treatment of tumours, in particular cancers. When the compounds of formula (I) are used as reductively-activated prodrugs fo cytotoxins, they can be used in association with nitroreductase enzymes, including the nitroreductases described in Application PCT/GB92/01947 , and so provide a system of cancer chemotherapy where the extent of exposure of the patient to the cytotoxic agent is limited, so far as possible, to those regions where there is the inter-reaction between the prodrug and the nitroreductase. The most or one of the most convenient ways of utilising such a system of cancer chemotherapy is to conjugate the nitroreductase to a targetting agent such as a monoclonal antibody that will bind with a tumour-associated antigen. The term "monoclonal antibody" will be understood by those of skill in the art not simply to refer to antibodies produced by traditional hybridoma techniques, but also to cover antibodies and variants thereof produced by recombinant means. These include, for example, humanised antibodies such as those with a constant region from a human antibody grafted onto a non-human antibody variable region (see for example EP-A-0 120 694), chimeric antibodies such as those with non-human complementarity determining regions (CDRs) grafted into a human variable region framework (see for example EP-A-0 239 400) and single chain antibodies. Fragments of such monoclonal antibodies which retain their tarset binding activity are also included by the general term "monoclonal antibody". This includes Fab' and F(ab'), fragments. The selection of monoclonal antibody will clearly be influenced by the nature of the target tumour but for the purposes of illustration, reference may be made to the anti-CEA antibody A 5 B-,. With this system, it is possible in a course of cancer chemotherapy to administer to the patient requiring the treatment the nitroaniline compound which is the prodrug for a cytotoxic compound and the enzyme/targeting agent conjugate. The prodrug and the conjugate can be administered simultaneously but it is often found preferable, in clinical practice, to administer the enzyme/agent conjugate before the prodrug; e.g. up to 72 hours before, in order to give the enzyme/agent conjugate an opportunity to localise in the region of the tumour target. By operating in this way, when the prodrug is administered, conversion of the prodrug to the cytotoxic agent tends to be confined to the regions where the enzyme/agent conjugate is localised, i.e. the region of the target tumour and damage to healthy cells caused by the premature release of the cytotoxic agent is minimised. The degree of localisation of the enzyme/agent conjugate (in terms of the ratio of localized to freely circulating active conjugate) can be further enhanced using the clearance and/or inactivation systems described in WO89/10140. This involves, usually following administration of the conjugate and before administration of the prodrug, the administration of a component (a "second component") which is able to bind to the such part of the conjugate so as to inactivate the enzyme and/or accelerate the clearance of the conjugate from the blood. Such a component may include an antibody to the nitroreductase which is capable of inactivating the enzyme. The second component may be linked to a macromolecule such as dextran. a iiposome, albumin, macroglobulin or a blood group O erythroc, ie so that the second component is restrained from leaving the vascular compartment. In addition or as an alternative, the second component may include a sufficient number of covalentlv bound galactose residues, or residues of other sugars such as lactose or mannose, so that it can bind the conjugate in plasma but be removed together with the conjugate from plasma by receptors for galactose or other sugars in the liver. The second component should be administered and designed for use such that it will not, to any appreciable extent, enter the extravascular space of the tumour where it could inactivate localised conjugate prior to and during administration of the prodrug. The exact dosage regime will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact nature of the prodrug and the cytotoxic agent to be released from the prodrug but some general guidance can be given. Chemotherapy of this type will normall involve parenteral administration of both the prodrug and the enzyme/age conjugate and administration by the intravenous route is frequently found to be th ■ most practical. - The active compounds may be orally administered, for example, with an iner diluent or with an assimilable edible carrier, or they may be enclosed in hard or sof shell gelatin capsules, or they may be compressed into tablets, or they may b incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form o ingestibie tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 and about 60% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 5 and about 200 mg of active compound. The tablets, troches, pills, capsules and the Uke may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course any material used in preparing any dosage unit form should be pharmaceutically pur and substantially non-toxic in the amounts employed. In addition, the activ compounds may be incorporated into sustained-release preparations an formulations. The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compound as a free base or pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixmres thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against be contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixmres thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens. clorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compound i the required amount in the appropriate solvent with various of the other ingredient enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is weE known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitabale as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired. The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically-acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from about 0.1 to about 400 mg, with from about 1 to about 30 mg being preferred. Expressed in proportions, the active compound is generally present in from about 0.1 to 400 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Claims: Tim WHAT WE CLAIM IS : 1. A compound represented by the general formula (I), where the nitro group is substituted at any one of the available benzene positions 2-6; where R and A separately represent the groups NO-,, CN, COOR 1 , CONR -R 2 , CSNR- R 2 or SO^M^R 2 and A is substituted at any one of the avaEable benzene positions 2-6; where B represents N(CH 2 CH 2 halogen) 2 or N(CH,CH 2 0S0 2 R 3 ) 2 substituted at any one of the avaEable benzene positions; and where R 1 , R 2 and R J separately represent H, or lower alkyl optionaEy substituted with hydroxyl, ether, carboxv or amino functions, including cyclic structures, or R 1 and R 2 together with the nitrogen form a hetero cycUc structure. 2. A compound of formula (I) according to claim 1, where N0 2 is in the 2-positioπ, R represents CONH 2 , A represents 4-NO, and B represents 5-N(CH 2 CH,Cl) 2 . 3. A compound of formula (I) according to claim 1, where N0 2 is in the 2-posItion, R represents CONH 2 , A represents 4-N0 2 and B represents 5-N(CH 2 CH,Br) 2 . 4. A compound of formula (I) according to claim 1, where N0 2 is in the 2-posϊtion, R represents CONH 2 , A represents 4-N0 2 and B represents 5-N(CH 2 CH,I) 2 . 5. A compound of formula (I) according to claim 1, where N0 2 is in th 2-position, R represents CONHCH 2 CH 2 NMe 2 , A represents 4-N0 2 and B represent 5-N(CH 2 CH 2 Cl) 2 . 6. A compound of formula (I) according to claim 1, where NO, is in the 2-position, R represents CONHCH 2 CH,N(0)Me 2 , A represents 4-N0 2 and B represents 5-N(CH 2 CH 2 Cl) 2 . 7. A compound of formula (I) according to claim 1, where N0 2 is in the 2-position, R represents CONH 2 , A represents 6-NO, and B represents 5-N(CH 2 CH 2 Cl) 2 . 8. A compound of formula (I) according to claim 1, where N0 2 is in the 2-position,R represents CONHCH 2 CH,NMe 2 , A represents 6-N0 2 and B represents 5-N(CH 2 CH 2 Cl) 2 . 9. A compound of formula (I) according to claim 1, where NO-, is in the 3-position,R represents CONH 2 , A represents 5-N0 2 and B represents 2-N(CH 2 CH 2 Cl) 2 . 10. A compound of formula (I) according to claim 1, where NO, is in the 3-position,R represents CONHCH 2 CH 2 NMe 2 , A represents 5-N0 2 and B represents 2-N(CH 2 CH 2 Cl) 2 . 11. A process for the preparation of a compound represented by the general formula (I) as defined in claim 1, which process is outlined at any one of Schemes 1-7 herein. 12. A compound represented by the general formula (I) as defined in claim 1, whenever prepared by a process outlined in claim 11, or by an obvious chemical equivalent thereof. 13. A pharmaceutical composition with comprises at least one compoun represented by the general formula (I) as defined in claim 1, and one or mor pharmaceutically-acceptable carriers or diluents. 14. The use of at least one compound represented by the general formula (I) as defined in claim 1 for the manufacmre of pharmaceutical compositions for the treatment of humans.

Inventors: DENNY WILLIAM ALEXANDER, PALMER BRIAN DESMOND, WILSON WILLIAM ROBERT, DENNY, WILLIAM, ALEXANDER, PALMER, BRIAN, DESMOND, WILSON, WILLIAM, ROBERT
Applicants: CANCER RES CAMPAIGN TECH, CANCER RESEARCH CAMPAIGN TECHNOLOGY LIMITED
Application year: 1992
Main classification: A61K31
IPC codes: A61K31, A61P35, A61P43, C07C211, C07C229, C07C237, C07C255, C07C291, C07C309, C07C311, C07C327, C07D295