AU4421100A - N-protected amines and their use as prodrugs - Google Patents

N-protected amines and their use as prodrugs Download PDF

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AU4421100A
AU4421100A AU44211/00A AU4421100A AU4421100A AU 4421100 A AU4421100 A AU 4421100A AU 44211/00 A AU44211/00 A AU 44211/00A AU 4421100 A AU4421100 A AU 4421100A AU 4421100 A AU4421100 A AU 4421100A
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William Alexander Denny
Michael Patrick Hay
William Robert Wilson
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Auckland Uniservices Ltd
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    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/42—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms with nitro or nitroso radicals directly attached to ring carbon atoms
    • C07D333/44—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms with nitro or nitroso radicals directly attached to ring carbon atoms attached in position 5
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    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C271/26—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring
    • C07C271/28—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring to a carbon atom of a non-condensed six-membered aromatic ring
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    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D277/62—Benzothiazoles
    • C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
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    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
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    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Description

WO 00/64864 PCT/GB00/01612 -1 N-PROTECTED AMINES AND THEIR USE AS PRODRUGS The present invention relates to methods and compounds for providing amines with N-protecting groups. It further relates to the protected amines themselves and their use as 5 prodrugs. The amines are protected as nitroaromatic carbamates (where "aromatic" includes "heteroaromatic"). They include nitro ,roun- , .-.-.- . to reduction, leading to loss of the protecting .te is desirably biologically active, the ? rotection. Thus it 10 may be an amine-based -io' n STP ae, an aniline mustard or an enediyne. Thus suitable prc arr agents, and/or may be useful as prodrugs for ADEPT) or gene directed enzyme prodrug reductase enzymes. 15 BACKGROUND TO TH The use of prodrul - converted into more active compounds in vivo) particularly in cancer therapy. For example a prodrug may be converted into an anti-tumour agent under the influence of an enzyme that is linkable to a monoclonal antibody that will bind to a tumour 20 associated antigen. The combination of such a prodrug with such an enzyme monoclonal/antibody conjugate represents a very powerful clinical agent. This approach to cancer therapy, often referred to as "antibody directed enzyme/prodrug therapy" (ADEPT), is disclosed in WO88/07378. A further therapeutic approach termed "virus-directed enzyme prodrug therapy" 25 (VDEPT) has been proposed as a method for treating tumour cells in patients using prodrugs. Tumour cells are targeted with a viral vector carrying a gene encoding an enzyme capable of activating a prodrug. The gene may be transcriptionally regulated by tissue specific promoter or enhancer sequences. The viral vector enters tumour cells and expresses the enzyme, in order that a prodrug is converted to an active drug within the tumour cells (Huber et al., Proc. 30 Natl. Acad. Sci. USA (1991) 88, 8039). Alternatively, non-viral methods for the delivery of genes have been used. Such methods include calcium phosphate co-precipitation, microinjection, liposomes, direct DNA uptake, and receptor-mediated DNA transfer. These WO 00/64864 PCT/GB00/01612 -2 are reviewed in Morgan & French, Annu. Rev. Biochem., 1993, 62;191. The term "GDEPT" (gene-directed enzyme prodrug therapy) is used to include both viral and non-viral delivery systems. 4-Nitrobenzyl carbamates (A) undergo multi-electron reduction to produce amines. 5 The mechanism probably involves the formation of electron-donating 4-hydroxylamine (B;Q=OH) or 4-amine (B;Q=H) species, which then fragment to generate a quinoneimine methide (C) and an amine (D) [P.L. Carl, P.K. Charkravarty, and J.A. Katzenellenbogen, J. Med. Chem., 1981, 24, 479].
NO
2 NHQ NQ + C02 + RNH 2 D S CH 2 O NHR O NHR C A B Despite a low reduction potential (ca. -490 mV) [P. Wardman, Environ. Health 10 Perspect., 1985, 64, 309] the 4-nitrobenzyl carbamate moiety undergoes facile reduction by the E. coli NR enzyme, and has been used as a prodrug "trigger" to deactivate highly cytotoxic amine "effectors" [M.P. Hay and W.A. Denny, Drugs Future,1996, 21, 917]. The E. coli enzyme has been shown to activate 4-nitrobenzyl carbamate derivatives of a limited number of amine-based cytotoxins, including actinomycin D and anthracyclines [A.B. 15 Mauger, P.J. Burke, H.H. Somani, F. Friedlos and R.J. Knox, J. Med. Chem., 1994, 37, 3452], aniline mustards [A.B. Mauger, P.J. Burke, H.H. Somani, F. Friedlos and R.J. Knox, J. Med. Chem., 1994, 37, 3452; M. Lee, J.E. Simpson Jnr, S. Woo, C. Kaenzig, G.M. Anlezark, E. Eno-Amooquaye, and P.J. Burke, Bioorg. Med. Chem. Lett., 1997, 7, 1065] and enediynes [M.P. Hay, W.R. Wilson, and W.A. Denny, Bioorg. Med. Chem. Lett., 1995, 5, 20 2829]. All of these studies have used the otherwise unsubstituted 4-nitrobenzyl carbamate moiety. To be fully effective, such prodrugs must be activated efficiently by the enzyme, and the resulting reduced species must fragment rapidly to release the cytotoxic amine effector. Kinetic structure-activity relationships (SAR) have been extensively studied for the one 25 electron reduction of nitrobenzyl halides [D.L. Kirkpatrick, K.E. Johnson, and A.C. Sartorelli, J. Med Chem., 1986, 29, 2048] and quaternary salts [M. Tercel, W.R. Wilson, WO 00/64864 PCT/GB00/01612 -3 R.F. Anderson, and W.A. Denny, J Med. Chem., 1996, 39, 1084 and refs therein], but not for 4-nitrobenzyl carbamates. We have found that suitable substituents on the 4-nitrobenzyl ring and/or alpha-carbon result in more rapid fragmentation of the 4-hydroxylamine intermediates, and can also serve as sites for attaching solubilising functionalities. 5 For a series of substituted 4-nitrobenzyl carbamate model compounds (X), fragmentation rates of the corresponding 4-hydroxylamines (Y) to release amines (Z) correlated with electron-donating properties (,p) of the substituent, as shown in Table 1. The maximum half-lives (Mti 2 ) of the hydroxylamine derivatives were measured by HPLC, following 4-fold stoichiometry radiolytic reduction of the corresponding substituted 4 10 nitrobenzyl carbamates. Assuming first order conditions, the half-life (tl/ 2 ) of species R is calculated from the equation ln([R]J/[R],) = t(In2/tl/2). The ratio [R]/[R], was taken as the fraction of nitrobenzyl carbamate which had not released the amine (Z) after 4-fold reduction. This method yields a maximum value for the half-life of fragmentation. E NHOH D D + H 2
H
2 N W A _ N A Z X (E = NO2) Y (E = NH-IOH) Table 1 Half-lives for fragmentation (Mtl/ 2 ) and percent of amine released (to) for 15 substituted 4-hydroxylaminobenzyl carbamates (derived from the corresponding 4 nitrobenzyl carbamates by radiolytic reduction).
WO 00/64864 PCT/GB00/01612 -4 D A nP Mt, (min) t o (%) 2-NO 2 H 0.78 88 18 5 3-NO, H 0.71 65 22 3-CO2Me H 0.37 20 44 3-OMe H 0.12 17 37 H H 0.0 16 40 2-OMe H -0.27 12 48 10 2-NHMe H -0.84 7 65 H Me 0.0 9.5 Table 1 shows that the unsubstituted hydroxylaminobenzyl carbamate normally used as a trigger has a half-life of 16 minutes. This is relatively long and, under biological 15 conditions, may result in substantial loss of material by side reactions not involving (activating) amine release. The half-life can be lowered significantly by the use of electron-donating substituents, and/or by the use of a-substituents (A). DISCLOSURE OF THE INVENTION In a first aspect, the invention provides a method of providing an amine with a 20 protecting group comprising (i) providing a plurality of different compounds selected from compounds of formulae (I) and (II)
S,NO
2 o Az No' 2
<Q
5 Z6 0 X.~ OO E 0a 0 E ~ 0 Yn -E O I II n wherein: X represents H, C.
6 alkyl or C6 alkoxy, said alkyl or alkoxy being optionally 25 substituted with one or more of the following groups: hydroxy (OH), ether (OR), amino
(NH
2 ), mono-substituted amino (NRH), di-substituted amino (NR'R 2 ), cyclic C 1
-
5 alkylamino, imidazolyl, Cl- 6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SR), tetrazole, carboxy (COOH), carboxylate (COOR, sulphoxy (S(=0) 2 0H), sulphonate WO 00/64864 PCT/GB00/01612 -5 (S(=0) 2 ORx), sulphonyl (S(=O) 2 R,), sulphixy (S(=O)OH), sulphinate (S(=O)ORx), sulphinyl (S(=O)R.), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORx) 2 ), where R,, Rx' and
R
2 are selected from a C, 6 alkyl group, a C 3
-
20 heterocyclyl group or a C 5
-
20 aryl group, preferably a C 1 6 alkyl group; a is 0,1,2,3 or 4; Y represents H or C 1
-
6 alkyl; 1, 2 or 3 of the 5 members Z of the 5-membered aromatic ring are independently selected from -O-,-S-,-N = or NR-, (where R is H or C-6 alkyl optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORR), amino (NH 2 ), mono-substituted amino (NRRH), di substituted amino (NRR'RR 2 ), C 1
.-
5 cyclic amino, imidazolyl, alkylpiperazinyl, morpholino, thiol (SH), thioether (SRR), tetrazole, carboxy (COOH), carboxylate (COORR), sulphoxy 10 (S(=0) 2 0H), sulphonate (S(=O) 2 ORR), sulphonyl (S(=0) 2 RR), sulphixy (S(=O)OH), sulphinate (S(=O)ORR), sulphinyl (S(=O)RR), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORR)2), where RR, RR' and RR 2 are selected from a C- 6 alkyl group, a C 3
-
20 heterocyclyl group or a C 5
-
20 aryl group, preferably a Cl.
6 alkyl group), the other ring atoms being C; n is 0 or 1; and E represents a moiety such that EH is an amine; 15 (ii) measuring the rates of fragmentation of the compounds to release EH when the nitro group is reduced and selecting a compound having a desired rate of decomposition; and (iii) providing the amine to be protected with a protecting group corresponding to that in the selected compound. In this aspect, the step of selecting the compound is preferably carried out in order to 20 provide a protecting group with a faster rate of fragmentation than unsubstituted 4-nitrobenzyl carbamate. However selecting a compound bearing a protecting group with a slower rate of fragmentation than 4-nitrobenzyl carbamate may be preferred. This particularly applies in situations in which it is desired to provide a prodrug which can diffuse away from the site of actuation by the appropriate enzyme, and thus kill tumour cells further away from the site of 25 actuation (the "bystander" effect). In a second aspect, the present invention relates to a compound represented by the general formula (I) or (II) as shown above, wherein X, Y, Z, E, a and n are as defined above; provided that in formula (I) if a = 0 then Y * H. EH is preferably a cytotoxic amine. E may be selected from formulae (III-XIII). 30 In (III-XIII), R, represents H or CI6 alkyl, being optionally substituted with one or more of the following groups: one or more of the following groups: hydroxy (OH), ether (ORE), amino (NH 2 ), mono-substituted amino (NREH), di-substituted amino (NRE RE 2 ), cyclic WO 00/64864 PCT/GB00/01612 -6
C
1
,
5 alkylamino, imidazolyl, Cz 6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRE), tetrazole, carboxy (COOH), carboxylate (COORE), sulphoxy (S(=O) 2 0H), sulphonate
(S(=O)
2 ORE), sulphonyl (S(=O) 2 RE), sulphixy (S(=O)OH), sulphinate (S(=O)ORE), sulphinyl (S(=O)RE), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORE) 2 ), where RE, RE 1 and 5 RE 2 are selected from a Cl- 6 alkyl group, a C 3
-
20 heterocyclyl group or a C 5
.
20 aryl group, more preferably from a C- 6 alkyl group ; R 2 represents H, C 1
.
6 alkyl, Cl- 6 alkoxy, OH, halogen,
NO
2 , NH 2 , NHMe, NMe 2 , SO 2 Me, CF 3 , CN, CONH 2 or CONHMe; each R 3 is independently selected from Cl, Br, I and OMS; and R 4 is selected from -C(=O)Me and -C(=O)CH 2 OH; Q represents substituted indole, substituted benzofuran or substituted cinnamoyl; in (IX) and 10 (X), each n is independently from 2-4, and each m is independently from 2-4, and p = 0 or 1.
WO 00/64864 PCT/GB00/01612 -7 Cl Cl CI C NR, NQ NQ 5 R3 R3 (III) NR N ' R N
R
3 R3 (V) (VI) R2 (IV)
R
1 10 OH O HN N S OH O HN R
R
2 OH 0 HN OH O HN Rj 15 (Vlla R (VIIb) R(VI) 0 N N -LN {VIIN R 00L K K ( R2 XI) where L = N N
NH
2 O OH R2 R4 N 20 Me OMe O OH Me 0 ooR, NH( XII) H o I' L (XIII) WO 00/64864 PCT/GB00/01612 -8 Compounds of formula V are described in EP 0 938 474, which is incorporated herein by reference. Compounds of formula VI are described in EP 0 850 220, which is incorporated herein by reference. A compound of formula (I) or (II) may be basic or acidic and may thus form 5 pharmaceutically acceptable salts with both organic and inorganic acids and bases. These are included within the scope of the second aspect. In a first type of preferred embodiment, X represents C1-6 alkyl or Cl- 6 alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy, ether (ORx), amino, alkylamino (NXH), dialkylamino (NRIRx 2 ), cyclic C 1 -5 10 alkylamino, imidazolyl, C 16 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRx), tetrazole and -CO 2 X' where X' is selected from the possibilities listed for X and Rx, R, 1 and R 2 are selected from C 1
-
6 alkyl; a is 0,1,2,3 or 4; Y represents H or lower alkyl; 1, 2 or 3 of the members Z of the 5-membered aromatic ring are independently selected from -O-,-S-,-N = or NR-, (where R is H or lower alkyl optionally substituted with one or more of the following 15 groups: hydroxy, ether (ORp), amino, alkylamino (NRRH), dialkylamino (NRRlRR 2 ), cyclic C 1 -5 alkylamino, imidazolyl, C 1 6 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRR), tetrazole and -CO 2 R' where R' is selected from the possibilities listed for R and RR, RRI and R 2 are selected from C- 6 alkyl); and E represents R 1 represents H or C 1 6 alkyl, being optionally substituted with one or more of the following groups hydroxy, ether (ORE), amino, alkylamino 20 (NREH), dialkylamino (NRE1RE2), cyclic C 1 5 alkylamino, imidazolyl, C,6 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRE), tetrazole and -CO 2 X' where X' is selected from the possibilities listed for X and RE, RE 1 and RE 2 are selected from C 1 -6 alkyl. In a second type of preferred embodiment, the compound is of formula (I) where a is 1. It is further preferred in this embodiment that X represents optionally substituted C-6 25 alkoxy and more preferably in the 2 position. The alkoxy group is preferably selected from methyl, ethyl and n-propyl, and the substituents from hydroxy, methoxy, phosphonoxy, NMe 2 , Nmorph, OCO 2 -tBu, and OCO 2 H. In more preferred embodiments the ethyl or n-propyl group is singly substituted, most preferably with hydroxy, whereas the Me group is unsubstituted. In this type of embodiment Y is preferably selected from H or Me. E is preferably 30 selected from III - XIII, most preferably V or XIII. If E is selected from XIII, n is preferably 1. In a third preferred type of embodiment, the compound is of formula (II) with one Z WO 00/64864 PCT/GB00/01612 -9 being -N= and another Z being -NR-, R being preferably Me or Et, the other members of the heterocyclic ring being C. If R is Et, it is preferably substituted with hydroxy. A is preferably 0. More preferably the -N= and -NR- are not adjacent in the heterocyclic ring. The most preferred arrangement is 2 (-NMe-) and 5 (-N=) , with the NO 2 at the 3 position. In this type 5 of embodiment, Y is preferably selected from H or Me. E is preferably selected from V or XIII. If E is selected from XIII, than n is preferably 1. In a fourth preferred type of embodiment, the compound is of formula (II), a is 0, and Z is either O or S. It is further preferred that the O or S is in the 2 position in the ring, and the
NO
2 is attached to the 3 position. In this type of embodiment, Y is preferably selected from H 10 or Me. In a fifth preferred embodiment, the compound is of formula (II), and Z is NR, preferably NMe. There may be one further substituent (a--l), or there may be no further substituents on the ring (a=0). The further substituent is preferably CO 2 Et. In this type of embodiment, Y is preferably selected from H or Me. 15 In a third aspect the invention provides a compound according to the second aspect for pharmaceutical use. In a fourth aspect the invention provides the use of a compound according to the second aspect for the manufacture of a composition for use in the treatment of a hyper proliferative disease, particularly a neoplastic disease. The composition may also include 20 activating means for simultaneous or separate administration, the activating means typically comprising an enzyme or means for providing an enzyme, for performing ADEPT or VDEPT therapy. The activating means typically leads to liberation of the amine EH. In a fifth aspect the invention provides a compound of the formula XVI or XVII where X, n, Z and Y are as defined for the second aspect and T is OH or an activated alcohol 25 functionality (such as -O.CO.L where L is a leaving group such as Cl) suitable for reaction with an amine EH to produce a compound according to the second aspect. In a sixth aspect the invention provides the use of a compound of formula (XVI) or
NO
2 NO 2 Xa Xa Z - 0 i-NHY NH-- Y O NH 'T 0 T XVI n nVI WO 00/64864 PCT/GB00/01612 -10 (XVII) in protecting an amine. This may include activation of an alcohol (XVI or XVII where T is OH) with a reagent such as phosgene, diphosgene or triphosgene or a chloroformate, e.g. 4-nitrophenylchloroformate or pentafluorophenylchloroformate, optionally 5 in conjunction with HOBT(1-hydroxybenzotriazole). In a further aspect, the present invention relates to a method of preparing compounds of the general formula (I); examples of the methods are outlined in Schemes 1-24. Thus (Scheme 1), reaction of the amine 7 [D-F. Shi, T. D. Bradshaw, S. Wrigley, C. J. McCall, P. Lelieveld, I. Fitchner, M. F. G. Stevens. J. Med. Chem., 1996, 39, 3375] with 4 10 nitrobenzylchloroformate gave carbamate 8. Scheme I
NO
2
H
2 NC)_/ N- \/, 0 S 7 Reagents: (i 4-nitrobenzlchboroformate, pyridine. 8 Reaction of the 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione 22 with amine 15 in pyridine gave the monocarbamate 23 and biscarbamate 24 (Scheme 7). Further reaction of 23 with 2-(2 aminoethylamino)ethanol gave carbamate 26. 15 In another example (Scheme 2), 4-nitrosalicylic acid (27) was methylated using a solution of diazomethane in ether and the methyl ester 28 reduced with DIBALH in THF to give the nitrobenzyl alcohol 29. Activation of the alcohol 29 with triphosgene (or alternatively phosgene or diphosgene) in the presence of pyridine, and reaction with N',N'-bis(2 hydroxyethyl)-1,4-benzenediamine (57) gives the carbamate 31, which was elaborated to the 20 mustard 32 using standard methods.
WO 00/64864 PCT/GB00/01612 -11 Scheme 2
HO
2
H
2
H
2 (ii) (iv),(v)
OR
2 OMe Oe 27 R, = CO 2 H, R 2 = H O> 0 NH 0 NH 28 R 1 = CO 2 Me, R 2 =Me 29 R, = CH20H, R 2 = Me N Reagents: (i) CH 2
N
2 ,Et 2 0; (i) DIBALH, THF; N Na (ii) triphosgene, pyidine, THF, 57 (iv) MsCI, HO OH 0 32 31 32 pyridine; (v) LiCI, DMF. Similarly (Scheme 3), activation of alcohol 29 with triphosgene in the presence of pyridine and reaction with amine 33 [M. Tercel and W. A. Denny. J. Chem. Soc. Perkin Trans. 1, 1998, 509] or amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 5 1997, 7, 1483] gave carbamates 34 and 35, respectively. Scheme 3 OMe a O ea NO7 - e N /- OO N / OOMe 0w 0 OMe OMe O . NHR NHR 33R= H 1 R= H 34 R = CO 2
CH
2 (2-OMe)Ph-4-NO2 35 R = CO 2 CH2(2-OMe)Ph-4-NO 2 Reagents: (i) tnphosgene, 29, pyridrie. Similarly (Scheme 4), activation of alcohol 29 with triphosgene in the presence of pyridine and reaction with amine 7 [D-F. Shi, T. D. Bradshaw, S. Wrigley, C. J. McCall, P. 10 Lelieveld, I. Fitchner, M. F. G. Stevens. J. Med Chem., 1996, 39, 3375]] gave carbamate 36.
WO 00/64864 PCT/GB00/01612 -12 Scheme 4 RHN 7R=H () 36 R = CO 2
CH
2 (2-OMe)Ph-4-NO2 Reagents: (i) triphosgene, 29, pyndine. In another example (Scheme 5), activation of alcohol 29 with 4 nitrophenylchloroformate gave the carbonate 37 which was reacted with doxorubicin (13) and triethylamine in DMF to give the doxorubicin carbamate 38. 0 OH 0 Scheme 5 N 2 OH 29 -ROMe OMe O OH O C)"U -N0) 2 0m 00o 37 0 Reagents: (I) 4-nitrophenylchloroformate, pyridine; 38 5 (i) 13, Et 3 N, DMF. 0 2 N " OMe In another example (Scheme 6), carbonate 37 was coupled to amine 9 using 1 hydroxybenzotriazole (HOBT), 4A molecular sieves and triethylamine to give protected carbamate 39. Removal of the TBDMS protecting group with aqueous acid gave the alcohol 40 which was activated with 4-nitrophenylchloroformate to give the carbonate 41. Reaction of 10 41 with doxorubicin (13) and triethylamine in DMF gave the carbamate 42.
WO 00/64864 PCT/GB00/01612 - 13 Scheme 6N0N2 Scheme NH 2
NO
2
NO
2
NO
2 ()(ii) (iv) + 3 OMe OMe OMe OTBDMS O 0 0 0 OH 0 O-NH 0 NH O< NH ~OHR DOX = OH OMeO OH 0 R O NO 2 O MeTO 0RZBEOO OANHDOX 39 R = TBDMSO (i) HO 40 R = OH 41 42 Reagents: (I) HOBT, moL sieves, Et 3 N, THF; (ii) HCI, aq. MeOH; (ii) NO 2 PhOCOCI, Et 3 N, THF; (iv) DOX, Et 3 N, DMF. In another example (Scheme 7), reaction of alcohol 29 with triphosgene and triethylamine, and coupling to 2,2,2-trifluoro-N-[2-(methylamino)ethyl]acetamide trifluoroacetate gave the trifluoroacetamide 43 which was deprotected under basic conditions 5 to give amine 44. Activation of the 5-methyl-9-oxo-9,10-dihydro-4-acridinecarboxylic acid (16) with thionyl chloride and coupling of the intermediate 9-chloroacridinyl acid chloride with amine 44 gave amide 45 which was converted to carbamate 46 using ammonia in phenol. Scheme 7 R Scheme 7 NO 2
NO
2 29 "I SOMe OMe Me PH NOM Me o2H O O N 16 ON--N NHR O N Me Me 43 R = COCF3 (i) 45 R = Cl 44R=H 46R=NH 2 ( ) Reagents: (I) triphosgene, CF 3
CONHCI-
2
CH
2 NHMe, DIEA; (ii) CS 2
CO
3 ; (iii) SOCI 2 , DMF; (iv) PhOH, NH 3
.
WO 00/64864 PCT/GB00/01612 -14 In another example (Scheme 8), the bistrifluoroacetamide 47 was coupled to alcohol 29 to give bisamide 48 which was deprotected under basic conditions to give the amine 49. The amine 49 was coupled to 4-(1H-imidazol-l-ylcarbonyl)-5-methylacridine (50) [S. A. Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J Med Chem., 5 1999, 42, 2383-2393] to give the carbamate 51. Scheme 8 NO 2
NO
2 N NH 2 2 N N 2(i)C OM e () OM e (i) 0 HN N O Me JN O N NHCOC 2 H 2 51 47 48 R = COCF 3 2 49 R = H Reagents: (i) EtOCOCF 3 , H 2 0, MeCN; (i) triphosgene, 29, Me O N DIEA, DCM; (ii) Cs2CO3, H 2 0, MeOH; (iv) 50, THF. 50 Reaction of the 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione 22 with amine 44 gave the monocarbamate 52 and biscarbamate 53 (Scheme 9). Further reaction of 52 with 2-(2 10 aminoethylamino)ethanol gave carbamate 55.
WO 00/64864 PCT/GB00/01612 -15 ye j .N02 Scheme 9 No NO 2 HO H OMe H+O | 53 OH O F OH O HN OH HN A 22 52
NO
2 O NO 2 2252 Me N0Me H HN H H OH 52 (i) OH5 + 555 OH O HN
O
H O HN-M 54 N 0O N'IQ MeO NO 2 Me NO 2 Reagents:(i) 44, pyridine; (i) NH 2
(CH
2 )2NH(CH 2
)
2 0H, pyridine. In another example (Scheme 10), reaction of 4-nitrophenylethan-1-ol (56) with triphosgene and pyridine, with the subsequent addition of M,N-bis(2-hydroxyethyl)-1,4 benzenediamine (57) gave the carbamate 58 which was elaborated to the dichloride 59. 5 WO 00/64864 PCT/GB00/01612 -16 Scheme 10
NO
2
NO
2
NO
2
NH
2 Me O 0 (i), (ii) Me 0 + O NH 0 NH N Me OH HO OH 56 H 7 OH N N Reagents: (i) tfiphosgene,THF; (ii) MsCI, HO OH Cl Cl pyridine; (iii) LiCI, DMF. 58 59 In another example (Scheme 11), coupling of the alcohol 56 with amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] using triphosgene and pyridine in THF gave the carbamate 60. 5 Scheme 11 c, CI OMe
NH
2 eMe 60 N +0 H OMe HN ~ IOMe HN 1110 OMe
NH
2 1 / 60 Reagents: (i) tiphosgene, pyridine, THF. NO Oxidation of alcohol 29 with pyridinium chlorochromate (PCC) in DCM gave the aldehyde 61 (Scheme 12). Reaction of aldehyde 61 with methyl magnesium bromide in THF gave the alcohol 62 which was coupled to amine 57 to give carbamate diol 63. The diol 63 was 10 elaborated to the dichloride 64 using standard methods.
WO 00/64864 PCT/GB00/01612 -17 Scheme 12
NO
2 N2 N2 (i) (ii) (iv), (v) 29 O OMe OMe R Me 0 Me O 61 R = CHO (i) ;NH O NH 62 R = C(CH 3 )OH N N Reagents: () PCC, DCM; (ii) MeMgBr, THF; (ii) N N triphosgene, pyridine, 57, THF; (iv) MsCI, HO OH CI C3 pyridine; (iii) LiCI, DMF. 63 64 In another example (Scheme 13), alkylation of methyl 2-hydroxy-4-nitrobenzoate 66 with bromide 65 under basic conditions gave the ester 67 which was reduced to alcohol 68 using DIBALH in THF. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, 5 Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 68 using catalytic dibutyltin diacetate to give carbamate 69. Deprotection of 69 under acidic conditions gave 70. Scheme 13 OMe ON 7 OMe O OH0 OTB DMS o-OTB DMS C CO2Me CO2Me OH HN O 66 67 68 O Br , OTB DMS O O R 65
NO
2 Reagents: (i 65, K 2 C0 3 , DMF; (ii) DIBALH, THF; (ii) 1, triphosgene, Et 3 N, 69 R = TBDMS> (iv) DCM, then 68, nBu 2 Sn(OAc) 2 ; (iv) HCI, MeOH. 70 R = 10 WO 00/64864 PCT/GB00/01612 -18 Similarly (Scheme 14), reaction of phenol 66 with 2-bromoethyl methyl ether gave ester 71 which was reduced to alcohol 72. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 72 using catalytic dibutyltin 5 diacetate to give carbamate 73. Scheme 14 2a OMe
O
2
NO
2
NO
2 e 66 - ' O (/ H O OMe O-- OMe OMe
CO
2 Me OH HN. 0 71 72 O Reagents: (i) BrCH 2
CH
2 OMe, K 2
CO
3 , DMF; (ii) DIBALH, THF; (iii) 1, triphosgene, Et 3 N, DCM, then 72, nBu 2 Sn(OAc) 2 . NO 2 73 In another example (Scheme 15), phenol 66 was alkylated with 3-iodopropyl tetrahydropyranyl ether under basic conditions to give ester 74 which was reduced to alcohol 10 75 using DIBALH in THF. Activation of the alcohol 75 with triphosgene and triethylamine (or pyridine, or another organic base) in THF and subsequent reaction with amine 57 gave the carbamate diol 76. The diol 76 was converted to the dichloride 77 using standard methods and the tetrahydropyranyl ether deprotected under acidic conditions to give carbamate 78. Scheme 15 NO 2 2 NC) 2
NO
2
NO
2 /J ( (i) (ii) 66 ) O" "OTHP O''OTHP O- OR 2 C2i OH 74 75 o-L- NH Reagents: (i) BrCH 2
CH
2
CH
2 OTHP, K 2
CO
3 , DMF; (ii) DIBALH, THF; (ii) triphosgene, Et 3 N, THF, then 57; (iv) MsC, pyridine; N (v) LiCI, DMF; (vi) HCI, MeOH. R, R, 76 R 1 = OH, R 2 = THP'. (iv), (v) 77R 1 = C1, R 2 = THP (v 15 78 R, = aCI, R 2 = H WO 00/64864 PCT/GB00/01612 -19 In another example (Scheme 16), alcohol 75 was activated using triphosgene and triethylamine (or pyridine, or another organic base) and coupled to amine 33 [M. Tercel and W. A. Denny. J. Chem. Soc. Perkin Trans. 1, 1998, 509] to give carbamate 79 which was deprotected under acidic conditions to give 80. 5 Scheme 16
NO
2 a OMe (i) N OMe O .OTHP OMe OH HN.. 0 75 O0 O - ,OR Reagents: (i) 33, triphosgene, pyridine, THF; 79 R = THP (i) (i) HCL MeOH.
NO
2 80 R = H In another example (Scheme 17), alkylation of phenol 66 with 3-bromopropyl tert butyl(dimethyl)silyl ether under basic conditions gave the ester 81 which was reduced to alcohol 82 using DIBALH in THF. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. 10 Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 82 using catalytic dibutyltin diacetate to give carbamate 83. Deprotection of 83 under acidic conditions gave carbamate 84. Scheme 17 N O 2a
NO
2
NO
2 ( I) .( i ) -i 66 0 O OMe O--- OTBDMS O- "OTBDMS HN 0 CO2iKe OH 81 82 83 R = TBDMS (iv) O0 OR 84R=H Reagents: (i) BrCH 2
CH
2
CH
2 OTBDMS, K 2
CO
3 , DMF; (ii) DIBALH, THF; (ii) 1, triphosgene, Et 3 N, DCM, then 82, nBu 2 Sn(OAc) 2 ; (iv) HCI, NO 2 MeOH.
WO 00/64864 PCT/GB00/01612 - 20 In another example (Scheme 18), activation of alcohol 75 with 4 nitrophenylchlorofonnrmate gave the carbonate 37 which was deprotected under acidic conditions to give carbonate 86. Reaction of 86 with doxorubicin (13) and triethylamine in 5 DMF gave the doxorubicin carbamate 87. Scheme 18 NO 2 O OH O OH H'OH (i) O OR (ii) 75 -- 0- --- " 75 OMeO OH O 0 O
NO
2 Me 0 HOJ 85R=THP 86R= H ;i) 87 Reagents: (i) NO 2 PhOCOCI, DIEA, THF (i) HCI 0 - O O THF; (ii) 13, Et 3 N, DMF. 0 2 N In another example (Scheme 19) alcohol 75 was activated with triphosgene and coupled to amine 47 to give trifluoroacetamide 88 which was deprotected under basic 10 conditions to give bis-amine 89. Coupling of the bis-amine 89 with the imidazolide 50 [S. A. Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J. Med. Chem., 1999, 42, 2383-2393] gave the carbamate 90 which was deprotected under acidic conditions to give carbamate 91. Scheme 19 NO 2
NO
2 () O OTHP 0 0 75 " \ O O O NHR 2 N OR 88 R = COCF3 (i) 89 R = H 90 R = THP Reagents: (i~ 47, DIEA, DCM; (i) Cs2CO 3 , aq. MeOH; (ii) 50, THF; (iv) H , 91 R = H 15 aq. MeOH.
WO 00/64864 PCT/GB00/01612 -21 In another example (Scheme 20), the alcohol 84 was reacted with di-tert-butyl 5 diethylphosphoramidite and tetrazole inTHF and the intermediate oxidised with MCPBA to give ester 92. Deprotection of 92 with trifluoroacetic acid (TFAA) gave the phosphate 93. OMe Scheme 20 Me N / OMe 0 OMe 84R= H (, (ii) 0 92 R = P(0)(OtBu) 2 O0 93R= P(O)(OH 2 (ii) O OR Reagents: (i) Et 2 NP(OtBu) 2 , tetrazole, THF; (i) MCPBA, DCM; (iii)TFAA, DCM
NO
2 In another example (Scheme 21), phenol 66 was alkylated with epichlorohydrin 10 under basic conditions to give epoxide 94. Hydrolysis of 94 with perchloric acid gave diol 95 which was protected as the acetonide 96. Reduction of 96 with DIBALH in THF gave the alcohol 97. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 97 using catalytic dibutyltin diacetate to give 15 carbamate 98. Deprotection of 98 under acidic conditions gave carbamate 99.
WO 00/64864 PCT/GB00/01612 - 22 Scheme 21 NO 2 2 2 N2 0) 0) 0 0- iv . 66 ) O)-- 0 O O - -OH O- O0 C0 2 Me
CO
2 Me OH C 2 Me O Me OH O Me 94 95 96 Me 97 Me OMe OMe N OMe (vi) N OMe HN 0 HN O Me (v) S OH O O I 0 _ OHI 'k 2 99 NO 98 Reagents: (i) epichlorohydrin, K 2
CO
3 , DMF; (i) HCO 4 ,THF; (iii) diethoxyacetone, PPTS, DMF; (iv) DIBALH, THF; (v) 1, tiphosgene, Et 3 N, DCM, then 97, nBu 2 Sn(OAc); (vi) HCI, aq. THF. In another example (Scheme 22), ), phenol 66 was alkylated with N-(3-chloropropyl) N,N-dimethylamine under basic conditions to give amine 100. Reduction of 100 with 5 DIBALH in THF gave the alcohol 101. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 101 using catalytic dibutyltin diacetate to give carbamate 102. Similarly (Scheme 22), phenol 66 was alkylated with 4-(3-chloropropyl)morpholine 10 under basic conditions to give amine 103. Reduction of 103 with DIBALH in THF gave the alcohol 104. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 104 using catalytic dibutyltin diacetate to give carbamate 105.
WO 00/64864 PCT/GB00/01612 - 23 Scheme 22
N
2 a OMe
NO
2 NON / )(-(ii) \ NOMe 66 0 R OMe HN 0
CO
2 Me OH 100 R = NMe 2 101 R = rNMve 2 O 103 R = Nmorph 104 R = Nmorph Reagents: (i) BrCH 2
CH
2
CH
2 NRR, K 2 CO3, DMF; (i) DIBALH, THF; (ii) 1, trphosgene, Et 3 N, DCM, then 101 or 104, nBu 2 Sn(OAc) 2 . 102 R = NMe 2
NO
2 105 R = Nmorph 5 In another example (Scheme 23), reaction of alcohol 91 with methanesulphonyl chloride gave the mesylate 106 which was reacted with morpholine to give carbamate 107. Scheme 23
NO
2 O --- R 91 R=OH oj 106 R = OMs ) (i) O 107 R= Nmorph Me o 'N-O - 2 Reagents: (i) MsC DCM; (ii) morpholine. 10 In another example (Scheme 24), phenol 66 was alkylated with tert-butyl 4 bromobutanoate under basic conditions to give ester 108. Hydrolysis of 108 under basic conditions gave acid 109 which was reduced with borane.dimethylsufide in THF to give 15 alcohol 110. Activation of alcohol 110 with triphosgene and diisopropylethylamine and subsequent coupling with N,N-bis[3-(5-methylacridine-4-carboxamido)propyl]amine (111) [S. A. Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J. Med WO 00/64864 PCT/GB00/01612 - 24 Chem., 1999, 42, 2383-2393] gave carbamate 112. Carbamate 112 was deprotected under acidic conditions to give acid 113. Scheme 24
NO
2
NO
2
NO
2 66 r O CO 2 tBu O CO 2 tBu O
CO
2 R OH 108 R= Me= (i) 110 Me 0 109 R = HO N O R 2 Reagents: (i) BrCH 2
CH
2
CH
2
CO
2 tBu, K 2 00 3 , DMF; (ii) NaOH, aq. MeOH 112 R = CO 2 tBu (ii) BH3.DMS, THF; (iv)tnphosgene, DIEA, DCM, then 111; (v) HCI, MeOH. 113 R = CO 2 H v) 5 In a further aspect, the present invention relates to a method of preparing compounds of the general formula (II); examples of the methods are outlined in Schemes 25-. Thus (Scheme 25), (2-nitro-1H-imidazol-5-yl)methanol (115) is obtained from the known ethyl 2-nitro-1H-imidazol-5-ylcarboxylate (113) [B. Cavalleri, R. Ballotta, G.C Lancini. J.1 Heterocyclic Chem. 1972, 9, 979.] by basic hydrolysis to the acid 114 and 10 reduction of an intermediate imidazolide with sodium borohydride. This procedure is a major improvement upon the above published methods. Reaction of 115 with 4-nitrophenyl chloroformate gives the activated carbonate 116 which reacts with N,N-bis-(2 chloroethyl)amine to give carbamate 117. Scheme 25
NO
2
NO
2
NO
2
NO
2
NO
2 Njj
(
e 4j_ (i)_ (ii) 00 M Il me -). N' Me - . Me :_lr - me CO2Me CO2H OH O OO- O 113 114 115 116 117 Reagents: (i) NaOH, aq. MeOH; (ii) CDI, NaBH 4 , THF; \ a (ii) NO2PhOCOCI, pyridine, THF; (iv)HN(CH 2
CH
2
CI)
2 . Cl 15 pyridine.
NO
2 WO 00/64864 PCT/GB00/01612 -25 Similarly (Scheme 26), reaction of 116 with the protected phenyldiamine diol 120, derived from the nitrophenylamino diol 118, gives carbamate 121. Deprotection of the bis-silyl alcohol 121 with TBAF gives the diol 122 which can be converted to the dichloride 123 under standard conditions. 5 Scheme 26
NO
2
NH
2
NO
2 (i) N Me , (121R = OTBDMS 0 N 0 122 R = OH H 123 R= CI (v) RO OR TBDMSO OTBDMS / \ 118R= H 119 R = OTBDMS) (' 120 R Reagents: (i) TBDMSCI, imidazole (ii) Pd/C, H 2 , EtOH; (ii) 116, pyridine; (iv) TBAF, THF; (v) (a) MsCI, pyridine, R (b) LiCI, DMF. In another example (Scheme 27), activation of amine 1 [G. J. Atwell, W. R. Wilson, 10 W.A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine (or pyridine, or another organic base) gave an intermediate isocyanate which was coupled with alcohol 115 using catalytic dibutyltin diacetate to give carbamate 124. O Scheme 27 c0 0 02 N OMe N NMe OMe -- )"- O Me OHHNN Oe -N 115 N O2-NO2 Reagents: (i) 1, triphosgene, Et 3 N, DCM, 12 Me then 115, nBu 2 Sn(OAc) 2 . 15 WO 00/64864 PCT/GB00/01612 - 26 In another example (Scheme 28), reaction of the carbonate 116 with doxorubicin (13) and triethylamine in DMF gave the carbamate 125. 0 OH 0 Scheme 28 OH 'OH 116 . OMe 0 OH 0 Me 0 / NH Me HO 5 Reagents: (i) 13, Et 3 N, DMF. 0 2 N N 125 Similarly (Scheme 29), reaction of carbonate 116 with amine 9 using HOBT, molecular sieves, and triethylamine gave the silyl ether 126. Deprotection of silyl ether 126 under acidic conditions gave alcohol 127 which was reacted with 4-nitrophenyl chloroformate to give carbonate 128. Reaction of the carbonate 128 with doxorubicin (13) and triethylamine 10 in DMF gave the carbamate 129. Scheme 29 NO 2 0 OH 0 OH N 'OH 116 (i) v) OMe 0 OH 0 HN O Me e O H 0 OR HN 126 R = TBDMS (i) 129 127 R= H N 128 CO 2 PhNO 2 (ii) Reagents: (i) 9, HOBT, Et 3 N, mol. sieves, THF; (ii) HCI, aq. MeOH; (iii) 4-NO 2 PhOCOCI, THF; (vi) 13, Et 3 N, DMF. In another example (Scheme 30), ozonolysis of the styrene 130 [D. C. Baker, S. R. Putt, H. D. H. Showalter, J. Heterocyclic Chem., 1983, 30, 629-634.] gave the alcohol 131.
WO 00/64864 PCT/GB00/01612 - 27 Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 131 using catalytic dibutyltin diacetate to give carbamate 132. Scheme 30 0 NNe/N>e N NMe te NP>sve (i) N HN OMe 130 131 O 132 NO 2 Reagents: (i) 03, DCM, MeOH; (ii) NaBH 4 , EtOH; (iii) 1, triphosgene, Et3N, DCM, then 131, 5 nBu 2 Sn(OAc) 2 . In another example (Scheme 31), treatment of (N-methyl-5-nitro-1H-imidazol-2 yl)methanol (133) [C. Rufer, H. J. Kessler, E. Schroder. J. Med Chem. 1971, 14, 94.] with 4 nitrophenylchloroformate gave the carbonate 134, which was displaced with N,N-bis(2 chloroethyl)amine to give the carbamate 135. 10 Scheme 31 OH 0O O 7 0 Oe (i) NO 2 (i) O N_ ----- . -- 1 ~
NO
2 N7Me 0
N
1 e 133 13 4
NO
2 NLMe Reagents: (I) NO 2 PhOCOCI, pyridine,THF; \ (i) HN(CH 2
CH
2 CI)2, pyridine. 135 NO2 Similarly (Scheme 32), activation of 133 with diphosgene and subsequent reaction with N',N'-bis(2-chloroethyl)-1,4-benzenediamine hydrochloride (136) gave the carbamate 137.
WO 00/64864 PCT/GB00/01612 -28 Scheme 32 O OH O N Me (1) N e
NO
2
NO
2 133 137 Reagents: (I) diphosgene, Et 3 N, THF; (i) 136, pyridine. In another example (Scheme 33), activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 133 using catalytic dibutyltin 5 diacetate to give carbamate 138. Scheme 33 CI 0
NO
2 N / Ome A () /I H N'- NMe- OMe OMe HN 0 115 ONH H-NO 2 0 N Reagents: (i) 1, triphosgene, Et 3 N, DCM, Me then 115, nBu 2 Sn(OAc) 2 . 124 In another example (Scheme 34), reaction of carbonate 134 with amine 9 using HOBT, molecular sieves, and triethylamine gave the silyl ether 139. Deprotection of silyl ether 10 139 under acidic conditions gave alcohol 140 which was reacted with 4-nitrophenyl chloroformate to carbonate 141. Reaction of the carbonate 141 with doxorubicin (13) and triethylamine in DMF gave the carbamate 142.
WO 00/64864 PCT/GB00/01612 - 29 Scheme 34 N02 H OH Me |"OH (___ __ _ OMe O OH HN O Me 0 0 HOJ_\ e 0_ 0 OR HO 139 R = TBDMS (i) 02 142 140 R = H N 141 CO 2 PhNO 2 (ii) Reagents: () 9, HOBT, Et 3 N, mol. sieves, THF; (ii) HCI, aq. MeOH; 4-NO 2 PhOCOCI, THF; (vi) 13, Et 3 N, DMF. In another example (Scheme 35), condensation of metronidazole (143) and benzaldehyde gave the styrene 144 which was protected with TBDMS triflate to give 145. Ozonolysis of styrene 145 gave alcohol 146. Activation of amine 1 [G. J. Atwell, W. R. 5 Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 146 using catalytic dibutyltin diacetate to give carbamate 147. Deprotection under acidic conditions gave the carbamate 148. CI Scheme 35 C OH N < ome e __OMe e. OV VH N m N OH N OR N N OTBDMS OMe IN O
NO
2 NO 2 NO 2 N
~~NO
2 143 144 R = H 146 145 R = TBDMS (i) "OR Reagents: (i) NaOMe, PhCHO, DMSO; (i) TBDMSTf, pyridine, DCM; 147 R = TBDMS (vi) (ii) 0 3 , DCM, MeOH; (vi) NaBH 4 , EtOH; (v) 1, triphosgene, Et 3 N, DCM, 10 then 146, nBu 2 Sn(OAc) 2 ; (vi) HCI, aq. MeOH.
WO 00/64864 PCT/GB00/01612 -30 Similarly (Scheme 36), activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 149 [D. C. Baker, S.R. Putt, H. D. H. Showalter, J. Heterocyclic Chem., 1983, 20, 629-634.] using catalytic dibutyltin diacetate to 5 give carbamate 150. CI Scheme 36 .Me (___ - N\ ¢ O - OMe H N2OMe NO, HN. Oe 149 149 NMe Reagents: (i 1, triphosgene, Et 3 N, DCM, 1NO then 149, nBu 2 Sn(OAc) 2 . N2 10 In another example (Scheme 37), reaction of the 5-nitrofuran-1-methanol (151) [J. M. Berry, C. Y. Watson, W. J. D. Whish, and M. D. Threadgill, J Chem. Soc. Perkin Trans. I, 1997, 1147.] with 4-nitrophenylchloroformate gave carbonate 152, which was displaced with N',N'-bis(2
-
hydroxyethyl)-1,4-benzenediamine (57) to give the carbamate diol 153. The diol 153 was converted to the dichloride 154 using standard methods. 15 Similarly (Scheme 37), reaction of (5-nitrothien-2-yl)methanol (156) [P. J. Narcombe, R. K. Norris. Aust. J. Chem. 1979, 32, 2647] with 4-nitrophenylchloroformate gave carbonate 157, which was displaced with N,N-bis(2-hydroxyethyl)-1,4-benzenediamine (57) to give the carbamate diol 158. The diol 158 was converted to the dichloride 159 using standard methods. The same technique was used on 5-nitrofuran-1-methanol (151) [J.M. 20 Berry, C.Y. Watson, W.J. Whish, and M.D. Threadgill, J. Chem. Soc. Perkin Trans. I, 1997, 1147].
WO 00/64864 PCT/GB00/01612 -31 Scheme 37 2 2 0 2 N X OH (i) XX 151 X= 0 0 0 NH 156 X= S 152 XNO 157 X= S R R 153 X= 0, R = OH Reagents: ( NO2PhOCOCI, pyridine,THF, 154 X = O, R=CI (ii) (i) 57, pyridine, (ii) (a) MsCI, pyridie, 158 X= S, R = OH (b) LCI, DMF. 159 X = S, R = CI In another example (Scheme 38), activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 151 [J. M. Berry, C. Y. 5 Watson, W. J. D. Whish, and M. D. Threadgill, J. Chem. Soc. Perkin Trans. I, 1997, 1147.] using catalytic dibutyltin diacetate to give carbamate 155. Similarly (Scheme 38), activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 156 [P. J. Narcombe, R. K. Norris. 10 Aust. J. Chem. 1979, 32, 2647] using catalytic dibutyltin diacetate to give carbamate 160. a Scheme 38 0 0 2 N X OH N OW ___ HN OWe OMe 151X=O0 HN 0 156 X= S Reagents: (i) 1, triphosgene, Et 3 N, DCM, 155 X = O X NO 2 then akohol, nBu 2 Sn(OAc) 2 . 160 X = S WO 00/64864 PCT/GB00/01612 - 32 In another example (Scheme 39), 1-methyl-5-nitro- 1H-pyrazole-4-carboxylic acid (161) [C. C. Cheng, J. Heterocyclic Chem. 1968, 5, 195-197] was reduced with borane.dimethyl sulfide to give alcohol 162. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and 5 triethylamine gave an intermediate isocyanate which was coupled with alcohol 162 using catalytic dibutyltin diacetate to give carbamate 163. Scheme 39 ci Me(i) Me (ii) eN" N O 2 N NO 2 -NINe 1 NO- / 0 OMe
CO
2 H L -OH HNL /TOMe 161 162 OMe HN O Reagents: (i) BH 3 .DMS, THF; (ii) 1, triphosgene, Et 3 N, 163 NMe DCM, then 162, nBu 2 Sn(OAc) 2
.
163 0 2 N In another example (Scheme 40), ethyl 4-formyl-5-nitro-1H-pyrrole-2-carboxylate 10 (164) [P. Fomrnari, M. Famrnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-291] was alkylated with dimethyl sulfate to give pyrrole 165. Reduction of 165 with sodium borohydride gave the alcohol 166. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 166 using catalytic dibutyltin diacetate to give 15 carbamate 167. In another example (Scheme 40), hydrolysis of ester 166 followed by decarboxylation with copper in quinoline gave alcohol 168. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 168 using catalytic dibutyltin 20 diacetate to give carbamate 169.
WO 00/64864 PCT/GB00/01612 -33 CI Scheme 40 ONe N Z OMe
O
2 N CO 2 Et (i) O 2 N R N
__},.HO--
/ __. O~ OHC HO OMe HN O R 164 R H 166 R = CO 2 Et 165 R =Me 168 R = H (v O NMe 02N Reagents: (i) Me 2
SO
4 , K 2 C0 3 , DMSO; (i) NaBH 4 , EtOH; 0 2 N (ii) 1, triphosgene, Et 3 N, DCM, then alcohol, nBu 2 Sn(OAc) 2 ; 167 R = CO 2 Et (iv)NaOH, aq. EtOH; (v)Cu, quinoline. 169 R = H Similarly (Scheme 41), ethyl 5-formyl-4-nitro- 1H-pyrrole-2-carboxylate (170) [P. Fornari, M. Farnier, C. Foumrnier, Bull. Soc. Chim. Fr. 1972, 283-291] was alkylated with dimethyl sulfate to give pyrrole 171. Reduction of 171 with sodium borohydride gave the 5 alcohol 172. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 172 using catalytic dibutyltin diacetate to give carbamate 173. In another example (Scheme 41), hydrolysis of ester 172 followed by decarboxylation 10 with copper in quinoline gave alcohol 174. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate isocyanate which was coupled with alcohol 174 using catalytic dibutyltin diacetate to give carbamate 175. Cl Scheme 41 H eOe HO .OMe OHC C 2Et R N(ii P(i) (ii)| HN OMe
O
2 N 0 2 N OMe HN 0 R 170 R = H 172 R = CO2Et OMeN RI 171 R = Me ( 174 R =c (iv), (v) O 02N Reagents: (i) Me 2
SO
4 , K 2
CO
3 , DMSO; (ii) NaBH 4 , EtOH; 0 2 N (ii) 1, trphosgene, Et 3 N, DCM, then alcohol, nBu 2 Sn(OAc} 2 ; 173 R = CO 2 Et (iv)NaOH, aq. EtOH; (v)Cu, quinolire. 17 R = H WO 00/64864 PCT/GB00/01612 - 34 In another example (Scheme 42), 1-methyl-5-nitro-1H-pyrrole-2-carbaldehyde (176) [P. Foumari, Bull. Soc. Chim. Fr. 1963, 488-491] was reduced with sodium borohydride to give alcohol 177. Activation of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] with triphosgene and triethylamine gave an intermediate 5 isocyanate which was coupled with alcohol 177 using catalytic dibutyltin diacetate to give carbamate 178. Cl Scheme 42 O2N ' F (iOMe e OH N 7 OMe 0 2 N CHO (i) 0 2 N (i) HN OMe OMe HN O 176 177 1 Reagents: (I) NaBH 4 , EtOH; (i) 1, trphosgene, 178N NO 2 Et 3 N, DCM, then 177,nBu 2 Sn(OAc) 2
.
Me In a further preferred aspect, the present invention relates to the use of a compound of formula (I) or (II) as defined in the second aspect of the invention, in conjunction with a 10 nitroreductase enzyme (for example, isolated from E. coli) in a method of ADEPT or GDEPT therapy. A drug produced by the action of the nitroreductase enzyme on a compound of formula (I) or (HI) may be used for the selective killing of oxic and hypoxic tumour cells in methods of treatment of cancers, for example leukemias and particularly solid cancers including breast, bowel and lung tumours, including small cell lung carcinoma. 15 The invention also provides a pharmaceutical composition comprising a compound of the formula (I) or (H) as defined in the second aspect of the invention together with a pharmaceutically acceptable carrier or diluent. Detailed Description of the Invention GDEPT 20 - Vector systems In general, the vector for use in GDEPT therapies may be any suitable DNA or RNA vector. Suitable viral vectors include those which are based upon a retrovirus. Such vectors are widely available in the art. Huber et al. (ibid) report the use of amphotropic retroviruses 25 for the transformation of hepatoma, breast, colon or skin cells. Culver et al. (Science (1992) WO 00/64864 PCT/GB00/01612 -35 256; 1550-1552) also describe the use of retroviral vectors in GDEPT. Such vectors or vectors derived from them may also be used. Other retroviruses may also be used to make vectors suitable for use in the present invention. Such retroviruses include rous sarcoma virus (RSV). Englehardt et al. (Nature Genetics (1993) 4; 27-34) describe the use of adenovirus 5 based vectors in the delivery of the cystic fibrosis transmembrane conductance product (CFTR) into cells, and such adenovirus based vectors may also be used. Vectors utilising adenovirus promoter and other control sequences may be of use in delivering a system according to the invention to cells in the lung, and hence useful in treating lung tumours. Other vector systems including vectors based on the Molony murine leukaemia virus 10 are known (Ram, Z et al. Cancer Research (1993) 53; 83-88; Dalton & Treisman, Cell (1992) 68; 597-612). These vectors contain the Murine Leukaemia virus (MLV) enhancer cloned upstream at a P-globin minimal promoter. The P3-globin 5' untranslated region up to the initiation ATG is supplied to direct efficient translation of the enzyme. Suitable promoters which may be used in vectors described above, include MLV, 15 CMV, RSV and adenovirus promoters. Preferred adenovirus promoters are the adenovirus early gene promoters. Strong mammalian promoters may also be suitable. An example of such a promoter is the EF-1 a promoter which may be obtained by reference to Mizushima and Nagata ((1990), Nucl. Acids Res. 18; 5322). Variants of such promoters retaining substantially similar transcriptional activities may also be used. 20 - Nitroreductase Compounds of the formula (I) or (II) can be activated by reduction of one (or more) of the available nitro groups by nitroreductase. Preferably, the enzyme is a non-mammalian nitroreductase enzyme, such as a bacterial nitroreductase. An E.coli nitroreductase as disclosed in WO93/08288 is particularly preferred. 25 The enzyme may be modified by standard recombinant DNA techniques, e.g. by cloning the enzyme, determining its gene sequence and altering the gene sequence by methods such as truncation, substitution, deletion or insertion of sequences for example by site-directed mutagenesis. Reference may be made to "Molecular Cloning" by Sambrook et al. (1989, Cold Spring Harbor) for discussion of standard recombinant DNA techniques. The modification 30 made may be any which still leaves the enzyme with the ability to reduce the nitro group in formula I or II but alters other properties of the enzyme, for example its rate of reaction or selectivity.
WO 00/64864 PCT/GB00/01612 - 36 In addition, small truncations in the N- and/or C-terminal sequence may occur as a result of the manipulations required to produce a vector in which a nucleic acid sequence encoding the enzyme is linked to the various other vector sequences. ADEPT 5 For applications in ADEPT systems, an antibody directed against a tumour specific marker is linked to the nitroreductase enzyme, which may be modified as described above. The antibody may be monoclonal or polyclonal. For the purposes of the present invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a tumour target antigen. Such fragments include Fv, 10 F(ab') and F(ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragments thereof may be humanised antibodies, e.g. as described in EP-A-239400. The antibodies may be produced by conventional hybridoma techniques or, in the case of modified antibodies or fragments, by recombinant DNA technology, eg by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment 15 operably linked to a promoter. Suitable host cells include bacterial (eg. E.coli), yeast, insect and mammalian. When the antibody is produced by such recombinant techniques the enzyme may be produced by linking a nucleic acid sequence encoding the enzyme (optionally modified as described above) to the 3' or 5' end of the sequence of the construct encoding the antibody or fragment thereof. 20 Applications of the invention Compounds of the invention can be used in vitro or in vivo for a range of applications. For example, a number of vector systems for the expression of nitroreductase in a cell have been developed. The further development of such systems (e.g. the development of promoters suitable for specific cell types) requires suitable candidate prodrugs capable of killing cells 25 when activated by nitroreductase. Prodrug compounds of the present invention may be used in such model systems. The model systems may be in vitro model systems or xenograft model systems comprising for example human tumour cells implanted in nude mice. Compounds of the invention which are not activatable by an enzyme may be tested in vitro against panels of different tumour cells types to determine efficacy against such tumour 30 cells. The efficacy of compounds of the invention against a range of tumour cell types may be used as points of reference for the development of further antitumour compounds. Compounds of the present invention may also be tested in combination with additional anti- WO 00/64864 PCT/GB00/01612 -37 cancer compounds to determine potential combination drug systems, for example combinations which are synergistic. The compounds of the invention may also be used in a method of treatment of the human or animal body. Such treatment includes a method of treating the growth of neoplastic 5 cells in a patient with neoplastic disease which comprises administering to a patient in need of treatment a compound of formula (I) or (II) of the invention as part of an ADEPT or GDEPT therapy system. Neoplastic diseases include leukaemia and solid tumours such as breast, bowel and lung tumours including small cell lung carcinoma. It will be understood that where treatment of tumours is concerned, treatment includes 10 any measure taken by the physician to alleviate the effect of the tumour on a patient. Thus, although complete remission of the tumour is a desirable goal, effective treatment will also include any measures capable of achieving partial remission of the tumour as well as a slowing down in the rate of growth of a tumour including metastases. Such measures can be effective in prolonging and/or enhancing the quality of life and relieving the symptoms of the disease. 15 ADEPT therapy The antibody/enzyme conjugate for ADEPT 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 or even 1 week before, in order to give the enzyme/agent conjugate an opportunity to localise in the region of the tumour target. By operating in this 20 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 the premature release of the compound produced by the action of the nitroreductase on the compound of formula (I) or (II) is minimised. In ADEPT the degree of localisation of the enzyme/agent conjugate (in terms of the 25 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 part of the conjugate so as to inactivate the enzyme and/or accelerate the clearance of the conjugate from 30 the blood. Such a component may include an antibody to the enzyme component of the system which is capable of inactivating the enzyme. The second component may be linked to a macromolecule such as dextran, a liposome, WO 00/64864 PCT/GB00/01612 -38 albumin, macroglobulin or a blood group O erythrocyte 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 covalently bound galactose residues, or residues of other sugars such as lactose or mannose, so that it can bind the conjugate in plasma 5 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. In ADEPT systems, the dose of the prodrug and conjugate will ultimately be at the 10 discretion of the physician, who will take into account such factors as the age, weight and condition of the patient. Suitable doses of prodrug and conjugate are given in Bagshawe et al. Antibody, Immunoconjugates, and Radiopharmaceuticals (1991), 4, 915-922. A suitable dose of conjugate may be from 500 to 200,000 enzyme units/m 2 (e.g. 20,000 enzyme units/m 2 ) and a suitable dose of prodrug may be from about 0.1 to 200 mg/Kg, preferably about from 10 to 15 100 mg/Kg per patient per day. In order to secure maximum concentration of the conjugate at the site of desired treatment, it is normally desirable to space apart administration of the two components by at least 4 hours. The exact regime will be influenced by various factors including the nature of the tumour to be targeted and the nature of the prodrug, but usually there will be an adequate 20 concentration of the conjugate at the site of desired treatment within 48 hours. The ADEPT system when used with nitroreductase also preferably comprises a suitable cofactor for the enzyme. Suitable cofactors include a riboside or ribotide of nicotinic acid or nicotinamide. The antibody/enzyme conjugate may be administered by any suitable route usually 25 used in ADEPT therapy. This includes parenteral administration of the antibody in a manner and in formulations similar to that described below. GDEPT therapy For use of the vectors in therapy, the vectors will usually be packaged into viral particles and the particles delivered to the site of the tumour, as described in for example Ram 30 et al. (ibid). The viral particles may be modified to include an antibody, fragment thereof (including a single chain) or tumour-directed ligand to enhance targeting of the tumour. Alternatively the vectors may be packaged into liposomes. The liposomes may be targeted to WO 00/64864 PCT/GB00/01612 - 39 a particular tumour. This can be achieved by attaching a tumour-directed antibody to the liposome. Viral particles may also be incorporated into liposomes. The particles may be delivered to the tumour by any suitable means at the disposal of the physician. Preferably, the viral particles will be capable of selectively infecting the tumour cells. By "selectively 5 infecting" it is meant that the viral particles will primarily infect tumour cells and that the proportion of non-tumour cells infected is such that the damage to non-tumour cells by administration of a prodrug will be acceptably low, given the nature of the disease being treated. Ultimately, this will be determined by the physician. One suitable route of administration is by injection of the particles in a sterile solution. 10 Viruses, for example isolated from packaging cell lines may also be administered by regional perfusion or direct intratumoral direction, or direct injection into a body cavity (intracaviterial administration), for example by intra-peritoneum injection. The exact dosage regime for GDEPT will, of course, need to be determined by individual clinicians for individual patients and this, in turn, will be controlled by the exact 15 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 normally involve parenteral administration of modified virus and administration by the intravenous route is frequently found to be the most practical. In GDEPT systems the amount of virus or other vector delivered will be such as to 20 provide a similar cellular concentration of enzyme as in the ADEPT system mentioned above. Typically, the vector will be administered to the patient and then the uptake of the vector by transfected or infected (in the case of viral vectors) cells monitored, for example by recovery and analysis of a biopsy sample of targeted tissue. This may be determined by clinical trials which involve administering a range of trial doses to a patient and measuring the degree of 25 infection or transfection of a target cell or tumour. The amount of prodrug required will be similar to or greater than that for ADEPT systems. In using a GDEPT system the prodrug will usually be administered following administration of the vector encoding an enzyme. Suitable doses of prodrug are from about 0.1 to 200 mg/Kg, preferably about from 10 to 100 mg/Kg per patient per day. 30 Administration of prodrug While it is possible for a compound of formula (I) or (II) to be administered alone it is preferable to present it as a pharmaceutical formulation. Suitable formulations comprise the WO 00/64864 PCT/GB00/01612 - 40 compounds, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier or carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof, for example, liposomes. Suitable liposomes include, for example, those comprising 5 the positively charged lipid (N[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those comprising dioleoylphosphatidylethanolamine (DOPE), and those comprising 3 [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). Formulations suitable for parenteral or intramuscular administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, 10 bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose containers, 15 for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Injection solutions and suspensions may be prepared extemporaneously from sterile powders, granules and tablets of the kind previously described. It should be understood that in addition to the ingredients particularly mentioned above 20 the formulations may include other agents conventional in the art having regard to the type of formulation in question. Of the possible formulations, sterile pyrogen-free aqueous and non aqueous solutions are preferred. The doses may be administered sequentially, eg. at daily, weekly or monthly intervals, or in response to a specific need of a patient. Preferred routes of administration are oral 25 delivery and injection, typically parenteral or intramuscular injection or intratumoral injection. 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 compound of formula (I) or (II) but some general guidance can be given. Typical dosage ranges generally will be those described above which may be administered in single or 30 multiple doses. Other doses may be used according to the condition of the patient and other factors at the discretion of the physician.
WO 00/64864 PCT/GB00/01612 -41 The following Examples illustrate the invention. General procedures Analyses were carried out in the Microchemical Laboratory, University of Otago, Dunedin, 5 NZ. Melting points were determined on an Electrothermal 2300 Melting Point Apparatus. IR spectra were recorded on a Midac FT-IR as KBr discs, unless otherwise stated. NMR spectra were obtained on a Bruker AM-400 spectrometer at 400 MHz for 'H and 100 MHz for 3 C spectra. Spectra were obtained in CDC13 unless otherwise specified, and are referenced to Me 4 Si. Chemical shifts and coupling constants were recorded in units ofppm and Hz, 10 respectively. Assignments were determined by APT, COSY, HSQC, and HMBC experiments. Mass spectra were determined on a VG-70SE mass spectrometer using an ionizing potential of 70 eV at a nominal resolution of 1000. High resolution spectra were obtained at nominal resolutions of 3000, 5000, or 10000 as appropriate. All spectra were obtained as electron impact (EI) using PFK as the reference unless otherwise stated. Solutions 15 in organic solvents were dried with anhydrous Na 2
SO
4 . Solvents were evaporated under reduced pressure on a Buchi rotary evaporator. Thin-layer chromatography was carried out on aluminium-backed silica gel plates (Merck 60 F 254 ) with visualisation of components by UV light (254 nm) or exposure to 12 . Column chromatography was carried out on silica gel, (Merck 230-400 mesh). All compounds designated for biological testing were analyzed at 20 >99% purity by reverse phase HPLC using a Philips PU4100 liquid chromatograph, a Phenomenex BondClone 10-C18 stainless steel column (300mm x 3.9 mm i.d.)and a Philips PU4120 diode array detector. Chromatograms were run using various gradients of aqueous (1 M NaH 2
PO
4 , 0.75 M heptanesulfonic acid, 0.5 M dibutylammonium phosphate, and MilliQ water in a 1:1:1:97 ratio) and organic (80% MeOH/MilliQ water) phases. DCM refers to 25 dichloromethane; DIEA refers to diisopropylethylamine, DMF refers to dry dimethyl formamide; DMSO refers to dimethylsulphoxide; ether refers to diethyl ether; EtOAc refers to ethyl acetate; EtOH refers to ethanol; iPr 2 0 refers to diisopropyl ether; light petroleum refers to petroleum ether, boiling range 40-60 oC; MeOH refers to methanol; THF refers to tetrahydrofuran dried over sodium benzophenone ketyl. All solvents were freshly distilled. 30 Example 1. Preparation of 4-nitrobenzyl 4-(1,3-benzothiazol-2-yl)phenylcarbamate (8). 4-Nitrophenyl chloroformate (0.15 g, 0.46 mmol) was added to a stirred solution of 2- WO 00/64864 PCT/GB00/01612 -42 (4-aminophenyl)benzthiazole (7) [D-F. Shi, T.D. Bradshaw, S. Wrigley, CJ. McCall, P. Lelieveld, I. Fitchner, M.F.G.Stevens. J. Med. Chem., 1996, 39, 3375] in pyridine (5 mL) and the solution stirred at 20 oC for 2 h. The solution was dilute with water (10 mL) and the mixture stirred for 40 min, filtered and the solid triturated with hot EtOH to give 8 (157 5 mg, 87%) as a pale green powder, mp 232-234 oC; 'H NMR [(CD 3
)
2 SO]8 10.31 (s, 1 H, OCONH), 8.29 (ddd, J= 8.7, 3.2, 2.2 Hz, 2 H, H 3", H 5"), 8.11 (d, J= 8.3 Hz, 1 H, H 4), 8.04 (br d, J= 8.7 Hz, 2 H, H 2", H 6"), 8.02 (d, J 8.3 Hz, 1 H, H 7), 7.72 (br d, J = 8.7 Hz, 2 H, H 2', H 6'), 7.68 (br d, J= Hz, 2 H, H 3', H 5'), 7.51-7.55 (m, 1 H, H 5), 7.40-7.46 (min, 1 H, H 6), 5.32 (s, 2 H, CH 2 0); Anal. (C 2 1 H1 5
N
3 0 4 S) C, H, N. 10 Example 2A. Preparation of 2-methoxy-4-nitrobenzyl 4-[bis(2 chloroethyl)aminolphenylcarbamate (32). Methyl 2-methoxy-4-nitrobenzoate (28). An ethereal solution of diazomethane (CAUTION) was added to a solution of 4-nitrosalicylic acid (27) (1.0 g, 5.46 mmol) in 15 ether (50 mL) until a yellow colour persisted and the solution stood at 20 oC for 4 h. The reaction was quenched with glacial acetic acid (2 mL), poured into sat. aq. NaHCO 3 solution and extracted with ether (2 x 50 mL). The combined organic fractions were dried and the solvent evaporated to give 28 (1.11 g, 96%) as white needles, mp (ether) 89-90 oC; IRN 1740, 1526, 1252, and 1086 cm-'; 'H NMR 6 7.89 (d, J= 8.3 Hz 1 H, H 5), 7.82-7.85 20 (m, 2 H, H 3, H 6), 4.01 (s, 3 H, OCH 3 ), and 3.94 (s, 3 H, OCH 3 ); 1 3 C NMR 165.2 (CO 2 ), 159.2 (C 2), 150.7 (C 4), 132.0 (C 1), 126.0 (C 6), 115.0 (C 5), 106.9 (C 3), 56.6 (OCH 3 ), and 52.6 (OCH 3 ); Anal. (C 9
H
9
NO
5 ) C, H, N. 2-Methoxy-4-nitrobenzyl alcohol (29). A solution of 28 (0.9 g, 4.26 mmol) in THF (20 25 mL) was added dropwise to a stirred solution of DIBALH (1 M solution in toluene, 13.4 mL, 13.4 mmol) in THF (20 mL) at 2 oC and the solution stirred at 2 oC for 15 min. The solvent was evaporated and residue partitioned between EtOAc (100 mL) and water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL) and the combined organic fraction dried and the solvent evaporated. The residue was purified by 30 chromatography, eluting with 50% EtOAc/light petroleum, to give 29 (0.74 g, 93%) as cream needles, mp (EtOAc/light petroleum) 103-104 oC; IR n 3310, 1523, 1250, and 1036 cm-'; 'HNMR 8 7.86 (dd, J= 8.3, 2.1 Hz, 1 H,H 5), 7.71 (d,J= 2.1 Hz, 1 H, H 3), 7.52 (d, WO 00/64864 PCT/GB00/01612 - 43 J= 8.3 Hz, 1 H, H 6), 4.76 (d, J= 5.5 Hz, 2 H, CH 2 0), 3.96 (s, 3 H, OCH 3 ), and 2.27 (br s, 1 H, OH); ' 3 C NMR 8 157.1, 148.3, 136.6, 127.9, 116.0, 105.0, 60.7, and 55.9; Anal. (CsH 9
NO
4 ) C, H, N. 5 2-Methoxy-4-nitrobenzyl 4-[bis(2-hydroxyethyl)amino ]phenylcarbamate (31). Pyridine (91 pL, 1.13 mmol) was added dropwise to a stirred solution of 29 (207 mg, 1.13 mmol) and triphosgene (117 mg, 0.40 mmol) in THF (10 mL) at 5 oC and the suspension stirred at 5 oC for 1 h. A solution of Ni,N'-bis(2-hydroxyethyl)-1,4-benzenediamine (57) [prepared by catalytic hydrogenation of NN-bis-(2-hydroxyethyl) 4-nitroaniline (30)] (244 mg, 1.24 10 mmol) with Pd/C under H 2 (60 psi) in EtOH) in THF (10 mL) and DMF (10 mL) was added and the mixture stirred at 20 oC for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc to give 31 (251 mg, 55%) as orange prisms, mp (EtOAc) 153-154 oC; 'H NMR 6 9.45 (br s, 1 H, OCONH), 7.90 (dd, J= 8.3, 2.0 Hz, 1 H, H 5'), 7.80 (d, J= 2.0 Hz, 1 H, H 3'), 7.60 (br d, J= 8.3 Hz, 1 H, H 6'), 7.21 15 (br d, J= 9.0 Hz, 2 H, H 2, H 6), 6.61 (d, J= 9.0 Hz, 2 H, H 3, H 5), 5.17 (s, 2 H, CH 2 0), 4.71 (t, J= 5.4 Hz, 2 H, 2 x OH), 3.97 (s, 3 H, OCH 3 ), 3.48-3.53 (m, 4 H, 2 x CH 2 0), 3.32 3.37 (m, 4 H, 2 x CH 2 N); 13C NMR 8 157.4, 153.7, 148.6, 144.6, 133.2, 129.0, 127.8, 120.7 (2), 116.0, 111.9 (2), 105.9, 60.6, 58.7 (2), 56.7, 53.9 (2); Anal. (C, 9
H
23
N
3 0 7 ) C, H, N. 20 2-Methoxy-4-nitrobenzyl 4-[bis(2-chloroethyl)amino]phenylcarbamate (32). Methanesulphonyl chloride (129 /zL, 1.67 mmol) was added dropwise to a stirred solution of 31 (226 mg, 0.55 mmol) in pyridine (10 mL) at 20 oC and the solution stirred for 1 h. The solvent was evaporated and the residue partitioned between DCM/water (100 mL). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction 25 washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiCl (0.15 g, 3.34 mmol) added, and the mixture stirred at 80 oC for 2 h. The solvent was evaporated and the residue partitioned between EtOAc/water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 25 mL). The combined organic fraction was washed with brine (30 mL), dried, and the solvent evaporated. The residue 30 was purified by chromatography, eluting with 25% EtOAc/light petroleum, to give 32 (230 mg, 93%) as pale green needles, mp (EtOAc/light petroleum) 129-130 oC; 'H NMR 8 7.84 (dd, J
=
8.3, 2.1 Hz, 1 H, H 5'), 7.72 (d,J
=
2.1 Hz, 1 H, H 3'), 7.51 (d,J
=
8.3 Hz, 1 H, H WO 00/64864 PCT/GB00/01612 - 44 6'), 7.27 (br d, J= 9.0 Hz, 2 H, H 2, H 6), 6.65 (ddd, J= 9.0, 3.5, 2.1 Hz, 2 H, H 3, H 5), 5.29, (s, 2 H, CH20), 3.96 (s, 3 H, OCH 3 ), 3.68-3.72 (min, 4 H, 2 x CH 2 N), 3.58-3.63 (min, 4 H, 2 x CH 2 C); 1 3 C NMR 6 157.2, 153.4, 148.6, 142.9, 132.2, 128.5, 128.1, 121.4 (2), 115.7, 112.7 (2), 105.3, 61.3, 56.0, 53.7 (2), 40.5 (2); Anal. (Cl 9
H
21 C1 2
N
3 0 5 ) C, H, N. 5 Example 2B. Preparation of 2-methoxy-4-nitrobenzyl 3-(chloromethyl)-1-[(5,6,7 trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-indol-6-ylcarbamate (34). Pyridine (20 pL, 0.25 mmol) was added dropwise to a stirred solution of 2-methoxy-4 nitrobenzyl alcohol (29) (45 mg, 0.25 mmol) and triphosgene (26 mg, 0.09 mmol) in THF 10 (10 mL) at 5 oC and the suspension stirred at 5 oC for 1 h. A solution of 6-amino-3 (chloromethyl)-1-[(5,6,7-trimethoxyindol-2-yl)carbonyl]indoline (33) [M. Tercel and W. A. Denny. J. Chem. Soc. Perkin Trans. 1, 1998, 509] (102 mg, 0.25 mmol) in THF (10 mL) was added and the mixture stirred at 20 'C for 16 h. The suspension was filtered and the solvent evaporated. The residue was purified by chromatography, eluting with 15 40%EtOAc/DCM, to give 34 (102 mg, 65%) as a tan powder, mp (DCM/pet. ether) 144 150 'C; 1 H NMR 8 9.74 (s, 1 H, indole-NH), 8.26 (d, J= 0.8 Hz, 1 H, H 7), 7.62-7.68 (min, 3 H, H 5, H 3", H 5"), 7.58 (br s, 1 H, OCONH), 7.35 (br d, J= 8.1 Hz, 1 H, H 6"), 7.20 (d, J = 8.3 Hz, 1 H, H 4), 6.91 (d, J= 2.1 Hz, 1 H, H 3'), 6.83 (s, 1 H, H 4'), 5.21 (s, 2 H, CH20), 4.58 (dd, J= 10.6, 8.9 Hz, 1 H, H 2), 4.41 (d, J= 10.6, 4.3 Hz, 1 H, H 2), 4.03, (s, 3 20 H, OCH 3 ), 3.92 (s, 3 H, OCH 3 ), 3.90 (s, 3 H, OCH 3 ), 3.89 (s, 3 H, OCH 3 ), 3.76-3.80 (min, 2 H, H 3, CH 2 C1), 3.51 (dd, J= 11.7, 10.6 Hz, 1 H, CH 2 Cl); 1 3 C NMR 6 160.5, 157.1, 153.1, 150.2, 148.4, 144.0, 140.5, 138.8, 138.7, 132.0, 129.5, 128.3, 126.0, 125.6, 124.5, 123.5, 115.6, 115.5, 108.8, 106.7, 105.5, 97.5, 61.4, 61.3, 61.1, 56.2, 55.9, 54.8, 46.9, 43.2; MS (FAB') m/z 627 (MH', 4%), 625 (MH', 12), 234 (25), 149 (100); HRMS (FAB') calc. for 25 C 30
H
30 3 5 C1N 4 0 1 0 (MH') m/z 625.1701, found 625.1690; C 3 0
H
30 37 C1N 4 0 1 0
(MH
+
) m/z 627.1672, found 627.1623; Anal. (C 30
H
29 C1N 4 0 9 ) C, H, N. Example 2C. Preparation of 2-methoxy-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7 trimethoxy-lH-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate 30 (35). Phosgene (300 gL, 0.3 mmol, 1M in toluene) was added to a stirred solution of 2 methoxy-4-nitrobenzyl alcohol (29) (20 mg, 0.11 mmol) in THF (10 mL) and stirred at 20 WO 00/64864 PCT/GB00/01612 - 45 oC for 16 h. The solvent was evaporated, the residue dissolved in THF (10 mL), a solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (50 mg, 0.11 mmol) in THF ( 10 mL) was added and the solution stirred at 20 oC for 4 days. The solvent was evaporated and the residue purified by chromatography, eluting 5 with 50% EtOAc/light petroleum to give 35 (31 mg, 43%) as a tan solid, mp (EtOAc/light petroleum) 162-165 oC; 'H NMR 6 9.52 (s, 1 H, indole-NH), 8.90 (s, 1 H, OCONH), 7.90 (d, J= 8.7 Hz, 1 H, H 6), 7.80 (d, J = 8.7 Hz, 1 H, H 5"), 7.77 (d, J= 8.4 Hz, 1 H, H 9), 7.70 (br s, 1 H, H 3"), 7.50-7.57 (min, 2 H, H 8, H 6"), 7.42-7.47 (min, 1 H, H 7), 7.25 (br s, 1 H, H 4), 6.99 (d, J= 2.2 Hz, 1 H, H 3'), 6.87 (s, 1 H, H 4'), 5.34 (d, J = 1.9 Hz, 2 H, 10 CH 2 0),4.78 (dd, J= 10.7, 1.6 Hz, 1 H, H 2), 4.64 (dd, J = 10.7, 8.8 Hz, 1 H, H 2), 4.07 4.17 (min, 5 H, H 1, CH 2 C1, OCH 3 ), 3.95 (s, 3 H, OCH 3 ), 3.94 (s, 3H, OCH 3 ), 3.91 (s, 3 H,
OCH
3 ), 3.45 (t, J = 10.9 Hz, 1 H, CH 2 Cl); ' 3 C NMR 8 160.3, 157.2, 154.0, 150.2, 148.6, 141.6, 140.6, 138.9, 133.9, 132.0, 129.7, 129.6, 128.8, 127.4, 127.2, 125.6, 125.4, 125.0, 123.6, 123.1, 123.0, 121.8, 122.4, 115.7, 106.5, 105.1, 97.6, 61.8, 61.5, 61.1, 56.2, 56.0, 15 54.9, 45.8, 43.4; MS (FAB') m/z 675 (MH', 10%), 677 (4), 659 (1), 639 (1), 517 (5), 234 (25); HRMS (FAB') calc. for C 35
H
32 35 ClN 4 0 9 (MHI) m/z 675.1858, found 674.1832; calc for C 35
H
3 2 3 7 C1N 4 0 9 (MH*) m/z 677.1828, found 677.1834; Anal. (C 34
H
3 1 C1N 4 0 9
.H
2 0) C, H, N. 20 Example 2D. Preparation of 2-methoxy-4-nitrobenzyl 4-(1,3-benzothiazol-2 yl)phenylcarbamate (36). Pyridine (36 mL, 0.44 mmol) was added dropwise to a stirred solution of alcohol of 2-methoxy-4-nitrobenzyl alcohol (29) (81 mg, 0.44 mmol) and triphosgene (66 mg, 0.22 mg) in DCM (10 mL) and the mixture was stirred at 20oC for 20 min. A solution of 2-(4-aminophenyl)benzthiazole (7) [D-F. Shi, T. D. Bradshaw, S. 25 Wrigley, C. J. McCall, P. Lelieveld, I. Fitchner, M. F. G. Stevens. J. Med. Chem., 1996, 39, 3375] (100 mg, 0.44 mmol) in DCM (5 mL) and the mixture stirred at 20 oC for 4 h. The mixture was partitioned between EtOAc (100 mL) and sat. aq. KHCO 3 solution (50 mL), the organic fraction dried and the solvent evaporated. The residue was slurried in warm EtOAc/MeOH (1:1, 20 mL), filtered and the solvent evaporated to give 36 (123 mg, 64%) 30 as a pale green powder mp (EtOH) 213-21 4 0 C; 'H NMR [(CD 3
)
2 SO] 8 10.31 (s, 1 H, OCONH), 8.11 (d, J= 7.8 Hz, 1 H, H 4), 8.01-8.06 (m, 3 H, H 7, H 2', H 6'), 7.92 (dd, J= 8.3, 2.2 Hz, 1 H, H 5"), 7.81 (d, J= 2.2 Hz, 1 H, H 3"), 7.65-7.69 (min, 3 H, H 3', H 5' H 6"), WO 00/64864 PCT/GB00/01612 - 46 7.51-7.55 (min, 1 H, H 5), 7.40-7.46 (min, 1 H, H 6), 5.27 (s, 2 H, CH 2 0) 3.98 (s, 3 H,
OCH
3
);'
3 C NMR [(CD3) 2 SO] 8 166.9, 157.1, 153.6, 152.9, 148.3, 141.8, 134.2, 132.0, 129.0 (2), 128.1, 127.0, 126.5, 125.1, 122.4, 122.1, 118.3 (2), 115.5, 105.5, 60.7, 56.2; Anal. (C 22
H,
7
N
3 5 OsS) C, H, N. 5 Example 2E. Preparation of 2-methoxy-4-nitrobenzyl doxorubicin carbamate (38). 2-Methoxy-4-nitrobenzyl 4-nitrophenyl carbonate (37). A solution of 4-nitrophenyl chloroformate (1.00 g, 4.97 mmol) in pyridine (4 mL) was added dropwise to a stirred solution of 2-methoxy-4-nitrobenzyl alcohol (29) (617 mg, 3.31 mmol) in pyridine (15 mL) 10 at 20 oC and the solution stirred for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (20-50%) EtOAc/light petroleum, to give 37 (928 mg, 80%) as pale yellow solid, mp (EtOAc/light petroleum) 105-106 oC; 'H NMR 8 8.28 (ddd, J= 9.2, 3.1, 2.1 Hz, 2 H, H 3'), 7.89 (dd, J= 8.3, 2.1 Hz, 1 H, H 5), 7.77 (d, J= 2.1 Hz, 1 H, H 3), 7.58 (d, J= 8.3 Hz, 1 H, H 6), 7.40 (ddd, J= 8.3, 3.1, 2.1 Hz, 2 H, 15 H 2'), 5.41 (s, 2 H, CH 2 0), 4.00 (s, 3 H, OCH 3 ); 13C NMR 8 157.6 (C 2), 155.4 (OCO 2 ), 152.3 (C 1), 149.2 (C 4), 145.5 (C 4'), 129.8 (C 1), 129.3 (C 6), 125.3 (C 2'), 121.7 (C 3'), 115.8 (C 5), 105.5 (C 3), 65.3 (CH 2 0), 56.2 (OCH 3 ); Anal. (C 15
HI
2
N
2 0s) C, H, N. 2-Methoxy-4-nitrobenzyl doxorubicin carbamate (38). A solution of carbonate 37 (23 20 mg, 66 mmol) in DMF (2 mL) was added to a solution of doxorubicin (13) (30 ming, 55 mmol) and Et 3 N (9 mL 66 mmol) in DMF (5 mL) at 20 oC and the solution stirred for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-5%) of MeOH/DCM, to give 38 (37 mg, 88%) as a red solid, mp (DCM) 159 161 oC; 'H NMR 5 13.97 (s, 1 H, 6-OH), 13.22 (s, 1 H, 11-OH), 8.02 (dd, J= 8.0, 1.0 Hz, 1 25 H, H 1), 7.77-7.81 (min, 2 H, H 2, H 5"), 7.66 (br s, 1 H, H 3"), 7.41 (d, J= 8.0 Hz, 1 H, H 6"), 7.39 (dd, J= 8.0, 1.0 Hz, 1 H, H 3), 5.52 (br d, J= 3.3 Hz, 1 H, H 1'), 5.29 (br s, 1 H, H 7), 5.25 (d, J= 8.7 Hz, 1 H, OCONH), 5.13 (2 d, J= 14.0 Hz, 2 H, CH 2 0), 4.75 ( s, 2 H, H 14), 4.51 (s, 1 H, 9-OH), 4.13-4.17 (m, 1 H, H 5'), 4.08 (s, 3 H, 4-OCH 3 ), 3.90 (s, 3 H, 2"-OCH 3 ), 3.84-3.88 (min, 1 H, H 3'), 3.69 (s, 1 H, H 4'), 3.24 (dd, J= 18.9, 1.3 Hz, 1 H, H 30 10), 3.03 (s, 1 H, 14-OH), 3.01 (d, J= 18.9 Hz, 1 H, H 10), 2.34 (br d, J= 14.7 Hz, 1 H, H 8), 2.18 (dd, J= 14.7, 4.0 Hz, 1 H, H 8), 2.02 (br s, 1 H, 4'-OH), 1.90 (dd, J= 13.2, 4.7 Hz, 1 H, H 2'), 1.79 (dd, J= 13.2, 3.3 Hz, 1 H, H 2'), 1.30 (d, J= 6.5 Hz, 3 H, H 6'); 1 3 C NMR WO 00/64864 PCT/GB00/01612 - 47 8 213.7 (C 13), 187.1 (C 5), 186.7 (C 12), 161.0 (C 4), 157.0 (C 2"), 156.1 (C 6), 155.6 (C 11), 155.2 (OCONH), 148.5 (C 4"), 135.8 (C 2), 135.5 (C12a), 133.5 (C 6a), 133.4 (C 10a), 132.5 (C 1"), 128.4 (C 6"), 120.8 (C 4a), 119.9 (C 1), 118.5 (C 3), 115.7 (C 5"), 111.6 (C 5a), 111.4 (C 1 la), 105.1 (C 3"), 100.7 (C 1'), 76.6 (C 9), 69.8 (C 7), 69.6 (C 4'), 67.2 (C 5 5'), 65.5 (C 14), 61.1 (CH 2 0), 56.7 (4-OCH 3 ), 56.0 (2'-OCH 3 ), 47.1 (C 3'), 35.6 (C 8), 34.0 (C 10), 30.2 (C 2), 16.8 (C 6'); MS (FAB') m/z 753 (MH', 0.3%); HRMS (FAB') calc. for
C
36
H
3 7
N
2 01 6 (MH) m/z 753.2143, found 753.2100; Anal (C 36
H
3 6
N
2 0 1 6 ) C, H, N. Example 2F. Preparation of 4-({[(2-methoxy-4 10 nitrobenzyl)oxy]carbonyl}amino)benzyl doxorubicin carbamate (42). 2-Methoxy-4-nitrobenzyl 4-( {[tert-butyl(dimethyl)silylloxy}methyl)phenylcarbamate (39). Et 3 N (0.40 mL, 2.84 mmol) was added to a stirred suspension of carbonate 37 (0.90 g, 2.58 mmol), 4-({[tert-butyl(dimethyl)silyl]oxy}methyl)aniline (9) (0.64 g, 2.71 mmol), HOBT (0.35 g, 2.58 mmol), and 4 A molecular sieves (900 mg) in THF (80 mL) and the 15 mixture stirred at 20 oC for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with 1 M HC1 (2 x 40 mL), water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 39 (0.89 g, 77%) as a white solid, mp (EtOAc/light petroleum) 120-122 'C; 'H NMR 8 20 7.84(dd, J=8.3,2.1 Hz, 1 H, H 5'),7.72(d,J=2.1 Hz, 1 H, H3'),7.51 (d,J=8.3Hz, 1 H, H 6'), 7.35 (d, J = 8.3 Hz, 2 H, H 2, H 6), 7.26 (d, J = 8.3 Hz, 2 H, H 3, H 5), 6.76 (br s, 1 H, OCONH), 5.30 (s, 2 H, CH 2 0), 4.69 (s, 2 H, CH 2 OSi), 3.93 (s, 3 H, OCH 3 ), 0.92 (s, 9 H, SiC(CH 3
)
3 ), 0.09 (s, 6 H, Si(CH 3 )2); 13C NMR 6 157.3 (C 2'), 153.0 (OCONH), 148.7 (C 4'), 137.0 (C 4), 136.4 (C 1), 132.1 (C 1'), 128.7 (C 6'), 126.9 (C 3, C 5), 118.6 (C 2, C 6), 25 115.7 (C 5'), 105.2 (C 3'), 64.6 (CH 2 0), 61.4 (CH 2 0), 56.0 (OCH 3 ), 26.9 (SiC(CH 3
)
3 ), 18.4 (SiC(CH 3
)
3 ), -5.2 (Si(CH 3
)
2 ); Anal. (C 22
H
30
N
2 06Si) C, H, N. 2-Methoxy-4-nitrobenzyl 4-(hydroxymethyl)phenylcarbamate (40). 1 M HCI (4 mL, 4 mmol) was added to a stirred solution of silyl ether 39 (0.89 g, 0.2 mmol) in MeOH (10 30 mL) and stirred at 20 oC for 1 h. The solution was poured into brine (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic fraction was washed with water (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting WO 00/64864 PCT/GB00/01612 -48 with a gradient (20-50%) EtOAc/light petroleum, to give 40 (628 mg, 95%) as a white solid, mp (EtOAc/light petroleum) 164-165 0 C; 'H NMR [(CD 3
)
2 SO] 6 9.83 (br s, 1 H, OCONH), 7.90 (dd, J= 8.3, 2.1 Hz, 1 H, H 5'), 7.80 (d, J= 2.1 Hz, 1 H, H 3'), 7.63 (d, J= 8.3 Hz, 1 H, H 6'), 7.41 (d, J= 8.4 Hz, 2 H, H 2, H 6), 7.22 (d, J= 8.4 Hz, 2 H, H 3, H 5), 5 5.21 (s, 2 H, CH 2 0), 5.07 (t, J= 5.6 Hz, 1 H, OH), 4.41 (t, J= 5.6 Hz, 2 H, CH 2 0), 3.97 (s, 3 H, OCH 3 ); 13 C NMR [(CD 3
)
2 SO] 8 157.0 (C 2'), 153.0 (OCONH), 148.2 (C 4'), 137.4 (C 4), 136.7 (C 1), 132.3 (C 1'), 128.8 (C 6'), 127.0 (C 3, C 5), 117.9 (C 2, C 6), 115.5 (C 5'), 105.4 (C 3'), 62.5 (CH 2 0), 60.4 (CH 2 0), 56.0 (OCH 3 ); Anal. (C 1 6
H
16
N
2 0 6 ) C, H, N. 10 4-({[(2-Methoxy-4-nitrobenzyl)oxyl]carbonyl}amino)benzyl 4-nitrophenyl carbonate (41). A solution of 4-nitrophenylchloroformate (205 mg, 1.02 mmol) in THF (5 mL) was added dropwise to a stirred solution of alcohol 40 (282 mg, 0.85 mmol) and Et 3 N (142 ,L, 1.02 mmol) in THF/DMF (1:1, 30 mL) the solution stirred at 20 oC for 16 h. The solvent was evaporated and the residue was purified by chromatography, eluting with 10% 15 EtOAc/DCM, to give 41 (238 mg, 56 %) as a white powder mp (EtOAc/DCM) 144-146 .C; 'H NMR [(CD 3
)
2 SO] 6 10.01 (s, 1 H, OCONH), 8.31 (ddd, J= 9.1, 3.4, 2.2 Hz, 2 H, H 3, H 5), 7.91 (dd, J= 8.3, 2.2 Hz, 1 H, H 5"), 7.81 (d, J= 2.2 Hz, 1 H, H 3"), 7.64 (d, J= 8.3 Hz, 1 H, H 6"), 7.56 (ddd, J= 9.1, 3.4, 2.2 Hz, 2 H, H 2, H 6), 7.53 (br d, J= 8.6 Hz, 2 H, H 3', H 5'), 7.41 (br d, J= 8.6 Hz, 2 H, H 2', H 6'), 5.24 (s, 4 H, 2 x CH 2 0), 3.98(s,3 20 H, OCH 3 ); ' 3 C NMR [(CD 3
)
2 SO] 5 157.0 (C 2"), 155.2 (OCO 2 ), 153.0 (OCONH), 151.9 (C 1), 148.2 (C 4"), 145.1 (C 4), 139.4 (C 1), 132.2 (C 1'), 129.6 (C 2', C 6'), 128.9 (C 6"), 128.5 (C 4'), 125.3 (C 2, C 6), 122.6 (C 3, C 5), 118.0 (C 3', C 5'), 115.5 (C 5"), 105.5 (C 3"), 70.2 (CH 2 0), 60.5 (CH 2 0), 56.2 (OCH 3 ); MS (FAB ) m/z 498 (MH+,0.5%); HRMS (FAB') calc. for C 2 3
H
20
N
3 010 (MH') m/z 498.1149, found 498.1151. Anal. (C 23
H
19 gN 3 0, 0 ) C, 25 H, N. 4-({[(2-Methoxy-4-nitrobenzyl)oxy]carbonyl}amino)benzyl doxorubicin carbamate (42). A solution of carbonate 41 (52 mg, 103 umol) in DMF (2 mL) was added dropwise to a stirred solution of doxorubicin (13) (45 mg, 86 gmol) and Et 3 N (15 gL, 103 gmol) in 30 DMF (5 mL) at 20 oC and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-5%) of MeOH/DCM, to give 42 (61 mg, 80%) as a red solid, mp (DCM) 128-131 oC; 'H NMR WO 00/64864 PCT/GB00/01612 - 49 [(CD 3
)
2 SO] 8 14.01 (s, 1 H, 6-OH), 13.25 (s, 1 H, 11-OH), 9.88 (s, 1 H, OCONH), 7.87 7.90 (m, 3 H, H 1, H 2, H 5"'), 7.79 (d, J = 2.2 Hz, I H, H 3"'), 7.59-7.63 (m, 2 H, H 3, H 6"'), 7.41 (d, J= 8.3 Hz, 2 H, H 3", H 5"), 7.22 (d, J= 8.3 Hz, 2 H, H 2", H 6"), 6.81 (d, J = 8.0 Hz, 1 H, OCONH), 5.44 (s, 1 H, H 7), 5.21 (d, J= 3.0 Hz, 1 H, H 1'), 5.19 (s, 2 H, 5 CH 2 0), 4.91-4.94 (m, 1 H, 9-OH), 4.87 (s, 2 H, CH 2 0), 4.83 (dd, J= 6.3, 5.9 Hz, 1 H, 14 OH), 4.69 (d, J = 5.7 Hz, 1 H, 4-OH), 4.58 (d, J = 6.0 Hz, 2 H, H 14), 4.12-4.18 (m, 1 H, H 5'), 3.97 (s, 3 H, OCH 3 ), 3.95 (s, 3 H, OCH 3 ), 3.68-3.75 (m, 1 H, H 3'), 3.43-3.47 (m, 1 H, H 4'), 2.99 (d, J= 18.4 Hz, 1 H, H 10), 2.92 (d, J= 18.4 Hz, 1 H, H 10), 2.20 (br d, J= 14.1 Hz, 1 H, H 8), 2.12 (dd, J= 14.1 Hz, 1 H, H 8), 1.85 (dt, J= 12.8, 3.7 Hz, 1 H, H 2'), 10 1.47 (dd, J= 12.8, 4.1 Hz, 1 H, H 2'), 1.13 (d, J= 6.5 Hz, 3 H, H 6'); 3 C NMR [(CD 3
)
2 SO] 8 213.7 (C 13), 186.4 (C 5), 186.3 (C 12), 160.7 (C 4), 157.0 (C 2'"), 156.0 (C 6), 155.2 (C 11), 154.4 (OCONH), 152.9 (OCONH), 148.2 (C 4'"), 138.4 (C 4"), 136.1 (C 2), 135.4 (C 12a), 134.6 (C 6a), 134.0 (C 10a), 132.2 (C 1"), 131.0 (C 1'"), 128.9 (C 2'"), 128.6 (C 2", C 6"), 119.9 (C 4a), 119.6 (C 1), 118.9 (C 3), 117.9 (C 3", C 5"), 115.4 (C 5'"), 110.7 (C 15 5a), 110.6 (C 1la), 105.4 (C 3'"), 100.2 (C 1'), 74.9 (C 9), 69.8 (C 7), 67.9 (C 4'), 66.6 (C 5'), 64.8 (C 14), 63.6 (CH 2 0), 60.4 (CH 2 0), 56.5 (OCH 3 ), 56.2 (OCH 3 ), 47.0 (C 3'), 36.5 (C 8), 32.0 (C 10), 29.7 (C 2'), 16.9 (C 6'); MS (FAB') m/z 902 (MH', 0.2%); Anal.
(C
44
H
4 3
N
3 0, 8
.H
2 0) C, H. N, calc 4.6, found 5.6%. 20 Example 2G. Preparation of 2-methoxy-4-nitrobenzyl 2-{[(9-amino-5-methyl-4 acridinyl)carbonyll amino}ethyl(methyl)carbamate (46). 2-Methoxy-4-nitrobenzyl methyl{2-[(trifluoroacetyl)aminol ethyl}carbamate (43). A solution of 2-methoxy-4-nitrobenzyl alcohol (29) (183 mg, 1.0 immol) and DIEA (0.19 mL, 1.2 mmol) in DCM (2 mL) was added dropwise to a solution of triphosgene (104 mg, 0.35 25 mmol) in DCM (1.5 mL) over 30 min at oC. The reaction was stirred at 5 oC for 1 h, then a solution of 2,2,2-trifluoro-N-[2-(methylamino)ethyl]acetamide trifluoroacetate (282 mg, 1.0 mmol) and DIEA (0.38 mL, 2.4 mmol) in DCM (2 mL) was added and the solution stirred at 20 oC for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/petroleum ether, to give 43 (371 mg, 94%) as a white solid, mp 30 107-109 C; 'H NMR 8 7.86 (dd,J= 8.4, 2.2 Hz, 1 H, H 5), 7.72 (d,J= 2.2Hz, 1 H, H 3), 7.56 (br s, 1 H, CONH), 7.44 (d, J = 8.4 Hz, 1 H, H 6), 5.23 (s, 2 H, CH 2 0), 3.95 (s, 3 H,
OCH
3 ), 3.58 (br s,4 H, 2 CH 2 N), 3.03 (s, 3 H, NCH 3 ); 13C NMR 5 157.7 (C 2), 157.4 (q, J WO 00/64864 PCT/GB00/01612 - 50 =37 Hz, COCF 3 ), 157.1 (OCON), 148.6 (C 4), 132.2 (C 1), 128.1 (C 6), 115.7 (C 5), 115.7 (q, J= 288 Hz, CF 3 ), 105.2 (C 3), 62.3 (CH 2 0), 56.0 (OCH 3 ), 47.9 (CH 2 N), 39.5 (CH 2 N), 35.0 (NCH 3 ); Anal. (C, 4
HI
6
F
3
N
3 0 6 ) C, H, N. 5 2-Methoxy-4-nitrobenzyl 2-aminoethyl(methyl)carbamate (44). A solution of carbamate 43 (948 mg, 2.5 mmol), Cs 2
CO
3 (4.0 g, 12.0 mmol) and water (5 mL) in methanol (20 mL) was stirred at 20 oC for 8 h. The pH was adjusted to 10 with 1 M HC1, water (50 mL) was added, and the solution was extracted with DCM (3 50 mL). The combined organic phase was dried and the solvent was evaporated to give 44 (578 mg, 83%) as a colorless oil which 10 was used directly, 'H NMR 8 7.85 (dd, J= 2.0, 8.2 Hz, 1 H, H 5), 7.72 (d, J= 2.0 Hz, 1 H, H 3), 7.47 (d, J= 8.2 Hz, 1 H, H 6), 5.23 (s, 2 H, CH 2 0), 3.95 (s, 3 H, OCH 3 ), 3.37-3.40 (m, 2 H, CH 2 ), 2.99-3.02 (m, 3 H, CH 3 N), 2.87-2.90 (m, 2 H, CH 2 ), NH 2 not observed; 1 3 C NMR 8 153.0, 148.5, 132.9, 128.3, 127.8, 115.7, 105.1, 61.7, 56.0, 52.2, 40.1, 35.2. 15 2-Methoxy-4-nitrobenzyl 2-{[(9-chloro-5-methyl-4 acridinyl)carbonyl]amino}ethyl(methyl)carbamate (45). A stirred suspension of 5 methyl-9-oxo-9,10-dihydro-4-acridinecarboxylic acid (16) (507 mg, 2.0 mmol) in SOC12 (30 mL) containing DMF (2 drops) was heated gently under reflux until homogeneous and then for a further 45 min. The solution was evaporated below 40 oC, and the residue 20 azeotroped with benzene. The residue was dissolved in DCM (30 mL), cooled to 5 oC, DIEA (1 mL, 6 mmol) and 44 added, and the solution stirred at 20 oC for 30 min. The solvent was evaporated and the residue purified by chromatography, eluting with 75% EtOAc/light petroleum, to give 45 (255 mg, 50 %) as a yellow solid, mp (EtOAc/light petroleum) 60-65 0 C; 'H NMR [(CD 3
)
2 SO] 6 11.92-11.96 (m, 1 H, NH), 8.91-9.00 (m, 1 H), 25 8.53-8.61 (m, 1 H), 8.26-8.29 (m, 1 H), 7.66-7.75 ( m, 2 H), 7.51-7.60 (m, 2 H), 7.17-7.30 (m, 2 H), 5.15 (s, 2 H, CH 2 0), 3.89 (s, 3 H, OCH 3 ), 3.72-3.75 (m, 2 H, CH 2 ), 3.62-3.65 (m, 2 H, CH 2 ), 3.09 (s, 3 H, CH 3 N), 2.75 (s, 3 H, CH 3 ); '3C NMR [(CD 3
)
2 SO] 6 165.9, 156.3, 146.6, 145.0, 143.3, 135.9, 135.6, 132.4, 131.6, 128.8, 128.7, 128.4, 128.2, 127.7, 127.5, 126.5, 124.2, 123.8, 123.0, 115.5, 104.3, 61.5, 55.8, 49.0, 38.0, 35.2, 18.9. 30 2-Methoxy-4-nitrobenzyl 2- { [(9-amino-5-methyl-4 acridinyl)carbonyl]amino}ethyl(methyl)carbamate (46). A solution of chloride 45 (100 WO 00/64864 PCT/GB00/01612 -51 mg, 0.17 mmol) in dry phenol (1.2 g, 13 mmol) was heated at 50 oC. Dry ammonia was bubbled through the solution while the temperature was raised from 50 to 120 0 C. Addition of ammonia was continued for 15 min, then the mixture was cooled and diluted with excess 40% aqueous NaOH. Prolonged cooling gave a solid that was purified by chromatography, 5 eluting with 20%MeOH/DCM, to give 46 (80 mg, 92 %) as a yellow solid, mp (MeOH/DCM) 245-248 °C; 'H NMR [(CD 3
)
2 SO] 8 12.72-12.74 (m, 1 H, NH), 8.55-8.66 (min, 2 H), 8.28-8.24 (min, 1 H), 8.10-8.14 (m, 2 H), 7.66-7.09 (min, 4 H), 5.00 (s, 2 H, CH 2 0), 3.73 (s, 3 H, OCH 3 ), 3.65 (min, 4 H, 2 CH 2 ), 2.95 (s, 3 H, CH 3 N), 2.51 (s, 3 H, CH 3 ); 1 3 C NMR [(CD 3
)
2 SO] 8 165.9, 155.7, 155.2, 152.2, 147.1, 146.2, 145.4, 134.3, 134.0, 132.2, 10 130.9, 127.2, 126.8, 126.6, 121.7, 121.1, 120.4, 114.7, 113.0, 111.7, 104.4, 60.8, 55.7, 48.4, 36.7, 34.3, 18.5. Anal. (C 2 7
H,
7 NsO 5 0 6 ) C, H, N. Example 2H. Preparation of 2-methoxy-4-nitrobenzyl bis(3-{[(5-methyl-4 acridinyl)carbonyl]amino}propyl)carbamate (51). 15 2,2,2-Trifluoro-N-[3-({3-[(trifluoroacetyl)amino]lpropyl}amino)propyllacetamide trifluoroacetate (47). Water (1.2 mL, 70 mmol) was added to a stirred solution of N-(3 aminopropyl)-1,3-propanediamine (4.0 g, 30.5 mmol) and ethyl trifluoroacetate (15.0 g, 105 mmol) in MeCN (60 mL) and the solution heated at reflux for 3 h. The solution was cooled, the solvent evaporated, and the residue was triturated with DCM (100 mL). The 20 suspension was filtered to give 47 (1.20 g, 90%) as white solid, mp (DCM) 175-178 oC; 'H NMR [(CD 3
)
2 SO] 8 9.55 (s, 2 H, NH 2 +), 8.45 (br s, 2 H, 2 CONH), 3.24-3.28 (m, 4 H, 2
CH
2 N), 2.90-2.94 (min, 4 H, 2 CH 2 N), 1.76-1.84 (m, 4 H, 2 CH 2 ); Anal. (Cl 2
H,
6
F
9
N
3 0 4 ) C, H,N. 25 2-Methoxy-4-nitrobenzyl 3-[(trifluoroacetyl)aminolpropyl(6,6,6-trifluoro-5 oxohexyl)carbamate (48). A solution of 2-methoxy-4-nitrobenzyl alcohol (29) (183 mg, 1.0 mmol) and DIEA (0.19 mL, 1.2 mmol) in DCM (2 mL) was added dropwise to a solution of triphosgene (104 mg, 0.35 mmol) in DCM (1.5 mL) over 30 minutes at 5 oC and the solution stirred for 1 h. The solution was added dropwise to a suspension of 30 bistrifluoroacetamide 47 (437 mg, 1.0 mmol) and DIEA (0.38 mL, 2.4 mmol) in DCM (2 mL) and the solution stirred at 20 'C for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/light petroleum, to give 48 (373 mg, WO 00/64864 PCT/GB00/01612 - 52 70%) as a white solid, mp (EtOAc/light petroleum) 133-135 0 C; 'H NMR [(CD 3
)
2 SO] 8 9.41 (s, 2 H, 2 CONH), 7.85 (dd, J= 8.0, 2.0 Hz, 1 H, H 5), 7.78 (d, J= 2.0 Hz, 1 H, H 3), 7.50 (d,J= 8.0 Hz, 1 H, H 6), 5.12 (s, 2 H, CH 2 0), 3.95 (s, 3 H, OCH 3 ), 3.15-3.35 (m, 8 H, 4
CH
2 N), 1.74 (m, 4 H, 2 CH 2 ). Anal. (C, 9
H
22
F
6
N
4 0 7 ) C, H, N. 5 2-Methoxy-4-nitrobenzyl bis(3-{[(5-methyl-4 acridinyl)carbonyll amino}propyl)carbamate (51). A solution of carbamate 48 (107 mg, 0.2 mmol), Cs 2
CO
3 (1.0 g, 3.0 mmol) and water (1 mL) in methanol (4 mL) was stirred at 20 oC for 8 hrs. The pH was adjusted to 10 with IM HC1, water (50 mL) added, and the 10 solution was extracted with DCM (3 50 mL). The combined organic fraction was dried, the solvent was evaporated to give crude 2-methoxy-4-nitrobenzyl bis(3 aminopropyl)carbamate 49). 4-(1H-Imidazol-1-ylcarbonyl)-5-methylacridine (50) [S. A. Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J. Med. Chem., 1999, 42, 2383-2393] (104 mg, 0.36 mmol) was added to a solution of the crude 15 carbamate 49 in THF (10 mL) at 5 oC and the reaction mixture was stirred at 20 oC for 8 hrs. The solvent was evaporated, and the residue was purified by chromatography on alumina-90, eluting with 2%MeOH/45%EtOAc/DCM, to give 51 (85 mg, 64%) as a yellow solid, mp (EtOAc/DCM) 88-90 oC; 'H NMR 8 11.87 (s, 1 H, NH), 11.81 (s, 1 H, NH), 8.90 (s, 2 H), 8.70 (s, 1 H), 8.67 (s, 1 H), 8.03 (m, 2 H), 7.78 (m, 2 H), 7.58 (m, 4 H), 7.40 (m, 2 20 H), 7.28 (d, J= 2.0 Hz, 1 H, H 3'"), 7.05 (d, J= 8.4 Hz, 1 H, H 6'"), 6.97 (dd, J= 8.4, 2.0 Hz, 1 H, H 5'"), 5.01 (s, 2 H, CH 2 0), 3.71 (m, 7 H), 3.58 (m, 4 H), 2.81 (s, 3 H, CH 3 ), 2.70 (s, 3 H, CH 3 ), 2.11 (m, 4 H); " 3 C NMR 8 166.1, 156.3, 155.6, 147.8, 146.9 (2), 146.7, 145.2, 145.1, 137.8, 135.7 (2), 135.3,135.1 (2), 132.3 (2), 132.2 (2), 130.9, 128.2, 127.9, 126.9 (2), 126.5, 126.3, 126.2, 126.1 (2), 125.7 (2), 125.3 (2), 115.1, 104.2, 61.6, 55.6, 45.8, 45.1, 25 37.7, 37.1, 29.2, 28.5, 18.9, 18.7; Anal. (C 45
H
42
N
6 0 7 . 2H 2 0) C, H, N. Example 21. Preparation of 2-methoxy-4-nitrobenzyl 2-[(5,8-dihydroxy-4-{[2 (methyl{ [(2-methoxy-4-nitrobenzyl)oxy] carbonyl} amino)ethyll amino}-9,10-dioxo 9,10-dihydro-1l-anthracenyl)amino]ethyl(methyl)carbamate (53). A solution of 1,4 30 difluoro-5,8-dihydroxyanthracene-9,10-dione (22) (1.0 g, 3.6 mmol) and 2-methoxy-4 nitrobenzyl 2-aminoethyl(methyl)carbamate (44) (0.8 g, 2.7 mmol) in pyridine (20 mL) was stirred at 20 oC for 48 h. The solvent was evaporated and residue was purified by WO 00/64864 PCT/GB00/01612 -53 column chromatography, eluting with a gradient (50-85%) of EtOAc/DCM, to give: (i) starting material (22) (0.15 g, 15%) and: (ii) 2-methoxy-4-nitrobenzyl 2-[(4-fluoro-5,8-dihydroxy-9,10-dioxo-9,10-dihydro-1 anthracenyl)amino]ethyl(methyl)carbamate (52) (0.54 g, 37%) as a purple solid mp 5 (EtOAc/DCM) 122-125 oC; 'H NMR 8 13.05-13.77 (m, 2 H, 2 x OH), 9.84-10.07 (br d, 1 H, NH), 6.99-7.83 (m, 7 H), 5.27 (s, 2 H, CH 2 0), 3.95 (s, 3 H, OCH 3 ), 3.63 (m, 4 H), 3.09 (s, 3 H, NCH 3 ); HRMS (FAB') calc. for C 26
H
22
FN
3 0 9 (M') m/z 539.1340, found 539.1331; and: (iii) 53 (120 mg, 5%) as a blue solid, 'H NMR 8 13.15-13.36 (m, 2 H, 2 x OH), 10.29 10 10.42 (m, 2 H, 2 x NH), 6.97-7.82 (m, 10 H), 5.26 (s, 2 H, CH 2 0), 5.12 (s, 2 H, CH 2 0), 3.95 (s, 3 H, OCH 3 ), 2.78 (s, 3 H, OCH 3 ), 3.50 (br, 8 H), 3.05 (s, 6 H, 2 x NCH 3 ); HRMS (FAB') calc. for C 38
H
3 8
N
6 014 (M
+
) m/z 802.2446, found 802.2446. Example 2J. Preparation of 2-methoxy-4-nitrobenzyl 2- { [5,8-dihydroxy-4-({2-[(2 15 hydroxyethyl)aminol ethyl}amino)-9,10-dioxo-9,10-dihydro-1 anthracenyll amino}ethyl(methyl)carbamate (55). A solution of fluoride 52 (0.54 g, 1.0 mmol) and 2-(2-aminoethylamino)ethanol (2.0 g, 19 mmol) in pyridine (20 mL) was stirred at 20 oC for 54 h. The solvent was evaporated and residue was purified by column chromatography, eluting with a gradient (50-85%) of 20 EtOAc/light petroleum followed by (2-10%) MeOH/EtOAc, to give: (i) 2-methoxy-4-nitrobenzyl 2- { [8,11-dihydroxy-4-(2-hydroxyethyl)-7,12-dioxo 1,2,3,4,7,12-hexahydronaphtho[2,3-J]quinoxalin-6-yl]amino } ethyl(methyl)carbamate (54) (0.1 g, 16%) as a blue solid; mp (DCMIlight petroleum) 221-224 oC; 'H NMR 5 13.43 14.25 (m, 2 H, 2 x OH), 10.89-11.29 (m, 2 H, 2 x NH), 6.96-7.77 (m, 6 H), 6.20 (s, 1 H), 25 4.80 (s, 2 H, CH20), 3.94 (s, 3 H, OCH 3 ), 3.65 (m, 12 H), 2.97 (s, 3 H, NCH 3 ); and: (ii) 55 (0.45 g, 72%) as a blue oil, 'H NMR 8 13.26-13.51 (m, 2 H, 2 x OH), 10.44-10.50 (m, 2 H, 2 x NH), 7.02-7.88 (m, 7 H), 5.28 (s, 2 H, CH 2 0), 3.96 (s, 3 H, OCH 3 ), 3.39-3.65 (m, 12 H), 3.07 (s, 3 H, NCH 3 ), NH, OH not observed; HRMS (FAB') calc. for
C
3 0
H
34 N501O 0
(MH
+
) m/z 624.2306, found 624.2297.. 30 Example 3A. Preparation of 1-(4-nitrophenyl)ethyl 4-[bis(2 chloroethyl)aminol ]phenylcarbamate (59).
WO 00/64864 PCT/GB00/01612 - 54 1-(4-Nitrophenyl)ethyl 4-[bis(2-hydroxyethyl)amino] phenylcarbamate (58). Pyridine (320 mL, 3.95 mmol) was added dropwise to a stirred solution of 1-(4-nitrophenyl)ethanol 56 (660 mg, 3.95 mmol) and triphosgene (410 mg, 1.38 mmol) in THF (50 mL) at 5 oC and the suspension stirred at 5 oC for 1 h. A solution ofN ,N-bis(2-hydroxyethyl)-1,4 5 benzenediamine (57) (prepared by catalytic hydrogenation of N,N-bis-(2-hydroxyethyl)-4 nitroaniline (30) (0.85 g, 4.34 mmol) with Pd/C under H 2 (60 psi) in EtOH) in THF (10 mL) and DMF (20 mL) was added and the mixture stirred at 20 oC for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc to give 58 (1.01 g, 66 %) as a brown oil, 'H NMR 6 8.19 (d, J= 8.8 Hz, 2 H, H 3', H 5'), 7.52 (br d, J 10 = 8.8 Hz, 2 H, H 2', H 6'), 7.16 (d, J= 9.0 Hz, 2 H, H 2, H 6), 6.69 (br s, 1 H, OCONH), 6.62 (d, J= 9.0 Hz, 2 H, H 3, H 5), 5.90 (q, J= 6.6 Hz, 1 H, CHO), 3.76-3.80 (min, 4 H, 2 x
CH
2 0), 3.55 (br s, 2 H, 2 x OH), 3.48-3.51 (min, 4 H, 2 x CH 2 N), 1.58 (d, J= 6.6 Hz, 3 H,
CH
3 ); ' 3 C NMR 8 153.1, 149.4, 147.4, 144.9, 127.3, 126.6 (2), 123.8 (2), 121.4 (2), 113.3 (2), 71.9, 60.6 (2), 55.3 (2), 22.5; MS (FAB') m/z 390 (MH', 25%); HRMS (FAB') calc. 15 for Cl 9
H
2 4
N
3 0 6 (MH') m/z 390.1665, found 390.1656. 1-(4-nitrophenyl)ethyl 4-[bis(2-chloroethyl)amino]phenylcarbamate (59). Methane sulphonyl chloride (600 pL, 7.7 mmol) was added dropwise to a stirred solution of 58 (1.0 g, 2.57 mmol) in pyridine (20 mL) at 20 'C and the solution stirred for 1 h. The solvent 20 was evaporated and the residue partitioned between DCM/water (100 mL). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in DMF (15 mL), LiC1 (0.65 g, 15.4 mmol) added, and the mixture stirred at 80 oC for 3 h. The solvent was evaporated and the residue partitioned between EtOAc/water (200 mL). The 25 aqueous fraction was extracted with EtOAc (2 x 50 mL). The combined organic fraction was washed with brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 25% EtOAc/light petroleum, to give 59 (1.0 g, 92 %) as a tan oil, 'H NMR 8 8.20 (ddd, J= 8.9, 2.2, 1.9 Hz, 2 H, H 3', H 5'), 7.53 (br d, J= 8.9 Hz, 2 H, H 2', H 6'), 7.23 (br d, J= 9.0 Hz, 2 H, H 2, H 6), 6.64 (ddd, J= 9.0, 3.4, 2.1 30 Hz, 2 H, H 3, H 5), 6.61 (br s, 1 H, OCONH), 5.92 (q, J= 6.6 Hz, 1 H, CHO), 3.67-3.71 (min, 4 H, 2 x CH 2 N), 3.58-3.62 (min, 4 H, 2 x CH 2 C1), 1.59 (d, J= 6.6 Hz, 3 H, CH 3 ); 13 C NMR 6 152.9, 149.3, 147.4, 142.9, 128.0, 126.6 (2), 123.8 (2), 121.4 (2), 112.7 (2), 71.9, WO 00/64864 PCT/GB00/01612 -55 53.6 (2), 40.4 (2), 22.4; MS (FAB') m/z 429 (M+, 5%), 427 (10), 425 (15); HRMS (FAB+) calc. for Cl 9
H
21 35 C1 2
N
3 0 4
(M
+
) m/z 425.0909, found 425.0901; calc. for C 19
H
21 35 C1 3 7 C1N 3 0 4 (M ) m/z 427.0880, found 427.0882; calc. for Cl 9
H
21 37 C1 2
N
3 0 4 (M ) m/z 429.0850, found 429.0868. 5 Example 3B. Preparation of 1-(4-nitrophenyl)ethyl 1-(chloromethyl)-3-[(5,6,7 trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (60). A solution of of 1-(4-nitrophenyl)ethanol (56) (18 mg, 0.11 mmol) in DCM (2 mL) was added dropwise to a stirred solution of triphosgene (16 mg, 0.054 mmol) and pyridine 10 (9 zL, 0.11 mmol) in DCM (2 mL) at 20 oC. The mixture was stirred at 20 oC for 2 h, the solvent evaporated and the residue dissolved in THF (5 mL). A solution of 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (50 mg, 0.11 mmol) in THF ( 5 mL) was added and the solution stirred at 20 oC for 16 h. The mixture was partitioned between EtOAc (50 mL) and sat. aq. KHCO 3 solution, the organic fraction dried 15 and the solvent evaporated. The residue was purified by chromatography, eluting with 25% EtOAc/light petroleum to give: (i) 60 (23 mg, 32%) as a tan solid mp (EtOAc/light petroleum) 175-178 'C; 'H NMR 8 9.49 (s, 1 H, indole-NH), 8.88 (s, 1 H, OCONH), 8.18 (d, J= 7.6 Hz, 2 H, H 3", H 5"), 7.88 (d, J =8.3 Hz, 1 H, H 6), 7.78 (d, J= 8.3 Hz, 1 H, H 9), 7.52-7.58 (min, 3 H, H 8, H 2", H 6"), 7.45 (dd, J = 7.8, 7.5 Hz, 1 H, H 7), 7.16 (br s, 1 H, 20 H 4), 7.00 (d, J= 1.90 Hz, 1 H, H 3'), 6.87 (s, 1 H, H 4'), 6.00 (q, J= 6.6 Hz, 1 H, CHO), 4.80 (dd, J= 10.7, 1.2 Hz, 1 H, H 2), 4.65 (dd, J= 10.7, 8.8 Hz, 1 H, H 2), 4.11-4.17 (m, 1 H, CH 2 Cl), 4.08 (s, 3 H, OCH 3 ), 3.93-3.97 (m, 4 H, OCH 3 , CH 2 Cl), 3.91 (s, 3 H, OCH 3 ), 3.45 (dt, J= 10.7, 3.3 Hz, 1 H, H 1), 1.65 (br d, J= 6.6 Hz, 3 H, CH 3 ); 1 3 C NMR 8 160.3, 153.4, 150.2, 149.0, 147.5, 141.6, 140.6, 138.9, 133.8, 130.4, 129.7, 129.6, 127.4, 126.8 25 (2), 125.6, 125.0, 123.9, 123.8 (2), 123.6, 123.1, 122.3, 121.7, 106.5, 97.6, 72.6, 61.5, 61.1, 56.3, 54.9, 45.8, 43.4, 22.6; MS (FAB') m/z 659 (MH', 6%), 658 (6), 510 (1), 234 (10); HRMS calc. for C 3 4
H
3 2 35 C1N 4 0 8 (MH ) m/z 659.1909, found 659.1881; calc for
C
34
H
3237 C1N 4 0 8 (MH ) m/z 661.1879, found 661.1882; Anal. (C 34
H
3 ,C1N 4 0s)C, H, N: and (ii) starting material (1) (30 mg, 60 %). 30 Example 3C. Preparation of 1-(2-methoxy-4-nitrophenyl)ethyl 4-[bis(2 chloroethyl)amino] phenylcarbamate (64).
WO 00/64864 PCT/GB00/01612 - 56 2-Methoxy-4-nitrobenzaldehyde (61). PCC (0.76 g, 3.52 mmol) and 4A molecular sieves (1.0 g) were added to a stirred solution of 2-methoxy-4-nitrobenzyl alcohol 29 (0.43 g, 2.35 mmol) in DCM (100 mL) and the suspension stirred at 20 oC for 1 h. The suspension was diluted with diethyl ether (150 mL) and the suspension filtered through Celite®, washed 5 with diethyl ether (2 x 20 mL). The combined organic fraction was evaporated and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 61 (0.42 g, 98%) as white crystals, mp (EtOAc/light petroleum) 117-119 oC; 'H NMR 6 10.52 (s, 1 H, CHO), 7.98 (br d, J= 8.2 Hz, 1 H, H 5), 7.85-7.89 (m, 2 H, H 3, H 6), 4.07 (s, 3 H,
OCH
3 ); ' 3 C NMR 8 188.2 (CHO), 161.8 (C 2), 152.2 (C 4), 129.5 (C 6), 128.6 (C 1), 115.6 10 (C 5), 107.2 (C 5), 56.4 (OCH 3 ); Anal. (CsH 7
NO
4 ) C, H, N. 1-(2-Methoxy-4-nitrophenyl)ethanol (62). A solution of MeMgBr (3 M in diethyl ether: 3.64 mL, 10.9 mmol) was added dropwise to a stirred solution of 61 (1.80 g, 9.93 mmol) in THF (100 mL) at -78 oC and the solution stirred at -78 oC for 20 min. The solution was 15 quenched with sat. aq NH4C1 solution (5 mL) and allowed to warm to 20 oC. The solvent was evaporated and the residue purified by chromatography, eluting with 20% EtOAc/light petroleum, to give (i) starting material (0.38 g, 21%) and (ii) 62 (0.88 g, 45%) as a white solid, mp (EtOAc/light petroleum) 63-65 oC; 'H NMR 6 7.86 (dd, J= 8.4, 2.1 Hz, 1 H, H 5), 7.70 (d,J= 2.1 Hz, 1 H, H 3), 7.58 (d,J = 8.4 Hz, 1 H, H 6), 5.19 (dq, J= 6.4, 4.7 Hz, 1 20 H, CHOCO), 3.96 (s, 3 H, OCH 3 ), 2.34 (d, J= 4.7 Hz, 1 H, OH), 1.48 (d, J= 6.4 Hz, 3 H,
CH
3 ); ' 3 C NMR 6 156.4 (C 2), 148.0 (C 4), 141.3 (C 1), 126.3 (C 6), 116.2 (C 5), 105.3 (C 3), 65.5 (CHOCO), 55.9 (OCH 3 ), 23.1 (CH 3 ); Anal. (C 9
HNO
4 ) C, H, N. 1-(2-Methoxy-4-nitrophenyl)ethyl 4-[bis(2-hydroxyethyl)aminolphenylcarbamate 25 (63). Pyridine (178 gL, 2.20 mmol) was added dropwise to a stirred solution of ethanol 62 (430 mg, 2.20 mmol) and triphosgene (229 mg, 0.77 mmol) in THF (50 mL) at 5 oC and the suspension stirred at 5 oC for 1 h. A solution of N',N'-bis(2-hydroxyethyl)-1,4 benzenediamine 57 (476 mg, 2.42 mmol) with Pd/C under H 2 (60 psi) in EtOH) in THF (10 mL) and DMF (10 mL) was added and the mixture stirred at 20 oC for 10 days. The solvent 30 was evaporated and the residue purified by chromatography, eluting with EtOAc to give 63 (860 mg, 93%) as a tan foam, 'H NMR 6 9.42 (br s, 1 H, OCONH), 7.91 (dd, J= 8.4, 2.1 Hz, 1 H, H 5), 7.79 (d, J= 2.1 Hz, 1 H, H 3), 7.60 (d, J= 8.4 Hz, 1 H, H 6), 7.18 (d, J= 9.0 WO 00/64864 PCT/GB00/01612 -57 Hz, 2 H, H 2', H 6'), 6.59 (d, J = 9.0 Hz, 2 H, H 3',H 5'), 6.00 (q, J = 6.5 Hz, 1 H, CHOCO), 4.69 (br s, 2 H, 2 x OH), 3.98 (s, 3 H, OCH 3 ), 3.48-3.53 (min, 4 H, 2 x CH 2 0), 3.32-3.37 (min, 4 H, 2 x CH 2 N), 1.46 (d, J = 6.5 Hz, 3 H, CH 3 ); 13 C NMR 5 155.7 (C 2), 152.6 (OCONH), 147.4 (C 4), 144.0 (C 4'), 138.7 (C 1'), 127.3 (C 1), 125.7 (C 6), 120.1 (C 5 2', C 6'), 115.8 (C 5), 111.4 (C 3', C 5'), 105.7 (C 3), 66.0 (CHOCO), 59.7 (2 x CH20), 58.2 (OCH 3 ), 53.4 (2 x CHzN), 20.8 (CH 3 ); MS (FAB') m/z 420 (MH
+
, 30%); HRMS (FAB') calc. for C 20
H
2 6
N
3 0 7 (MH') m/z 420.1771, found 420.1761. 1-(2-Methoxy-4-nitrophenyl)ethyl 4-[bis(2-chloroethyl)amino]phenylcarbamate (64). 10 Methanesulphonyl chloride (404 zL, 5.2 mmol) was added dropwise to a stirred solution of 63 (0.73 g, 1.74 mmol) in pyridine (20 mL) at 20 oC and the solution stirred for 1 h. The solvent was evaporated and the residue partitioned between DCM/water (100 mL). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in 15 DMF (20 mL), LiCl (0.44 g, 10.4 mmol) added, and the mixture stirred at 80 'C for 3 h. The solvent was evaporated and the residue partitioned between EtOAc/water (200 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL). The combined organic fraction was washed with brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 64 (0.53 g, 20 67 %) as a tan foam, 'H NMR 5 7.84 (dd, J= 8
.
4 , 1.9 Hz, 1 H, H 5), 7.71 (d, J= 1.9 Hz, 1 H, H 3), 7.50 (d, J= 8.4 Hz, 1 H, H 6), 7.24 (br d, J= 9.0 Hz, 2 H, H 2', H 6'), 6.64 (d, J= 9.0 Hz, 2 H, H 3', H 5'), 6.62 (br s, 1 H, OCONH), 6.19 (q, J= 6.5 Hz, 1 H, CHOCO), 3.95 (s, 3 H, OCH 3 ), 3.69 (dd, J= 7.0, 6.4 Hz, 4 H, 2 x CH 2 N), 3.60 (dd, J= 7.0, 6.4 Hz, 4 H, 2 x CH 2 C1), 1.52 (d, J= 6.5 Hz, 3 H, CH 3 ); 1 3 C NMR 5 156.0 (C 2), 152.8 (OCONH), 148.1 25 (C 4), 142.9 (C 4'), 138.5 (C 1'), 128.3 (C 1), 125.8 (C 6), 121.3 (C 2', C 6'), 116.0 (C 5), 112.8 (C 3', C 5'), 105.5 (C 3), 67.6 (CHOCO), 56.0 (OCH 3 ), 53.7 (2 x CH 2 N), 40.5 (2 x
CH
2 C1), 21.2 (CH 3 ); MS m/z 459 (M', 2%), 457 (M', 12), 455 (M', 16), 276 (20), 231 (100); HRMS calc. for C 20
H
23 35 C1 2
N
3 0 5 (M ) m/z 455.1015, found 455.1017; calc. for
C
20
H
23 35 C1 37 C1N 3 0 5
(M
+
) m/z 457.0985, found 457.0990; calc. for C 20
H
23 37 C1 2
N
3 0 5 (M ) m/z 30 459.0956, found 459.0972. Example 4A. Preparation of 2-(2-hydroxyethoxy)-4-nitrobenzyl 1-(chloromethyl)-3- WO 00/64864 PCT/GB00/01612 - 58 [(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5 ylcarbamate (70). 2-Bromoethyl tert-butyl(dimethyl)silyl ether (65). TBDMS triflate (5.0 mL, 21.8 mmol) was added dropwise to a stirred solution of bromoethanol (1.40 mL, 19.8 mmol) and 5 pyridine (2.4 mL, 29.7 mmol) in DCM (50 mL) at 5 'C and the solution stirred at 20 oC for 16 h. MeOH (5 mL) was carefully added, the solution stirred for 5 min and poured into sat. aq. KHCO 3 (150 mL). The mixture was extracted with DCM (3 x 80 mL), the combined organic fraction dried, and the solvent evaporated. Chromatography of the residue, eluting with 10% EtOAc/light petroleum, gave 65 (3.95 g, 83%) as a colourless oil, 'H NMR 6 10 3.88 (t, J= 6.6 Hz, 2 H, CH 2 0), 3.40 (t, J= 6.6 Hz, 2 H, CH 2 Br), 0.90 (s, 9 H, SiC(CH 3
)
3 ), 0.08 (s, 6 H, Si(CH 3
)
2 ); 1 3 C NMR 8 63.5 (CH20), 33.3 (CH 2 Br), 25.8 (SiC(CH 3
)
3 ), 18.3 (SiC(CH 3
)
3 ), -5.3 (Si(CH 3
)
2 ); MS (CI, NH 3 ) m/z 241 (MH', 1%), 239 (MH', 1%), 225 (2), 223 (2), 183 (55), 181 (55), 139 (100); HRMS (CI, NH 3 ) calc for C 8
H
20 79 BrOSi (MH
+
) m/z 239.0467; found 239.0460; calc for CsH 20 81 BrOSi (MH
+
) m/z 241.0446; found 241.0450. 15 Methyl 2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethoxy)-4-nitrobenzoate (67). A mixture of methyl 2-hydroxy-4-nitrobenzoate (66) (0.55 g, 2.79 mmol) and K 2
CO
3 (0.58 g, 4.19 mmol) in DMF (15 mL) was stirred at 20 oC for 30 min. A solution of 65 (1.00 g, 4.19 mmol) in DMF (5 mL) was added and the mixture was stirred at 100 'C for 4 h. The 20 solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography,eluting with 20% EtOAc/light petroleum, to give 67 (0.76 g, 77%) as a white solid, mp (EtOAc/light petroleum) 47-48 oC; 'H NMR 6 7.89 (d, J= 1.3 Hz, 1 H, H 3), 7.81-7.85 (m, 2 H, H 5, H 25 6), 4.24 (t, J= 5.0 Hz, 2 H, CH 2 0), 4.03 (t, J= 5.0 Hz, 2 H, CH 2 0), 3.92 (s, 3 H, OCH 3 ), 0.88 (s, 9 H, SiC(CH 3
)
3 ), 0.08 (s, 6 H, Si(CH 3
)
2 ); 13C NMR (CDCl 3 ) 8 165.5 (CO 2 ), 158.6 (C 2), 150.4 (C 4), 131.8 (C 6), 126.7 (C 1), 115.0 (C 5), 108.3 (C 3), 71.1 (CH 2 0), 61.7
(CH
2 0), 52.5 (OCH 3 ), 25.7 (SiC(CH 3
)
3 ), 18.3 (SiC(CH 3
)
3 ), -5.5 (Si(CH 3
)
2 ); Anal. (Cl 6
H
25
NO
6 Si) C, H, N. 30 [2-(2- { [tert-Butyl(dimethyl)silyl]oxy}ethoxy)-4-nitrophenyl]methanol (68). DIBALH (1M in DCM, 6.7 mL, 6.7 mmol) was added dropwise to a stirred solution of ester 67 (0.72 WO 00/64864 PCT/GB00/01612 - 59 g, 2.02 mmol) in THF (50 mL) at 5 oC and the solution stirred at 5 oC for 1 h. The solution was poured into a solution of potassium sodium tartrate (1 M, 50 mL) and stirred vigorously for 30 min. The mixture was extracted with EtOAc (3 x 50 mL), the combined organic fraction washed with water (50 mL), brine (50 mL), dried and the solvent was 5 evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 68 (0.64 g, 97%) as a white solid, mp (EtOAc/light petroleum) 89-90 oC; 'H NMR 8 7.85 (dd, J= 8.2, 2.1 Hz, 1 H, H 5), 7.74 (d, J= 2.1 Hz, 1 H, H 3), 7.46 (d, J = 8.2 Hz, 1 H, H 6), 4.75 (s, 2 H, CH 2 0), 4.21 (dd, J= 4.9, 4.4 Hz, 2 H, CH 2 0), 4.01 dd, J = 4.9, 4.4 Hz, 2 H, CH 2 0), 2.84 (br s, 1 H, OH), 0.90 (s, 9 H, SiC(CH 3
)
3 ), 0.06 (s, 6 H, 10 Si(CH 3
)
2 ); 13C NMR 6 157.0 (C 2), 148.3 (C 4), 137.1 (C 1), 128.4 (C 6), 116.3 (C 5), 106.8 (C 3), 70.5 (CH20), 61.6 (CH 2 0), 61.3 (CH 2 0), 25.8 (SiC(CH 3
)
3 ), 18.3 (SiC(CH 3
)
3 ), -5.4 (Si(CH 3
)
2 ); Anal. (C 1 5
H
25 NOsSi) C, H, N. 2-(2- {[tert-Butyl(dimethyl)silylloxy}ethoxy)-4-nitrobenzyl 1-(chloromethyl)-3- [(5,6,7 15 trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (69). A solution of triphosgene (24 mg, 80 Mmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (107 mg, 230 /zmol) and Et 3 N (64 gL, 460 gmol) in DCM (20 mL) and stirred at 20 oC for 2 h. A solution of [2-(2- {[tert-butyl(dimethyl)silyl]oxy}ethoxy)-4 20 nitrophenyl]methanol (68) (83 mg, 253 gmol) in DCM (5 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-20%) EtOAc/DCM, to give 69 (177 mg, 94%) as a white solid, mp (EtOAc/light petroleum) 182 185 oC; 'H NMR 8 9.45 (s, 1 H, indole-NH), 8.93 (s, 1 H, OCONH), 7.92 (d, J= 8.5 Hz, 1 25 H, H 6), 7.76-7.83 (min, 3 H, H 9, H 3", H 5"), 7.53-7.60 (min, 2 H, H 8, H 6"), 7.47 (ddd, J= 8.5, 7.1, 0.8 Hz, 1 H, H 7), 7.13 (br s, 1 H, H 4), 7.01 (d, J= 2.2 Hz, 1 H, H 3'), 6.88 (s, 1 H, H 4'), 5.39 (s, 2 H, CH 2 0), 4.81 (dd, J = 10.7, 1.8 Hz, 1 H, H 2), 4.67 (dd, J= 10.7, 8.7 Hz, 1 H, H 2), 4.21 (br dd, J= 5.0, 4.8 Hz, 2 H, CH 2 0), 4.15-4.18 (m, 1 H, H 1), 4.09 (s, 3 H, OCH 3 ), 4.02 (br d, J1= 5.0, 4.8 Hz, 2 H, CH 2 0), 3.97 (d, J= 11.5, 3.1 Hz, 1 H, CH 2 CI), 30 3.95 (s, 3 H, OCH 3 ), 3.92 (s, 3 H, OCH 3 ), 3.48 (dd, J= 11.5, 10.9 Hz, 1 H, CH 2 C1), 0.90 (s, 9 H, SiC(CH 3
)
3 ), 0.10 (s, 6 H, Si(CH 3
)
2 ); 1 3 C NMR 6 160.3 (CO), 156.7 (C 2"), 154.0 (OCONH), 150.2 (C 5'), 148.5 (C 4"), 141.7 (C 3a), 140.6 (C 6'), 138.9 (C 7'), 133.8 (C WO 00/64864 PCT/GB00/01612 - 60 5), 132.3 (C 1"), 129.7 (C 2'), 129.6 (C 9a), 128.7 (C 6"), 127.5 (C 8), 125.6 (C 7a'), 125.0 (C 7), 123.6 (C 3a'), 123.1 (C 9), 122.4 (C 6, C 9b), 121.8 (C 5a), 115.8 (C 5"), 112.8 (C 4), 106.5 (C 3', C 3"), 97.7 (C 4'), 70.7 (CH 2 0), 61.8 (CH 2 0), 61.7 (CH 2 0), 61.5 (OCH 3 ), 61.1 (OCH 3 ), 56.3 (OCH 3 ), 54.9 (C 2), 45.8 (CH 2 C1), 43.5 (C 1), 25.8 (SiC(CH 3
)
3 ), 18.3 5 (SiC(CH 3
)
3 ), -5.4 (Si(CH 3
)
2 ); MS (FAB') m/z 819 (MH
+
, 25%), 821 (MH
+
, 12); HRMS (FAB') calcd for C 41
H
48 35 C1N 4 OioSi (MH) m/z 819.2828, found 819.2804; calc. for
C
41
H
48 37 C1N 4 01 0 Si (MH
+
) m/z 821.2799, found 821.2803; Anal. (C 41
H
47 C1N 4 01 0 Si) C, H, N. 10 2-(2-Hydroxyethoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2 yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (70). 1 M HC1 (0.4 mL, 400 gmol) was added to a stirred solution of silyl ether 69 (157 mg, 192 /mol) in MeOH (5 mL) and the solution stirred at 20 oC for 1 h. The solvent was evaporated and the residue partitoned between EtOAc (50 mL) and water (50 mL). The organic fraction was washed 15 with water (50 mL), brine (25 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) of EtOAc/light petroleum, to give 70 (119 mg, 88%) as a hygroscopic white solid, 'H NMR 8 9.72 (s, 1 H, indole-NH), 8.80 (s, 1 H, OCONH), 7.86 (d, J= 8.5 Hz, 1 H, H 6), 7.79 (br d, J= 8.1 Hz, 1 H, H 5"), 7.67-7.73 (m, 2 H, H 9, H 3"), 7.47-7.53 (m, 3 H, H 4, H 8, H 6"), 7.37 (ddd, J= 8.5, 7.1, 20 0.8 Hz, 1 H, H 7), 6.97 (d, J= 2.2 Hz, 1 H, H 3'), 6.87 (s, 1 H, H 4'), 5.40 (s, 2 H, CH20), 4.73 (dd, J= 10.7, 1.6 Hz, 1 H, H 2), 4.59 (dd, J= 10.7, 8.7 Hz, 1 H, H 2), 4.21 (br dd, J= 4.6, 4.0 Hz, 2 H, CH20), 4.07-4.11 (m, 4 H, H 1, OCH 3 ), 4.00-4.04 (m, 2 H, CH 2 0), 3.95 (s, 3 H, OCH 3 ), 3.92 (s, 3 H, OCH 3 ), 3.85 (d, J= 11.3, 3.0 Hz, 1 H, CH 2 C1), 3.39 (br s, 1 H, OH), 3.28 (dd, J= 11.3, 10.9 Hz, 1 H, CH 2 Cl); 13C NMR 5 160.5 (CO), 157.2 (C 2"), 154.4 25 (OCONH), 150.2 (C 5'), 148.9 (C 4"), 141.4 (C 3a), 140.6 (C 6'), 138.9 (C 7'), 133.8 (C 5), 131.9 (C 1"), 130.3 (C 6"), 129.6 (C 2', C 9a), 127.4 (C 8), 125.8 (C 7a'), 125.0 (C 7), 123.6 (C 3a'), 123.0 (C 9), 122.5 (C 6, C 9b), 121.9 (C Sa), 115.9 (C 5"), 112.8 (C 4), 106.7 (C 3'), 106.6 (C 3"), 97.7 (C 4'), 70.7 (CH 2 0), 62.0 (CH 2 0), 61.5 (OCH 3 ), 61.1
(OCH
3 ), 60.9 (CH 2 0), 56.3 (OCH 3 ), 55.1 (C 2), 45.6 (CH 2 C1), 43.3 (C 1); MS (FAB') m/z 30 707 (MH
+
, 5%), 705 (MH', 14); HRMS (FAB') called for C 3 5
H
34 35 C1N 4 0Io (MH
+
) m/z 705.1964, found 705.1919; calc. for C 35
H
34 37
CIN
4 01 0
(MH
+
) m/z 707.1934, found 707.1931; Anal. (C 35
H
3 3 C1N 4 0o) C, H, N.
WO 00/64864 PCT/GB00/01612 -61 Example 4B. Preparation of 2-(2-methoxyethoxy)-4-nitrobenzyl 1-(chloromethyl)-3 [(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5 ylcarbamate (73). 5 Methyl 2-(2-methoxyethoxy)-4-nitrobenzoate (71). A mixture of methyl 2-hydroxy-4 nitrobenzoate (66) (1.0 g, 5.07 mmol) and K 2
CO
3 (1.05 g, 7.61 mmol) in DMF (25 mL) was stirred at 20 oC for 30 min. A solution of 2-bromoethyl methyl ether (0.72 mL, 7.61 mmol) in DMF (3 mL) was added and the mixture was stirred at 100 oC for 4 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The 10 organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography,eluting with 30% EtOAc/light petroleum, to give 71 (1.27 g, 98%) as a white solid, mp (EtOAc/light petroleum) 45-46 0 C; 'H NMR 8 7.90 (d, J= 8.3 Hz, 1 H, H 6), 7.82-7.86 (m, 2 H, H 3, H 5), 4.28-4.30 (m, 2 H, CH 2 0), 3.93 (s, 3 H, OCH 3 ), 3.82-3.87 (m, 2 H, CH 2 0), 3.48 (s, 3 H, OCH 3 ); 1 3 C NMR 8 15 165.1 (CO 2 ), 158.6 (C 2), 150.6 (C 4), 132.1 (C 6), 126.4 (C 1), 115.2 (C 5), 108.4 (C 3), 70.5 (CH 2 0), 69.4 (CH 2 0), 59.4 (OCH 3 ), 52.5 (OCH 3 ); Anal. (ClH, 3
NO
6 ) C, H, N. 2-[2-(Methoxy)ethoxy]-4-nitrobenzyl alcohol (72). DIBALH (1 M in DCM, 16.4 mL, 16.4 mmol) was added dropwise to a stirred solution of ester 71 (1.27 g, 4.97 mmol) in 20 THF (100 mL) at 5 oC and the solution stirred at 5 oC for 1 h. The solution was poured into a solution of potassium sodium tartrate (1 M, 100 mL) and stirred vigorously for 30 min. The mixture was extracted with EtOAc (2 x 100 mL), the combined organic fraction washed with water (50 mL), brine (50 mL), dried and the solvent was evaporated. The residue was purified by chromatography, eluting with a gradient (30-50%) of EtOAc/light 25 petroleum, to give 72 (1.03 g, 91%) as a white solid, mp (EtOAc/light petroleum) 89-90.5 0 C; 1 H NMR 8 7.86 (dd, J= 8.2, 2.1 Hz, 1 H, H 5), 7.72 (d, J= 2.1 Hz, 1 H, H 3), 7.47 (d, J = 8.2 Hz, 1 H, H 6), 4.74 (br s, 2 H, CH 2 0), 4.26-4.29 (m, 2 H, CH 2 0), 3.78-3.80 (m, 2 H, CHzO), 3.45 (s, 3 H, OCH 3 ), 3.10 (br s, 1 H, OH); 1 3 C NMR 6 156.8 (C 2), 148.2 (C 4), 137.5 (C 1), 128.6 (C 6), 116.5 (C 5), 107.0 (C 3), 70.5 (CH 2 0), 68.4 (CH 2 0), 61.1 (CH20), 30 59.1 (OCH 3 ); Anal. (Cl 0
H
1 3 NOs) C, H, N. 2-(2-Methoxyethoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2- WO 00/64864 PCT/GB00/01612 - 62 yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (73). A solution of triphosgene (12 mg, 40 gmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (53 mg, 114 kmol) and Et 3 N (32 pL, 228 pmol) in DCM (10 mL) and stirred at 20 5 'C for 2 h. A solution of 2-[2-(methoxy)ethoxy]-4-nitrobenzyl alcohol 72 (28 mg, 125 /lmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20% EtOAc/DCM, to give 73 (75 mg, 91%) as a tan gum, 'H NMR 5 9.49 (s, 1 H, indole-NH), 8.19 (s, 1 H, OCONH), 7.92 (d, J= 8.5 Hz, 1 H, H 6), 10 7.80-7.82 (m, 1 H, H 5"), 7.78 (d, J= 8.3 Hz, 1 H, H 9), 7.71 (d, J= 1.8 Hz, 1 H, H 3"), 7.56 (ddd, J= 8.3, 7.1, 0.8 Hz, 1 H, H 8), 7.49-7.54 (m, 1 H, H 6"), 7.45 (ddd, J= 8.5, 7.1, 0.8 Hz, 1 H, H 7), 7.27 (br s, 1 H, H 4), 7.00 (d, J= 2.3 Hz, 1 H, H 3'), 6.87 (s, 1 H, H 4'), 5.39 (s, 2 H, CH 2 0), 4.79 (dd, J= 10.7, 1.7 Hz, 1 H, H 2), 4.66 (dd, J= 10.7, 8.7 Hz, 1 H, H 2), 4.23 (dd, J= 4.6, 4.4 Hz, 2 H, CH20), 4.15-4.20 (m, 1 H, H 1), 4.08 (s, 3 H, OCH 3 ), 15 3.94-3.98 (m, 4 H, OCH 3 , CH 2 C1), 3.91 (s, 3 H, OCH 3 ), 3.80 (dd, J= 4.6, 4.4 Hz, 2 H,
CH
2 0), 3.47 (d, J= 10.9 Hz, 1 H, CH 2 C1), 3.44 (s, 3 H, OCH 3 ); 13C NMR 6 160.3 (CO), 156.5 (C 2"), 154.0 (OCONH), 150.2 (C 5'), 148.5 (C 4"), 141.7 (C 3a), 140.6 (C 6'), 138.9 (C 7'), 133.9 (C 5), 132.4 (C 1"), 129.7 (C 2'), 129.6 (C 9a), 128.7 (C 6"), 127.5 (C 8), 125.6 (C 7a'), 125.0 (C 7), 123.6 (C 3a'), 123.1 (C 9), 122.5 (C 6, C 9b), 121.8 (C 5a), 20 116.0 (C 5"), 112.7 (C 4), 106.5 (C 3'), 106.3 (C 3"), 97.7 (C 4'), 70.7 (CH 2 0), 68.5 (CH20), 61.9 (CH20), 61.5 (OCH 3 ), 61.1 (OCH 3 ), 59.3 (OCH 3 ), 56.3 (OCH 3 ), 54.9 (C 2), 45.9 (CH 2 C1), 43.1 (C 1); MS (FAB') m/z 721 (MH
+
, 1.5%), 719 (MH', 3.5); HRMS (FAB') calc. for C 3 6
H
3 6 37 C1N 4 0 1 0
(MH
+
) m/z 721.2091, found 721.2131; calc. for
C
36
H
36 35 C1N 4 01 0
(MH
+
) m/z 719.2120, found 719.2133; Anal. (C 36
H
35 C1N 4 01 0 ) C, H, N. 25 Example 4C. Preparation of 1-[2-(3-hydroxypropoxy)-4-nitrophenyl] ethyl 4-[bis(2 chloroethyl)amino]phenylcarbamate (78). Methyl 4-nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzoate (74). A mixture of methyl 4-nitrosalicylate (66) (2.3 g, 11.7 mmol) and K 2
CO
3 (2.42 g, 17.5 mmol) in DMF 30 (25 mL) was stirred at 20 oC for 20 min. A solution of 3-iodopropyl tetrahydropyranyl ether (4.7 g, 17.5 mmol) in DMF (5 mL) was added and the mixture stirred at 100 oC for 2 h. The mixture was poured into water, extracted with EtOAc (3 x 100 mL), the combined WO 00/64864 PCT/GB00/01612 - 63 organic extracts washed with water (2 x 50 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 74 (3.66 g, 92 %) as a colourless oil, 'H NMR 8 7.89 (d, J= 8.5 Hz, 1 H, H 6), 7.80-7.84 (min, 2 H, H 3, H 5), 4.60-4.62 (min, 1 H, OCHO), 4.27 (t, J = 6.2 Hz, 2 H, 5 CH 2 0), 3.95-4.00 (min, 1 H CH 2 0), 3.94 (s, 3 H, OCH 3 ), 3.79-3.86 (min, 1 H, CH 2 0), 3.59 3.66 (min, 1 H, CH 2 0), 3.47-3.52 (min, 1 H, CH 2 0), 2.13-2.17 (min, 2 H, CH 2 ), 1.78-1.84 (min, 1 H, CH 2 ), 1.68-1.75 (min, 1 H, CH 2 ), 1.47-1.62 (min, 4 H, 2 CH 2 ); 1 3 C NMR 5 164.5, 158.6, 150.7, 132.0, 126.2, 114.8, 107.9, 99.0, 66.5, 63.4, 62.4, 52.5, 30.6, 29.3, 25.4, 19.6; MS m/z 339 (M-, 2%), 322 (12), 239 (20), 222(40), 85 (100); HRMS calc. for C, 6
H
21
NO
7
(M
+
) 10 m/z 339.1318, found 339.1317. {4-Nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]phenyl}methanol (75). DIBALH (1 M in DCM, 34 mL, 34 mmol) was added dropwise to a solution of 74 (3.46 g, 10.2 mmol) in THF (100 mL) at 5 oC and the solution stirred at 5 oC for 1 h. The solution was 15 poured into a solution of sodium potassium tartrate (1 M, 100 mL) and stirred for 30 min. The mixture was extracted with EtOAc (3 x 100 mL), the combined organic fraction washed with water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50%EtOAc/light petroleum, to give 75 (3.11 g, 98 %) as a pale yellow solid, mp (EtOAc/light petroleum) 64-65.5 oC; 'H NMR 6 7.84 20 (dd, J= 8.2, 2.1 Hz, 1 H, H 5), 7.72 (d, J= 2.1 Hz, 1 H, H 3), 7.50 (d, J= 8.2 Hz, 1 H, H 6), 4.74 (dd, J= 14.8, 4.2 Hz, 2 H, CH 2 0), 4.58-4.61 (m, 1 H, OCHO), 4.24 (t, J= 6.1 Hz, 2 H, CH 2 0), 3.96 (dt, J= 10.0, 5.8 Hz, 1 H, CH20), 3.80-3.86 (min, 1 H, CH 2 0), 3.62 (dt, 10.0, 5.8 Hz, 1 H, CH20), 3.46-3.51 (m, 1 H, CH 2 ), 2.30 (br s, 1 H, OH), 2.08-2.11 (min, 2 H,
CH
2 ), 1.79-1.85 (m, 1 H, CH 2 ), 1.69-1.77 (m, 1 H, CH 2 ), 1.48-1.62 (min, 4 H, 2 x CH 2 ); 1 3 C 25 NMR 8 156.5, 148.2, 136.8, 128.1, 115.9, 105.8, 99.3, 65.9, 63.9, 62.8, 60.8, 30.6, 29.3, 25.3, 19.7; MS (CI, NH 3 ) m/z 312 (MH', 0.5%), 294 (1), 245 (15), 227(30), 85 (100); HRMS (CI, NH 3 ) calc. for C, 5
H
2 2
NO
6 (MH ) m/z 312.1447, found 312.1438. Anal.
(C
15
H
21 NO6) C, H, N. 30 4-Nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzyl 4-[bis(2 hydroxyethyl)aminolphenylearbamate (76). Pyridine (135 /L, 1.67 mmol) was added dropwise to a stirred solution of alcohol 75 (521 mg, 1.67 mmol) and triphosgene (174 mg, WO 00/64864 PCT/GB00/01612 - 64 0.59 mmol) in THF (20 mL) at 5 oC and the suspension stirred at 5 oC for 1 h. A solution of N',N'-bis(2-hydroxyethyl)-1,4-benzenediamine 57 [prepared by catalytic hydrogenation of NN-bis-(2-hydroxyethyl) 4-nitroaniline (360 mg, 1.84 mmol) with Pd/C under H 2 (60 psi) in EtOH] in THF (10 mL) and DMF (10 mL) was added and the mixture stirred at 20 5 oC for 16 h. The solvent was evaporated and the residue partitioned between EtOAc/water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 80-100% EtOAc/light petroleum to give 76 (220 mg, 25 %) as a colourless oil, 'H NMR [(CD 3
)
2 SO] 8 9.42 (s, 1 H, OCONH), 7.88 (dd, 10 J= 8.3, 2.0 Hz, 1 H, H 5'), 7.79 (d, J = 2.0 Hz, 1 H, H 3'), 7.61 (br d, J= 8.3 Hz, 1 H, H 6'), 7.72 (br d, J= 9.1 Hz, 2 H, H 2, H 6), 6.61 d, J = 9.1 Hz, 2 H, H 3, H 5), 5.19 (s, 2 H,
CH
2 0), 4.73 (t, J= 5.4 Hz, 1 H, CH 2 0), 4.54-4.58 (min, 1 H, OCHO), 4.23 (t, J= 6.1 Hz, 2 H, CH 2 0), 3.80 (dt, J= 9.9, 6.4 Hz, 1 H, CH 2 0), 3.67-3.71 (min, 1 H, CH 2 0), 3.55 (t, J= 5.4 Hz, 1 H, CH 2 0), 3.50 (t, J= 6.0 Hz, 4 H, 2 x CH 2 0), 3.32-3.35 (min, 4 H, 2 x CH 2 N), 1.99 15 2.06 (min, 2 H, CH 2 ), 1.67-1.73 (min, 1 H, CH 2 ), 1.56-1.62 (min, 1 H, CH 2 ), 1.40-1.49 (min, 4 H, 2 x CH 2 ); 1 3 C NMR [(CD 3
)
2 SO] 8 156.2, 153.2, 148.1, 144.1, 132.9, 128.6, 127.4, 120.2 (2), 115.4, 111.4 (2), 106.0, 98.0, 65.7, 63.0, 61.3, 60.1, 58.1 (2), 53.4 (2), 30.2, 28.8, 25.0, 19.1; MS (FAB') m/z 533 (M', 20 %); HRMS (FAB') calc. for C 2 6
H
35
N
3 0 9
(M
+
) m/z 533.2373, found 533.2365. 20 2-(3-Hydroxypropoxy)-4-nitrobenzyl 4-[bis(2-chloroethyl)amino]phenylcarbamate (78). Methanesulphonyl chloride (85 4L, 1.1 mmol) was added dropwise to a stirred solution of 76 (195 mg, 0.36 mmol) in pyridine (10 mL) at 20 oC and the solution stirred for 1 h. The solvent was evaporated and the residue partitioned between DCM/water (100 25 mL). The aqueous fraction was extracted with DCM (2 x 50 mL) and the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiCl (93 mg, 2.2 mmol) added, and the mixture stirred at 80 oC for 3 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL). The 30 combined organic fraction was washed with brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 25% EtOAc/light petroleum, to give (i) 4-nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzyl 4-[bis(2- WO 00/64864 PCT/GB00/01612 - 65 chloroethyl)amino]phenylcarbamate (77) (46 mg, 22 %) as a colourless oil, 'H NMR 8 7.82 (dd, J= 8.3, 2.1 Hz, 1 H, H 5'), 7.73 (d, J= 2.1 Hz, 1 H, H 3'), 7.50 (br d, J= 8.3 Hz, 1 H, H 6'), 7.26-7.29 (m, 3 H, OCONH, H 2, H 6), 6.65 (ddd, J= 9.1, 3.4, 1.9 Hz, 2 H, H 3, H 5), 5.25 (s, 2 H, CH 2 0), 4.67 (br s, 1 H, OCHO), 4.20-4.27 (m, 2 H, CH 2 0), 4.01 (dt, J= 5 9.7, 6.1 Hz, 1 H, CH 2 0), 3.65-3.75 (m, 6 H, CH 2 0, 2 x CH 2 N), 3.58-3.63 (m, 4 H, 2 x
CH
2 C1), 3.44-3.50 (m, 1 H, CH 2 0), 2.11-2.15 (m, 2 H, CH 2 ), 1.66-1.72 (m, 1 H, CH 2 ), 1.55 1.62 (m, 1 H, CH 2 ), 1.40-1.50 (m, 4 H, 2 x CH 2 ); 13C NMR 6 157.2, 153.6, 148.8, 142.6, 132.0, 129.8, 128.7, 121.3 (2), 115.5, 112.8 (2), 105.9, 98.4, 65.3, 63.0, 61.7 (2), 53.7 (2), 40.5 (2), 30.5, 29.3, 25.4, 19.0; MS (FAB') m/z 569 (M', 3%); HRMS (FAB') calc for 10 C 26
H
3 3 3 5 C1 2
N
3 0 7 (M') m/z 569.1696, found 569.1689; calc. for C 26
H
33 35 C1 3 7 C1N 3 0 7
(M
+
) m/z 571.1666, found 569.1680; calc. for C 26
H
33 3 7 C1 2
N
3 0 7 (M ) m/z 573.1637, found 569.1654. Further elution gave 78 (99 mg, 57 %) as a white powder, mp (DCM/pet. ether) 104-105 oC; 'H 8 7.84 (dd, J= 8.3, 2.0 Hz, 1 H, H 5'), 7.74 (d, J= 2.0 Hz, 1 H, H 3'), 7.51 (br d, J= 15 8.3 Hz, 1 H, H 6'), 7.24 (br d, J= 9.0 Hz, 2 H, H 2, H 6), 6.87 (br s, 1 H, OCONH), 6.64 (d, J= 9.0 Hz, 2 H, H 3, H 5), 5.27 (s, 2 H, CH 2 0), 4.27 (t, J= 5.8 Hz, 2 H, CH 2 0), 3.89 (t, J= 5.7 Hz, 2 H, CH 2 0), 3.67-3.72 (m, 4 H, 2 x CH 2 N), 3.58-3.64 (m, 4 H, 2 x CH 2 C1), 2.08 2.12 (m, 2 H, CH 2 ); 3 C NMR 8 156.8, 153.4, 148.7, 145.6, 132.0, 129.4, 128.2, 123.4 (2), 115.6, 112.7 (2), 106.1, 66.6, 61.5, 59.9, 53.6 (2), 40.5 (2), 31.6; Anal. (C 21
H
25 C1 2
N
3 0 6 ) C, 20 H, N, Cl. Example 4D. Preparation of 2-(3-hydroxypropoxy)-4-nitrobenzyl 3-(chloromethyl)-1 [(5,6,7-trimethoxy-1H-indol-2-yl)carbonyll-2,3-dihydro-1H-indol-6-ylcarbamate (80). Pyridine (35 gL, 0.44 mmol) was added dropwise to a stirred solution of alcohol 75 (45 25 mg, 0.25 mmol) and triphosgene (45 mg, 0.15 mmol) in THF (10 mL) at 5 oC and the suspension stirred at 5 oC for 1 h. A solution of 3-(chloromethyl)-1-[(5,6,7-trimethoxy-1H indol-2-yl)carbonyl]-2,3-dihydro-1H-indol-6-ylamine (33) [M. Tercel and W. A. Denny. J. Chem. Soc. Perkin Trans. 1, 1998, 509] (199 mg, 0.48 mmol) in THF (10 mL) was added and the mixture stirred at 20 oC for 16 h. The suspension was filtered and the solvent 30 evaporated. The residue was purified by chromatography, eluting with 40% EtOAc/DCM, to give (i) 4-nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzyl 3-(chloromethyl)-1 [(5,6,7-trimethoxy- 1H-indol-2-yl)carbonyl]-2,3-dihydro- 1H-indol-6-ylcarbamate 79 (116 WO 00/64864 PCT/GB00/01612 - 66 mg, 35%) as an oil, 'H NMR 8 9.54 (s, 1 H, NH), 8.24 (s, 1 H, H 7), 7.84 (s, 1 H, H 4), 7.77 (dd, J= 8.3, 2.0 Hz, 1 H, H 5"), 7.71 d, J= 2.0 Hz, 1 H, H 3"), 7.52 (br s, 1 H, OCONH), 7.48 (d, J= 8.3 Hz, 1 H, H 6"), 7.22 (d, J= 8.3 Hz, 1 H, H 4), 6.93 (d, J= 2.2 Hz, 1 H, H 3'), 6.85 (s, 1 H, H 4'), 5.27 (s, 2 H, CH 2 0), 4.69 (br s, 1 H, OCHO), 4.62 (dd, J= 10.6, 9.4 5 Hz, 1 H, H 2), 4.45 (dd, J= 10.6, 3.8 Hz, 1 H, H 2), 4.20-4.24 (m, 2 H, CH 2 0), 4.08 (s, 3 H,
OCH
3 ), 3.94-3.98 (m, 1 H, CH 2 0), 3.93 (s, 3 H, OCH 3 ), 3.91 (s, 3 H, OCH 3 ), 3.71-3.82 (m, 3 H, H 3, CH 2 0, CH 2 C1), 3.49-3.61 (m, 3 H, CH 2 C1, CH 2 0), 2.10-2.15 (m, 2 H, CH 2 ), 1.65 1.78 (m, 2 H, CH 2 ), 1.45-1.60 (m, 4 H, 2 x CH 2 ); 13C NMR 6 160.3, 157.2, 153.3, 150.2, 148.8, 144.1, 140.5, 138.9, 138.8, 131.8, 129.6, 129.5, 125.9, 125.5, 124.5, 123.6, 115.5, 10 114.6, 108.7, 107.9, 105.9,98.4,97.6,67.6,65.4,63.0,61.8,61.4,61.1, 56.2,54.7,46.9, 43.2, 30.6, 29.2, 25.4, 19.0; MS (FAB') m/z 752 (M', 8%), 669 (20), 234 (30); HRMS (FAB') calc. for C 3 7
H
4 1 35 C1N 4 0,, (M
+
) m/z 752.2460, found 752.2455; calc. for
C
37
H
4 1 37 C1N 4 011,, (M
+
) m/z 754.2431, found 754.2424. 15 Further elution gave 80 (44 mg, 15%) as a tan solid, mp (EtOAc/light petroleum) 166-168 oC; 'H NMR 8 9.47 (s, 1 H, indole-NH), 8.24 (d, J= 1.8 Hz, 1 H, H 7), 7.79 (dd, J= 8.3, 2.1 Hz, 1 H, H 5"), 7.71 (d, J= 2.1 Hz, 1 H, H 3"), 7.48 (d, J= 8.3 Hz, 1 H, H 6"), 7.42 (br s, 1 H, OCONH), 7.33 (s, 1 H, H 5), 7.21 (d, J= 8.2 Hz, 1 H, H 4), 6.93 (d, J= 2.3 Hz, 1 H, H 3'), 6.85 (s, 1 H, H 4'), 5.29 (s, 2 H, CH 2 0), 4.62 (dd, J= 10.8, 9.8 Hz, 1 H, H 2), 4.46 20 (dd, J= 10.8, 4.5 Hz, 1 H, H 2), 4.25 (t, J= 5.8 Hz, 2 H, CH 2 0), 4.07 (s, 3 H, OCH 3 ), 3.94 (s, 3 H, OCH 3 ), 3.88-3.93 (m, 5 H, OCH 3 , CH 2 0), 3.79-3.85 (m, 2 H, H 3, CH 2 C1), 3.55 (dd, J= 12.1, 10.3 Hz, 1 H, CH 2 C1), 2.28 (br t, J= 4.9 Hz, 1 H, OH), 2.09-2.14 (m, 2 H,
CH
2 ); 1 3 C NMR 5 160.5, 156.8, 153.1, 150.1, 148.5, 144.0, 140.5, 138.7, 138.6, 131.7, 130.8, 129.5, 129.3, 126.0, 125.8, 124.5, 123.5, 115.5, 114.6, 108.5, 106.7, 106.0, 66.0, 25 61.7, 61.5, 61.1, 59.4, 56.1, 54.7, 46.9, 43.2, 31.6; MS (FAB') m/z 671 (MH', 1%), 669 (MH', 3%), 391 (15), 149 (100); HRMS (FAB') calc. for C 32 H34 3 5 C1N 4 0 10 (MH') m/z 669.1964, found 669.1921; calc. for C 32
H
3 437CN 4 01 0 (MH') m/z 669.1934, found 671.1875; Anal. (C 32
H
3 3
CN
4 01 0
.
2
H
2 0) C, H, N. 30 Compound 80 was also prepared by treating a solution of 79 (96 mg, 0.13 mmol) in MeOH (5 mL) with 0.1 M HCI (2 mL) and stirring at 20 'C for 16 h. The solvent was evaporated and the residue partitioned between DCM (50 mL) and water (50 mL). The organic fraction WO 00/64864 PCT/GB00/01612 - 67 was washed with water (10 mL), brine (10 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 40% EtOAc/DCM, to give 80 (69 mg, 79%) as a tan solid, spectroscopically identical with the sample prepared above. 5 Example 4E. Preparation of 2-(3-hydroxypropoxy)-4-nitrobenzyl 1-(chloromethyl)-3 [(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5 ylcarbamate (84). Methyl 2-(3-{[tert-butyl(dimethyl)silyl]oxy}propoxy)-4-nitrobenzoate (81). A mixture of methyl 2-hydroxy-4-nitrobenzoate (66) (1.82 g, 9.23 mmol) and K 2
CO
3 (1.91 g, 13.85 10 mmol) in DMF (30 mL) was stirred at 20 oC for 30 min. A solution of 3-bromopropyl tert butyl(dimethyl)silyl ether (3.50 g, 13.85 mmol) in DMF (10 mL) was added and the mixture stirred at 100 oC for 3 h. The mixture was poured into water (300 mL), extracted with EtOAc (3 x 100 mL) and the combined organic extract washed with water (2 x 100 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by 15 chromatography, eluting with 10% EtOAc/light petroleum, to give 81 (3.19 g, 93%) as a pale yellow solid, mp (EtOAc) 36.5-37 oC; 'H NMR 5 7.88 (d, J= 8.9 Hz, 1 H, H 6), 7.80 7.84 (min, 2 H, H 3, H 5), 4.24 (t, J= 6.0 Hz, 2 H, CH 2 0), 3.92 (s, 3 H, OCH 3 ), 3.85 (t, J= 5.9 Hz, 2 H, CH 2 0), 2.04-2.09 (min, 2 H, CH 2 ), 0.88 (s, 9 H, OSi(CH 3
)
3 ), 0.04 (s, 6 H, OSi(CH 3
)
2 ); ' 3 C NMR 6 165.4 (CO 2 ), 158.7 (C 2), 150.7 (C 4), 132.0 (C 6), 126.1 (C 1), 20 114.8 (C 5), 107.7 (C 3), 64.0 (CH 2 0), 59.0 (CH 2 0), 52.5 (OCH 3 ), 32.0 (CH 2 ), 25.9 (SiC(CH 3
)
3 ), 18.3 (SiC(CH 3
)
3 ), -5.5 (Si(CH 3
)
2 ); Anal. (C1 7
H
27
NO
6 Si) C, H, N. [2-(3-{ [tert-Butyl(dimethyl)silyl]oxy} propoxy)-4-nitrophenyl] methanol (82). DIBALH (1 M in DCM, 16.5 mL, 16.5 mmol) was added to a stirred solution of ester 81 (1.85 g, 5.0 25 mmol) in THF (100 mL) at 5 oC and the solution stirred at 5 oC for 1 h. The solution was poured into a solution of potassium sodium tartrate (1 M, 100 mL) and the mixture stirred vigorously for 20 min. The mixture was extracted with EtOAc (3 x 100 mL), the combined organic fraction washed with water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light 30 petroleum, to give 82 (1.64 g, 94%) as a pale yellow solid, mp (EtOAc/light petroleum) 48 49 oC; 'H NMR 8 7.84 (dd, J= 8.3, 2.1 Hz, 1 H, H 5), 7.71 (d,J= 2.1 Hz, 1 H, H 3), 7.51 (d,J= 8.3 Hz, 1 H, H 6), 4.76 (d, J= 6.3 Hz, 2 H, CH20), 4.21 (t, J= 6.1 Hz, 2 H, CH 2 0), WO 00/64864 PCT/GB00/01612 - 68 3.82 (t, J= 5.9 Hz, 2 H, CH 2 OSi), 2.40 (t, J = 6.3 Hz, 1 H, OH), 2.02-2.08 (m, 2 H, CH 2 ), 0.89 (s, 9 H, OSiC(CH 3
)
3 ), 0.06 (s, 6 H, OSi(CH 3
)
2 ); 3 C NMR 6 156.5 (C 2), 148.2 (C 4), 136.7 (C 1), 127.8 (C 6), 115.9 (C 5), 105.8 (C 3), 65.5 (CH20), 60.8 (CH20), 59.3 (CH 2 0), 32.0 (CH 2 ), 25.9 (OSiC(CH 3
)
3 ), 18.3 (OSiC(CH 3
)
3 ), -5.4 (OSi(CH 3
)
2 ; Anal. (CI 6
H
27 NOsSi) 5 C, H,N. 2-(3-{[tert-Butyl(dimethyl)silyl]loxy}propoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7 trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (83). A solution of triphosgene (15 mg, 51 4mol) in DCM (2 mL) was added dropwise to a 10 stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (60 mg, 129 pmol) and Et 3 N (40 .L, 289 mol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of alcohol 82 (54 mg, 159 umol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient 15 (0-10%) MeOH/EtOAc, to give 83 (72 mg, 67%) as a yellow solid mp (MeOH) 149-151 0 C; 'H NMR 8 9.42 (s, 1 H, indole-NH), 8.96 (s, 1 H, OCONH), 7.91 (d, J= 8.4 Hz, 1 H, H 6), 7.78-7.85 (m, 2 H, H 9, H 5"), 7.75 (d, J, 1.7 Hz, 1 H, H 3"), 7.53-7.59 (m, 2 H, H 8, H 6"), 7.47 (ddd, J= 8.4, 7.4, 0.8 Hz, 1 H, H 7), 7.08 (br s, 1 H, H 4), 7.02 (d, J= 2.2 Hz, 1 H, H 3'), 6.89 (s, 1 H, H 4'), 5.38 (s, 2 H, CH 2 0), 4.82 (dd, J= 10.7, 1.7 Hz, 1 H, H 2), 4.69 20 (dd, J= 10.7, 8.7 Hz, 1 H, H 2), 4.21 (t, J= 6.0 Hz, 2 H, CH 2 0), 4.17-4.20 (m, 1 H,
CH
2 C1), 4.09 (s, 3 H, OCH 3 ), 3.99 (dd, J= 11.3, 2.9 Hz, 1 H, H 1), 3.95 (s, 3 H, OCH 3 ), 3.92 (s, 3 H, OCH 3 ), 3.83 (t, J= 5.9 Hz, 2 H, CH20), 3.49 (t, J= 11.0 Hz, 1 H, CH 2 C1), 2.02-2.08 (m, 2 H, CH 2 ), 0.88 (s, 9 H, OSiC(CH 3
)
3 ), 0.04 (s, 6 H, OSi(CH 3
)
2 ; MS (FAB') m/z 833 (MH
+
, 25%), 835 (MH', 12), 775 (5), 599 (5); HRMS (FAB') calc. for 25 C 42
H
50 35 C1N 4 0 Si (MH) m/z 833.2985, found 833.3008; calc. for C 42 Hs 5 0 37 C1N 4 01oSi
(MH
+
) m/z 835.2955, found 835.2982; Anal. (C 42
H
49 ClN 4 0oSi) C, H, N. 2-(3-Hydroxypropoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol 2-yl)carbonyll-2,3-dihydro-1H-benzo[e]lindol-5-ylcarbamate (84). 1 M HCI (0.2 mL, 30 200 pmol) was added to a stirred solution of silyl ether 83 (64 mg, 77 pmol) in MeOH (5 mL) and the solution stirred at 20 oC for 30 min. The solvent was evaporated and the residue dissolved in EtOAc (50 mL), washed with water (2 x 50 mL), brine (25 mL), dried WO 00/64864 PCT/GB00/01612 - 69 and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (50-100%) of EtOAc/light petroleum, to give 84 (52 mg, 94%) as a tan solid, mp (EtOAc) 122-126 oC; 'H NMR 8 9.51 (s, 1 H, indole-NH), 8.90 (s, 1 H, OCONH), 7.92 (d, J= 8.5 Hz, 1 H, H 6), 7.80 (d, J= 8.2 Hz, 1 H, H 5"), 7.77 (d, J= 8.3 Hz, 1 H, H 9), 7.73 5 (d, J, 1.8 Hz, 1 H, H 3"), 7.50-7.57 (min, 2 H, H 8, H 6"), 7.40-7.46 (min, 2 H, H 4, H 7), 6.99 (d, J= 2.2 Hz, 1 H, H 3'), 6.87 (s, 1 H, H 4'), 5.37 (d, J= 13.1 Hz, 1 H, CH 2 0), 5.32 (d, J= 13.1 Hz, 1 H, CH 2 0), 4.77 (dd, J= 10.8, 1.6 Hz, 1 H, H 2), 4.64 (dd, J= 10.8, 8.6 Hz, 1 H, H 2), 4.27 (t, J= 5.7 Hz, 2 H, CH 2 0), 4.11-4.18 (min, 1 H, CH 2 C1), 4.09 (s, 3 H, OCH 3 ), 3.96 (s, 3 H, OCH 3 ), 3.91-3.95 (min, 3 H, H 1, CH 2 0), 3.90 (s, 3 H, OCH 3 ), 3.44 (t, J= 10.9 Hz, 1 10 H, CH 2 C1), 2.75 (br s, 1 H, OH), 2.12-2.18 (min, 2 H, CH 2 ); 13C NMR 6 160.4 (CO), 157.2 (C 2"), 153.8 (OCONH), 150.2 (C 5'), 148.9 (C 4"), 141.6 (C 3a), 140.6 (C 6'), 138.9 (C 7'), 134.0 (C 5), 131.6 (C 1"), 130.1 (C 6"), 129.7 (C 2'), 129.6 (C 9a), 127.5 (C 8), 125.7 (C 7a'), 125.0 (C 7), 123.6 (C 3a'), 123.1 (C 9), 122.4 (C 6, C 9b), 121.6 (C 5a), 115.7 (C 5"), 112.2 (C 4), 106.6 (C 3'), 106.1 (C 3"), 97.7 (C 4'), 66.8 (CH 2 0), 62.2 (CH 2 0), 61.5 15 (OCH 3 ), 61.1 (OCH 3 ), 60.1 (CH 2 0), 56.3 (OCH 3 ), 55.0 (C 2), 45.8 (CH 2 C1), 43.4 (C 1), 31.6 (CH 2 ); MS (FAB
+
) m/z 721 (MH', 2%), 719 (MH
+
, 4); HRMS (FAB') calc. for
C
3 6
H
35 35 C1N 4 010 (MH
+
) m/z 719.2120, found 719.2107; calc. for C 3 6
H
35 37 C1N 4 01 0 (MH') m/z 721.2091, found 721.2093; Anal. (C 36
H
35 C1N 4 0 1 0 ) C, H, N. 20 Example 4F. Preparation of 2-(3-hydroxypropoxy)-4-nitrobenzyl doxorubicin carbamate (87). 4-Nitrophenyl 4-nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzyl carbonate (85). A solution of 4-nitrophenylchloroformate (0.43 g, 2.1 mmol) in THF (10 mL) was added dropwise to a stirred solution of alcohol 75 (0.44 g, 1.4 mmol) and DIEA (0.49 mL, 25 2.8 mmol) in THF (40 mL) and the mixture stirred at 20 oC for 48 h. The solution was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water (3 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (10-50%) EtOAc/light petroleum, to give (i) starting material (176 mg, 40%); and (ii) 85 (0.38 g, 30 56%) as a pale yellow oil, 'H NMR 6 8.30 (ddd, J= 9.2, 3.1, 2.1 Hz, 2 H, H 3, H 5), 7.87 (dd, J= 8.4, 2.1 Hz, 1 H, H 5'), 7.79 (d, J = 2.1 Hz, 1 H, H 3'), 7.57 (d, J= 8.4 Hz, 1 H, H 6'), 7.41 (ddd, J= 9.2, 3.1, 2.1 Hz, 2 H, H 2, H 6), 5.42 (s, 2 H, CH 2 0), 4.58-4.61 (m, 1 H, WO 00/64864 PCT/GB00/01612 - 70 OCHO), 4.28 (t, J= 6.3 Hz, 2 H, CH 2 0), 3.96 (dt, J= 10.0, 6.0 Hz, 1 H, CH 2 0), 3.78-3.83 (m, 1 H, CH 2 0), 3.59 (dt, J = 10.0, 6.0 Hz, 1 H, CH 2 0), 3.45-3.52 (m, 1 H, CH 2 0), 2.13 2.18 (m, 2 H, CH 2 0), 1.79-1.86 (m, 1 H, CH 2 ), 1.67-1.76 (m, 1 H, CH 2 ), 1.48-1.60 (m, 4 H, 2 x CH 2 ); 13 C NMR 8 157.0 (C 1), 155.4 (C 2'), 153.4 (OCONH), 149.2 (C 4'), 145.5 (C 5 4), 129.9 (C 1), 129.2 (C 6'), 125.3 (C 3, C 5), 121.7 (C 2, C 6), 115.6 (C 5'), 106.3 (C 6'), 99.1 (OCO), 66.1 (CH 2 0), 65.3 (CH 2 0), 63.5 (CH 2 0), 60.4 (CH 2 0), 30.6 (CH 2 ), 29.4
(CH
2 ), 25.4 (CH 2 ), 19.7 (CH 2 ); MS m/z 476 (M', 2%), 459 (5), 392 (2), 210(30), 85 (100); HRMS calc. for C 2 2
H
24
N
2 010 (M') m/z 476.1431, found 476.1425. 10 2-(3-Hydroxypropoxy)-4-nitrobenzyl 4-nitrophenyl carbonate (86). A solution of ether 85 (207 mg, 0.47 mmol) in THF (20 mL) and 1 M HC1 (5 mL) was stirred at 20 oC for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (50 mL) and water (50 mL). The organic fraction was dried, the solvent evaporated, and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 86 (125 mg, 15 68%) as a white solid, mp (EtOAc/light petroleum) 116-117 oC; 'H NMR 8 8.29 (ddd, J= 9.1, 3.2, 2.1 Hz, 2 H, H 3, H 5), 7.88 (dd, J= 8.3, 2.1 Hz, 1 H, H 5'), 7.80 (d, J= 2.1 Hz, 1 H, H 3'), 7.58 (d, J= 8.3 Hz, 1 H, H 6'), 7.40 (ddd, J= 9.1, 3.2, 2.1 Hz, 2 H, H 2, H 6), 5.41 (s, 2 H, CH20), 4.30 (t, J= 6.0 Hz, 2 H, CH20), 3.90 (dt, J= 5.4, 4.6 Hz, 2 H, CH 2 0), 2.10-2.15 (m, 2 H, CH 2 ), 1.65 (br s, 1 H, OH); 3 C NMR 8 157.0 (C 2'), 155.3 (C 1), 152.3 20 (OCONH), 149.3 (C 4'), 145.5 (C 4), 129.8 (C 1'), 129.6 (C 6'), 125.4 (C 2, C 6), 121.7 (C 3, C5), 115.8 (C 5'), 106.4 (C 3'), 66.2 (CH 2 0), 65.3 (CH 2 0), 59.5 (CH 2 0), 31.7 (CH 2 ); Anal. (C, 7
H,
6
N
2 0 9 ) C, H, N. 2-(3-Hydroxypropoxy)-4-nitrobenzyl doxorubicin carbamate (87). A solution of 25 carbonate 86 (41 mg, 104 pmol) in DMF (2 mL) was added dropwise to a stirred solution of doxorubicin 13 (46 mg, 86 gmol) and Et 3 N (15 gL, 104 gmol) in DMF (5 mL) at 20 oC and the solution stirred for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-10%) of MeOH/DCM, to give 87 (69 mg, 84%) as a red solid, mp (DCM) 154-160 oC; 'H NMR [(CD 3
)
2 SO] 6 14.00 (s, 1 H, 6-OH), 13.24 30 (s, 1 H, 11-OH), 7.85-7.89 (m, 2 H, H 1, H 3), 7.80 (dd, J= 8.3, 1.8 Hz, 1 H, H 5"), 7.71 (d, J= 1.8 Hz, 1 H, H 3"), 7.62 (dd, J= 6.6, 2.8 Hz, 1 H, H 2), 7.50 (d, J= 8.3 Hz, 1 H, H 6"), 7.07 (d, J= 8.0 Hz, 1 H, OCONH), 5.42 (s, 1 H, 9-OH), 5.14 (br s, 1 H, H 1'), 5.05 (d, J= WO 00/64864 PCT/GB00/01612 -71 18.4 Hz, 1 H, CH 2 0), 4.99 (d, J 18.4 Hz, 1 H, CH 2 0), 4.92 (br s, 1 H, H 7), 4.85 (t, J= 6.0 Hz, 1 H, 14-OH), 4.74 (d, J= 5.8 Hz, 1 H, 4'-OH), 4.58 (d, J= 6.0 Hz, 2 H, H 14), 4.55 (t, J= 5.3 Hz, 1 H, H 5'), 4.14-4.20 (min, 2 H, CH 2 0), 3.97 (s, 3 H, 4-OCH 3 ), 3.69-3.76 (min, 1 H, H 3'), 3.54 (dt, J= 6.0, 5.7 Hz, 2 H, CH 2 0), 3.48 (br s, 1 H, H 4'), 3.30 (br s, 1 H, OH), 5 2.98(d,J= 18.2 Hz, 1 H, H 10), 2.90 (d, J = 18.2Hz, 1 H,H 10), 2.22 (brd, J= 14.4 Hz, 1 H, H 8), 2.09 (dd, J= 14.4, 5.5 Hz, 1 H, H 8), 1.88-1.92 (min, 1 H, H 2'), 1.82-1.87 (min, 2 H,
CH
2 ), 1.50 (dd, J= 12.4, 3.7 Hz, 1 H, H 2'), 1,13 (d, J= 6.4 Hz, 3 H, H 6'); " 3 C NMR
[(CD
3
)
2 SO] 8 213.7 (C 13), 186.4 (C 5), 186.3 (C 12), 160.7 (C 4), 156.0 (C 2"), 155.9 (C 6), 154.9 (C 11), 154.4 (OCONH), 147.8 (C 4"), 136.1 (C 2), 135.4 (C 12a), 134.5 (C 6a), 10 134.0 (C 10a), 133.2 (C 1"), 127.8 (C 6"), 119.9 (C 4a), 119.6 (C 1), 118.9 (C 3), 115.3 (C 5"), 110.6 (C 5a), 110.5 (C 1la), 105.8 (C 3"), 100.2 (C 1'), 74.8 (C 9), 69.8 (C 7), 67.9 (C 4'), 66.6 (C 5'), 65.6 (CH 2 0), 63.6 (C 14), 59.8 (CH 2 0), 57.0 (CH20), 56.5 (4-OCH 3 ), 47.2 (C 3'), 36.5 (C 8), 32.0 (C 10), 31.7 (CH 2 ), 29.7 (C 2'), 16.9 (C 6'); MS (FAB') m/z 797
(MH
+
, 0.3%); HRMS (FAB') calc. for C 38
H
40
N
2 017 (MH ) m/z 797.2405, found 797.2953; 15 Anal. (C 38
H
40
N
2 01 7 . 2H 2 0) C, H, N. Example 4G. Preparation of 2-(3-hydroxypropoxy)-4-nitrobenzyl bis(3-{[(5-methyl-4 acridinyl)carbonyll amino}propyl)carbamate (91). 4-Nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxylbenzyl bis{3 20 [(trifluoroacetyl)amino]propyl}carbamate (88). A solution of alcohol 75 (623 mg, 2.0 mmol) and DIEA (0.40 mL, 2.4 mmol) in DCM (8 mL) was added dropwise to a solution of triphosgene (208 mg, 0.70 mmol) in DCM (6 mL) over 30 minutes at 5 oC and stirred for 1 h. The reaction mixture was added dropwise to a suspension of bistrifluoroacetamide 47 (880 mg, 2.0 mmol) and DIEA (0.76 mL, 4.8 mmol) in DCM (8 mL) and the solution 25 stirred at 20 oC for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with 50% EtOAc/petroleum ether, to give 88 (804 mg, 61%) as a colorless oil, 'H NMR [(CD 3
)
2 SO] 8 7.85 (dd, J= 8.0, 2.0 Hz, 1 H, H 5'), 7.80 (br s, 1 H, CONH), 7.76 (d, J= 2.0 Hz, 1 H, H 3'), 7.44 (d, J= 8.0 Hz, 1 H, H 6'), 6.81 (br s, 1 H, CONH), 5.25 (s, 2 H, CH20), 4.58-4.61 (min, 1 H), 4.22-4.26 (mn, 2 H), 3.91-3.98 (min, 1 H), 30 3.79-3.86 (min, 1 H), 3.56-3.63 (m, 1 H), 3.46-3.53 (min, 1 H), 3.28-3.40 (m, 8 H), 2.04-2.16 (min, 2 H), 1.72-1.87 (min, 6 H), 1.50-1.63 (min, 4 H); 13C NMR [(CD 3
)
2 SO] 8 157.0, 156.9, 148.9, 131.5, 129.4, 115.7, 105.3, 99.2, 65.9, 63.7, 62.6, 62.5, 44.3 (2), 37.4, 36.1, 30.7, WO 00/64864 PCT/GB00/01612 - 72 29.4, 28.1, 27.1, 25.3, 19.6, 2 x CF 3 CO not observed; HRMS (FAB') calc. for
C
26
H
34
F
6
N
4 0 9 (M+) m/z 660.2230; found 660.2234 4-Nitro-2- [3-(tetrahydro-2H-pyran-3-yloxy)propoxy]benzyl bis(3- { [(5-methyl-4 5 acridinyl)carbonyl]amino}propyl)carbamate (90). A solution of carbamate 88 (165 mg, 0.25 mmol), Cs 2
CO
3 (1.0 g, 3.0 mmol) and water (1 mL) in methanol (4 mL) was stirred at 20 oC for 8 h. The pH was adjusted to 10, water (50 mL) added, the solution was extracted with DCM (3 x 50 mL). The combined organic fraction was dried, and the solvent was evaporated to give crude 4-nitro-2-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]benzyl bis(3 10 aminopropyl)carbamate (89). 4-(1H-Imidazol-1-ylcarbonyl)-5-methylacridine (50) [S. A. Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, . Med. Chem., 1999, 42, 2383-2393] (144 mg, 0.50 mmol) was added to a solution of carbamate (89) in THF (10 mL) at 5 oC and the reaction mixture was stirred at 20 oC for 8 h. The solvent was evaporated, and the residue was purified by chromatography on alumina-90, 15 eluting with 1%MeOH/55%EtOAc/DCM, to give 90 (183 mg, 88%) as a yellow solid, mp (EtOAc/DCM) 80-81 oC; 'H NMR 8 11.90 (s, 1 H, NH), 11.83 (s, 1 H, NH), 8.88-8.92 (m, 2 H), 8.62-8.72 (min, 2 H), 8.02-8.05 (m, 2 H), 7.74-7.83 (min, 2 H), 7.50-7.63 (min, 4 H), 7.36 7.45 (min, 2 H), 7.30 (d, J= 2.0 Hz, 1 H, H 3"'), 7.02 (d, J= 8.4 Hz, 1 H, H 6"'), 6.90 (dd, J = 8.4, 2.0 Hz, 1 H, H 5"'), 5.01 (s, 2 H, CH 2 0), 4.56 (s, 1 H), 4.00-3.50 (min, 14 H), 2.83 (s, 20 3 H, CH 3 ), 2.71 (s, 3 H, CH 3 ), 2.14-1.50 (m, 12 H); ' 3 C NMR 6 166.1, 155.6, 155.5, 147.5 (2), 147.0 (2), 145.2 (2), 137.9 (2), 135.8, 135.4, 135.2 (2), 132.6 (2), 132.3 (2), 131.0 (2), 128.3, 128.0, 126.5, 126.4, 126.2, 126.1 (2), 125.7 (2), 125.3 (2), 114.9, 104.9, 99.1, 65.6, 63.6, 62.5, 61.6, 45.9, 45.1, 37.7, 37.1, 30.7, 29.3, 29.4, 28.6, 25.4, 19.7, 18.9, 18.8, 14.2; Anal. (C 52
H
54
N
6 0 9 .V2H 2 0) C, H, N. 25 2-(3-Hydroxypropoxy)-4-nitrobenzyl bis(3-{[(5-methyl-4 acridinyl)carbonyl]amino}propyl)carbamate dihydrochloride (91). A solution of ether 90 (51 mg, 56 Mmol) and HCI (1 M, 1.5 mL) in MeOH (10 mL) was stirred at 20 oC for 4 hrs. The solvent was evaporated and the residue was recrystallized to give 91 (46 mg, 92%) 30 as a yellow solid, mp (MeOH/EtOAc/light petroleum) 143-145 0 C; 'H NMR [(CD 3
)
2 SO] 5 11.23 (s, 2 H, 2 x NH), 9.17 (s, 1 H), 9.11 (s, 1 H), 8.70 (br s, 2 H), 8.24 (br s, 2 H), 7.96 (br s, 2 H), 7.66 (br s, 4 H), 7.51 (br s, 2 H), 7.32 (d, J= 2.0 Hz, 1 H, H 3"), 7.02 (d, J= 8.4 WO 00/64864 PCT/GB00/01612 - 73 Hz, 1 H, H 6"), 6.97 (dd, J= 8.4, 2.0 Hz, 1 H, H 5"), 4.80 (s, 2 H, CH 2 0), 3.92-3.96 (min, 2 H), 3.50-3.53 (min, 10 H), 2.72 (s, 3 H, CH 3 ), 2.63 (s, 3 H, CH 3 ), 2.01-2.04 (min, 4 H, 2 x
CH
2 ), 1.75-1.82 (min, 2 H); HRMS (FAB') calc. for (C 4 7
H
4 6
N
6 0 8 ) (M
+
) m/z 823.3455, found 823.3467; Anal. (C 47
H
46
N
6 0s.2HC.2'2H 2 0) C, H, N. 5 Example 4H. Preparation of 4-nitro-2-[3-(phosphonooxy)propoxy]benzyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (93). 2-(3-{[Di(tert-butoxy)phosphoryl]oxy} propoxy)-4-nitrobenzyl 1-(chloromethyl)-3 10 [(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5 ylcarbamate (92). Tetrazole (40 mg, 567 /.zmol) was added to a stirred solution of alcohol 84 (Example 4E) 136 mg, 189 gmol) and di-tert-butyl diethylphosphoramidite (68 YL, 227 mmol) in THF (10 mL) under N 2 and the solution stirred at 20 oC for 4 h. The solution was cooled to -40 oC and a dried (Na 2
SO
4 ) solution of MCPBA (70 %, 65 mg, 265 4mol) in 15 DCM (3 mL) added. The solution was stirred at -40 oC for 10 min and a solution of 10% NaHSO 4 (10 mL) added and the mixture stirred for 10 min. The mixture was extracted with diethyl ether (80 mL), the organic fraction washed with 10% aq. NaHSO 4 (10 mL), sat. aq.
KHCO
3 (10 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (10-50%) EtOAc/light petroleum, to give 92 (160 20 mg, 93%) as an oil, 'H NMR 8 9.42 (s, 1 H, indole-NH), 8.89 (s, 1 H, OCONH), 8.15 (d, J = 8.5 Hz, 1 H, H 6), 7.83 (dd, J= 8.2, 2.1 Hz, 1 H, H 5"), 7.79 (s, 1 H, H 4), 7.75 (d, J= 8.2 Hz, 1 H, H 6"), 7.73 (d, J= 2.1 Hz, 1 H, H 3"), 7.60 (d, J= 8.3 Hz, 1 H, H 9), 7.53 (ddd, J = 8.3, 7.1, 0.8 Hz, 1 H, H 8), 7.39 (ddd, J= 8.5, 7.1, 0.8 Hz, 1 H, H 7), 7.01 (d, J= 2.1 Hz, 1 H, H 3'), 6.89 (s, 1 H, H 4'), 5.34 (s, 2 H, CH 2 0), 4.81 (dd, J= 10.7, 1.8 Hz, 1 H, H 2), 25 4.64 (dd, J= 10.7, 8.6 Hz, 1 H, H 2), 4.35 (dt, J= 5.6, 5.5 Hz, 2 H, CH 2 0), 4.29 (t, J= 5.6 Hz, 2 H, CH 2 0), 4.15-4.20 (min, 1 H, H 1), 4.10 (s, 3 H, OCH 3 ), 3.98 (dd, J= 11.2, 2.9 Hz, 1 H, CH 2 C1), 3.95(s, 3 H, OCH 3 ), 3.92 (s, 3 H, OCH 3 ), 3.45 (dd, J= 10.9, 10.8 Hz, 1 H,
CH
2 C1), 2.19-2.25 (min, 2 H, CH 2 ), 1.35 (2s, 18 H, 2 x OC(CH 3
)
3 ); MS (FAB') m/z 913
(MH
+
, 0.4%), 911 (MH
+
, 0.8); HRMS (FAB') calc. for C 44
H
53 35 C1N 4 0 1 3 P (MH
+
) m/z 30 911.3035, found 911.3003; calc. for C 44 H537C1N 4 0 1 3 P (MH) m/z 913.3006, found 913.3002.
WO 00/64864 PCT/GB00/01612 - 74 4-Nitro-2-[3-(phosphonooxy)propoxy]benzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy 1H-indol-2-yl)carbonyl]-2,3-dihydro-H-benzo[e]indol-5-ylcarbamate (93). Trifluoroacetic acid (130 4L, 1.64 /mol) was added to a stirred solution of ester 92 (150 mg, 165 /tmol) in DCM (5 mL) and the solution stirred at 20 oC for 1 h. The solvent was 5 evaporated, and the residue azeotroped with benzene (3 x 1 mL) to give 93 (88 mg, 66%) as a gum, 'H NMR 6 11.47 (s, 1 H, indole-NH), 9.94 (s, 1 H, OCONH), 8.57 (br s, 3 H, H 4, 2 x OH), 8.11 (d, J = 8.5 Hz, 1 H, H 6), 7.98 (d, J= 8.3 Hz, 1 H, H 9), 7.91 (d, J= 8.3 Hz, 1 H, H 5"), 7.81 (d, J= 1.8 Hz, 1 H, H 3"), 7.69 (d, J= 8.3 Hz, 1 H, H 6"), 7.58 (ddd, J = 8.3, 7.2, 0.7 Hz, 1 H, H 8), 7.47 (ddd, J= 8.5, 7.2, 0.7 Hz, 1 H, H 7), 7.10 (d, J= 2.2 Hz, 10 1 H, H 3'), 6.97 (s, 1 H, H 4'), 5.29 (s, 2 H, CH 2 0), 4.80 (dd, J= 10.8, 9.4 Hz, 1 H, H 2), 4.53 (dd, J= 10.8, 1.7 Hz, 1 H, H 2), 4.31 -4.37 (min, 1 H, H 1), 4.27 (t, J= 6.1 Hz, 2 H,
CH
2 0), 4.07 (dd, J = 11.2 Hz, 1 H, CH 2 C1), 4.03 (dt, J= 7.1, 6.2 Hz, 2 H, CH 2 0), 3.91-3.95 (min, 4 H, OCH 3 , CH 2 CI), 3.82 (s, 3 H, OCH 3 ), 3.80 (s, 3 H, OCH 3 ), 2.02-2.10 (min, 2 H, CH 2 );
'
3 C NMR 5 160.2 (CO), 156.2 (C 2"), 154.4 (OCONH), 149.1 (C 5'), 148.1 (C 4"), 141.4 (C 15 3a), 139.9 (C 6'), 139.0 (C 7'), 134.4 (C 5), 132.8 (C 1"), 130.8 (C 9a), 129.4 (C 2'), 128.5 (C 6"), 127.1 (C 8), 125.4 (C 5a, C 7a'), 124.3 (C 7), 123.8 (C 9), 123.2 (C 6), 123.1 (C 3a'), 122.0 (C 9b), 115.4 (C 5"), 113.0 (C 4), 106.2 (C 3'), 106.0 (C 3"), 98.0 (C 4'), 65.1 (CH20), 61.3 (CH 2 0), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 60.7 (CH 2 0), 55.9 (OCH 3 ), 54.9 (C 2), 47.5 (CH 2 CI), 41.2 (C 1), 29.6 (CH 2 ); MS (FAB') m/z 801 (MH
+
, 0.5%), 799 (MH
+
, 0.8); 20 HRMS (FAB
+
) calc. for C 3 6
H
37 35 C1N 4 0 1 3 P (MH ) m/z 799.1783, found 799.1757; calc. for
C
36
H
3 737CIN 4 0, 3 P (MH
+
) m/z 801.1754, found 801.1730. Example 4I. Preparation of 2-(2,3-dihydroxypropoxy)-4-nitrobenzyl 1-(chloromethyl) 25 3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5 ylcarbamate (99). Methyl 4-nitro-2-(2-oxiranylmethoxy)benzoate (94). A mixture of methyl 4 nitrosalicylate (66) (0.99 g, 5.02 mmol) and K 2 CO3 (1.04 g, 7.53 mmol) in DMF (25 mL) was stirred at 20 oC for 20 min. Epichlorohydrin (0.59 mL, 7.53 mmol) was added and the 30 mixture stirred at 100 oC for 2 h. The mixture was poured into water, extracted with EtOAc (3 x 100 mL), the combined organic extracts washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) EtOAc/light petroleum, to give (i) starting material (0.18 WO 00/64864 PCT/GB00/01612 - 75 g, 18%) and (ii) 94 (0.75 g, 59 %) as a colourless solid, mp (EtOAc/light petroleum) 62-63 0 C; 'H NMR 8 7.91 (dd, J= 7.7, 1.0 Hz, 1 H, H 5), 7.84-7.86 (min, 2 H, H 3, H 6), 4.49 (dd, 5 J= 11.2, 2.4 Hz, 1 H, H 3'), 4.14 (dd, J= 11.2, 5.2 Hz, 1 H, H 3'), 3.94 (s, 3 H, OCH 3 ), 3.40-3.44 (min, 1 H, H 2'), 2.91-2.97 (min, 2 H, H 1'); 13 C NMR 8 165.0 (CO 2 ), 158.1 (C 2), 150.6 (C 4), 132.3 (C 6), 126.1 (C 1), 115.6 (C 5), 108.4 (C 3), 69.6 (OCH 3 ), 52.6 (CH 2 0), 49.7 (CH 2 0), 44.3 (C 2'); MS (CI, NH 3 ) m/z 295 (M+CH 3 CN', 70%), 259 (MH', 100%); Anal. (CJIH, NO 6 ) C, H, N. 10 Methyl 2-(2,3-dihydroxypropoxy)-4-nitrobenzoate (95). Perchloric acid (1 mL) and water (3 mL) was added to a stirred solution of 94 (205 mg, 0.81 mmol) in THF (20 mL) and the solution stirred at 20 oC for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (50 mL) and water (50 mL). The organic fraction was washed 15 with water (50 mL), brine (25 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 70% EtOAc/light petroleum, to give 95 (172 mg, 78%) as an oil which solidified on standing, mp 60-65 oC; 'H NMR 6 8.02 (d, J= 8.5 Hz, 1 H, H 6), 7.87 (dd, J= 8.5, 2.0 Hz, 1 H, H 5), 7.84 (d, J= 2.0 Hz, 1 H, H 3), 4.38 (dd, J= 9.3, 5.4 Hz, 1 H, H 3'), 4.23 (dd, J = 9.3, 5.4 Hz, 1 H, H 3'), 4.10-4.14 (min, 1 H, H 2'), 3.95 20 (s, 3 H, OCH 3 ), 3.88 (br d, J= 4.1 Hz, 2 H, H 1'), 3.05 (br s, 1 H, OH), 1.95 (br s, 1 H, OH); 1 3 C NMR 8 164.8 (CO 2 ), 159.1 (C 2), 151.0 (C 4), 132.8 (C 6), 124.9 (C 1), 115.6 (C 5), 108.8 (C 3), 73.0 (CH 2 0), 69.2 (C 2'), 63.2 (CH 2 0), 52.8 (OCH 3 ); MS (CI, NH 3 ) m/z 272 (MH-, 1%), 240 (50%), 165 (100); HFIRMS (CI, NH 3 ) calc. for C,H, 4 N0 7
(MH
+
) m/z 272.0770, found 272.0766. Anal. (CIH 13
NO
7 ) C, H, N. 25 Methyl 2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-4-nitrobenzoate (96). 2,2 Dimethoxypropane (0.91 mL, 7.37 mmol) was added dropwise to a stirred solution of diol 95 (400 mg, 1.47 mmol) and PPTS (37 mg, 0.15 mmol) in DMF (20 mL) under N 2 and stirred at 20 oC for 24 h. The solvent was evaporated and the residue partitioned between 30 EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water (50 mL), brine (25 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 30% EtOAc/light petroleum, to give 96 (458 mg, 100%) as a yellow oil, 'H NMR 8 7.90 (d, J= 8.2 Hz, 1 H, H 6), 7.84-7.88 (min, 2 H, H 3, H 5), 4.49 4.54 (min, 1 H, H 4"), 4.25 (dd, J= 9.6, 4.6 Hz, 1 H, H 5"), 4.19 (dd, J= 8.5, 6.4 Hz, 1 H, H 35 2'), 4.15 (dd, J= 9.6, 4.6 Hz, 1 H, H 5"), 4.03 (dd, J= 8.5, 5.8 Hz, 1 H, H 2'), 3.94 (s, 3 H,
OCH
3 ), 1.46 (s, 3 H, CH 3 ), 1.41 (s, 3 H, CH 3 ); 1 3 C NMR 6 165.1 (CO 2 ), 158.2 (C 2), 150.6 WO 00/64864 PCT/GB00/01612 - 76 (C 4), 132.2 (C 6), 126.4 (C 1), 115.5 (C 5), 109.9 (C 2"), 108.4 (C 3), 73.6 (C 4"), 69.8
(CH
2 0), 66.5 (CH 2 0), 52.6 (OCH 3 ), 26.6 (CH 3 ), 25.3 (CH 3 ); MS (CI, NH 3 ) m/z 312 (MH', 15%), 296 (95), 101 (95), 71 (100); HRMS (CI, NH 3 ) calc. for C, 4
H,NO
7 (MH') m/z 312.1083, found 312.1092. 5 {2-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]-4-nitrophenyl}methanol (97). DIBALH (1 M in DCM, 5.1 mL, 5.1 mmol) was added to a stirred solution of ester 96 (457 mg, 1.47 mmol) in THF (50 mL) at 5 oC and the solution stirred at 5 oC for 1 h. The solution was poured into a solution of potassium sodium tartrate (1 M, 100 mL) and the mixture stirred 10 vigorously for 20 min. The mixture was extracted with EtOAc (3 x 50 mL), the combined organic fraction washed with water (50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 97 (385 mg, 92%) as a white solid, mp (EtOAc/light petroleum) 90-92 0 C; 'H NMR 6 7.87 (dd, J= 8.2, 2.1 Hz, 1 H, H 5), 7.72 (d, J= 2.1 Hz, 1 H, H 3), 7.50 (d, J 15 = 8.2 Hz, 1 H, H 6), 4.82 (d, J= 14.1 Hz, 1 H, CH 2 0), 4.70 (d, J= 14.1 Hz, 1 H, CH 2 0), 4.51-4.57 (m, 1 H, H 4"), 4.23 (dd, J= 9.8, 4.0 Hz, 1 H, H 5"), 4.19 (dd, J = 9.8, 5.4 Hz, 1 H, H 2'), 4.11 (dd, J= 9.8, 5.4 Hz, 1 H, H 5"), 3.95 (dd, J= 8.7, 5.4 Hz, 1 H, H 2'), 3.25 (br s, 1 H, OH), 1.48 (s, 3 H, CH 3 ), 1.41 (s, 3 H, CH 3 ); 1 3 C NMR 8 156.5 (C 2), 148.2 (C 4), 137.2 (C 1), 128.6 (C 6), 116.6 (C 5), 110.0 (C 2") 106.4 (C 3), 73.7 (CH20), 69.7 (CH 2 0), 20 66.0 (CH 2 0), 61.0 (CH 2 0), 26.6 (CH 3 ), 25.0 (CH 3 ); MS m/z 283 (M', 3%), 268 (20), 225 (30), 101 (100); HRMS calc for C 13
H,
7
NO
6 (M') m/z 283.1056, found 283.1055; Anal.
(C,
3
H,
7
NO
6 ) C, H, N. 2-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxyl-4-nitrobenzyl 1-(chloromethyl)-3- [(5,6,7 25 trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (98). A solution of triphosgene (22 mg, 75 gmol) in DCM (3 mL) was added dropwise to a stirred solution of amin 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (100 mg, 215 /.zmol) and Et 3 N (60 gL, 429 gImol) in DCM (10 mL) and stirred at oC for 2 h. A solution of alcohol 97 (73 mg, 256 /amol) in DCM (3 mL) was 30 added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20%EtOAc/DCM, to give 98 (160 mg, 96%) as a gum; 'H NMR 6 9.44 (s, 1 H, indole NH), 8.94 (s, 1 H, OCONH), 7.92 (d, J= 8.5 Hz, 1 H, H 6), 7.87 (dd, J= 8.2, 2.1 Hz, 1 H, WO 00/64864 PCT/GB00/01612 - 77 H 5"), 7.81 (d, J= 8.2 Hz, 1 H, H 9), 7.73 (d, J= 2.1 Hz, 1 H, H 3"), 7.58 (ddd, J= 8.2, 7.3, 0.7 Hz, 1 H, H 8), 7.45-7.51 (min, 2 H, H 7, H 6"), 7.13 (br s, 1 H, H 4), 7.02 (d, J= 2.2 Hz, 1 H, H 3'), 6.89 (s, 1 H, H 4'), 5.38 (s, 2 H, CH 2 0), 4.83 (dd, J= 10.8, 1.7 Hz, 1 H, H 2), 4.69 (dd, J= 10.8, 8.7 Hz, 1 H, H 2), 4.50-4.56 (min, 1 H, H 4""), 4.23 (dd, J= 9.8, 4.0 Hz, 1 5 H, H 5"" .... ), 4.15-4.20 (min, 2 H, H 1, H 2'"), 4.09-4.14 (min, 4 H, OCH 3 , H 5""), 3.95-4.00 (inm, 5 H, OCH 3
CH
2 C1, H 2'"), 3.92 (s, 3 H, OCH 3 ), 3.50 (dd, J = 10.9, 10.8 Hz, 1 H, CH 2 C), 1.45 (s, 3 H, CH 3 ), 1.39 (s, 3 H, CH 3 ); MS (FAB') m/z 777 (MH', 10%), 775 (MH', 35); HRMS (FAB') calc. for C 3 9
H
40 35
CIN
4 0 1 , (MH
+
) m/z 775.2381, found 777.2379; calc. for
C
39
H
4 037C1N 4 0,, (MH') m/z 777.2535, found 777.2354. 10 2-(2,3-Dihydroxypropoxy)-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H indol-2-yl)carbonyl] -2,3-dihydro-1H-benzo[e] indol-5-ylcarbamate (99). 1 M HCI (1 mL) was added to a stirred suspension of 98 (160 mg, 206 mol) in THF (20 mL) and the mixture stirred at 20 oC for 16 h. The mixture was evaporated and the residue partitioned 15 between DCM (50 mL) and water (50 mL). The organic fraction was washed with water (30 mL), brine (30 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50%EtOAc/DCM, to give 99 (87 mg, 56%) as a white solid, mp (MeOH/iPr 2 0) 147-149 oC; 'H NMR [(CD 3
)
2 SO] 8 11.46 (s, 1 H, indole-NH), 9.90 (s, 1 H, OCONH), 8.56 (s, 1 H, H 4), 8.12 (d, J= 8.5 Hz, 1 H, H 6), 7.98 (d, J= 8.3 Hz, 1 H, H 20 9), 7.92 (dd, J= 8.3, 1.9 Hz, 1 H, H 5"), 7.83 (d, J= 1.9 Hz, 1 H, H 3"), 7.69 (d, J= 8.3 Hz, 1 H, H 6"), 7.59 (dd, J= 8.2, 7.6 Hz, 1 H, H 8), 7.49 (dd, J= 8.5, 7.6 Hz, 1 H, H 7), 7.09 (d, J= 2.1 Hz, 1 H, H 3'), 6.98 (s, 1 H, H 4'), 5.33 (s, 2 H, CH 2 0), 5.07 (d, J= 5.2 Hz, 1 H, OH), 4.81 (dd, J= 11.0, 9.7 Hz, 1 H, H 2), 4.73 (t, J= 5.7 Hz, 1 H, H 3'"), 4.53 (dd, J= 11.0, 3.5 Hz, 1 H, H 2), 4.32-4.37 (min, 1 H, H 1), 4.24 (dd, J= 10.0, 3.9 Hz, 1 H, CH 2 C1), 25 4.09-4.13 (min, 1 H, H 2""), 4.02-4.06 (min, 1 H, H 3'), 3.93-3.96 (min, 4 H, OCH 3
CH
2 C1), 3.84-3.89 (min, 1 H, OH), 3.83 (s, 3 H, OCH 3 ), 3.81 (s, 3 H, OCH 3 ), 3.51 (t, J= 5.7 Hz, 2 H, H 1'"); 13C NMR [(CD 3
)
2 SO] 8 160.2 (CO), 156.2 (C 2"), 154.3 (OCONH), 149.2 (C 5'), 148.0 (C 4"), 141.5 (C 3a), 139.9 (C 6'), 139.0 (C 7'), 134.3 (C 5), 133.0 (C 1"), 130.7 (C 9a), 129.4 (C 2'), 128.0 (C 6"), 127.1 (C 8), 125.4 (C 5a, C 7a), 124.3 (C 7), 123.9 (C 9), 30 123.3 (C 6), 123.1 (C 3a'), 122.0 (C 9b), 115.4 (C 5"), 113.0 (C 4), 106.3 (C 3'), 106.2 (C 3"), 98.0 (C 4'), 70.7 (CH 2 0), 69.7 (CHOH) 62.4 (CH 2 0), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 60.7
(CH
2 0), 55.9 (OCH 3 ), 54.9 (C 2), 47.5 (CH 2 C1), 41.4 (C 1); MS (FAB') m/z 737 (MH , 3%), 735 (MH
+
, 8); HRMS (FAB') calc. for C 36
H
36 35 C1N 4 0, 1 (MH*) m/z 735.2069, found WO 00/64864 PCT/GB00/01612 - 78 735.2050; calc. for C 36
H
36 37 C1N 4 0,, (MH-) m/z 737.2040, found 737.2000; Anal.
(C
36
H
35 C1N 4 0 1
I
1
.CH
3 OH) C, H, N. Example 4J. Preparation of 2-[3-(dimethylamino)propoxy]-4-nitrobenzyl 1 5 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (102). Methyl 2-[3-(dimethylamino)propyloxy]-4-nitrobenzoate (100). A mixture of methyl 2 hydroxy-4-nitrobenzoate (66) (1.03 g, 5.22 mmol) and K 2
CO
3 (2.17 g, 15.67 mmol) in DMF (30 mL) was stirred at 20 oC for 30 min. A solution of N-(3-chloropropyl)-N,N 10 dimethylamine (1.24 g, 7.83 mmol) in DMF (10 mL) was added and the mixture stirred at 100 'C for 3 h. The mixture was poured into water (300 mL), extracted with EtOAc (3 x 100 mL) and the combined organic extract washed with water (2 x 100 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) of EtOAc/light petroleum, to give 100 (1.05 g, 71%) as a pale yellow 15 oil which was stored as the HCI salt, mp (EtOAc) 175-177 oC; 'H NMR [(CD 3
)
2 SO] 5 10.90 (br s, 1 H, NHC1), 7.87-7.93 (min, 3 H, H 3, H 5, H 6), 4.32 (t, J= 6.0 Hz, 2 H, CH 2 0), 3.89 (s, 3 H, OCH 3 ), 3.18-3.23 (min, 2 H, CH 2 N), 2.77 (d, J= 4.8 Hz, 6 H, N(CH 3
)
2 ), 2.18 2.24 (min, 2 H, CH 2 ); 1 3 C NMR [(CD 3
)
2 SO] 8 165.0 (CO 2 ), 157.3 (C 2), 150.2 (C 4), 131.6 (C 6), 126.0 (C 1), 115.3 (C 5), 108.4 (C 3), 66.5 (CH 2 0), 53.7 (CH 2 N), 52.6 (OCH 3 ), 42.0 20 (N(CH 3
)
2 ), 23.3 (CH 2 ); Anal. (C 13
H,
9 C1N 2 0 5 ) C, H, N, Cl. {2-[3-(Dimethylamino)propoxy]-4-nitrophenyl}methanol (101). DIBALH (1 M, in DCM, 13.0 mL, 13.0 mmol) was added to a stirred solution of ester 100 (1.05 g, 3.72 mmol) in THF (50 mL) at 5 oC and the solution stirred at 5 oC for 1 h. The solution was 25 poured into a solution of potassium sodium tartrate (1 M, 100 mL) and the mixture stirred vigorously for 20 min. The mixture was extracted with EtOAc (3 x 100 mL), the combined organic fraction washed with water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography on alumina, eluting with a gradient (0-10%) of MeOH/EtOAc, to give 101 (0.81 g, 86%) as a pale yellow solid, mp 30 (EtOAc) 104-105 oC; 1H NMR 8 7.87 (dd, J= 8.3,2.1 Hz, 1 H,H 5), 7.69 (d, J= 2.1 Hz, 1 H, H 3), 7.64 (d, J= 8.3 Hz, 1 H, H 6), 5.43 (br s, 1 H, OH), 4.58 (s, 2 H, CH 2 0), 4.14 (t, J = 6.5 Hz, 2 H, CH 2 0), 2.36 (t, J= 7.0 Hz, 2 H, CH 2 N), 2.15 (s, 6 H, N(CH 3
)
2 ), 1.85-1.91 (min, 2 H, CH 2 ); ' 3 C NMR 8 155.2 (C 2), 147.0 (C 4), 138.9 (C 1), 126.7 (C 6), 115.3 (C 5), WO 00/64864 PCT/GB00/01612 - 79 105.2 (C 3), 66.5 (CH 2 0), 57.6 (CH 2 0), 55.5 (NCH 2 ), 45.1 (N(CH 3
)
2 ), 26.5 (CH 2 ); Anal. (Cl 2
H
1 8
N
2 0 4 ) C, H, N. 2-[3-(Dimethylamino)propoxy]-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy 5 1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (102). A solution of triphosgene (17 mg, 55 4mol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (65 mg, 140 Mmol) and Et 3 N (44 gL, 313 /imol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of alcohol 101 (44 mg, 172 .mol) in DCM (2 mL) was 10 added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-20%) MeOH/EtOAc, to give 102 (57 mg, 55%) as a yellow solid which was converted to the hydrochloride salt, mp (MeOH) 176-180 oC; 'H NMR [(CD 3
)
2 SO] 6 11.43 (s, 1 H, indole-NH), 10.47 (br s, 1 H, NH'CI), 9.92 (s, 1 H, OCONH), 8.58 (s, 1 H, H 4), 8.10 (d, J 15 = 8.5 Hz, 1 H, H 6), 7.97 (d, J= 8.3 Hz, 1 H, H 9), 7.93 (dd, J= 8.4, 2.0 Hz, 1 H, H 5"), 7.82 (d, J= 2.0 Hz, 1 H, H 3"), 7.71 (br d, J= 8.4 Hz, 1 H, H 6"), 7.56-7.61 (min, 1 H, H 8), 7.46-7.51 (m, 1 H, H 7), 7.10 (d,J= 2.1 Hz, 1 H, H 3'), 6.97 (s, 1 H, H 4'), 5.34 (s, 2 H, CH20), 4.81 (dd, J= 10.8, 9.5 Hz, 1 H, H 2), 4.53 (dd, J= 10.8, 1.7 Hz, 1 H, H 2), 4.33 4.38 (min, 1 H, H 1), 4.30 (t, J = 5.9 Hz, 2 H, CH 2 0), 4.07 (dd, J = 11.1, 2.9 Hz, 1 H, CH 2 C), 20 3.94-3.97 (m, 4 H, CH 2 C1, OCH 3 ), 3.83 (s, 3 H, OCH 3 ), 3.81 (s, 3 H, OCH 3 ), 3.23-3.27 (t, J = 7.5 Hz, 2 H, CH 2 N), 2.75 (s, 6 H, N(CH 3
)
2 ), 2.17-2.23 (min, 2 H, CH 2 ); 13 C NMR
[(CD
3
)
2 SO] 5 160.2 (CO), 155.8 (C 2"), 154.3 (OCONH), 149.2 (C 5'), 148.0 (C 4"), 141.5 (C 3a), 139.9 (C 6'), 138.9 (C 7'), 134.3 (C 5), 132.9 (C 1"), 130.7 (C 2'), 129.5 (C 9a), 128.5 (C 6"), 127.2 (C 8), 125.4 (C 7a'), 124.4 (C 7), 123.7 (C 9), 123.3 (C 6), 123.1 (C 25 3a'), 122.1 (C 9b), 121.2 (C 5a), 115.7 (C 5"), 113.0 (C 4), 106.3 (C 3', C 3"), 98.0 (C 4'), 66.8 (CH20), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 60.7 (CH 2 0), 55.9 (OCH 3 ), 54.9 (C 2), 53.7
(CH
2 N), 47.5 (CH 2 C1), 42.0 (N(CH 3
)
2 ), 41.1 (C 1), 23.6 (CH 2 ); Anal. (C 38
H
4 C1N 5 0 9 .2HC1): C, H,N. 30 Example 4K. Preparation of 2-[3-(4-morpholinyl)propoxy]-4-nitrobenzyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-l1H-indol-2-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (105). Methyl 2-[3-(4-morpholinyl)propoxy]-4-nitrobenzoate (103). A mixture of methyl 2- WO 00/64864 PCT/GB00/01612 - 80 hydroxy-4-nitrobenzoate (1.0 g, 5.12 mmol) and K 2
CO
3 (1.06 g, 7.68 mmol) in DMF (20 mL) was stirred at 20 oC for 30 min. A solution of 4-(3-chloropropyl)morpholine (0.98 g, 7.68 mmol) in DMF (5 mL) was added and the mixture stirred at 100 'C for 6 h. The mixture was cooled to 20 oC and poured into water (300 mL) and extracted with EtOAc (3 5 x 100 mL). The combined organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with EtOAc, to give 103 (1.33 g, 80%) as an oil, 'H NMR 8 7.88 (d,J= 9.1 Hz, 1 H, H 6), 7.81 (min, 2 H, H 3, H 5), 4.21 (t, J= 6.3 Hz, 2 H, CH 2 0), 3.93 (s, 3 H, OCH 3 ), 3.69-3.74 (min, 4 H, 2 x CH 2 0), 2.57 (t, J = 7.0 Hz, 2 H, CH 2 N), 2.47-2.51 (min, 4 H, 2 x 10 CH 2 N), 2.02-2.07 (min, 2 H, CH 2 ); ' 3 C NMR 6 165.3 (CO 2 ), 158.6 (C 2), 150.6 (C 4), 132.0 (C 6), 126.2 (C 1), 114.8 (C 5), 107.8 (C 3), 67.6 (CH 2 0), 66.9 (2 x CH 2 0), 55.0 (CH 2 N), 53.7 (2 x CH 2 N), 52.5 (OCH 3 ), 25.9 (CH 2 ). Compound 103 was conveniently stored as the hydrochloride salt, mp (EtOAc) 160-163 oC; Anal. (C1 5
H
20
N
2 0 6 .HC1) C, H, Cl. N, calc. 7.8, found 9.1%. 15 {2-[3-(4-Morpholinyl)propoxy]-4-nitrophenyl}methanol (104). A solution of ester 103 (1.33 g, 4.10 mmol) in THF (100 mL) was added dropwise to a stirred solution of DIBALH (1 M in DCM, 13.5 mL, 13.5 mmol) at 5 oC and the solution stirred at 5 oC for 1 h. The solution was carefully poured into 1 M HCI (50 mL) and stirred for 10 min. The solution 20 was concentrated under reduced pressure, neutralised and extracted with EtOAc ( 3 x 100 mL). The combined organic fraction was dried and the solvent evaporated. The residue was purified by chromatography on alumina, eluting with a gradient (0-10%) MeOH/EtOAc, to give 104 (1.07 g, 88%) as a tan solid, mp (EtOAc) 105-106 oC; 'H NMR 8 7.83 (dd, J= 8.2, 2.1 Hz, 1 H, H 5), 7.70 (d, J= 2.1 Hz, 1 H, H 3), 7.46 (d, J= 8.2 Hz, 1 H, H 6), 4.71 25 (s, 2 H, CH 2 0), 4.18 (t, J= 6.0 Hz, 2 H, CH 2 0), 3.74-3.77 (min, 4 H, 2 x CH 2 0), 2.56 (dd, J = 6.7, 6.5 Hz, 2 H, CH 2 N), 2.45-2.49 (min, 4 H, 2 x CH 2 N), 2.01-2.06 (min, 2 H, CH 2 ); 3 C NMR 8 156.7 (C 2), 148.2 (C 4), 137.2 (C 1), 128.2 (C 6), 116.1 (C 5), 106.2 (C 3), 67.7
(CH
2 0), 66.5 (2 x CH 2 0), 60.7 (CH 2 0), 56.3 (CH 2 N), 53.9 (2 x CH 2 N) 25.5 (CH 2 ); Anal.
(C
1 4
H
2 0N 2 0 5 ) C, H, N. 30 2-[3-(4-Morpholinyl)propoxy]-4-nitrobenzyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy 1H-indol-2-yl)carbonyll-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (105). A solution of triphosgene (15.5 mg, 52 imol) in DCM (2 mL) was added dropwise to a stirred WO 00/64864 PCT/GB00/01612 -81 solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (52 mg, 133 /mol) and Et 3 N (42 gL, 299 ,umol) in DCM (10 mL) and stirred at oC for 2 h. A solution of alcohol 104 (49 mg, 164 gmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The 5 solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-10%) MeOH/EtOAc, to give 105 (96 mg, 92%) as a tan powder mp (EtOAc) 102-107 0 C; 'H NMR 8 9.48 (s, 1 H, indole-NH), 8.93 (s, 1 H, OCONH), 7.91 (d, J= 8.5 Hz, 1 H, H 6), 7.78-7.84 (m, 2 H, H 9, H 5"), 7.73 (d, J= 1.8 Hz, 1 H, H 3"), 7.57 (ddd, J= 8.2, 7.0, 1.0 Hz, 1 H, H 8), 7.50-7.53 (min, 1 H, H 6"), 7.45 (ddd, J= 8.5, 7.0, 1.0 Hz, 1 H, H 7), 7.28 10 (br s, 1 H, H 4), 7.00 (d, J= 2.2 Hz, 1 H, H 3'), 6.88 ( s, 1 H, H 4'), 5.36 (s, 2 H, CH 2 O), 4.81 (dd, J= 10.8, 1.8 Hz, 1 H, H 2), 4.68 (dd, J = 10.8, 8.7 Hz, 1 H, H 2), 4.16-4.22 (m, 3 H, CH 2 0, CH 2 C1), 4.08 (s, 3 H, OCH 3 ), 3.93-3.99 (min, 4 H, H 1, OCH 3 ), 3.92 (s, 3 H,
OCH
3 ), 3.69 (t, J= 4.6 Hz, 4 H, 2 x CH 2 0), 3.49 (t, J= 10.9 Hz, 1 H, CH 2 C1), 2.55 (t, J= 7.1 Hz, 2 H, CH 2 N), 2.43-2.49 (min, 4 H, 2 x CH 2 N), 2.00-2.08 (min, 2 H, CH 2 ); ' 3 C NMR 6 15 160.4 (CO), 156.7 (C 2"), 154.0 (OCONH), 150.2 (C 5'), 148.6 C 4"), 141.7 (C 3a), 140.6 (C 6'), 138.9 (C 7'), 133.9 (C 5), 132.2 (C 1"), 129.8 (C 2'), 129.6 (C 9a), 128.8 (C 6"), 127.5 (C 8), 125.7 (C 7a'), 125.0 (C 7), 123.6 (C 3a'), 123.2 (C 9), 122.4 (C 6, C 9b), 121.6 (C 5a), 115.7 (C 5"), 112.2 (C 4), 106.5 (C 3'), 106.1 (C 3"), 97.7 (C 4'), 67.1 (CH 2 0), 66.8 (2 x CH 2 0), 61.9 (CH 2 0), 61.5 (OCH 3 ), 61.1 (OCH 3 ), 56.3 (OCH 3 ), 55.2 (C 2), 54.9 20 (CH 2 N), 53.6 (2 x CH 2 N), 45.8 (CH 2 C1), 43.4 (C 1), 26.1 (CH 2 ); MS (FAB') m/z 788 (MH
+
, 6%), 790 (MH
+
, 3); HRMS (FAB') calc. for C 4 0H 4 3 35
CN
5 0 10
(MH
+
) m/z 788.2699, found 788.2721; calc. for C 4 0
H
4 3 3 7 C1N 5 010 (MH+) m/z 790.2699, found 790.2728; Anal.
(C
40
H
42 C1N 5 0, 0 .2H 2 0) C, H, N. 25 Example 4L. Preparation of 2-[3-(4-morpholinyl)propoxy]-4-nitrobenzyl bis(3- {[(5 methyl-4-acridinyl)carbonyl]amino}propyl)carbamate (107). DIEA (0.3 mL, 3 mmol) was added to a suspension of carbamate dihydrochloride 91 (44 mg, 0.5 mmol) in DCM (10 mL) at 5 oC and the mixture stirred for 10 min. Methanesulfonyl chloride (0.1 mL), was added and the mixture stirred for 30 min. MeOH (2 mL) was added, the mixture stirred for 30 10 min, and the solvent evaporated. The residue was purified by chromatography on alumina-90, eluting with 1%MeOH/40%EtOAc/DCM to give the crude mesylate 106 (42 mg, 95%) as a yellow solid, 'H NMR 8 11.94 (s, 1 H, NH), 11.85 (s, 1 H, NH), 8.94 (m, 2 H), 8.77 (m, 2 H), 8.07 (min, 2 H), 7.81 (min, 2 H), 7.62 (m, 4 H), 7.43 (min, 2 H), 7.30 (d, J= WO 00/64864 PCT/GB00/01612 - 82 2.0 Hz, 1 H, H 3'"), 7.05 (d, J= 8.4 Hz, 1 H, H 6'"), 6.95 (dd, J= 8.4, 2.0 Hz, 1 H, H 5'"), 5.02 (s, 2 H, CH 2 0), 4.39 (t, 2 H, CH 2 0), 3.98 (t, 2 H), 3.73-3.58 (m, 8 H), 3.02 (s, 3 H,
CH
3
SO
2 ), 2.85 (s, 3 H, CH 3 ), 2.76 (s, 3 H, CH 3 ), 2.20-2.05 (min, 6 H). A solution of mesylate 106 (42 mg, 0.047 mmol) in morpholine (0.5 mL) was stirred at 20 5 oC for 20 hrs. The reaction mixture was purified by chromatography on alumina-90, eluting with 1%MeOH/40%EtOAc/DCM to give 107 (38 mg, 91%) as a yellow solid, 'H NMR 5 11.94 (s, 1 H, NH), 11.86 (s, 1H, N H), 8.89-8.91 (min, 2 H), 8.73-8.77 (m, 2 H), 8.06-8.08 (min, 2 H), 7.80-7.84 (m, 2 H), 7.59-7.64 (m, 4 H), 7.42-7.46 (min, 2 H), 7.32 (d, J= 2.0 Hz, 1 H, H 3'"), 7.06 (d, J= 8.4 Hz, 1 H, H 6'"), 6.95 (dd, J= 8.4, 2.0 Hz, 1 H, H 5'"), 5.03 (s, 2 10 H, CH20), 3.92 (t, 2 H), 3.75-3.67 (min, 8 H, 4 x CH 2 N), 3.59 (min, 4 H, 2 x CH 2 ), 2.86 (s, 3 H, CH 3 ), 2.74 (s, 3 H, CH3), 2.46 (br s, 6 H, CH 2 N), 2.18-2.04 (m, 4 H), 1.93 (br s, 2 H); HRMS (FAB') calc. for (C 5 1
H
54
N
7 0 8 ) (MH
+
) m/z 892.4034, found 892.4055. Example 4M. Preparation of 4-{2-[({[bis(3-{[(5-methyl-4 15 acridinyl)carbonyl]amino}propyl)aminol carbonyl}oxy)methyl]-5 nitrophenoxy}butanoic acid (111). Methyl 2-(4-tert-butoxy-4-oxobutoxy)-4-nitrobenzoate (108). A mixture of methyl 2 hydroxy-4-nitrobenzoate (66) (0.61 g, 3.09 mmol) and K 2
CO
3 (0.64 g, 4.64 mmol) in DMF (20 mL) was stirred at 20 oC for 30 min. A solution of tert-butyl 4-bromobutanoate (1.04 g, 20 4.64 mmol) in DMF (5 mL) was added and the mixture stirred at 100 oC for 6 h. The mixture was cooled to 20 oC, poured into water (300 mL) and extracted with EtOAc (3 100 mL). The combined organic fraction was washed with water (2 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 20%EtOAc/light petroleum, to give 108 (0.86 g, 82%) as an oil, IR N 2978, 25 1728, 1532, 1350, 1285, 1250, 1155 cm'; 'H NMR 8 7.89 (d, J= 8.5 Hz, 1 H, H 6), 7.85 (dd, J= 8.5, 2.0 Hz, 1 H, H 5), 7.79 (d, J= 2.0 Hz, 1 H, H 3), 4.17 (t, J= 6.2 Hz, 2 H, CH20), 3.93 (s, 3 H, OCH 3 ), 2.47 (t, J= 7.3 Hz, 2 H, CH2CO), 2.11-2.16 (m, 2 H, CH2), 1.49 (s, 9 H, CH3); ' 3 C NMR 8 172.3 (CO 2 ), 165.4 (CO 2 ), 158.5 (C 2), 150.7 (C 4), 132.1 (C 6), 126.2 (C 1), 115.0 (C 5), 107.9 (C 3), 80.6 (OC(CH 3
)
3 ), 68.4 (CH20), 52.5 (OCH3), 30 31.5 (CH 2 ), 28.1 (OC(CH 3
)
3 ), 24.4 (CH 2 ); MS m/z 339 (M', 1%), 308 (2), 197 (50), 87 (90), 57 (100); HRMS calc. for C, 6
H
21
NO
7
(M
+
) m/z 339.1318, found 339.1314. 2-(4-tert-Butoxy-4-oxobutoxy)-4-nitrobenzoic acid (109). A mixture of ester 108 (0.61 g, WO 00/64864 PCT/GB00/01612 - 83 1.80 mmol) and NaOH (1 M, 9 mL, 9 mmol) in MeOH (20 mL) was stirred at 20 oC for 2 h. The mixture was extracted with diethyl ether (20 mL) and the pH adjusted to 4 with 1 M HC1. The mixture was extracted with EtOAc (3 50 mL), the combined organic fraction washed with brine (50 mL), dried and the solvent evaporated. The residue was crystallized 5 to give 109 as a white solid, mp (EtOAc/light petroleum) 76-77 oC; IR N 1723, 1678, 1532, 1350, 1260, 1154 cm-'; 'H NMR [(CD 3
)
2 SO] 6 13.0 (br s, 1 H, CO 2 H), 7.80-7.86 (inm, 3 H, H 3, H 5, H 6), 4.20 (t, J= 6.2 Hz, 2 H, CH 2 0), 2.40-2.46 (min, 2 H, CH 2 CO), 1.92-1.99 (min, 2 H, CH 2 ), 1.40 (s, 9 H, CH 3 ); " 3 C NMR [(CD 3
)
2 SO] 8 174.0 (CO 2 ), 171.8 (C0 2 ), 166.2 (C 2), 157.0 (C 4), 130.9 (C 6), 128.3 (C 1), 115.1 (C 5), 107.9 (C 3), 79.7 (OC(CH 3
)
3 ), 10 68.0 (CH 2 0), 31.0 (CH 2 ), 27.7 (OC(CH 3
)
3 ), 24.0 (CH 2 ); Anal. (C ,5
H
1
,
9
NO
7 .1AH 2 0) C, H, N. tert-Butyl 4-[2-(hydroxymethyl)-5-nitrophenoxyl butanoate (110). BH 3 .DMS (0.28 mL, 2.8 mmol) was added to a stirred solution of acid 109 and trimethyl borate (0.64 mL, 5.66 mmol) in THF (50 mL) and the solution heated at reflux temperature under N 2 for 3 h. The 15 solution was cooled to 20 oC, MeOH (2 mL) added carefully and the mixture stirred for 5 min. Water (2 mL) was carefully added, the solution stirred for 5 min, 1 M aq. citric acid solution added and the mixture stirred for 30 min. The mixture was extracted with EtOAc (3 60 mL), the combined organic fraction washed with water (50 mL), brine (30 mL), dried, and the solvent evaporated. The residue was purified by chromatography, elutiing 20 with a gradient (30-50%) of EtOAc/light petroleum, to give 110 (0.30 g, 68%) as a white solid, mp (EtOAc/light petroleum) 42-43 oC; IRN 3434, 1726, 1524, 1346, 1248, 1155 cm '; 'H NMR 8 7.85 (dd, J= 8.2, 2.1 Hz, 1 H, H 5), 7.68 (d, J= 2.1 Hz, 1 H, H 3), 7.53 (d, J= 8.2 Hz, 1 H, H 6), 4.76 (d, J = 4.6 Hz, 2 H, CH20), 4.12 (t, J = 6.2 Hz, 2 H, CH 2 0), 2.54 (br t, J= 5.0 Hz, 1 H, OH), 2.45 (t, J= 7.2 Hz, 2 H, CH 2 CO), 2.11-2.18 (min, 2 H, CH 2 ), 1.50 (s, 25 9 H, CH 3 ); ' 3 C NMR 8 172.3 (CO 2 ), 156.2 (C 2), 148.1 (C 4), 136.8 (C 1), 127.0 (C 6), 116.0 (C 5), 105.7 (C 3), 80.9 (OC(CH 3
)
3 ), 67.7 (CH 2 0), 60.6 (CH 2 0), 31.9 (CH 2 ), 28.1
(OC(CH
3
)
3 ), 24.3 (CH 2 ); MS (CI, NH 3 ) m/z 329 (M'+NH 3 , 5%), 312 (MH', 2), 273 (100); HRMS (CI, NH 3 ) calc. for C 15
H
2 2
NO
6
(MH
+
) m/z 312.1447, found 312.1448. Anal.
(C
15
H
22 NO6) C, H, N. 30 4- {2-[({ [Bis(3-{ [(5-methyl-4 acridinyl)carbonyll amino} propyl)aminol carbonyl) oxy)methyl] -5 nitrophenoxy}butanoic acid (113). A solution of alcohol 110 (19 mg, 0.06 mmol) and WO 00/64864 PCT/GB00/01612 - 84 DIEA (25 4L, 140 tmol) in DCM (2 mL) was added dropwise to a solution of triphosgene (10 mg, 35 ptmol) in DCM (1.5 mL) over 30 minutes at 5 oC and the solution stirred for I hr. A solution of N,N-bis[3-(5-methylacridine-4-carboxamido)propyl]amine (111) [S. A. Gamage, J. A. Spicer, G. J. Atwell, G. J. Finlay, B. C. Baguley, W. A. Denny, J. Med. 5 Chem., 1999, 42, 2383-2393] (24 mg, 42 4mol) and DIEA (4 ML, 240 p/mol) in DCM (2 mL) was added and the solution stirred at 20 oC for 16 h. The solvent was evaporated and the residue was purified by chromatography on alumina-90, eluting with 1%MeOH/60%EtOAc/DCM to give crude ester 112 (34 mg, 89%) as a yellow foam, 'H NMR 8 11.90 (s, 1 H, NH), 11.86 (s, 1 H, NH), 8.85-8.95 (min, 2 H), 8.66-8.76 (min, 2 H), 10 8.02-8.08 (min, 2 H), 7.78-7.87 (min, 2 H), 7.50-7.64 (min, 4 H), 7.38-7.46 (min, 2 H), 7.25 (d, J= 2.0 Hz, 1 H, H 3'), 7.01 (d, J= 8.3 Hz, 1 H, H 6'), 6.89 (dd, J= 8.3, 2.0 Hz, 1 H, H 5'), 4.77 (s, 2 H, CH 2 0), 3.84-3.88 (min, 2 H), 3.58-3.73 (min, 8 H), 2.83 (s, 3 H, CH 3 ), 2.73 (s, 3 H, CH 3 ), 2.42-3.46 (min, 2 H), 2.10-2.17 (m, 4 H), 2.00-2.02 (m, 2 H), 1.45 (s, 9 H); ' 3 C NMR 8 172.2, 166.2, 156.2, 155.6, 155.3, 147.4, 147.0 (2), 145.2 (2), 137.9 (2), 136.9, 15 135.8, 135.4 (2), 135.2, 132.6, 132.2 (2), 131.0 (2), 127.9 (2), 126.5, 126.4, 126.2, 126.1 (2), 125.8 (2), 125.3, 116.0, 115.1, 105.7, 104.8, 67.7, 61.6, 60.5, 45.9, 45.1, 37.7, 37.0, 31.9, 31.6, 29.2, 28.6, 28.1, 24.3, 24.3, 18.9, 18.8. Ester 112 (80 mg, 88 ptmol) was added to a HCI saturated solution of MeOH (20 mL) and DCM (20 mL) at 5 oC and the solution stood for 3 days at 5 oC. The solvent was 20 evaporated and the residue crystallized to give 113 (45 mg, 58%) as a yellow solid, mp (MeOH/EtOAc/light petroleum) 128-130 0 C; 'H NMR [(CD 3
)
2 SO] 8 11.39 (s, 2 H, 2 CONH), 9.15 (s, 1 H), 9.08 (s, 1 H), 8.68 (br s, 2 H), 8.25 (br s, 2 H), 7.95 (br s, 2 H), 7.65 (br s, 4 H), 7.49 (br s, 2 H), 7.32 (d, J= 2.0 Hz, 1 H), 7.00 (d, J= 8.3 Hz, 1 H), 6.93 (dd, J -= 8.3, 2.0 Hz, 1 H), 4.77 (s, 2 H, CH 2 0), 3.88-3.91 (m, 2 H), 3.50-3.58 (m, 8 H), 2.77 (s, 3 25 H, CH 3 ), 2.66 (s, 3 H, CH 3 ), 2.01 (br s, 4 H), 1.87-1.93 (min, 2 H); Anal.
(C
4 8
H
46
N
6 0 9 .2HC1.2H 2 0) C, H, N. Example 5A. Preparation of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl bis(2 chloroethyl)carbamate (117). 30 1-Methyl-2-nitro-1H-imidazole-5-carboxylic acid (114). Sodium hydroxide solution (1 M, 125 ml, 125 mmol) was added slowly to a stirred suspension of ethyl 1-methyl-2-nitro 1H-imidazole-5-carboxylate (114) [B. Cavalleri, R. Ballotta, G.C. Lancini. J. Heterocyclic Chem. 1972, 9, 979] (5.0 g, 25.1 mmol) in water (50 mL) and the mixture stirred at 20 oC WO 00/64864 PCT/GB00/01612 - 85 until complete dissolution occurred. The pH of the solution was adjusted to 3 with 5 N HCI and the mixture extracted with EtOAc (3 x 100 mL). The combined organic fractions were dried and the solvent evaporated to give 114 (4.29 g, 100%), as white crystals, mp 160-161 oC (lit. (B. Cavalleri, R. Ballotta, V. Arioli, G.C. Lancini, J. Med. Chem. 1973, 16, 557) 5 (EtOAc) 161-163 oC); 'H NMR [(CD 3
)
2 SO] 8 13.60 (br s, 1 H, CO 2 H), 7.37 (s, 1 H, H 4), 4.20 (s, 3 H, NCH 3 ); 13C NMR [(CD 3
)
2 SO] 5 160.3, 147.2, 133.7, 127.0, 35.0. (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (115). A solution of CDI (7.0 g, 43.1 mmol) and 114 was stirred at 20 oC for 30 min and then added to a stirred solution of 10 NaBH 4 (4.07 g, 108 mmol) in EtOH (10 mL) and the mixture stirred at 20 oC for 1 h. 5 M HCI (20 mL) was added carefully and the mixture stirred for 30 min. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc, to give 115 (2.23 g, 68%) as a white solid, mp 138-140 'C (lit. [B. Cavalleri; R. Ballotta; V. Arioli; G.C. Lancini, J. Med. Chem. 1973, 16, 557] 142-144 oC); 1 H NMR [(CD 3
)
2 SO] 87.12 (s, 1 15 H, H 4), 5.49 (br s, 1 H, OH), 4.55 (s, 2 H, CH 2 0), 3.92 (s, 3 H, NCH 3 ); " 3 C NMR
[(CD
3
)
2 SO] 5 145.6, 138.6, 126.5, 52.9, 34.0. (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-nitrophenyl carbonate (116). A solution of 4-nitrophenylchloroformate (0.67 g, 3.34 mmol) in THF (5 mL) was added to a stirred 20 solution of alcohol 115 (0.50 g, 3.18 mmol) and pyridine (283 /L, 3.50 mmol) in THF (50 mL) at 20 oC under N 2 . The solution was stirred at 20 oC for 16 h, the solvent evaporated and the residue dissolved in EtOAc (100mL). The solution was washed with water (2 x 50 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 116 (0.87 g, 84%) as a 25 white solid, mp (EtOAc) 156.5-157.5 'C; IR N 1771, 1537, 1359 cm-'; 'H NMR
[(CD
3
)
2 SO] 8 8.33 (ddd, J= 9.1, 3.2, 2.1 Hz, 2 H, H 3, H 5), 7.59 (ddd, J= 9.1, 3.2, 2.1 Hz, 2 H, H 2, H 6), 7.37 (s, 1 H, H 4'), 5.48 (s, 2 H, CH 2 0), 4.00 (s, 3 H, NCH 3 ); 13C NMR
[(CD
3
)
2 SO] 8 155.1, 151.4, 146.3, 145.2, 131.5, 129.6, 125.4 (2), 122.5 (2), 59.4, 34.3; Anal. (C 12 HIoN 4 0 7 ) C, H. N calc. 17.4, found 16.7%. 30 (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl bis(2-chloroethyl)carbamate (117). A solution of carbonate 116 (0.68 g, 2.11 mmol) in pyridine (3 mL) was added to a solution of bis-(2-chloroethyl)amine hydrochloride (0.75 g, 4.22 mmol) in pyridine (30 mL) under WO 00/64864 PCT/GB00/01612 - 86 N 2 . The solution was stirred at 20 oC for 16 h and the solvent evaporated. The residue was dissolved in DCM (100 mL) and washed with 2% citric acid solution (2 x 50 mL), water (50 mL), brine (50 mL), dried and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 117 (0.51 g, 74%) as 5 white crystals, mp (EtOAc) 100-101 oC; IR N 1703, 1489, 1344 cm 1 ; 'H NMR 8 7.23 (s, 1 H, H 4), 5.21 (s, 2 H, CH 2 0), 4.05 (s, 3 H, NCH 3 ), 3.58-3.70 (m, 8 H, 2 x CH 2 N, 2 x
CH
2 C); 13C NMR 8 154.8, 144.5, 132.1, 129.7, 56.2, 50.8 (2), 41.6 (2), 34.3; Anal.
(C,
0
H,
4 C1 2
N
4 0 4 ) C, H, N. 10 Example 5B. Preparation of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 4-[bis(2 chloroethyl)amino]phenylcarbamate (123). N,N-Bis(2- { [tert-butyl(dimethyl)silyl]oxy}ethyl)-4-nitroaniline (119). A solution of TBDMSC1 (4.20 g, 27.9 mmol) in DMF (15 mL) was added to a stirred solution of NN bis(2-hydroxyethyl)-4-nitroaniline (118) (3.0 g, 13.26 mmol) and imidazole (3.79 g, 55.7 15 mmol) in DMF (50 mL) and the solution stirred at 20 oC for 48 h. The solvent was evaporated and the residue partitioned between EtOAc (150 mL) and water (150 mL). The organic fraction was washed with water (2 x 200 mL), dried, and the solvent evaporated.The residue was purified by chromatography, eluting with 10% EtOAc/light petroleum, to give 119 (5.72 g, 95%) as a white solid, mp (pet. ether) 48-49 oC; IR N 1597, 20 1520, 1300, 1202, 1107 cm-'; 'H NMR 5 8.07 (ddd, J= 9.5, 3.5, 2.1 Hz, 2 H, H 3, H 5), 6.67 (ddd, J = 9.5, 3.5, 2.1 Hz, 2 H, H 2, H 6), 3.80 (dd, J = 6.0, 5.7 Hz, 4 H, 2 x CH 2 N), 3.63 (dd, J= 5.9, 5.7 Hz, 4 H, 2 x CH20), 0.86 (s, 18 H, 2 x SiC(CH 3
)
3 ), 0.01 (s, 12 H, 2 x Si(CH 3
)
2 ); 1 3 C NMR 8 153.0, 138.6, 126.2 (2), 110.4 (2), 60.2 (2), 53.6 (2), 25.8 (6), 18.2 (2), -5.5 (4); MS (DEI) m/z 454 (M-, 10%), 439 (5), 397 (10), 309 (100); HRMS (DEI) 25 calc. for C 2 2
H
4 2
N
2 0 4 Si 2
(M
+
) m/z 454.2683, found 454.2668; Anal. (C 2 2
H
4 2 N20 4 Si 2 ) C, H, N. Ns,N'-Bis(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-1,4-benzenediamine (120). A mixture of 119 (1.54 g, 3.39 mmol) and Pd/C (50 mg) in EtOAc/EtOH (1:1) (50 mL) was stirred 30 under hydrogen (60 psi) for 30 minutes, filtered through celite, washed with EtOH (2 x 10 mL) and the solvent evaporated to give crude benzenediamine (120) as an oil that was used directly without further purification or characterization.
WO 00/64864 PCT/GB00/01612 - 87 (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-[bis(2- { [tert butyl(dimethyl)silyl]oxy}ethyl)amino]phenylcarbamate (121). A solution of carbonate 116 (0.87 g, 2.68 mmol), 120 (3.39 mmol), and pyridine (217 /L, 2.68 mmol) in THF (50 mL) was stirred at 20 oC for 48 h. The solvent was evaporated and the residue partitioned 5 between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (20-50%) of EtOAc/light petroleum to give 121 (1.37 g, 84%) as a white solid, mp 143-144 oC; IRN 3258, 1721, 1539, 1257, 1103 cm-'; 1 H NMR [(CD 3
)
2 SO] 5 7.23 (s, 1 H, H 4'), 7.15 (br d, J= 8.9 Hz, 2 H, H 3, H 5), 10 6.63 (d, J= 8.9 Hz, 2 H, H 2, H 6), 6.52 (br s, 1 H, OCONH), 5.20 (s, 2 H, CH 2 0), 4.06 (s, 3 H, NCH 3 ), 3.73 (dd, J= 6.5, 6.3 Hz, 4 H, 2 x CH 2 0), 3.47 (dd, J= 6.5, 6.3 Hz, 4 H, 2 x
CH
2 N), 0.88 (s, 18 H, 2 x Si(C(CH 3
)
3 ), 0.02 (s, 12 H, 2 x Si(CH 3
)
2 ); 1 3 C NMR [(CD 3
)
2 SO] 5 152.9, 146.1, 145.4, 132.5, 129.6, 125.4, 121.7 (2), 111.8 (2), 60.3 (2), 55.2, 53.6 (2), 34.3, 26.0 (6), 18.2 (2), -5.4 (4); Anal. (C 28
H
49
N
5 sO 6 Si 2 ) C, H, N. 15 (1-Methyl-2-nitro-lH-imidazol-5-yl)methyl 4-[bis(2 hydroxyethyl)amino]phenylcarbamate (122). TBAF (1 M in THF, 3.9 mL, 3.9 mmol) was added to dropwise to a stirred solution of 121 (1.07 g, 1.76 mmol) in THF (30 mL) at 5 oC. The solution was stirred for 30 minutes and the solvent evaporated. The residue was 20 purified by chromatography, eluting with a gradient (0-10%) of MeOH/EtOAc, to give 122 (0.59 g, 88%) as a white solid, mp (MeOH) 171-174 oC; IR N 3445, 3329, 3266, 1717, 1609, 1549, 1491, 1375, 1248 cm-'; 'H NMR [(CD 3
)
2 SO] 6 9.37 (s, 1 H, OCONH), 7.28 (s, 1H,H 4'), 7.19 (br d,J= 9.1Hz, 2 H, H 3, H 5), 6.61 (d,J= 9.1Hz, 2 H, H 2, H 6), 5.23 (s, 2 H, CH20), 4.71 (t, J= 5.4 Hz, 2 H, 2 x OH), 3.96 (s, 3 H, NCH 3 ), 3.51 (dd, J= 6.4, 5.9 25 Hz, 4 H, 2 x CH20), 3.34 (dd, J= 6.2, 5.9 Hz, 4 H, 2 x CH 2 N); 13 C NMR [(CD 3
)
2 SO] 5 152.8, 146.0, 144.2, 133.6, 128.6, 127.1, 120.3 (2), 111.4 (2), 59.7, 58.2 (2), 53.4 (2), 34.2; MS (FAB') m/z 380 (MH
+
, 10%), 348 (5); HRMS (FAB') m/z calc. for C 16
H
2 2
N
5 0 6 (MH') 380.1570, found 380.1579; Anal. (CI 6
H
21
N
5 0 6 ) C, H, N. 30 (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-[bis(2 chloroethyl)amino]phenylcarbamate (123). Methanesulfonyl chloride (191 UL, 2.47 mmol) was added dropwise to a stirred solution of diol 122 (312 mg, 0.82 mmol) in pyridine (10 mL) at 5 oC and the solution stirred at 20 'C for 1 h. The solvent was WO 00/64864 PCT/GB00/01612 -88 evaporated and the residue partitioned between DCM (100 mL) and water (100 mL). The aqueous fraction was washed with DCM (2 x 50 mL), the combined organic extracts dried, and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiCI (210 mg, 4.9 mmol) added and the mixture stirred at 80 oC for 3 h. The solvent was evaporated and the 5 residue partitioned between EtOAc (100 mL) and water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL), the combined extracts dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 123 (227 mg, 66%) as a white solid, mp (MeOH) 156-157 oC; IR N 3408, 3246, 1725, 1531, 1354, 1221 cm-'; 'H NMR [(CD 3
)
2 SO] 6 9.49 (s, 1 H, OCONH), 10 7.26-7.29 (m, 3 H, H 4', H 3, H 5), 6.70 (d, J= 9.1 Hz, 2 H, H 2, H 6), 5.24 (s, 2 H, CH 2 0), 3.96 (s, 3 H, NCH 3 ), 3.64-3.70 (m, 8 H, 2 x CH 2 N, 2 x CH 2 C); ' 3 C NMR [(CD 3
)
2 SO] 6 152.8, 146.0, 142.3, 133.5, 128.7, 128.6, 120.3 (2), 112.3 (2), 54.9, 52.2 (2), 41.1 (2), 34.1. Anal. (C 6 H1 9 C1 2
N
5 0 4 ) C, H, N, Cl. 15 Example 5C. Preparartion of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-yl)carbonyl]-2,3-dihydro-lH benzo[e]indol-5-ylcarbamate (124). A solution of triphosgene (14.5 mg, 49 /mol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (58 mg, 124 /Zmol) and 20 Et 3 N (39 mL, 280 gmol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of alcohol 115 ( 24 mg, 154 /tmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 48 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/light petroleum, to give 124 (55 mg, 68%) as a tan powder, mp (EtOAc) 202-204 oC; 'H NMR 6 9.49 (br s, 1 H, indole 25 NH), 8.81 (br s, 1 H, H 4), d, J= 8.5 Hz, 1 H, H 6), 7.78 (d, J= 8.3 Hz, 1 H, H 9), 7.57 (m, 1 H, H 8), 7.43 (m, 1 H, H 7), 7.25 (s, 1 H, H 4"), 7.21 (br s, 1 H, OCONH), 7.00 (d, J= 1.6 Hz, 1 H H 3'), 6.87 (s, 1 H, H 4'), 5.31 (d, J = 13.6 Hz, 1 H, CH 2 0), 5.25 (d, J= 13.6 Hz, 1 H, CH 2 0), 4.80 (dd, J= 10.5, 1.6 Hz, 1 H, H 2), 4.65 (dd, J = 10.5, 8.7 Hz, 1 H, H 2), 4.13 4.20 (m, 1 H, H 1), 4.11 (s, 3 H, OCH 3 ), 4.01 (br s, 3 H, NCH 3 ), 3.94-3.98 (m, 4 H, CH 2 CI, 30 OCH 3 ), 3.92 (s, 3 H, OCH 3 ), 3.47 (dd, J= 10.8, 10.8 Hz, 1 H, CH 2 C1); ' 3 C NMR 8 160.4 (CO), 153.5 (OCONH), 150.2 (C 5'), 146.4 (C 2"), 141.6 (C 3a), 140.7 (C 6'), 138.9 (C 7'), 133.3 (C 5), 132.1 (C 5"), 129.8 (C 4"), 129.7 (C 9a), 129.5 (C 2'), 127.6 (C 8), 125.7 (C 7a'), 125.1 (C 7, C 5a), 123.6 (C 3a'), 123.2 (C 9), 122.3 (C 6, C 9b), 113.0 (C 4), 106.6 WO 00/64864 PCT/GB00/01612 - 89 (C 3'), 97.6 (C 4'), 61.5 (OCH 3 ), 61.2 (OCH 3 ), 56.3 (OCH 3 ), 55.8 (CH 2 0), 54.9 (C 2), 45.8
(CH
2 Cl), 43.4 (C 1), 34.3 (NCH 3 ); MS (FAB*) m/z 649 (MH, 2%); HRMS (FAB') calc. for
C
31
H
3 0 35 C1N 6 0 8 (MH) m/z 649.1814, found 649.1767; calc. for C 31
H
30 3 7 C1N 6 0 8
(MH
+
) m/z 651.1784, found 651.1819; Anal (C 31 H2 9 C1N 6 0 8 .2H 2 0) C, H, N. 5 Alternative preparation of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-yl)carbonyl]l-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (124). A solution of alcohol 115 (17 mg, 0.11 mmol) in DCM (2 mL) was added dropwise to a stirred solution of triphosgene (12 mg, 0.04 mmol) 10 and pyridine (9 ML, 0.11 mmol) in DCM (2 mL) at 20 oC. The mixture was stirred at 20 oC for 2 h, the solvent evaporated and the residue dissolved in THF (5 mL). A solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (50 mg, 0.11 mmol) in THF ( 5 mL) was added and the solution stirred at 20 oC for 16 h. The mixture was partitioned between EtOAc (50 mL) and sat. aq. KHCO 3 solution, 15 the organic fraction dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (50-100%) of EtOAc/light petroleum to give 124 (23 mg, 33%) as a tan solid, mp 200-205 'C (dec); spectroscopically identical with an authentic sample prepared above. 20 Example 5D. Preparation of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl doxorubicin carbamate (125). A solution of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 4-nitrophenyl carbonate (116) (33 mg, 104 gmol) in DMF (2 mL) was added dropwise to a stirred solution of doxorubicin (13) (46 mg, 86 gmol) and Et 3 N (15 zL, 104 /mol) in DMF (5 mL) 25 at 20 oC and the solution stirred for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-5%) of MeOH/DCM, to give 125 (44 mg, 70%) as a red solid, mp (DCM) 162-166 oC; 'H NMR [(CD 3
)
2 SO] 6 13.96 (s, 1 H, 6-OH), 13.21 (s, 1 H, 11-OH), 7.82-7.87 (m, 2 H, H 1, H 3), 7.58 (dd, J = 7.5, 2.1 Hz, 1 H, H 2), 7.18 (s, 1 H, H 4"), 7.02 (d, J= 7.9 Hz, 1 H, OCONH), 5.42 (s, 1 H, 9-OH), 5.21 (d, J 30 = 2.6 Hz, 1 H, H 1'), 5.07 (s, 2 H, CH 2 0), 4.86-4.91 (m, 2 H, H 7, 14-OH), 4.73 (d, J= 5.9 Hz, 1 H, 4-OH), 4.58 (d, J= 5.9 Hz, 2 H, H 14), 4.13-4.17 (m, 1 H, H 5'), 3.96 (s, 3 H, 4
OCH
3 ), 3.88 (s, 3 H, NCH 3 ), 3.66-3.74 (m, 1 H, H 3'), 3.41-3.46 (m, 1 H, H 4'), 2.97 (d, J = 18.3 Hz, 1 H, H 10), 2.87 (d, J= 18.3 Hz, 1 H, H 10), 2.21 (d, J= 14.0 Hz, 1 H, H 8), WO 00/64864 PCT/GB00/01612 - 90 2.17 (dd, J= 14.0, 5.4 Hz, 1 H, H 8), 1.84 (dt, J= 12.8, 3.5 Hz, 1 H, H 2'), 1.57 (dd, J= 12.8, 3.8 Hz, 1 H, H 2'), 1.12 (d, J= 6.4 Hz, 3 H, H 6'); 13 C NMR [(CD 3
)
2 SO] 6 213.7 (C 13), 186.3 (C 5), 186.2 (C 12), 160.7 (C 4), 156.0 (6), 154.6 (C 11), 154.4 (OCONH), 145.8 (C 2"), 136.1 (C 2), 135.4 (C 12a), 134.5 (C 6a), 134.0 (C 10a), 133.8 (C 5"), 128.3 (C 4"), 5 119.8 (C4a), 119.6(C 1), 118.9 (C3), 110.6 (C 5a), 110.5 (C 11a), 100.3 (C 1'),74.9 (C 9), 69.8 (C 7), 67.8 (C 4'), 66.6 (C 5'), 63.7 (C 14), 56.5 (4-OCH 3 ), 54.7 (CH 2 0), 47.3 (C 3'), 38.4 (C 8), 34.1 (NCH 3 ), 32.0 (C 10), 29.7 (C 2'), 17.0 (C 6'); MS (FAB') m/z 727 (MH', 0.2%); HRMS (FAB') calc. for C 33
H
3 4
N
4 0, 5 (MH) m/z 727.2099, found 727.2075; Anal (C 3 3
H
34
N
4 0, 5 s.-V2H 2 0) C, H, N. 10 Example 5E. Preparation of 4-({[(1-methyl-2-nitro-1H-imidazol-5 yl)methoxy]carbonyl}amino)benzyl doxorubicin carbamate (129). (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-( { [tert butyl(dimethyl)silylloxy}methyl)phenylcarbamate (126). Et 3 N (0.26 mL, 1.86 mmol) 15 was added to a stirred suspension of (1-methyl-2-nitro-1H-imidazol-5-yl)methyl 4 nitrophenyl carbonate (116) (0.50 g, 1.55 mmol), 4-({[tert butyl(dimethyl)silyl]oxy}methyl)aniline (9) (0.40 g, 1.71 mmol), HOBT (0.21 g, 1.55 mmol), and 4 A molecular sieves (500 mg) in THF (80 mL) and the mixture stirred at 20 oC for 16 h. The solvent was evaporated and the residue partitioned between EtOAc (100 20 mL) and water (100 mL). The organic fraction was washed with 1 M HCI (2 x 40 mL), water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 40% EtOAc/light petroleum, to give 126 (0.43 mg, 66%) as a white solid, mp (EtOAc/light petroleum) 131-132 oC; 'H NMR 8 7.33 (br d, J= 8.8 Hz, 2 H, H 3, H 5), 7.27 (d, J =8.8 Hz, 2 H, H 2, H 6), 7.23 (s, 1 H, H 4'), 6.83 (br s, 1 H, 25 OCONH), 5.22 (s, 2 H, CH 2 0), 4.69 (s, 2 H, CH 2 0), 4.05 (s, 3 H, NCH 3 ), 0.93 (s, 9 H, SiC(CH 3
)
3 ), 0.09 (s, 6 H, Si(CH 3
)
2 ); 1 3 C NMR 8 152.3 (OCONH), 146.1 (C 2'), 137.4 (C 1), 135.8 (C 4), 132.5 (C 5'), 129.6 (C 4'), 126.9 (C 2, C 6), 118.8 (C 3, C 5), 64.5 (CH20), 55.4 (CH 2 0), 34.3 (NCH 3 ), 25.9 (SiC(CH 3
)
3 ), 18.4 (SiC(CH) 3 ), -5.3 (Si(CH 3 )); Anal.
(CWH
2 8 N40sSi) C, H, N. 30 (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl 4-(hydroxymethyl)phenylcarbamate (127). 1 M HCI (2 mL, 2 mmol) was added to a stirred solution of silyl ether 126 (0.39 g, 0.93 mmol) in MeOH (10 mL) and stirred at 20 oC for I h. The solution was poured into brine WO 00/64864 PCT/GB00/01612 -91 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic fraction was washed with water (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (50-100%) EtOAc/light petroleum, to give 127 (247 mg, 87%) as a pale yellow solid, mp (EtOAc) 180-181 0 C; 'H NMR [(CD 3
)
2 SO] 8 5 9.77 (br s, 1 H, OCONH), 7.40 (d, J= 8.5 Hz, 2 H, H 3, H 5), 7.31 (s, 1 H, H 4'), 7.22 (d, J = 8.5 Hz, 2 H, H 2, H 6), 5.27 (s, 2 H, CH20), 5.08 (t, J= 5.6 Hz, 1 H, OH), 4.42 (d, J= 5.6 Hz, 2 H, CH 2 0), 3.97 (s, 3 H, NCH 3 ); ' 3 C NMR [(CD 3
)
2 SO] 5 152.6 (OCONH), 146.0 (C 2'), 137.2 (C 1), 136.8 (C 4), 133.3 (C 5'), 128.7 (C 4'), 127.0 (C 2, C 6), 118.0 (C 3, C 5), 62.4 (CH 2 0), 55.0 (CH 2 0), 34.2 (NCH 3 ); Anal. (Cz 3
H,
4 4 0) C, H, N. 10 4-({[(1-methyl-2-nitro-lH-imidazol-5-yl)methoxy]ecarbonyl}amino)benzyl 4 nitrophenyl carbonate (128). A solution of 4-nitrophenylchloroformate (216 mg, 1.07 mmol) in THF (5 mL) was added dropwise to a stirred solution of alcohol 127 (219 mg, 0.72 mmol) in THF (40 mL) and the solution stirred at 20 oC for 96 h. The solvent was 15 evaporated and the residue partitioned between EtOAc (100 ml) and water (100 mL). The organic fraction was washed with water (2 x 50 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 128 (62 mg, 18%) as a white solid; 'H NMR [(CD 3
)
2 SO] 5 9.96 (s, 1 H, OCONH), 8.31 (ddd J= 9.2, 3.3, 2.2 Hz, 2 H, H 3", H 5"), 7.56 (ddd, J= 9.2, 3.3, 2.2 Hz, 2 20 H, H 2",H 6"), 7.51 (d, J = 8.6Hz, 2 H,H 3,H 5), 7.40 (d,J =8.6Hz, 2H, H 2, H 6), 7.31 (s, 1 H, H 4'), 5.33 (s, 2 H, CH 2 0), 5.24 (s, 2 H, CH 2 0), 3.98 (s, 3 H, NCH 3 ); 3 C NMR
[(CD
3
)
2 SO] 8 155.2 (OCO2), 152.6 (OCONH), 151.9 (C 1"), 145.1 (C 2'), 139.2 (C 1), 133.2 (C 4), 129.6 (C 3", C 5"), 128.8 (C 4'), 128.7 (C 5'), 125.6 (C 2, C 6), 122.2 (C 2", C 6"), 118.8 (C 2, C 6), 70.2 (CH20), 55.2 (CH 2 0), 34.2 (NCH 3 ); MS (FAB
+
) m/z 472 25 (MH',1%), 443 (0.5); HRMS (FAB') calc. for C 20
H
8 NsO 5 0 9
(MH
+
) m/z 472.1105, found 472.1106. 4-({[(1-Methyl-2-nitro-lH-imidazol-5-yl)methoxy]carbonyl} amino)benzyl doxorubicin carbamate (129). A solution of carbonate 128 (81 mg, 172 Mmol) in DMF (2 mL) was 30 added dropwise to a stirred solution of doxorubicin (45 mg, 86 Mmol) and Et 3 N (15 UL, 103 ymol) in DMF (5 mL) at 20 oC and the solution stirred for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 5% MeOH/DCM, to give 129) (57 mg, 75%) as a red solid, mp (DCM) 160-162 oC; 'H NMR [(CD 3
)
2 SO] 8 WO 00/64864 PCT/GB00/01612 - 92 13.99 (s, 1 H, 6-OH), 13.24 (s, 1 H, 11-OH), 9.82 (s, 1 H, OCONH), 7.84-7.89 (mn, 2 H, H 1, H 2), 7.60-7.63 (min, 1 H, H 3), 7.40 (d, J= 8.3 Hz, 2 H, H 3", H 5"), 7.29 (s, 1 H, H 4"'), 7.23 (d, J= 8.3 Hz, 2 H, H 2", H 6"), 6.81 (d, J= 8.0 Hz, 1 H, OCONH), 5.43 (s, 1 H, H 7), 5.25 (s, 2 H, CH 2 0), 5.21 (d, J= 2.9 Hz, 1 H, H 1'), 4.89-4.91 (min, 1 H, 9-OH), 4.87 (s, 2 H, 5 CH 2 0), 4.84 (dd, J= 6.3, 5.8 Hz, 1 H, 14-OH), 4.69 (d, J= 5.7 Hz, 1 H, 4-OH), 4.58 (d, J= 6.0 Hz, 2 H, H 14), 4.13-4.17 (min, 1 H, H 5'), 3.97 (s, 3 H, OCH 3 ), 3.95 (s, 3 H, NCH 3 ), 3.68-3.75 (min, 1 H, H 3'), 3.43-3.46 (min, 1 H, H 4'), 2.99 (d, J= 18.3 Hz, 1 H, H 10), 2.91 (d, J= 18.3 Hz, 1 H, H 10), 2.21 (br d, J= 14.1 Hz, 1 H, H 8), 2.10 (dd, J= 14.1 Hz, 1 H, H 8), 1.84 (dt, J= 12.9, 3.6 Hz, 1 H, H 2'), 1.47 (dd, J= 12.9, 3.8 Hz, 1 H, H 2'), 1.13 (d, J 10 = 6.5 Hz, 3 H, H 6'); 1 3 C NMR [(CD 3
)
2 SO] 6 213.7 (C 13), 186.4 (C 5), 186.3 (C 12), 160.7 (C 4), 156.0 (C 6), 155.2 (C 11), 154.4 (OCONH), 152.6 (OCONH), 146.0 (C 2'"), 138.2 (C 4"), 136.1 (C 2), 135.4 (C 12a), 134.6 (C 6a), 134.0 (C 10a), 13.2 (C 5'"), 131.2 (C 1"), 128.7 (C 4'"), 128.6 (C 2", C 6"), 119.9 (C 4a), 119.6 (C 1), 118.9 (C 3), 118.0 (C 3", C 5"), 110.7 (C 5a), 110.5 (C 11a), 100.2 (C 1'), 74.9 (C 9), 69.8 (C 7), 67.9 (C 4'), 66.6 (C 15 5'), 64.8 (C 14), 63.6 (CH20), 56.5 (OCH 3 ), 55.1 (CH 2 0), 47.0 (C 3'), 36.5 (C 8), 34.1
(NCH
3 ), 32.0 (C 10), 29.8 (C 2'), 16.9 (C 6'); MS (FAB') m/z 876 (MH
+
, 0.2%); Anal.
(C
41
H
4 1
N
5 0 17
.H
2 0) C, H, N. Example 5F. Preparation of (1-methyl-4-nitro-1H-imidazol-5-yl)methyl 1 20 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-l1-yl)carbonyl]-2,3-dihydro-1H benzol[e]indol-5-ylcarbamate (132). (1-Methyl-4-nitro-lH-imidazol-5-yl)methanol (131). Ozone was bubled through a solution of 1-methyl-4-nitro-5-[(E)-2-phenylethenyl]-1H-imidazole (130) [ D. C. Baker, S. R. Putt, H. D. H. Showalter, J. Heterocyclic Chem., 1983, 30, 629-634.] (1.0 g, 4.36 mmol) 25 in DCM/MeOH (1:1, 120 mL) at -78 oC until a blue colour persisted. The solution was warmed to -40 oC with a nitrogen purge to remove excess ozone. A solution of NaBH 4 (165 mg, 4.36 mmol) in EtOH (10 mL) was added dropwise over 15 min and the mixture stirred for 30 min. Acetic acid (0.5 mL) was added and the solvent evaporated. The residue was partitioned between water (50 mL) and light petroleum (50 mL). The aqueous fraction 30 was evaporated and the residue triturated with hot acetone (60 mL). The mixture was filtered and the solution concentrated to give 131 (523 mg, 78%) as a tan powder, mp (acetone) 135-137 oC; 1 H NMR 65 7.78 (s, 1 H, H 2), 5.48 (t, J= 5.6 Hz, 1 H, OH), 4.85 (d, J= 5.6 Hz, 2 H, CH 2 0), 3.75 (s, 3 H, NCH 3 ); 13 C NMR 5 143.5 (C 4), 136.8 (C 2), 133.2 (C WO 00/64864 PCT/GB00/01612 - 93 5), 51.4 (CH20), 32.5 (NCH 3 ); Anal. (C 5
H
7
N
3 0 3 ) C, H, N. (1-Methyl-4-nitro-1H-imidazol-5-yl)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H indol-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (132). A solution of 5 triphosgene (13 mg, 43 gmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (51 mg, 109 pmol) and Et 3 N (34 pL, 246 pmol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of (1-methyl-4-nitro-1H-imidazol-5-yl)methanol 131 (23 mg, 147 kzmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution 10 stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc, to give 132 (40 mg, 56%) as a white powder, mp (EtOAc/light petroleum) 229-231 oC; 'H NMR 5 9.48 (s, 1 H, indole-NH), 8.87 (s, 1 H, OCONH), 7.87 (d, J= 8.5 Hz, 1 H, H 6), 7.76 (d, J = 8.3 Hz, 1 H, H 9), 7.55 (ddd, J= 8.3, 7.4, 0.7 Hz, 1 H, H 8), 7.44 (ddd, J= 8.5, 7.4, 0.7 Hz, 1 H, H 7), 7.40 (s, 1 H, H 2"), 7.33 15 (br s, 1 H, H 4), 7.00 (d, J= 2.3 Hz, 1 H, H 3'), 6.87 ( s, 1 H, H 4'), 5.61 (s, 2 H, CH20), 4.78 (dd, J= 10.7, 1.7 Hz, 1 H, H 2), 4.65 (dd, J= 10.7, 8.7 Hz, 1 H, H 2), 4.15-4.20 (m, 1 H, H 1), 4.05-4.10 (m, 4 H, OCH 3 , CH 2 C1), 3.95 (s, 3 H, OCH 3 ), 3.92 (s, 3 H, OCH 3 ), 3.83 (br s, 3 H, NCH 3 ), 3.44 (dd, J = 10.9, 10.7 Hz, 1 H, CH 2 C); 13 C NMR 8 160.4 (CO), 153.8 (OCONH), 150.2 (C 5'), 146.3 (C 4"), 141.5 (C 3a), 140.6 (C 6'), 138.8 (C 7'), 136.3 (C 2"), 20 133.4 (C 5), 129.7 (C 9a), 129.5 (C 2'), 127.6 (C 8), 126.9 (C 5"), 125.7 (C 7a'), 125.2 (C 7), 125.2 (C 5a), 123.6 (C 3a'), 123.1 (C 9), 122.3 (C 6), 121.9 (C 9b), 112.5 (C 4), 106.6 (C 3'), 97.7 (C 4'), 61.5 (OCH 3 ), 61.1 (OCH 3 ), 56.3 (OCH 3 ), 54.9 (C 2), 54.4 (CH 2 0), 45.8
(CH
2 C1), 43.4 (C 1), 33.2 (NCH 3 ); MS (FAB') m/z 651 (MH, 1%), 649 (MH', 2); HRMS (FAB') calc. for C 31
H
30 35
CIN
6 0 8 (MH-) m/z 649.1814, found 649.1818; calc. for 25 C 31
H
30 37 C1N 6 0 8 (MH') m/z 651.1784, found 651.1805; Anal. (C 3
,
1
H
29 C1N 6 0 8 ) C, H, N. Example 5G. Preparation of (1-methyl-5-nitro-1H-imidazol-2-yl)methyl bis(2 chloroethyl)carbamate (135). (1-Methyl-5-nitro-1H-imidazol-2-yl)methanol (133). A mixture of 1-methyl-5-nitro- 1 H 30 imidazole [C.E. Hazeldine, F.L. Pyman, J. Winchester. J. Chem. Soc. 1924, 1431] (1.0 g, 7.9 mmol) and paraformaldehyde (1.4 g, 15.7 mmol) in DMSO (10 mL) was heated in a sealed tube at 100 'C for 24 h. The mixture was cooled to 20 oC, EtOH (50 mL) was added, and the suspension was filtered. The filtrate was concentrated and the residue was purified WO 00/64864 PCT/GB00/01612 - 94 by chromatography on alumina, eluting with a gradient of MeOH/CHCl 3 (0-10%), to give 133 (0.71 g, 57%) as a white solid, mp (CHCl 3 ) 116-118 oC (lit. [C. Rufer, H. J. Kessler, E. Schroder. J. Med. Chem. 1971, 14, 94] mp 111 oC); 'H NMR [(CD 3
)
2 SO] 8 7.90 (s, 1 H, H 4), 5.62 (t, J= 5.8 Hz, 1 H, OH), 4.62 (d, J= 5.8 Hz, 2 H, CH 2 0), 3.99 (s, 3 H, NCH 3 ); 1 3 C 5 NMR [(CD 3
)
2 SO] 8 151.8, 138.8, 130.9, 56.1, 32.9. (1-Methyl-5-nitro-1H-imidazol-2-yl)methyl 4-nitrophenyl carbonate (134). A solution of 4-nitrophenyl chloroformate (1.48 g, 7.4 mmol) in THF (8 mL) was added slowly to a stirred solution of alcohol 133 (1.10 g, 7.0 mmol) and pyridine (0.62 mL, 7.7 mmol) in 10 THF (50 mL) at 20 oC under N 2 . The mixture was stirred at 20 oC for 16 h, then partitioned between EtOAc/H 2 0. The organic fraction was washed with saturated aqueous NaHCO 3 (50 mL), and the solvent evaporated to give 134 (2.04g, 94%) as a tan solid, mp (EtOAc/light petroleum) 168-171 'C; 'H NMR [(CD 3
)
2 SO] 8 8.33 (ddd, J= 9.1, 3.2, 2.2 Hz, 2 H, H 3, H 5), 8.11 (s, 1 H, H 4'), 7.59 (ddd, J= 9.1, 3.2, 2.2 Hz, 2 H, H 2, H 6), 5.48 15 (s, 2 H, CH 2 0), 3.97 (s, 3 H, NCH 3 ); 1 3 C [(CD 3
)
2 SO] 6 155.0, 151.3, 145.9, 145.2, 139.5, 131.6, 125.3 (2), 122.4 (2), 61.7, 33.5. (1-Methyl-5-nitro-1H-imidazol-2-yl)methyl bis(2-chloroethyl)carbamate (135). A solution of carbonate 134 (2.0 g, 6.5 mmol) in pyridine (5 mL) was added to a stirred 20 solution of N,N-bis-(2-chloroethyl)amine hydrochloride (1.5 g, 8.4 mmol) in pyridine (30 mL) at 0 'C. The solution was stirred at 20 oC for 16 h, then solvent was evaporated and the residue was partitioned between DCM (100 mL) and 10% aqueous citric acid (100 mL). The organic fraction was dried, the solvent evaporated, and the residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 135 (2.0 g, 95%) as an 25 oil; 'H NMR 6 7.99 (s, 1 H, H 4') 5.27 (s, 2 H, CH 2 0), 4.03 (s, 3 H, NCH 3 ), 3.60-3.71 (m, 8 H, 2 x CH 2 N, 2 x CH 2 C1); 1 3 C NMR 5 154.8, 146.9, 139.6, 132.2, 58.7, 51.1 (2), 41.8 (2), 39.7; HRMS calc. for CoHI 4 Cl 2 N404 (M') m/z 324.1392; found 324.1381. Example 5H. Preparation of (1-methyl-5-nitro-1H-imidazol-2-yl)methyl 4-[bis(2 30 chloroethyl)amino]phenylcarbamate (137). Diphosgene (85mL, 0.7 mmol) was added dropwise to a stirred solution of (1-methyl-5-nitro- 1 H-imidazol-2-yl)methanol (133) (0.2 g, 1.27 mmol) and Et 3 N (98 mL, 0.7 mmol) in THF (10 mL) at 5 oC. The suspension was stirred at 5 oC for 30 min and a mixture of N'X,N'-bis(2-chloroethyl)- 1,4-benzenediamine WO 00/64864 PCT/GB00/01612 - 95 hydrochloride (136) (J. L. Everett, W. C. J. Ross. J. Chem. Soc. 1949, 1972] (0.38 g, 1.40 mmol) and Et 3 N (195 mL, 1.40 mmol) in THF (4 mL) was added dropwise to the above suspension. The mixture was stirred at 20 oC for 4 h, the solvent evaporated and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 137 (0.19 g, 5 36%), mp (CHCl 3 /pet. ether) 164-164.5 'C; IR n 3250, 3185, 3127, 1723, 1603, 1547, 1516, 1381 cm-'; 'H NMR ([(CD 3
)
2 SO] 8 9.57 (br s, 1 H, OCONH), 8.08 (s, 1 H, H 4'), 7.27 (br d, J =8.0 Hz, 2 H, H 3, H 5), 6.70 (d, J =9.1 Hz, 2 H, H 2, H 6), 5.26 (s, 2 H, CH 2 0), 3.95 (s, 3 H, NCH 3 ), 3.65-3.72 (m, 8 H, 2 x CH 2 N, 2 x CH 2 C1); 1 3 C NMR [(CD 3
)
2 SO] 6 152.7, 148.0, 142.3, 139.3, 131.7, 128.7, 120.3 (2), 112.3 (2), 57.4, 52.2 (2), 41.1 (2), 33.4; 10 MS (DEI) m/z 415 (M', 1%), 366 (2), 316 (2), 258 (20), 211 (30), 209 (100); HRMS (DEI) m/z calc. for C 16
H
19 3 5 C1 2
N
5 0 4 (M') 415.0814, found 415.0808; calc. for C 16
HI
9 35 C1 37 C1N 5 0 4
(M
+
) 417.0785, found 417.0781; calc. for Cl 6 HI9 37 C1 2 N50 4
(M
+
) 419.0755, found 419.0769; Anal. (C 1 6
H
1 9 C1 2
N
5 0 4 ) C, H, N. 15 Example 51. Preparation of (1-methyl-5-nitro-1H-imidazol-2-yl)methyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (138). A solution of triphosgene (12 mg, 41 zmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (48 mg, 103 p~mol) and Et 3 N (32 4L, 20 231 gmol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of (1-methyl-5-nitro 1H-imidazol-2-yl)methanol (133) (20 mg, 127 Mmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/DCM, to give 138 (23 mg, 34%) as a tan powder mp (EtOAc) 169-170 oC; 1 H NMR 8 9.47 (s, 1 H, 25 indole-NH), 8.85 (s, 1 H, OCONH), 7.98 (s, 1 H, H 4"), 7.86 (d, J= 8.5 Hz, 1 H, H 6), 7.77 (d, J= 8.3 Hz, 1 H, H 9), 7.57 (br dd, J= 8.3, 7.4 Hz, 1 H, H 8), 7.44 (ddd, J= 8.5, 7.4, 0.7 Hz, 1 H, H 7), 7.37 (br s, 1 H, H 4), 6.99 (d, J= 2.3 Hz, 1 H, H 3'), 6.87 ( s, 1 H, H 4'), 5.38 (d, J= 13.4 Hz, 1 H, CH 2 0), 5.34 (d, J= 13.4 Hz, 1 H, CHzO), 4.79 (dd, J= 10.7, 1.6 Hz, 1 H, H 2), 4.65 (dd, J= 10.7, 8.7 Hz, 1 H, H 2), 4.15-4.19 (m, 1 H, H 1), 4.09 (s, 3 H, OCH 3 ), 30 4.02 (br s, 3 H, NCH 3 ), 3.95 (s, 3 H, OCH 3 ), 3.92-3.94 (m, 4 H, OCH 3 , CH 2 C1), 3.45 (dd, J = 10.9, 10.7 Hz, 1 H, CH 2 Cl); 1 3 C NMR 8 160.4 (CO), 153.4 (OCONH), 150.2 (C 5'), 146.9 (C 5"), 141.6 (C 3a), 140.7 (C 6'), 139.6 (C 2"), 138.9 (C 7'), 133.3 (C 5), 132.1 (C 4"), 129.7 (C 9a), 129.6 (C 2'), 127.6 (C 8), 125.7 (C 7a'), 125.2 (C 7 and C 5a), 123.6 (C 3a'), WO 00/64864 PCT/GB00/01612 - 96 123.2 (C 9), 122.3 (C 6), 122.2 (C 9b), 112.8 (C 4), 106.6 (C 3'), 97.7 (C 4'), 61.5 (OCH 3 ), 61.2 (OCH 3 ), 58.4 (CH 2 0), 56.3 (OCH 3 ), 54.9 (C 2), 45.8 (CH 2 C1), 43.4 (C 1), 33.8
(NCH
3 ); MS (FAB') m/z 649 (MH', 3%), 651 (1.5); HRMS (FAB') calc. for
C
31
H
30 35 C1N 6 0 8 (MH-) m/z 649.1814, found 649.1797; calc. for C 3 1
H
30 37 C1N 6 0 8
(MH
+
) m/z 5 651.1784, found 651.1802; Anal. (C 31
H
29 C1N 6 0 8 ) C, H, N. Example 5J. Preparation of 4-({I[(1-methyl-5-nitro-1H-imidazol-2 yl)methoxy]carbonyl}amino)benzyl doxorubicin carbamate (142). 1-Methyl-5-nitro-1H-imidazol-2-yl 4-({[tert 10 butyl(dimethyl)silyl]oxy}methyl)phenylcarbamate (139). Et 3 N (1.10 mL, 7.87 mmol) was added to a stirred suspension of (1-methyl-5-nitro-1H-imidazol-2-yl)methyl 4 nitrophenyl carbonate (134) (2.31 g, 7.17 mmol), 4-({[tert butyl(dimethyl)silyl]oxy}methyl)aniline (120) (1.79 g, 7.87 mmol), HOBT (0.97 g, 7.17 mmol), and 4 A molecular sieves (2.5 g) in THF (100 mL) and the mixture stirred at 20 oC 15 for 48 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with 1 M HCI (2 x 40 mL), water (100 mL), brine (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 139 (2.57 g, 85%) as a white solid, mp (EtOAc/light petroleum) 145-146 oC; 'H NMR 5 7.99 (s, 1 H, H 4'), 7.32 20 (brd, J= 8.1 Hz, 2 H, H 3,H 5), 7.27 (d, J = 8.1 Hz, 2H, H 2,H 6), 6.96 (brs, 1 H, OCONH), 5.30 (s, 2 H, CH 2 0), 4.69 (s, 2 H, CH 2 0), 4.05 (s, 3 H, NCH 3 ), 0.93 (s, 9 H, SiC(CH 3
)
3 ), 0.09 (s, 6 H, Si(CH 3
)
2 ); 1 3 C NMR 6 152.3 (OCONH), 147.0 (C 5'), 139.6 (C 2'), 137.4 (C 1), 135.8 (C 4'), 129.6 (C 4), 126.9 (C 2, C 6), 118.8 (C 3, C 5), 64.5 (CH 2 0), 58.0 (CH 2 0), 33.7 (NCH 3 ), 25.9 (SiC(CH 3
)
3 ), 18.4 (SiC(CH 3
)
3 ), -5.3 (Si(CH 3
)
2 ); Anal. 25 (Ct 9
H
28
N
4 05Si) C, H, N. (1-Methyl-5-nitro-1H-imidazol-2-yl)methyl 4-(hydroxymethyl)phenylcarbamate (140). 1 M HCI (16 mL, 16 mmol) was added to a stirred solution of silyl ether 139 (1.36 g, 3.22 mmol) in MeOH (50 mL) and stirred at 20 oC for 1 h. The solution was poured into brine 30 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic fraction was washed with water (50 mL), dried, and the solvent evaporated. The residue was crystallized from EtOAc/light petroleum, to give 140 (0.86g, 47%) as a white solid, mp (EtOAc/light petroleum) 181-183 oC; 'H NMR [(CD 3
)
2 SO] 5 9.85 (br s, 1 H, OCONH), 8.09 (s, 1 H, H WO 00/64864 PCT/GB00/01612 - 97 4'), 7.40 (d, J= 8.5 Hz, 2 H, H 3, H 5), 7.22 (d, J= 8.5 Hz, 2 H, H 2, H 6), 5.29 (s, 2 H,
CH
2 0), 4.42 (s, 2 H, CH 2 0), 3.96 (s, 3 H, NCH 3 ), 3.79 (br s, 1 H, OH); 13C NMR
[(CD
3
)
2 SO] 8 152.5 (OCONH), 147.8 (C 5'), 139.3 (C 2'), 137.4 (C 1), 136.8 (C 4), 131.7 (C 2, C 6), 127.0 (C 3, C 5), 118.0 (C 4), 62.4 (CH 2 0), 57.5 (CH 2 0), 33.4 (NCH 3 ); Anal. 5 (C, 3
HI
4
N
4 0 5 ) C, H, N. 4-({[(1-Methyl-5-nitro-lH-imidazol-2-yl)methoxy]carbonyl} amino)benzyl 4 nitrophenyl carbonate (141). A solution of 4-nitrophenylchloroformate (0.72 g, 3.55 mmol) in THF (10 mL) was added dropwise to a stirred solution of alcohol 140 (0.73 g, 10 2.37 mmol) and Et 3 N (0.66 mL, 4.73 mmol) in THF (40 mL) and the solution stirred at 20 oC for 16 h. The solvent was evaporated and the residue was purified by chromatography, eluting with 20% EtOAc/DCM, to give 141 (0.71 g, 63%) as a white solid; 'H NMR
[(CD
3
)
2 SO] 6 10.02 (s, 1 H, OCONH), 8.31 (dddJ = 9.1, 3.3, 2.1 Hz, 2 H, H 3, H 5), 8.08 (s, 1 H, H 4"), 7.57 (ddd, J= 9.1, 3.3, 2.1 Hz, 2 H, H2, H 6), 7.51 (d, J= 8.5 Hz, 2 H, H 3', 15 H 5'), 7.40 (d, J= 8.5 Hz, 2 H, H 2', H 6'), 5.32 (s, 2 H, CH20), 4.70 (s, 2 H, CH 2 0), 3.97 (s, 3 H, NCH 3 ); MS (FAB') m/z 472 (MH*,1.5%); HRMS (FAB') calc. for C 2 0
H
8 NsO 5 9 (MH') m/z 472.1105, found 472.1108. 4-({[(-Methyl-5-nitro-1H-imidazol-2-yl)methoxy]ecarbonyl}amino)benzyl doxorubicin 20 carbamate (142). A solution of carbonate 141 (61 mg, 129 gmol) in DMF (2 mL) was added dropwise to a stirred solution of doxorubicin (13) (45 mg, 86 Amol) and Et 3 N (18 /2L, 129 gmol) in DMF (5 mL) at 20 oC and the solution stirred for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 5% MeOH/DCM, to give 142 (50 mg, 66%) as a red solid, mp (DCM) 170-173 oC; 'H NMR [(CD 3
)
2 SO] 8 25 14.00 (s, 1 H, 6-OH), 13.24 (s, 1 H, 11-OH), 9.91 (s, 1 H, OCONH), 8.07 (s, 1 H, H 4'"), 7.86-7.90 (m, 2 H, H 1, H 2), 7.60-7.63 (m, 1 H, H 3), 7.40 (d, J= 8.4 Hz, 2 H, H 3", H 5"), 7.23 (d, J = 8.4 Hz, 2 H, H 2", H 6"), 6.81 (d, J = 8.0 Hz, 1 H, OCONH), 5.43 (s, 1 H, H 7), 5.27 (s, 2 H, CH 2 0), 5.21 (d, J= 2.9 Hz, 1 H, H 1'), 4.92-4.95 (m, 1 H, 9-OH), 4.87 (s, 2 H, CH20), 4.84 (dd, J= 6.3, 5.9 Hz, 1 H, 14-OH), 4.69 (d, J= 5.7 Hz, 1 H, 4-OH), 4.58 (d, J 30 6.0 Hz, 2 H, H 14), 4.12-4.18 (m, 1 H, H 5'), 3.97 (s, 3 H, OCH 3 ), 3.95 (s, 3 H, NCH 3 ), 3.68-3.75 (m, 1 H, H 3'), 3.43-3.46 (m, 1 H, H 4'), 2.98 (d, J= 18.3 Hz, 1 H, H 10), 2.91 (d, J= 18.3 Hz, 1 H, H 10), 2.20 (br d, J= 14.1 Hz, 1 H, H 8), 2.11 (dd, J= 14.1 Hz, 1 H, H 8), 1.84 (dt, J
=
12.9, 3.7 Hz, 1 H, H 2'), 1.47 (dd, J
=
12.9, 4.0 Hz, 1 H, H 2'), 1.12 (d,J WO 00/64864 PCT/GB00/01612 - 98 = 6.5 Hz, 3 H, H 6'); 3 C NMR [(CD 3
)
2 SO] 8 213.7 (C 13), 186.4 (C 5), 186.3 (C 12), 160.7 (C 4), 156.0 (C 6), 155.2 (C 11), 154.4 (OCONH), 152.4 (OCONH), 147.8 (C 5'"), 139.3 (C 2"'), 138.2 (C 4"), 136.1 (C 2), 135.4 (C 12a), 134.6 (C 6a), 134.0 (C 10a), 131.7 (C 4'"), 131.2 (C 1"), 128.6 (C 2", C 6"), 119.9 (C 4a), 119.6 (C 1), 118.9 (C 3), 118.0 (C 3", 5 C 5"), 110.7 (C 5a), 110.5 (C 1 la), 100.2 (C 1'), 74.9 (C 9), 69.8 (C 7), 67.9 (C 4'), 66.6 (C 5'), 64.8 (C 14), 63.6 (CH 2 0), 57.6 (CH 2 0), 56.5 (OCH 3 ), 47.0 (C 3'), 36.5 (C 8), 33.4
(NCH
3 ), 32.0 (C 10), 29.7 (C 2'), 16.9 (C 6'); MS (FAB') m/z 876 (MH
+
, 0.6%); HRMS (FAB') calc. for C 4 1
H
42 N5017 (MH*) m/z 876.2576, found 876.2573; Anal.
(C
4 1
H
41
N
5 0 17
.H
2 0) C, H, N. 10 Example 5K. Preparation of [1-(2-hydroxyethyl)-5-nitro-1H-imidazol-2-yl]methyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (148). 2-{5-Nitro-2-[(E)-2-phenylethenyl]-1H-imidazol-1-yl}ethanol (144). A solution of Na 15 (2.0 g, 87.6 mmol) in dry MeOH (30 mL) was added in one portion to a stirred solution of metronidazole (143) (10.0 g, 58.4 mmol) and benzaldehyde (7.1 mL, 70 mmol) in DMSO (30 mL) at 20 oC. The mixture was stood at 20 'C for 24 h. Water (80 mL) was added and the resulting solid filtered. The solid was dissolved in EtOAc (100 mL) dried, and the solvent evaporated. The residue was purified by chromatography, eluted with 50% 20 EtOAc/light petroleum, to give 144 (4.0 g, 26%) as a yellow powder, mp 155 oC (lit mp 156-157 oC [ W. J. Ross, W. B. Jamieson, and M. C. McCowen, J. Med. Chem. 1972, 15, 1035-1039]); 'H NMR 8 8.06 (s, 1 H, H 4'), 7.83 (d, J= 15.8 Hz, 1 H, CH=), 7.52-7.58 (inm, 2 H, H 2", H 6"), 7.33-7.38 (min, 3 H, H 3", H 4", H 5"), 7.05 (d, J = 15.8 Hz, 1 H, CH=), 4.64 (dd, J = 5.1, 5.0 Hz, 2 H, H 1), 4.07 (dd, J = 5.1, 5.0 Hz, 2 H, H 2), 2.42 (br s, 1 H, 25 OH); 13C NMR 8 150.9 (C 5'), 140.0 (CH=), 138.5 (C 2'), 135.3 (C 1"), 134.6 (C 4'), 129.7 (C 4"), 128.9 (C 3", C 5"), 127.6 (C 2", C 6"); 112.1 (CH=), 61.8 (C 1), 47.7 (C 2). tert-Butyl(dimethyl)silyl 2- {5-nitro-2-[(E)-2-phenylethenyl]-1H-imidazol-1-yl}ethyl ether (145). TBDMS triflate (2.7 mL, 11.75 mmol) was added dropwise to a stirred 30 solution of alcohol 144 (2.77 g, 10.7 mmol) and pyridine (1.3 mL, 16.0 mmol) in DCM (100 mL) at-5 oC and the solution stirred at 20 oC for 16 h. The reaction was quenched with MeOH (5 mL) and poured in sat. aq. KHCO 3 (100 mL). The mixture was extracted with DCM (3 x 50 mL), the combined organic fraction dried and the solvent evaporated.
WO 00/64864 PCT/GB00/01612 - 99 The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to give 145 (4.00 g, 100%) as a yellow solid, mp (EtOAc/light petroleum) 99-100.5 oC; 'H NMR 8 8.13 (s, 1 H, H 4'), 7.87 (d, J = 15.8 Hz, 1 H, CH=), 7.57 (d, J= 6.8 Hz, 2 H, H 2", H 6"), 7.34-7.41 (min, 3 H, H 3", H 4", H 5"), 7.06 (d, J= 15.8 Hz, 1 H, CH=), 4.62 (dd, J= 5 5.0, 4.8 Hz, 2 H, H 1), 4.00 (dd, J= 5.0, 4.8 Hz, 2 H, H 2), 0.77 (s, 9 H, SiC(CH 3
)
3 ), 0.10 (s, 6 H, Si(CH 3
)
2 ); 13 C NMR 8 151.2 (C 5'), 139.3 (CH=), 138.4 (C 2'), 135.5 (C 1'), 134.8 (C 4'), 129.6 (C 4"), 128.9 (C 3", C 5"), 127.5 (C 2", C 6"), 112.9 (CH=), 62.3 (C 1), 47.8 (C 2), 25.7 (SiC(CH 3
)
3 ), 18.1 (SiC(CH 3
)
3 ), -5.8 (Si(CH 3
)
2 ); Anal. (CI 9
H
27
N
3 0 3 Si) C, H, N. 10 [1-(2-{[tert-Butyl(dimethyl)silylloxy}ethyl)-5-nitro-1H-imidazol-2-yl] methanol (146). Ozone was bubbled into a solution of imidazole 145 (1.3 g, 3.48 mmol) in DCM/MeOH (1:1, 120 mL) at -78 oC until a blue colour persisted. The solution was warmed to -40 0 C with a N 2 purge to remove excess ozone. A solution of NaBH 4 (132 mg, 3.48 mmol) in EtOH (10 mL) was added dropwise over 15 min and the mixture stirred for 30 min. The 15 mixture was treated with acetic acid (0.5 mL), stirred for 10 min and the solvent evaporated. The residue was partitioned between EtOAc (100 mL) and water (100 mL). The organic fraction was washed with water (50 mL), brine (25 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 146 (0.92 g, 88%) as a white solid, mp 104-105 oC; 1 H NMR 6 7.97 (s, 1 20 H, H 4), 4.79 (s, 2 H, CH 2 0), 4.62 (t, J= 4.8 Hz, 2 H, CH20), 3.97 (t, J= 4.8 Hz, 2 H, CHzN), 3.80 (br s, 1 H, OH), 0.81 (s, 9 H, SiC(CH 3
)
3 ), 0.10 (s, 6 H, Si(CH 3
)
2 ); 1 3 C NMR 5 157.2 (C 2), 138.8 (C 5), 132.3 (C 4), 62.0 (CH 2 0), 57.2 (CH 2 0), 48.3 (CH 2 N), 25.7 (SiC(CH 3
)
3 ), 18.2 (SiC(CH 3 )3), -5.8 (Si(CH 3
)
2 ); Anal. (C, 2
H
23 N30 4 Si) C, H, N. 25 l[1-(2-{ [tert-Butyl(dimethyl)silyl]oxy} ethyl)-5-nitro-1H-imidazol-2-yllmethyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-lH benzo[elindol-5-ylcarbamate (147). A solution of triphosgene (25 mg, 84 Iimol) in DCM (3 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (112 mg, 240 /zmol) and Et 3 N (67 IzL, 30 481 kimol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of alcohol 146 (78 mg, 264 kmol) in DCM (3 mL) was added, followed by nBuSn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20% EtOAc/DCM, to give 147 (93 mg, 49%) as a colourless WO 00/64864 PCT/GB00/01612 - 100 oil, 'H NMR 6 9.41 (s, 1 H, indole-NH), 8.88 (s, 1 H, OCONH), 8.06 (s, 1 H, H 4"), 7.87 (d, J= 8.5 Hz, 1 H, H 6), 7.79 (d, J= 8.3 Hz, 1 H, H 9), 7.57 (dd, J= 8.3, 7.5 Hz, 1 H, H 8), 7.46 (dd, J= 8.5, 7.5 Hz, 1 H, H 7), 7.17 (br s, 1 H, H 4), 7.00 (d, J= 2.2 Hz, 1 H, H 3'), 6.88 ( s, 1 H, H 4'), 5.44 (d, J= 13.5 Hz, 1 H, CH 2 0), 5.39 (d, J= 13.5 Hz, 1 H, CH 2 0), 5 4.81 (dd, J= 10.7, 1.5 Hz, 1 H, H 2), 4.65-4.74 (m, 3 H, H 2, CH 2 N), 4.16-4.22 (m, 1 H, H 1), 4.10 (s, 3 H, OCH 3 ), 3.95-3.99 (m, 4 H, CH 2 C1, OCH 3 ), 3.89-3.93 (m, 5 H, OCH 3 ,
CH
2 N), 3.48 (t, J= 10.9 Hz, 1 H, CH 2 C1) ), 0.81 (s, 9 H, SiC(CH 3
)
3 ), -0.08 (s, 6 H, Si(CH 3
)
2 ); MS (FAB') m/z 795 (MH
+
, 12%), 793 (25); HRMS (FAB') calc. for
C
38
H
46 35 C1N60 9 Si (MH
+
) m/z 793.2784, found 793.2762; calc. for C 38
H
46 37 C1N60 9 Si (MH +) 10 m/z 795.2755, found 795.2751. [I1-(2-hydroxyethyl)-5-nitro-1H-imidazol
-
2-yl] methyl 1-(chloromethyl)-3-[(5,6,7 trimethoxy-1H-indol-2-yl)carbonyl]-2,3-dihydro-1H-benzo[elindol-5-ylcarbamate 15 (148). IM HCI (0.23 mL, 230 Mmol) was added to a stirred solution of silyl ether 147 (91 mg, 115 pmol) in MeOH (5 mL) and the solution stirred at 20 oC for 4 h. The solvent was evaporated and the residue partitioned between EtOAc (40 mL) and water (40 mL). The organic fraction was washed with water (25 mL), brine (20 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (0 20 10%MeOH/40%EtOAc/DCM), to give 148 as a white solid, mp (EtOAc/light petroleum) 148-150 (dec.); 'H NMR 8 [(CD 3
)
2 SO] 11.45 (s, 1 H, indole-NH), 9.92 (s, 1 H, OCONH), 8.45 (s, 1 H, H 4), 8.16 (s, 1 H, H 4"), 8.06 (d, J= 8.5 Hz, 1 H, H 6), 7.97 (d, J= 8.3 Hz, 1 H, H 9), 7.58 (ddd, J= 8.3, 7.2, 0.8 Hz, 1 H, H 8), 7.46 (ddd, J= 8.5, 7.2, 0.8 Hz, 1 H, H 7), 7.09 (d, J= 2.0 Hz, 1 H, H 3'), 6.97 ( s, 1 H, H 4'), 5.37 (s, 2 H, CH 2 0), 5.12 (t, J= 5.4 25 Hz, 1 H, OH), 4.80 (dd, J= 10.7, 9.4 Hz, 1 H, H 2), 4.56-4.60 (m, 3 H, H 2, CH 2 0), 4.32 4.38 (m, 1 H, H 1), 4.06 (dd, J= 11.1, 3.2 Hz, 1 H, CH 2 C1), 3.91-3.95 (m, 4 H, OCH 3 ,
CH
2 C1), 3.83 (s, 3 H, OCH 3 ), 3.81 (s, 3 H, OCH 3 ), 3.70-3.75 (m, 2 H, CH 2 N); 13C NMR 8
[(CD
3
)
2 SO] 160.1 (CO), 154.0 (OCONH), 149.1 (C 5'), 148.7 (C 5"), 141.4 (C 3a), 139.9 (C 6'), 139.0 (C 7'), 138.9 (C 2"), 134.1 (C 5), 132.5 (C 4"), 130.7 (C 9a), 129.4 (C 2'), 30 127.1 (C 8), 125.4 (C 7a'), 125.3 (C 5a), 124.3 (C 7), 123.8 (C 9), 123.3 (C 6), 123.1 (C 3a'), 122.2 (C 9b), 113.2 (C 4), 106.2 (C 3'), 98.0 (C 4'), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 59.7 (CH20), 58.1 (CH 2 0), 55.9 (OCH 3 ), 54.9 (C 2), 48.2 (CH 2 N), 47.8 (CH 2 C1), 41.1 (C 1); MS (FAB') m/z 681 (MH
+
, 5%), 679 (MH
+
, 12%); HRMS (FAB') calc. for C 32
H
32 35 C1N 6 0 9 WO 00/64864 PCT/GB00/01612 - 101 (MH') m/z 679.1919, found 679.1797; calc. for C 3 2
H
32 37 C1N 6 0 9 (MH) m/z 681.1890, found 681.1892; Anal. (C 3 2
H
3 1 C1N 6 0 9 ) C, H, N. Example 5L. Preparation of (1-methyl-5-nitro-1H-imidazol-4-yl)methyl 1 5 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-l1-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (150). A solution of triphosgene (21 mg, 70 p/tmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (93 mg, 200 gmol) and Et 3 N (55 /L, 400 Mmol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of (1-methyl-5-nitro 10 1H-imidazol-4-yl)methanol 149 [ D. C. Baker, S.R. Putt, H. D. H. Showalter, J. Heterocyclic Chem., 1983, 20, 629-634.] (37 mg, 240 gmol) in DCM (3 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 5% MeOH/EtOAc, to give 150 (40 mg, 56%) as a tan powder, mp (EtOAc) 219-220 oC; 'H 15 NMR [(CD 3
)
2 SO] 8 11.47 (s, 1 H, indole-NH), 9.80 (s, 1 H, OCONH), 8.54 (br s, 1 H, H 4), 8.08-8.11 (m, 2 H, H 6, H 2"), 7.97 (d, J= 8.4 Hz, 1 H, H 9), 7.57 (ddd, J= 8.4, 7.2, 0.7 Hz, 1 H, H 8), 7.45 (ddd, J= 8.5, 7.2, 0.7 Hz, 1 H, H 7), 7.09 (d, J= 2.0 Hz, 1 H, H 3'), 6.97 ( s, 1 H, H 4'), 5.40 (s, 2 H, CH 2 0), 4.79 (dd, J = 10.8, 1.4 Hz, 1 H, H 2), 4.52 (dd, J= 11.0, 1.9 Hz, 1 H, H 2), 4.31-4.36 (m, 1 H, H 1), 4.07 (dd, J= 11.1, 3.0 Hz, 1 H, CH 2 CI), 20 3.89-3.95 (m, 7 H, OCH 3 , CH 2 C1, NCH 3 ), 3.83 (s, 3 H, OCH 3 ), 3.81 (s, 3 H, OCH 3 ); ' 3 C NMR [(CD 3
)
2 SO] 8 160.1 (CO), 154.3 (OCONH), 154.2 (C 5"), 149.1 (C 5'), 141.4 (C 2"), 141.2 (C 3a), 139.9 (C 6'), 139.0 (C 7'), 135.1 (C 4"), 134.4 (C 5), 130.8 (C 9a), 129.4 (C 2'), 127.0 (C 8), 125.5 (C Sa), 125.4 (C 7a'), 124.2 (C 7) 123.9 (C 9), 123.2 (C 6), 123.1 (C 3a'), 122.0 (C 9b), 113.1 (C 4), 106.2 (C 3'), 98.0 (C 4'), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 59.6 25 (CH 2 0), 55.9 (OCH 3 ), 54.8 (C 2), 47.5 (CH 2 CI), 41.1 (C 1), 35.1 (NCH 3 ); MS (FAB') m/z 651 (MH , 2%), 649 (MH
+
, 8); HRMS (FAB') calc. for C 31
H
30 35 C1N 6 0 8 (MH') m/z 649.1814, found 649.1802; calc. for C 31
H
3 0 37 C1N 6 0 8 (MH') m/z 651.1784, found 651.1761; Anal. (C 3
,H
2 9 C1N 6 0 8 ) C, H, N. 30 Example 6A. Preparation of (5-nitro-2-furyl)methyl 4-[bis(2 chloroethyl)amino]phenylcarbamate (154). (5-Nitro-2-furyl)methyl 4-nitrophenyl carbonate (152). A solution of 4-nitrophenyl chloroformate (4.17 g, 20.7 mmol) in dry THF (50 mL) was added slowly to a stirred WO 00/64864 PCT/GB00/01612 - 102 solution of (5-nitrofuran-2-yl)methanol (151) [J. M. Berry, C. Y. Watson, W. J. D. Whish, and M. D. Threadgill. J. Chem. Soc. Perkin Trans. I, 1997, 1147] (2.69 g, 18.7 mmol) and pyridine (1.67 mL, 20.7 mmol) in THF (100 mL) at 20 oC under N 2 . The mixture was stirred at 20 oC for 16 h, then partitioned between EtOAc (100 mL) and H 2 0 (100 mL). The 5 organic layer was washed with saturated aqueous NaHCO 3 (50 mL), dried, and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (25-50%) of EtOAc/light petroleum to give 152 (4.79 g, 83%) as a white powder, mp (EtOAc/light petroleum) 93-94 'C; IR N 1775, 1526, 1352, 1215 cm-'; 'H NMR [(CD 3
)
2 SO] 8 8.34 (ddd, J= 9.2, 3.2, 2.1 Hz, 2 H, H 3, H 5), 7.72 (d, J = 3.8 Hz, 1 H, H 4'), 7.61 (ddd, J= 9.2, 3.2, 10 2.1 Hz, 2 H, H 2, H 6), 7.07 (d, J= 3.8 Hz, 1 H, H 3'), 5.43 (s, 2 H, CH 2 0); " 3 C [(CD 3
)
2 SO] 8 155.0, 154.1, 151.8, 151.5, 145.2, 125.4(2), 122.5(2), 115.1, 113.3, 61.5; Anal.
(C
12
H
8
N
2 0 8 ) C, H, N. (5-Nitro-2-furyl)methyl 4-[bis(2-hydroxyethyl)amino]phenylcarbamate (153). A 15 solution of carbonate 152 (1.00 g, 3.24 mmol), N',N-bis(2-hydroxyethyl)-1,4 benzenediamine (57) (3.24 mmol), and pyridine (260 gL, 3.24 mmol) in THF (80 mL) was stirred at 20 oC for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (0-10%) of MeOH/EtOAc, to give 153 (0.74 g, 63%) as an oil; 'H NMR [(CD 3
)
2 SO] 5 9.44 (s, 1 H, OCONH), 7.68 (d, J= 3.7 Hz, 1 H, H 20 4'), 7.20 (br d, J= 9.1 Hz, 2 H, H 2, H 6), 6.93 (d, J= 3.7 Hz, 1 H, H 3'), 6.62 (d, J= 9.1 Hz, 2 H, H 3, H 5), 5.19 (s, 2 H, CHzO), 4.71 (t, J= 5.4 Hz, 2 H, 2 x OH), 3.48-3.54 (m, 4 H, 2 x CH20), 3.33-3.38 (min, 4 H, 2 x CH 2 N); 13C NMR [(CD 3
)
2 SO] 8 154.0, 152.7, 151.5, 144.2, 127.0, 120.4 (2), 113.8, 113.6, 111.4 (2), 58.1 (2), 57.1, 53.4 (2); MS (DEI) m/z 365 (M', 15%), 334 (70), 222 (20), 196 (40), 191 (100); HRMS (DEI) calc. for C, 6
H
19
N
3 0 7 25 (M') m/z 365.1223, found 365.1218. (5-Nitro-2-furyl)methyl 4-[bis(2-chloroethyl)aminolphenylcarbamate (154). Methane sulphonyl chloride (460 zL, 6.0 mmol) was added dropwise to a stirred solution of diol 153 (0.73 g, 2.0 mmol) in pyridine (30 mL) at 5 oC and the solution stirred at 20 'C for 2 h. 30 The solvent was evaporated and the residue partitioned between DCM (100 mL) and water (100 mL). The aqueous fraction was washed with DCM (2 x 50 mL), the combined organic extracts dried, and the solvent evaporated. The residue was dissolved in DMF (20 mL), LiCI (0.51 g, 12.0 mmol) added and the mixture stirred at 80 oC for 3 h. The solvent was WO 00/64864 PCT/GB00/01612 - 103 evaporated and the residue partitioned between EtOAc (150 mL) and water (150 mL). The aqueous fraction was extracted with EtOAc (2 x 80 mL), the combined extracts dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 154 (0.62 g, 77%) as an oil; 'H NMR 8 9.57 (br s, 1 H, 5 OCONH), 7.69 (d, J= 3.7 Hz, 1 H, H 4'), 7.28 (br d, J= 9.1 Hz, 2 H, H 2, H 6), 6.95 (d, J= 3.7 Hz, 1 H, H 3'), 6.70 (d,J = 9.1 Hz, 2 H, H 3, H 5), 5.21 (s, 2 H, CH 2 0), 3.63-3.72 (m, 8 H, 2 x CH 2 N, 2 x CH 2 C); 13C NMR (CDC13) 8 153.9, 152.7, 151.5, 1142.3, 128.7, 120.3 (2), 113.8, 113.6, 112.3 (2), 57.2, 52.3 (2), 41.1 (2); MS (DEI) m/z 401 (M', 50%), 403 (30), 405 (10) 354 (40), 352 (100); HRMS (DEI) calc. for C, 6
H,
7 35 C1 2 N305 (M
+
) m/z 10 401.0545, found 401.0546; calc. for C, 6
H
1 73C1 37
CIN
3 0 5
(M
+
) m/z 403.0516, found 403.0521; calc. for C, 6 HI7 37 C1 2
N
3 05 (M-) m/z 405.0486, found 405.0498. Example 6B. Preparation of (5-nitro-2-furyl)methyl 1-(chloromethyl)-3-[(5,6,7 trimethoxy-1H-indol-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate 15 (155). A solution of triphosgene (14 mg, 48 kimol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (57 mg, 122 Mmol) and Et 3 N (38 pL, 275 gmol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of (5-nitrofuran-2-yl)methanol (151) [J. M. Berry, C. Y. Watson, W. J. D. Whish, and M. D. Threadgill. J. Chem. Soc. Perkin Trans. I, 20 1997, 1147] (24 mg, 165 pmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 ( 2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 30% EtOAc/DCM, to give 155 (65 mg, 84%) as a white solid, mp (EtOAc/light petroleum) 185-187 oC; 'H NMR 8 11.46 (s, 1 H, indole-NH), 9.92 (s, 1 H, OCONH), 8.54 (s, 1 H, H 4), 8.04 (d, J= 8.5 Hz, 1 H, H 6), 7.98 25 (d, J= 8.3 Hz, 1 H, H 9), 7.72 (d, J= 3.8 Hz, 1 H, H 4"), 7.58 (ddd, J= 8.3, 7.2, 0.7 Hz, 1 H, H 8), 7.45 (ddd, J= 8.5, 7.2, 0.7 Hz, 1 H, H 7), 7.09 (d, J= 2.1 Hz, 1 H, H 3'), 6.98-7.00 (m, 2 H, H 4', H 3"), 5.30 (s, 2 H, CH 2 0), 4.80 (dd, J= 10.8, 9.5 Hz, 1 H, H 2), 4.53 (dd, J = 10.8, 1.9 Hz, 1 H, H 2), 4.32-4.37 (m, 1 H, H 1), 4.07 (dd, J= 11.1, 3.0 Hz, 1 H, CH 2 C1), 3.91-3.96 (m, 4 H, OCH 3 , CH 2 C1), 3.83 (s, 3 H, OCH 3 ), 3.80 (s, 3 H, OCH 3 ); ' 3 C NMR 8 30 160.1 (CO), 153.9 (OCONH), 153.8 (C 5"), 151.1 (C 2"), 149.2 (C 5'), 141.4 (C 3a), 139.8 (C 6'), 139.0 (C 7'), 134.0 (C 5), 130.7 (C 9a), 129.4 (C 2'), 127.1 (C 8), 125.4 (C 5a), 125.3 (C 7a'), 124.3 (C 7), 123.7 (C 9), 123.3 (C 6), 123.1 (C 3a), 122.2 (C 9b), 113.9 (C 3"), 113.6 (C 4"), 113.1 (C 4), 106.2 (C 3'), 98.0 (C 4'), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 57.7 WO 00/64864 PCT/GB00/01612 - 104 (CH 2 0), 55.9 (OCH 3 ), 54.8 (C 2), 47.5 (CH 2 C1), 41.1 (C 1); MS (FAB') m/z 635 (MH
+
, 6%), 637 (MH
+
, 3); HRMS (FAB') calcd for C 31
H
28 3 5 C1N 4 0 9
(MH
+
) m/z 635.1545, found 635.1552; calcd for C 3
,H
28 37 C1N 4 0 9 (MH') m/z 637.1515, found 637.1514; Anal.
(C
31
,H
2 7C1N 4 0 9 ) 5 Example 7A. Preparation of (5-nitro-2-thienyl)methyl 4-[bis(2 chloroethyl)amino]phenylcarbamate (159). 4-Nitrophenyl (5-nitro-2-thienyl)methyl carbonate (157). 4-Nitrophenyl chloroformate (2.58 g, 12.8 mmol) in dry THF (20 mL) was added slowly to a stirred solution of(5 10 nitrothien-2-yl)methanol (156) [P. J. Narcombe, R. K. Norris. Aust. J. Chem. 1979, 32, 2647] (1.85 g, 11.6 mmol) and pyridine (1.03 mL, 12.8 mmol) in THF (50 mL) at 20 oC under N 2 . The mixture was stirred at 20 oC for 16 h, then partitioned between EtOAc (100 mL) and H 2 0 (100 mL). The organic layer was washed with saturated aqueous NaHCO 3 (50 mL), and the solvent evaporated to give 157 (1.86g, 49%), mp (EtOAc/light petroleum) 15 121-122 oC; IRN 1763, 1522, 1345, 1231 cm-'; 'H NMR [(CD 3
)
2 SO] 8 8.33 (ddd, J= 9.2, 3.4, 2.2 Hz, 2 H, H 3', H 5'), 8.08 (d, J= 4.2 Hz, 1 H, H 4), 7.60 (ddd, J= 9.2, 3.4, 2.2 Hz, 2 H, H 2', H 6'), 7.4 (d, J= 4.2 Hz, 1 H, H 3), 5.56 (s, 2 H, CH20); 1 3 C NMR [(CD 3
)
2 SO] 5 155.0, 151.6, 148.2, 145.2, 144.8, 129.5, 129.0, 125.4 (2), 122.6 (2), 64.4; Anal.
(CI
2
H
8
N
2 0 7 S) C, H, N. 20 (5-Nitro-2-thienyl)methyl 4-[bis(2-hydroxyethyl)amino]phenylcarbamate (158). A solution of 157 (0.75 g, 2.3 mmol), N',N'-bis(2-hydroxyethyl)-1,4-benzenediamine (57) (2.5 mmol), and pyridine (206 pL, 2.5 mmol) in THF (50 mL) was stirred at 20 oC for 16 h. The solvent was evaporated and the residue purified by chromatography, eluting with 25 EtOAc to give 158 (0.56 g, 64%), mp (EtOAc/light petroleum) 139-140.5 oC; IR N 3360, 3208, 1730, 1530, 1337, 1215 cm'-; 'H NMR [(CD 3
)
2 SO] 8 9.46 (s, 1 H, OCONH), 8.02 (d, J= 4.2 Hz, 1 H, H 4'), 7.29 (d, J= 4.2 Hz, 1 H, H 3'), 7.21 (br d, J= 9.1 Hz, 2 H, H 2, H 6), 6.62 (d, J= 9.1 Hz, 2 H, H 3, H 5), 5.33 (s, 2 H, CH 2 0), 4.72 (t, J= 5.5 Hz, 2 H, 2 x OH), 3.49-3.56 (m, 4 H, 2 x CH 2 0), 3.36 (t, J= 6.2 Hz, 4 H, 2 x CH 2 N); 3 C NMR [(CD 3
)
2 SO] 8 30 153.0, 150.8, 148.1, 144.3, 129.6, 127.5, 126.9, 120.5 (2), 111.4 (2), 60.1, 58.2 (2), 53.4 (2); Anal. (C, 6
HI
9
N
3 0 6 S) C, H; N, calc 11.0, found 10.5%. (5-Nitro-2-thienyl)methyl 4-[bis(2-chloroethyl)amino]phenylcarbamate (159).
WO 00/64864 PCT/GB00/01612 - 105 Methane-sulphonyl chloride (260 4L, 3.4 mmol) was added dropwise to a stirred solution of diol 158 (0.43 g, 1.1 mmol) in pyridine (10 mL) at 5 'C and the solution stirred at 20 oC for 2 h. The solvent was evaporated and the residue partitioned between DCM (50 mL) and water (50 mL). The aqueous fraction was washed with DCM (2 x 50 mL), the combined 5 organic extracts dried, and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiCl (0.29 g, 6.8 mmol) added and the mixture stirred at 80 oC for 3 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). The aqueous fraction was extracted with EtOAc (2 x 50 mL), the combined extracts dried, and the solvent evaporated. The residue was purified by chromatography, eluting with 50% 10 EtOAc/light petroleum, to give 159 (0.35 g, 69%) as pale green needles, mp (EtOAc/light petroleum) 99-100 oC; IRN 3353, 1723, 1547, 1530, 1339, 1219 cm- 1 ; 'HNMR
[(CD
3
)
2 SO] 6 9.58 (br s, 1 H, OCONH), 8.04 (d, J= 4.2 Hz, 1 H, H 4'), 7.28-7.30 (min, 3 H, H 3', H 3, H 5), 6.71 (d, J =9.1 Hz, 2 H, H 2, H 6), 5.34 (s, 2 H, CH 2 0), 3.65-3.72 (m, 8 H, 2 x CH 2 N, 2 x CH 2 C); 13 C NMR [(CD 3
)
2 SO] 8 153.0, 150.8, 148.2, 142.4, 129.5, 128.6, 15 127.5, 120.4 (2), 112.3 (2), 60.1, 52.2 (2), 41.1 (2); Anal. (C 1 6
H
1 9 C1 2
N
5 0 4 ) C, H, N, Cl. Example 7B. Preparation of (5-nitro-2-thienyl)methyl 1-(chloromethyl)-3-[(5,6,7 trimethoxy-1H-indol-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (160). A solution of triphosgene (15 mg, 51 /tmol) in DCM (2 mL) was added dropwise to 20 a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (61 mg, 131 gmol) and Et 3 N (41 4L, 294 /mol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of (5-nitro-2-thienyl)methanol (156) [P. J. Narcombe, R. K. Norris. Aust. J. Chem. 1979, 32, 2647] (28 mg, 176 Mmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 25 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 10% EtOAc/DCM, to give 160 (76 mg, 89%) as a white solid, mp (EtOAc/light petroleum) 218-219 oC; 'H NMR [(CD 3
)
2 SO] 5 11.48 (br s, 1 H, indole-NH), 9.34 (br s, 1 H, OCONH), 8.55 (br s, 1 H, H 4), 8.08 (d, J= 4.1 Hz, 1 H, H 4"), 8.04 (d, J= 8.5 Hz, 1 H, H 6), 7.99 (d, J= 8.3 Hz, 1 H, H 9), 7.58 (dd, J= 8.3, 7.4 Hz, 1 H, H 8), 7.47 (dd, J= 8.5, 7.4 30 Hz, 1 H, H 7), 7.33 (d, J= 4.1 Hz, 1 H, H 3"), 7.10 (d, J= 1.9 Hz, 1 H, H 3'), 6.97 (s, 1 H, H 4'), 5.43 (s, 2 H, CH 2 0), 4.80 (dd, J= 11.0, 9.5 Hz, 1 H, H 2), 4.54 (dd, J= 11.0, 1.8 Hz, 1 H, H 2), 4.32-4.36 (min, 1 H, H 1), 4.07 (dd, J= 11.0, 3.0 Hz, 1 H, CH 2 C1), 3.92-3.96 (m, 4 H, CH 2 C1, OCH 3 ), 3.92 (min, 3 H, OCH 3 ), 3.81 (s, 3 H, OCH 3 ); 1 3 C NMR [(CD 3
)
2 SO] 6 160.3 WO 00/64864 PCT/GB00/01612 - 106 (CO), 154.4 (OCONH), 151.1 (C 5"), 149.3 (C 5'), 148.2 (C 2"), 141.6 (C 3a), 140.0 (C 6'), 139.1 (C 7'), 134.1 (C 5), 130.8 (C 9a), 129.7 (C 3"), 129.6 (C 2'), 127.8 (C 4"), 127.4 (C 8), 125.6 (C 5a), 125.5 (C 7a'), 124.6 (C 7), 123.8 (C 9), 123.3 (C 6), 123.2 (C 3a'), 122.5 (C 9b), 113.5 (C 4), 106.4 (C 3'), 98.1 (C 4'), 61.2 (OCH 3 ), 61.0 (OCH 3 ), 60.8 5 (CH20), 56.0 (OCH 3 ), 55.0 (C 2), 47.7 (CH 2 C1), 41.2 (C 1); MS (FAB') m/z 653 (MH +, 4%), 651 (MH
+
, 8); HRMS (FAB') calc. for C 31
H
28 3 5 C1N 4 08S (MH
+
) m/z 651.1316, found 651.1311; calc. for C 3
,H
28 37 C1N 4 0 8 S (MH
+
) m/z 653.1287, found 653.1307; Anal.
(C
31
H
27 C1N 4 0 8 S) C, H, N. 10 Example 8. Preparation of (1-methyl-5-nitro-1H-pyrazol-4-yl)methyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (163). (1-methyl-5-nitro-1H-pyrazol-4-yl)methanol (162). Borane dimethylsulfide (2 M solution in THF, 4.2 mL, 8.4 mmol) was added to a solution of 1-methyl-5-nitro-lH 15 pyrazole-4-carboxylic acid (161) [C.C. Cheng, J. Heterocyclic Chem. 1968, 5, 195-197] (1.11 g, 6.5 mmol) in THF (50 mL) under N 2 , and the mixture stirred at reflux temperature for 80 min, then cooled. MeOH (5 mL), then water (5 mL), then 2 M HCI (5 mL) were added, the THF was evaporated, and the residue was diluted with water and extracted with EtOAc (3 x 50 mL). The combined organic extract was dried, the solvent evaporated, and 20 the residue purified by chromatography, eluting with 50% EtOAc/petroleum ether, to give 162 (0.52 g, 51%) as a white solid, mp (benzene) 78-80 oC. 'H NMR 8 7.58 (s, 1 H, H 3), 4.82 (d, J= 3.4 Hz, 2 H, CH 2 0), 4.25 (s, 3 H, NCH 3 ), 2.39 (br s, 1 H, OH); Anal. (CsH 7
N
3 0 3 ) C, H, N. 25 (1-Methyl-5-nitro-1H-pyrazol-4-yl)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H indol-1-yl)carbonyll-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (163). A solution of triphosgene (14.3 mg, 48 lzmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny. Bioorg. Med. Chem. Lett., 1997, 7, 1483] (57 mg, 122 gmol) and Et 3 N (38 /.zL, 275 gmol) in DCM (10 mL) and stirred at 20 30 oC for 2 h. A solution of alcohol 162 (26 mg, 165 mol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20 % EtOAc/DCM, to give 163 (41 mg, 52%) as a white solid, mp (EtOAc/light petroleum) 201- WO 00/64864 PCT/GB00/01612 - 107 202 oC; 'H NMR [(CD 3
)
2 SO] 6 11.47 (br s, 1 H, indole-NH), 9.80 (br s, 1 H, OCONH), 8.56 (br s, 1 H, H 4), 8.06 (d, J= 8.5 Hz, 1 H, H 6), 7.98 (d, J= 8.3 Hz, 1 H, H 9), 7.74 (br s, 1 H, H 3"), 7.58 (dd, J= 8.3, 7.4 Hz, 1 H, H 8), 7.47 (dd, J= 8.5, 7.4 Hz, 1 H, H 7), 7.45 (d, J= 1.6 Hz, 1 H, H 3'), 6.98 (s, 1 H, H 4'), 5.33 (s, 2 H, CH20), 4.80 (dd, J= 11.0, 9.4 5 Hz, 1 H, H 2), 4.53 (dd, J= 11.0, 1.8 Hz, 1 H, H 2), 4.32-4.38 (m, 1 H, H 1), 4.17 (s, 3 H,
NCH
3 ), 4.07 (dd, J= 11.0, 3.1 Hz, 1 H, CH 2 C1), 3.91-3.96 (m, 4 H, OCH 3 , CH 2 C1), 3.82 (s, 3 H, OCH 3 ), 3.80 (s, 3 H, OCH 3 ); 13C NMR [(CD 3
)
2 SO] 8 160.2 (CO), 154.3 (OCONH), 149.1 (C 5'), 142.5 (C 5"), 141.5 (C 3a), 139.9 (C 6'), 139.0 (C 7'), 137.5 (C 3"), 134.2 (C 5), 130.7 (C 9a), 129.4 (C 2'), 127.1 (C 8), 125.4 (C 5a, C 7a), 124.3 (C 7), 123.7 (C 9), 10 123.3 (C 6), 123.1 (C 3a'), 122.0 (C 9b), 117.4 (C 4"), 113.0 (C 4), 106.2 (C 3'), 98.0 (C 4'), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 57.2 (CH 2 0), 55.9 (OCH 3 ), 54.8 (C 2), 47.5 (CH 2 C1), 41.1 (C 1), 40.8 (NCH 3 ); MS (FAB') m/z 650 (MH*, 2%), 648 (MH
+
, 5); HRMS (FAB') calc. for C 31
H
30 35 C1N 6 0 8
(MH
+
) m/z 649.1814, found 649.1803; calc. for C 31
H
30 37 C1N 6 0 8 (MH) m/z 651.1784, found 651.1796; Anal. (C 3 1
H
29 C1N 6 0 8 ) C, H, N. 15 Example 9A. Preparation of ethyl 4-({[({1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H indol-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e] indol-5-yl} amino)carbonyl] oxy} methyl) 1-methyl-5-nitro-1H-pyrrole-2-carboxylate (167). Ethyl 4-formyl-1-methyl-5-nitro-H-pyrrole-2-carboxylate (165). Dimethyl sulfate 20 (0.31 mL, 3.2 mmol) was added to a mixture of ethyl 4-formyl-5-nitro-1H-pyrrole-2 carboxylate (164) [P. Fornari, M. Farnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-291] (0.57 g, 2.7 mmol) and K 2 C0 3 (0.56 g, 4.0 mmol) in DMSO (4 mL) and the brown suspension was stirred at 20 oC for 1 h. The mixture was diluted with water (50 mL), acidified with HCl (2 N), and extracted with EtOAc (2 x 50 mL). The combined extract 25 was dried, the solvent evaporated. The residue was chromatograped, eluting with 10%EtOAc/light petroleum, to give 165 (0.53 g, 86%) as a pale green solid, mp (benzene/light petroleum) 59-60.5 'C; 'H NMR 8 10.32 (s, 1 H, CHO), 7.42 (s, 1 H, H 3), 4.37 (q, J= 7.1 Hz, 2 H, CH 2 ), 4.33 (s, 3 H, NCH 3 ), 1.39 (t, J= 7.1 Hz, 3 H, CH 3 ); Anal.
(C
9 HIoN 2 0 5 ) C, H, N. 30 Ethyl 4-(hydroxymethyl)-1l-methyl-5-nitro-1H-pyrrole-2-carboxylate (166). NaBH 4 (0.33 g, 8.7 mmol) was added in portions to a solution of aldehyde 165 (3.96 g, 17.5 mmol) in EtOH (100 mL) and the mixture was stirred at 20 oC for 20 min. Water (5 mL) was WO 00/64864 PCT/GB00/01612 - 108 slowly added, the EtOH was evaporated, and the residue was diluted with aq. NaCl and extracted with EtOAc (2 ' 50 mL). The combined extract was washed with aq. NaCl (50 mL), dried, and the solvent evaporated. The residue was recystallized to give 166 (1.40 g, 35%) as white needles, mp (benzene) 95.5-96.5 'C; 'H NMR 5 7.01 (s, 1 H, H 3), 4.80 (br 5 s, 2 H, CH 2 0), 4.36 (q, J= 7.1 Hz, 2 H, CH 2 ), 4.31 (s, 3 H, NCH 3 ), 2.49 (br s, 1 H, OH), 1.38 (t, J= 7.1 Hz, 3 H, CH 3 ); Anal. (C 9 H I 2
N
2 0 5 ) C, H, N. The mother liquor was evaporated and purified by chromatography 10% EtOAc/light petroleum to give more 166 (1.46 g, 37%). 10 Ethyl 4-({[({1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-yl)carbonyl]-2,3 dihydro-1H-benzo [e] indol-5-yl}amino)carbonyl] oxy} methyl)-1-methyl-5-nitro-1H pyrrole-2-carboxylate (167). A solution of triphosgene (13.8 mg, 46 4mol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (55 mg, 118 pmol) and Et 3 N (37 yL, 15 265 umol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of ethyl 4 (hydroxymethyl)- 1-methyl-5-nitro- 1H-pyrrole-2-carboxylate (166) (36 mg, 159 ymol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/DCM, to give 167 (26 mg, 31%) as a white solid, mp 20 (EtOAc/light petroleum) 248-250 oC; 'H NMR [(CD 3
)
2 SO] 6 11.45 (s, 1 H, indole-NH), 9.86 (s, 1 H, OCONH), 8.56 (br s, 1 H, H 4"), 8.09 (d, J= 8.5 Hz, 1 H, H 6"), 7.99 (d, J= 8.3 Hz, 1 H, H 9"), 7.59 (dd, J= 8.3, 7.7 Hz, 1 H, H 8"), 7.48 (dd, J = 8.5, 7.7 Hz, 1 H, H 7"), 7.10 (d, J= 2.0 Hz, 1 H, H 3'"), 7.06 (br s, 1 H, H 3), 6.97 (s, 1 H, H 4'"), 5.38 (s, 2 H, CH20), 4.80 (dd, J = 11.0, 9.6 Hz, 1 H, H 2"), 4.53 (dd, J= 11.0, 2.0 Hz, 1 H, H 2"), 4.35 25 4.40 (m, 1 H, H 1"), 4.29 (q, J= 7.1 Hz, 2 H, H 1'), 4.19 (s, 3 H, NCH 3 ), 4.07 (dd, J= 11.0, 3.0 Hz, 1 H, CH 2 C1), 3.92-3.96 (m, 4 H, CH 2 C1, OCH 3 ), 3.83 (m, 3 H, OCH 3 ), 3.81 (s, 3 H,
OCH
3 ), 1.31 (t, J= 7.1 Hz, 3 H, H 2'); ' 3 C NMR [(CD 3
)
2 SO] 8 160.2 (CO), 159.4 (CO 2 ), 154.3 (OCONH), 149.1 (C 5'"), 141.5 (C 3a"), 139.9 (C 6'"), 139.0 (C 7'"), 137.1 (C 5), 134.2 (C 5"), 133.9 (C 2), 130.7 (C 9a"), 129.5 (C 2'"), 127.1 (C 8"), 125.6 (C 5a"), 125.5 30 (C 4), 125.4 (C 7a'"), 124.3 (C 7"), 123.7 (C 9"), 123.3 (C 6"), 123.1 (C 3a'"), 122.1 (C 9b"), 114.6 (C 3), 113.0 (C 4"), 106.2 (C 3'"), 98.0 (C 4'"), 61.2 (C 1'), 61.0 (OCH 3 ), 60.9
(OCH
3 ), 59.6 (CH 2 0), 55.9 (OCH 3 ), 54.9 (C 2"), 47.5 (CH 2 C1), 41.1 (C 1"), 35.2 (NCH 3 ), 14.0 (C 2'); MS (FAB') m/z 722 (MH', 0.3%), 720 (MH', 0.6); HRMS (FAB') calc. for WO 00/64864 PCT/GB00/01612 - 109 C 35
H
3 5 35 C1NsO 5 1 0
(MH
+
) m/z 720.2073, found 720.2059; calc. for C 35
H
35 37 CINsO 5 10 (MH') m/z 722.2043, found 722.2031; Anal. (C 35
H
34 C1N 5 0t 0 ) C, H, N. Example 9B. Preparation of (1-methyl-2-nitro-1H-pyrrol-3-yl)methyl 1 5 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-yl)carbonyl]-2,3-dihydro-1H benzo[elindol-5-ylcarbamate (169). (1-methyl-2-nitro-1H-pyrrol-3-yl)methanol (168). A solution of NaOH (2.7 g, 68 mmol) in water (15 mL) was added to a solution of ester 166 (1.02 g, 4.47 mmol) in EtOH (30 10 mL), and the mixture was stirred at 20 oC for 1 h. The EtOH was evaporated, and the aqueous phase washed with EtOAc (20 mL) and then acidified (HC1). The aqueous mixture was extracted with EtOAc (3 ' 50 mL), the combined organic extract was dried and the solvent evaporated to give crude 4-(hydroxymethyl)-l1-methyl-5-nitropyrrole-2-carboxylic acid (0.84 g, 96%) as a red-brown solid. 15 The acid was suspended in quinoline (6 mL) with Cu powder (0.44 g) and the mixture was heated at 170-180 oC for 50 min. The cooled mixture was diluted with HCI (2N), extracted with EtOAc (3 ' 50 mL), the combined extract was dried, and the solvent evaporated. The residue was purified by chromatography, eluting 40% EtOAc/light petroleum, to give 168 as a pink solid (0.45 g, 64%), mp (benzene) 79-80.5 oC; 'H NMR 5 6.78 (d, J= 2.8 Hz, 1 20 H, H 5), 6.27 (d, J= 2.5 Hz, 1 H, H 4), 4.80 (d, J= 6.8 Hz, 2 H, CH 2 0), 4.00 (s, 3 H,
NCH
3 ), 2.75 (t, J= 6.8 Hz, 1 H, OH); 3 C NMR 6 131.6 (C 3), 129.1 (C 4), 109.0 (C 5), 58.8 (CH 2 0), 38.6 (NCH 3 ); Anal. (C 6
HN
2 0 3 ) C, H, N. (1-Methyl-2-nitro-1H-pyrrol-3-yl)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H 25 indol-1-yl)carbonyl] -2,3-dihydro-1H-benzo [e] indol-5-ylcarbamate (169). A solution of triphosgene (13.3 mg, 45 tmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny. Bioorg. Med. Chem. Lett., 1997, 7, 1483] (53 mg, 114 ymol) and Et 3 N (36 gL, 256 gmol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of (1-methyl-2-nitro-1H-pyrrol-3-yl)methanol (168) (24 mg, 153 30 pmol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (10-20%) of EtOAc/DCM, to give 169 (21 mg, 28%) as a white solid, mp (EtOAc) 218-220 'C; 'H NMR [(CD 3
)
2 S0] 8 11.46 (br s, 1 H, WO 00/64864 PCT/GB00/01612 -110 indole-NH), 9.80 (br s, 1 H, OCONH), 8.55 (br s, 1 H, H 4), 8.09 (d, J= 8.5 Hz, 1 H, H 6), 7.98 (d, J= 8.3 Hz, 1 H, H 9), 7.58 (dd, J= 8.3, 7.3 Hz, 1 H, H 8), 7.48 (dd, J= 8.5, 7.3 Hz, 1 H, H 7), 7.33 (d, J= 2.6 Hz, 1 H, H 4"), 7.09 (d, J= 1.9 Hz, 1 H, H 3'), 6.98 (s, 1 H, H 4'), 6.37 (br s, 1 H, H 3"), 5.37 (s, 2 H, CH 2 0), 4.80 (dd, J= 11.0, 9.3 Hz, 1 H, H 2), 4.52 5 (dd,J= 11.0, 1.9Hz, 1 H, H 2),4.31-4.37 (m, 1 H,H 1),4.07 (dd, J= 11.1, 3.0 Hz, 1 H,
CH
2 C1), 3.96 (s, 3 H, NCH 3 ), 3.91-3.94 (m, 4 H, CH 2 C1, OCH 3 ), 3.82 (s, 3 H, OCH 3 ), 3.80 (s, 3 H, OCH 3 ); " 3 C NMR [(CD 3
)
2 SO] 8 160.2 (CO), 154.4 (OCONH), 149.1 (C 5'), 141.5 (C 3a), 139.9 (C 6'), 139.0 (C 7'), 134.3 (C 5), 133.2 (C 2"), 130.7 (C 9a), 129.4 (C 2'), 127.1 (C 8), 125.4 (C 5a), 125.3 (C 7a'), 124.3 (C 7), 123.8 (C 9), 123.3 (C 6), 123.1 (C 10 3a'), 122.0 (C 9b), 113.0 (C 4), 108.1 (C 5"), 106.1 (C 3'), 98.0 (C 4'), 61.0 (OCH 3 ), 60.9
(OCH
3 ), 60.2 (CH 2 0), 55.9 (OCH 3 ), 54.8 (C 2), 47.5 (CH 2 C1), 41.1 (C 1), 37.9 (NCH 3 ); MS (FAB') m/z 650 (MH
+
, 1.5%), 648 (MH
+
, 3.5); HRMS (FAB') calc. for C 32
H
3 1 35 C1N 5 0 8 (MH') m/z 648.1861, found 648.1844; calc. for C 32
H
31 3 7 C1N 5 0 8
(MH
+
) m/z 650.1832, found 650.1826; Anal. (C 32
H
30 C1NsOs 5 0 8 ) C, H, N. 15 Example 9C. Preparation of ethyl 5-({[({1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H indol-1-yl)carbonyll-2,3-dihydro-H-benzol[elindol-5-yl}amino)carbonyl]oxy}methyl) 1-methyl-4-nitro-1H-pyrrole-2-carboxylate (173). Ethyl 5-formyl-l-methyl-4-nitro-1H-pyrrole-2-carboxylate (171). Dimethyl sulfate 20 (0.31 mL, 3.2 mmol) was added to a mixture of ethyl 5-formyl-4-nitro-1H-pyrrole-2 carboxylate (170) [P. Fomrnari, M. Famrnier, C. Foumrnier, Bull. Soc. Chim. Fr. 1972, 283-291] (0.57 g, 2.7 mmol) and K 2
CO
3 (0.56 g, 4.0 mmol) in DMSO (4 mL) and the brown suspension was stirred at 20 oC for 1 h. The mixture was diluted with water (50 mL), acidified with HC1 (2 N), and extracted with EtOAc (2 x 50 mL). The combined extract 25 was dried, the solvent evaporated. The residue was chromatograped, eluting with 10%EtOAc/light petroleum, to give 171 as a pale green solid (89%), mp (benzene/light petroleum) 70.5-71.5 'C; 'H NMR 6 10.55 (s, 1 H, CHO), 7.49 (s, 1 H, H 3), 4.38 (q, J= 7.2 Hz, 2 H, CH 2 ), 4.33 (s, 3 H, NCH 3 ), 1.40 (t, J= 7.2 Hz, 3 H, CH3); Anal. (C 9
HI
0
N
2 0 5 s) C, H,N. 30 Ethyl 5-(hydroxymethyl)-l-methyl-4-nitro-1H-pyrrole-2-carboxylate (172). NaBH 4 (0.33 g, 8.7 mmol) was added in portions to a solution of aldehyde 171 (3.96 g, 17.5 mmol) in EtOH (100 mL) and the mixture was stirred at 20 oC for 20 min. Water (5 mL) was WO 00/64864 PCT/GB00/01612 -111 slowly added, the EtOH was evaporated, and the residue was diluted with brine and extracted with EtOAc (2 ' 50 mL). The combined extract was washed with brine (50 mL), dried, and the solvent evaporated. The residue was recystallized to give 172 as a cream solid (81%), mp (benzene) 119-120.5 oC; 'H NMR 8 7.48 (s, 1 H, H 3), 4.97 (d,J= 6.8 Hz, 5 2 H, CH20), 4.32 (q, J= 7.1 Hz, 2 H, CH 2 ), 4.06 (s, 3 H, NCH 3 ), 2.72 (t, J= 7.1 Hz, 1 H, OH), 1.37 (t, J = 7.1 Hz, 3 H, CH 3 ); Anal. (C 9
H
12
N
2 0 5 ) C, H, N. Ethyl 5-({[1({1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-yl)carbonyl]-2,3 10 dihydro-1H-benzo[e]indol-5-yl}amino)carbonyl]oxy}methyl)-1-methyl-4-nitro-1H pyrrole-2-carboxylate (173). A solution of triphosgene (13.5 mg, 46 4mol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (54 mg, 116 /mol) and Et 3 N (36 gL, 260 bmol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of ethyl 5 15 (hydroxymethyl)-1-methyl-4-nitro-1H-pyrrole-2-carboxylate (172) (36 mg, 156 /mol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 40% EtOAc/DCM, to give 173 (52 mg, 62%) as a white solid, mp (EtOAc/light petroleum) 227-229 oC; 'H NMR [(CD 3
)
2 SO] 8 11.46 (s, 1 H, indole-NH), 20 9.83 (s, 1 H, OCONH), 8.56 (br s, 1 H, H 4"), 8.02 (d, J= 8.5 Hz, 1 H, H 6"), 7.97 (d, J= 8.3 Hz, 1 H, H 9"), 7.57 (dd, J= 8.3, 7.4 Hz, 1 H, H 8"), 7.46 (dd, J= 8.5, 7.4 Hz, 1 H, H 7"), 7.43 (s, 1 H, H 3), 7.10 (d,J = 2.0 Hz, 1 H, H 3'"), 6.98 (s, 1 H, H 4'"), 5.63 (s, 2 H,
CH
2 0), 4.80 (dd, J= 11.0, 9.4 Hz, 1 H, H 2"), 4.53 (dd, J= 11.0, 1.9 Hz, 1 H, H 2"), 4.33 4.37 (m, 1 H, H 1"), 4.29 (q, J= 7.1 Hz, 2 H, H 1'), 4.00-4.08 (m, 4 H, CH 2 C1, NCH 3 ), 25 3.91-3.95 (m, 4 H, CH 2 C1, OCH 3 ), 3.83 (m, 3 H, OCH 3 ), 3.80 (s, 3 H, OCH 3 ), 1.31 (t, J= 7.1 Hz, 3 H, H 2'); 1 3 C NMR [(CD 3
)
2 SO] 8 160.1 (CO), 159.3 (CO 2 ), 154.0 (OCONH), 149.1 (C 5'"), 141.4 (C 3a"), 139.9 (C 6'"), 139.0 (C 7'"), 134.1 (C 5"), 133.6 (C 4), 133.4 (C 2), 130.7 (C 9a"), 129.4 (C 2'"), 127.1 (C 8"), 125.4 (C 5a"), 125.3 (C 7a'"), 124.3 (C 7"), 123.7 (C 9"), 123.3 (C 6"), 123.1 (C 3a'"), 122.4 (C 5), 122.1 (C 9b"), 113.0 (C 4"), 30 111.2 (C 3), 106.2 (C 3'"), 98.0 (C 4'"), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 60.8 (CH 2 0), 55.9
(OCH
3 ), 54.8 (C 2"), 54.6 (C 1'), 47.5 (CH 2 C1), 41.1 (C 1"), 33.6 (NCH 3 ) 13.9 (C 2'); MS (FAB-) m/z 722 (MH', 2.5%), 720 (MH', 6); HRMS (FAB') calc. for C 3
H
35 35 C1N 5 0O 1 0 (MH') m/z 720.2073, found 720.2045; calc. for C 35
H
35 3 7 C1N 5 0O 1 0 (MH*) m/z 722.2043, WO 00/64864 PCT/GB00/01612 -112 found 722.2039; Anal. (C 35
H
34 C1N 5 0 0 ) C, H, N. Example 9D. Preparation of (1-methyl-3-nitro-1H-pyrrol-2-yl)methyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-lH-indol-1-yl)carbonyl]-2,3-dihydro-1H 5 benzo[e]indol-5-ylcarbamate (175). 2-(Hydroxymethyl)-1-methyl-3-nitropyrrole (174). A solution of NaOH (1 M, 16.7 mL, 16.7 mmol) was added to a solution of ester 172 (0.76 g, 3.34 mmol) in EtOH (50 mL), and the mixture was stirred at 20 oC for 1 h. The EtOH was evaporated, water (50 mL) added and the aqueous phase washed with diethyl ether (50 mL). The pH of the aqueous phase 10 was adjusted to 2 with 1 M HC1. The aqueous mixture was extracted with EtOAc (3 ' 50 mL), the combined organic extract was dried and the solvent evaporated to give crude 4 (hydroxymethyl)-1-methyl-5-nitropyrrole-2-carboxylic acid (0.65 g, 97%) as a red-brown solid. The acid was suspended in quinoline (10 mL) with Cu powder (0.50 g) and the mixture was 15 heated at 180-190 oC for 50 min under N 2 . The cooled mixture was diluted with EtOAc (50 mL) and 1 M HCI (50 mL), extracted with EtOAc (3 ' 50 mL), the combined extract was dried, and the solvent evaporated. The residue was purified by chromatography, eluting 50% EtOAc/light petroleum, to give 174 as a pale yellow solid (0.29 g, 57%), mp 63-64 oC (benzene); 'H NMR 6 6.73 (d, J= 3.4 Hz, 1 H, H 5), 6.50 (d, J= 3.4 Hz, 1 H, H 4), 4.89 (d, 20 J= 7.2 Hz, 2 H, CH 2 0), 3.73 (s, 3 H, NCH 3 ), 2.83 (t, J= 7.2 Hz, 1 H, OH); 1 3 C NMR 6 134.9 (C 3), 133.5 (C 2), 122.0 (C 4), 105.7 (C 5), 53.8 (CH20), 34.8 (NCH 3 ); Anal.
(C
6 HsN 2 0 3 ) C, H, N. (1-Methyl-3-nitro-1H-pyrrol-2-yl)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H 25 indol-1-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamate (175). A solution of triphosgene (15 mg, 50 gmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (59 mg, 127 pzmol) and Et 3 N (40 uL, 284 ymol) in DCM (10 mL) and stirred at 20 oC for 2 h. A solution of alcohol 174 (27 mg, 171 gmol) in DCM (2 mL) was added, 30 followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with a gradient (20 30%) of EtOAc/DCM, to give 175 (11 mg, 13%) as a white solid, mp (EtOAc) 218-220 oC; 'H NMR 6 9.42 (s, 1 H, indole-NH), 8.94 (s, 1 H, OCONH), 7.87 (d, J= 8.4 Hz, 1 H, H 6), WO 00/64864 PCT/GB00/01612 - 113 7.79 (d, J= 8.2 Hz, 1 H, H 9), 7.57 (ddd, J= 8.2, 7.4, 0.9 Hz, 1 H, H 8), 7.47 (ddd, J= 8.4, 7.4, 0.9 Hz, 1 H, H 7), 7.08 (br s, 1 H, H 4), 7.01 (d, J= 2.2 Hz, 1 H, H 3'), 6.89 (s, 1 H, H 4'), 6.80 (d, J= 3.3 Hz, 1 H, H 5"), 6.57 (d, J= 3.3 Hz, 1 H, H 4"), 5.65 (s, 2 H, CH 2 0), 4.81 (dd, J= 10.7, 91.7 Hz, 1 H, H 2), 4.67 (dd, J= 10.7, 8.6 Hz, 1 H, H 2), 4.15-4.20 (m, 1 5 H, H 1), 4.10 (s, 3 H, OCH 3 ), 3.95-3.99 (m, 4 H, OCH 3 , CH 2 C1), 3.92 (s, 3 H, OCH 3 ), 3.80 (br s, 3 H, NCH 3 ), 3.48 (dd, J= 11.0, 10.7 Hz, 1 H, CH 2 C; 13C NMR 6 160.4 (CO), 153.9 (OCONH), 150.2 (C 5'), 141.6 (C 3a), 140.6 (C 6'), 138.9 (C 7'), 133.7 (C 5), 130.9 (C 3"), 129.7 (C 9a), 129.6 (C 2'), 128.8 (C 2"), 127.5 (C 8), 125.6 (C 7a'), 125.1 (C 7, C Sa), 123.6 (C 3a'), 123.1 (C 9), 122.6 (C 6), 122.2 (C 4"), 121.6 (C 9b), 113.0 (C 4), 106.5 (C 10 3'), 106.1 (C 5"), 97.7 (C 4'), 61.5 (OCH 3 ), 61.1 (OCH 3 ), 56.3 (OCH 3 ), 55.3 (CH 2 0), 54.9 (C 2), 45.8 (CH 2 C1), 43.4 (C 1), 35.3 (NCH 3 ); MS (FAB') m/z 650 (MH', 0.6%), 648
(MH
+
, 1.5); HRMS (FAB') calc. for C 3 2
H
31 , 35 C1NsO 5 8
(MH
+
) m/z 648.1861, found 648.1850; calc. for C 3 2
H
31 37 C1N 5 0 8 (MH ) m/z 650.1832, found 650.1841; Anal. (C 32
H
30 C1NsOs 8 ) C, H, N. 15 Example 9E. Preparation of (1-methyl-5-nitro-1H-pyrrol-2-yl)methyl 1 (chloromethyl)-3-[(5,6,7-trimethoxy-1H-indol-1-yl)carbonyl]-2,3-dihydro-1H benzo[e]indol-5-ylcarbamate (178). (1-Methyl-5-nitro-lH-pyrrol-2-yl)methanol (177). NaBH 4 (0.19 g, 5.03 mmol) was 20 added to a stirred solution of 1-methyl-5-nitro-1H-pyrrole-2-carbaldehyde (176) [ P. Fournari, Bull. Soc. Chim. Fr. 1963, 488-491] (0.78 g, 5.07 mmol) in MeOH (40 mL) at room temperature under N 2 . After addition was complete, the reaction mixture was stirred for a further 20 min, then water (40 mL) was added and the mixture was saturated with solid K 2 C0 3 . The mixture was extracted with EtOAc (3 ' 50 mL), the combined organic 25 fraction dried, and the solvent evaporated to give 177 (0.77 g, 97%)as a white solid, mp (EtOAc/light petroleum) 76-77 oC; 'H NMR 8 7.16 (d, J= 4.3 Hz, 1 H, H 4), 6.17 (d, J= 4.3 Hz, 1 H, H 3), 4.68 (s, 2 H, CH 2 ), 4.02 (s, 3 H, CH 3 ); Anal. (C 6
HN
2 0 3 ) C, H, N. (1-Methyl-5-nitro-1H-pyrrol-2-yl)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-1H 30 indol-1-yl)carbonyll-2,3-dihydro-1H-benzo[elindol-5-ylcarbamate (178). A solution of triphosgene (16 mg, 52 pmol) in DCM (2 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med Chem. Lett., 1997, 7, 1483] (62 mg, 133 p/mol) and Et 3 N (42 4L, 299 gmol) in DCM (10 mL) and stirred at 20 WO 00/64864 PCT/GB00/01612 -114 oC for 2 h. A solution of alcohol 177 (28 mg, 179 /mol) in DCM (2 mL) was added, followed by nBu 2 Sn(OAc) 2 (2 drops) and the solution stirred at 20 oC for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20% EtOAc/DCM, to give 178 (54 mg, 63%) as a white solid, mp (EtOAc) 212-214 oC; 'H 5 NMR [(CD 3
)
2 SO] 6 11.45 (br s, 1 H, indole-NH), 9.82 (br s, 1 H, OCONH), 8.55 (br s, 1 H, H 4), 8.04 (d, J= 8.5 Hz, 1 H, H 6), 7.89 (d, J= 8.3 Hz, 1 H, H 9), 7.57 (dd, J= 8.3, 7.2 Hz, 1 H, H 8), 7.45 (dd, J= 8.5, 7.2 Hz, 1 H, H 7), 7.24 (d, J= 4.4 Hz, 1 H, H 4"), 7.09 (d, J= 2.0 Hz, 1 H, H 3'), 6.98 (s, 1 H, H 4'), 6.45 (d, J =4.4 Hz, 1 H, H 3"), 5.30 (s, 2 H,
CH
2 0), 4.80 (dd, J = 11.0, 9.4 Hz, 1 H, H 2), 4.53 (dd, J = 11.0, 1.8 Hz, 1 H, H 2), 4.32 10 4.37 (min, 1 H, H 1), 4.07 (dd, J= 11.1, 3.1 Hz, 1 H, CH 2 C1), 3.91-3.96 (min, 7 H, CH 2 C1,
NCH
3 , OCH 3 ), 3.83 (s, 3 H, OCH 3 ), 3.81 (s, 3 H, OCH 3 ); 1 3 C NMR [(CD 3
)
2 SO] 8 160.2 (CO), 154.0 (OCONH), 149.1 (C 5'), 142.1 (C 5"), 141.4 (C 3a), 139.9 (C 6'), 138.2 (C 7'), 136.1 (C 2"), 134.2 (C 5), 130.7 (C 9a), 129.4 (C 2'), 127.1 (C 8), 125.3 (C Sa), 125.2 (C 7a'), 124.3 (C 7), 123.7 (C 9), 123.3 (C 6), 123.1 (C 3a'), 122.1 (C 9b), 113.1 (C 3"), 15 113.0 (C 4), 110.6 (C 4"), 106.2 (C 3'), 98.0 (C 4'), 61.0 (OCH 3 ), 60.9 (OCH 3 ), 57.6
(CH
2 0), 55.9 (OCH 3 ), 54.8 (C 2), 47.5 (CH 2 C1), 41.1 (C 1), 33.9 (NCH 3 ); MS (FAB') m/z 650 (MH-, 1%), 648 (MH', 2); HRMS (FAB') calc. for C 32
H
31 35 C1N 5 0 8 (MH') m/z 648.1861, found 648.1852; calc. for C 32
H
31 3 7 C1NsO 5 8
(MH
+
) m/z 650.1832, found 650.1836; Anal. (C 32
H
30 C1NsO 5 8 ) C, H, N. 20 Elemental analysis data No Formula Calculated (%) Found (%) 1 8 C, 1 H,5N30 4 S C, 62.2; H, 3.7; N, 10.4 C, 62.3; H, 3.6; N, 10.7 25 2A 32 C,,H,,C1,NO, C, 51.6: H, 4.8: N, 9.5 C, 51.8; H, 4.9;N, 9.5 2B 34 CoH,, 29
CIN
4 0 9 C, 57.65; H, 4.7; N, 9.0 C, 57.45: H, 4.7; N, 8.7 2C 35 C 3 4 H3 1 CIN40O.H,O C, 59.1; H, 4.5; 8.1 59.1; H, 4.2; N, 7.9 2D 36 C,,H 17 N3OSS C, 60.7; H, 3.9; N, 9.7 C, 60.6; H, 4.0; N, 9.8 2E 38 C 16 H3 6
N
2 0 16 C, 57.4; H, 4.8; N, 3.7 C, 57.2; H, 5.1; N, 3.9 30 2F 42 C 4 4
H
43
N
3
O
1 g.HO0 C, 57.45; H, 4.9 C, 57.4; H, 5.1 2G 46 C,2 7
H
1 iNsO6 C, 62.7; H, 5.3; N, 13.5 C, 62.1; H, 5.5; N, 13.4 2H 51 C 4
,H
42
N
6 0 7 .2H,O C, 68.6; H, 5.5; N, 10.7 C, 68.4; H, 5.6; N, 10.65 21 53 CHIN 6
O,
4 (M*) 802.2446 802.2446 2J 55 C-oH 4
N
5
O
0 o (MH) 624.2306 624.2297 35 3A 59 C,,H,, 35 CI2,N 3 0 4 (M') 425.0909 425.0901 3B 60 C, 4
H,,CIN
4 0, C, 62.0; H, 4.7; N, 8.5 C, 61.9; H, 4.8; N, 8.3 3C 64 C, 0
H-,
3 5 CI2NO, (M ) 455.1015 455.1017 4A 70 C,,H,3CIN 4 0 o C, 59.6; H, 4.7; N, 7.95 C, 59.4; H, 4.9; N, 7.65 4B 73 C,, 6 H, sCIN 4 I0o C, 60.1; H, 4.9; N, 7.8 C, 60.3; H, 5.1; N, 7.5 40 4C 78 C 2 ,HsCIN,0 6 C, 51.9; H, 5.2; N, 8.6; CI, 14.6 C, 51.95; H, 5.2; N, 8.6; CI, 14.5 4D 80 C,,HCIN 4 0o. V 2
H
2 0 C, 56.7; H, 5.05; N, 8.3 C, 56.9: H, 4.8; N, 8.1 4E 84 CHsCIN40,n C, 60.1; H, 4.9; N, 7.8 C, 60.2; H, 5.2; N, 7.6 WO 00/64864 PCT/GB00/01612 -115 4F 87 CgH 4 oN 2
OI
7 . H 2 C, 56.6; H, 5.1; N, 3.5 C, 56.7; H, 5.1; N, 3.5 4G 91 C 4 7
H
4
,N
6 O.2HC1.2 H 2 C, 60.0; H, 5.7; N, 8.9 C, 59.8; H, 5.95; N, 9.0 4H 93 C 6 H,7 37
CINOI,
3 P (MH*) 799.1783 799.1757 41 99 C 3 6
HCIN
4 0,,.CHOH C, 57.9; H, 5.1; N, 7.3 C, 57.8; H, 4.8; N, 7.2 5 4J 102 C sH 41 CIN,0 9 .2HCI C, 55.7; H, 5.2; N, 8.55 C, 56.1; H, 5.5; N, 8.6 4K 105 C 4
H
42
CIN
5 OIo.'2HO C, 60.3; H, 5.4; N, 8.8 60.3; H, 5.5; N, 8.5 4L 107 C,,H, 4 NO, (M') 892.4034 892.4055 4M 113 C 4
,H
46
N
6 0,.2HCI.2H20,O C, 60.1; H, 5.45; N, 8.8 C, 59.8; H, 5.6; N, 8.8 5A 117 CIoH, 4 C1,N 4 0 4 C, 36.9; H, 4.3; N, 17.2; Cl, 21.8 C, 37.4; H, 4.1; N, 17.2; CI, 21.8 10 SB 123 C 1
,H,
9
CIN
s
O
4 C, 46.2; H, 4.6; N, 16.8; Cl, 17.0 C, 46.3; H, 4.8; N, 17.1; CI, 17.1 5C 124 CHH,,CIN 6
O.'H
2 ,O C, 56.6; H, 4.6; N, 12.8 C, 56.6; H, 4.4; N, 12.5 5D 125 C,,H, 4
N
4 0.'/2H,O0 C, 53.9; H, 4.8; N, 7.6 C, 53.7; H, 4.8; N, 7.3 5E 129 C 41
H
4 1 NsOi 7
.HO
2 0 C, 55.1; H, 4.85; N, 7.8 C, 54.7; H, 4.9; N, 7.5 5F 132 C,,H,gCIN 6 O C, 57.4; H, 4.5; N, 12.95 C, 57.7; H, 4.5; N, 12.9 15 5G 135 CH1 4 Cl 2
N
4 04(M') 324.1392 324.1381 5H 137 CisH,,C1,NO0 4 C, 46.2; H, 4.6; H, 16.8 C, 46.1; H, 4.6; N, 16.7 51 138 CH 29
,,CIN
6 0O C, 57.4; H, 4.5; N, 12.95 C, 57.5; H, 4.6; N, 12.9 5 142 C 4
,H
4 ,N ,O 7 .H,O C, 55.1; H, 4.85; N, 7.8 C, 55.2; H, 4.9; N, 7.9 5K 148 C, 2
H,,CIN
6 09 C, 56.6; H, 4.6; N, 12.4 C, 56.5; H, 49; N, 12.1 20 5L 150 C,H 2
,,CIN
6 O, C, 57.4; H, 4.5; N, 12.95 C, 57.2; H, 4.5; N, 12.9 6A 154 CI 6 Hi 7 35 CI,NOs(M
+
) 401.0545 401.0546 6B 155 CH 7
CIN
4 0, 9 C, 58.6; H, 4.3; N, 8.8 C, 58.5; H, 4.3; N, 8.9 7A 159 C,6H,,CIN O 4 C, 45.9; H, 4.1; N, 10.1; CI, 17.0 C, 46.2; H, 4.0; N, 9.9; CI, 17.2 7B 160 C,,H 27 CIN40,,S C, 57.2; H, 4.2; N, 8.6 C, 57.4; H, 4.0; N, 8.4 25 8 163 C,,H,,CIN 6 0, C, 57.4; H, 4.5; N, 12.95 C, 57.5; H, 4.6; N, 13.1 9A 167 C1H 34 CINsO 5 , C, 58.4; H, 4.8; N, 9.7 C, 58.2; H, 4.9; N, 9.7 9B 169 C3,HoCINsOx C, 59.3; H, 4.7; N, 10.8 C, 59.4; H, 4.8; N, 10.8 9C 173 C,,H, 4 CINO,o C, 58.4; H, 4.8; N, 9.7 C, 58.6; H, 4.9; N, 9.8 9D 175 CH OCIN 5 Og C, 59.3; H, 4.7; N, 10.8 C, 59.1; H, 4.7; N, 10.9 30 9E 178 C~,HoCINSO, C, 59.3; H, 4.7; N, 10.8 C, 59.4; H, 4.8; N, 10.7 Example 10 Biological activity 35 Selected compounds were evaluated for cytotoxicity (measured as IC 50 values in /M following and 18 h drug exposure) in pairs of mammalian cell lines, and the results are given in Table 2. The human ovarian carcinoma line (SKOV3) is wild-type, while the SC3.2 line is the NR+ transfectant. The human colon carcinoma line NR- line (WIDR) is wild-type, while the WC14.10 line is the NR+ transfectant. The murine mammary 40 carcinoma (EMT6-V) is wild-type, while the EN2A is the NR+ transfectant. Ratios (NR /NR+) provide a major measure of efficacy of action. Table 3. Biological activity for selected compounds. Example No SKOV3 SKOV/ WiDr WiDr/ EMT6-V EMT6/ SC3.2 WC14.10 EN2A 45 NR- IC 5 0 ratios NR- Ratio NR- Ratio
IC
5 (umol) ICo(4mol) ICo(4mol) 2C 35 0.059 16.6 0.14 47 2E 38 2.45 5.9 2.78 3.1 - 2F 42 >10 - >10 >16 3.33 >54 2G 46 1.81 3.9 1.21 3.3 0.76 49 50 2H 51 5.3 8.1 2.8 3.6 0.62 2.44 WO 00/64864 PCT/GB00/01612 -116 3B 60 0.157 5.6 0.15 6.6 - 4A 70 0.224 84 0.30 147 0.106 104 4B 73 0.105 67 0.109 45 0.033 78 4E 84 0.145 58 0.19 91 0.47 91 5 4G 91 9.4 >36 6.6 22 1.6 15.6 4I 99 0.114 40 0.13 25 0.44 13.4 4K 105 0.088 6.4 0.097 9 0.024 6.1 5B 123 2.3 28 1.7 6.7 - 5C 124 0.075 21 0.075 40 - 10 5D 125 3.74 14.5 3.59 10.3 0.46 6.4 5E 129 >1.5 >12 >1.5 >5.6 0.24 4.95 5I 138 0.15 50 0.23 99 0.078 71 5J 142 >3 >9.0 2.72 1.64 0.23 >1.22 5K 148 0.23 30 0.38 <38 0.067 <7 15 5L 150 0.061 5.6 0.024 3.4 0.063 6.5 7B 160 0.01 7.0 0.018 13.5 0.004 16.5 8 163 0.032 30 0.011 13.5 0.039 87 9A 167 0.097 9.6 0.094 13.2 0.028 11.6

Claims (36)

1. A compound of formula (I) or (II): wherein: 5 X represents H, Cl- 6 alkyl or C,- 6 alkoxy, said alkyl or alkoxy being optionally NO 2 Z Y OO O0E O O-O 0 II- n substituted with one or more of the following groups: hydroxy (OH), ether (ORx), amino (NH 2 ), mono-substituted amino (NRxH), di-substituted amino (NRxRx 2 ), cyclic C 1 5 alkylamino, imidazolyl, C 6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRx), tetrazole, carboxy (COOH), carboxylate (COORx), sulphoxy (S(=0) 2 0H), sulphonate 10 (S(=O) 2 ORx), sulphonyl (S(=O) 2 Rx), sulphixy (S(=0)OH), sulphinate (S(=O)ORx), sulphinyl (S(=O)Rx), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORx) 2 ), where Rx, R' and Rx 2 are selected from a Cl. 6 alkyl group, a C 3 . 20 heterocyclyl group or a C 5 - 2 0 aryl group; a is 0,1,2,3 or 4; Y represents H or C 1 . 6 alkyl; 1, 2 or 3 of the members Z of the 5-membered aromatic ring are independently selected from -O-,-S-,-N= or -NR-, 15 where R is H or Cz. 6 alkyl optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORR), amino (NH 2 ), mono-substituted amino (NRRH), di-substituted amino (NRRIRR 2 ), cyclic 1-5 alkylamino, imidazolyl, alkylpiperazinyl, morpholino, thiol (SH), thioether (SRR), tetrazole, carboxy (COOH), carboxylate (COORR), sulphoxy (S(=O) 2 0H), sulphonate (S(=O) 2 ORR), sulphonyl (S(=O) 2 RR), sulphixy (S(=O)OH), 20 sulphinate (S(=O)ORR), sulphinyl (S(=O)RR), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORR) 2 ), where RR, RR1 and RR 2 are selected from a C 1 . 6 alkyl group, a C 3 - 20 heterocyclyl group or a C 5 2 0 aryl group, the other ring atoms being C; n is 0 or 1; and E represents a moiety such that EH is an amine; provided that in formula (I) if a = 0 then Y H. 25 WO 00/64864 PCT/GB00/01612 -118
2. A compound according to claim 1 wherein EH is a cytotoxic amine.
3. A compound according to claim 2, wherein E is selected from formulae (III-XIII); wherein, R, represents H or CI_ 6 alkyl, being optionally substituted with one or more of the 5 following groups: one or more of the following groups: hydroxy (OH), ether (ORE), amino (NH 2 ), mono-substituted amino (NREH), di-substituted amino (NRE RE2), cyclic C 1 . 5 alkylamino, imidazolyl, C 1 - 6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRE), tetrazole, carboxy (COOH), carboxylate (COORE), sulphoxy (S(=O) 2 0H), sulphonate (S(=O) 2 ORE), sulphonyl (S(=O) 2 RE), sulphixy (S(=O)OH), sulphinate (S(=O)ORE), sulphinyl 10 (S(=O)RE), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORE) 2 ), where RE, REl and RE 2 are selected from a C,. 6 alkyl group, a C 3 . 20 heterocyclyl group or a C 5 . 2 0 aryl group; R 2 represents H, CI. 6 alkyl, Cl_6 alkoxy, OH, halogen, NO2, N2, NHMe, NMe2, SO2Me, CF 3 , CN, CONH 2 or CONHMe; each R 3 is independently selected from Cl, Br, I and OMS; and R 4 is selected from -C(=O)Me and -C(=O)CH 2 OH; Q represents substituted indole, substituted 15 benzofuran or substituted cinnamoyl; in (IX) and (X), each n is independently from 2-4, and each m is independently from 2-4, and p = 0 or 1. WO 00/64864 PCT/GB00/01612 -119 NR 1 Cl Cl NO NQ 5 RNN (III)R1 N R N 3 R3 N R3 R3 Ri Rl N (IV) (V) (VI) R. R1 10 OH O HN N S OH 0 HN R2 OH O HN OH 0 HN- R 15 (VIla R (VIIb) R (VIII) 0 -- L-- N R 2 R R 20 0 L 2 (iX) (x) S-R2 (XI) where L = R2 (XI) NH 2 O OH 25 R2 R4 N 9 OMe O OH S0 Me 0 30 Ri (xin) 30 (XII) HO R 1 N (XIII) WO 00/64864 PCT/GB00/01612 -120
4. A compound according to any one of the preceding claims, wherein R, R,, Rx 2 , RR, RR I and RR 2 are independently Cl- 6 alkyl groups.
5. A compound according to any one of the preceding claims, wherein the compound is 5 of formula (I) and a is 1.
6. A compound according to claim 5, wherein X represents optionally substituted C,-6 alkoxy. 10
7. A compound according to claim 6, wherein the optionally substituted C- 1 . 6 alkoxy is in the 2 position.
8. A compound according to claims 7 or 8, wherein the C 1 . 6 alkoxy group is selected from methyl, ethyl and n-propyl. 15
9. A compound according to any one of claims 6 to 8, wherein the C 1 - alkoxy group substituents are selected from hydroxy, methoxy, phosphonoxy, NMe 2 , Nmorph, OCO 2 -tBu, and OCO 2 H. 20
10. A compound according to claim 9, wherein the C 1 - 6 alkoxy group is ethoxy or n propyl group with a single substituent.
11. A compound according to claim 10, wherein the ethyl or n-propyl substituent is hydroxy. 25
12. A compound according to any one of claims 6 to 8, wherein the C,. alkoxy group is unsubstituted methyl.
13. A compound according to any one of claims 5 to 12, wherein Y is either H or Me. 30
14. A compound according to anyone of claims 5 to 13, wherein E is of formula V. WO 00/64864 PCT/GB00/01612 - 121
15. A compound according to any one of claims 5 to 13, wherein E is of formula XIII.
16. A compound according to claim 15, wherein n = 1. 5
17. A compound according to any one of claims 1 to 4, wherein the compound is of formula (II) with two Z, one Z being -N= and the other Z being -NR-.
18. A compound according to claim 17, wherein R is either Me or Et. 10
19. A compound according to claim 18, wherein R is Et substituted with hydroxy.
20. A compound according to any one of claims 17 to 19, wherein a is 0.
21. A compound according to any one of claims 17 to 20, wherein the -N= and -NR- are 15 not adjacent in the heterocyclic ring.
22. A compound according to any one of claims 17 to 21, wherein E is of formula V.
23. A compound according to any one of claims 17 to 21, wherein E is of formula XIII. 20
24. A compound according to claim 23, wherein n = 1.
25. A compound according to any one of claims 1 to 4, wherein the compound is of formula (H), a is 0, and Z is selected from O or S. 25
26. A compound according to any one of claims 1 to 4, wherein the compound is of formula (II), Z is NR, and a is either 0 or 1.
27. A compound according to claim 26, wherein Z is NMe. 30
28. A compound according to either claim 26 or 27, wherein a is 1 and X is CO 2 Et. WO 00/64864 PCT/GB00/01612 - 122
29. A compound according to any one of the preceding claims for pharmaceutical use.
30. A pharmaceutical composition comprising a compound according to any one of claims 1 to 28 and a pharmaceutically acceptable carrier or diluent. 5
31. A two component system for the treatment of neoplastic disease which comprises: (i) a vector encoding and capable of expressing a nitroreductase enzyme in a tumour cell; and (ii) a compound as defined in any one of claims 1 to 28. 10
32. A two component system for the treatment of neoplastic disease which comprises: (i) a tumour directed antibody linked to a nitroreductase enzyme; and (ii) a compound as defined in any one of claims 1 to 28. 15
33. A compound according to any one of claims 1 to 28, a composition according to claim 26, or a system according to claims 31 or 32 for use in a method of medical treatment.
34. A method of treating neoplastic disease which comprises administering to a patient in need of treatment an effective amount of a compound according to any one of claims 1 to 28, 20 a composition according to claim 30, or a system according to claims 31 or 32.
35. The use of a compound according to any one of claims 1 to 28 for the manufacture of a composition for use in the treatment of a hyper-proliferative disease. 25
36. A method of providing an amine with a protecting group comprising: ,NO 2 NO 2 Z - % _ E 0 Y O O O '0E O n E 0- n WO 00/64864 PCT/GB00/01612 - 123 (i) providing a plurality of different compounds selected from compounds of formulae (I) and (II) wherein: X represents H, C 1 - 6 alkyl or C,-6 alkoxy, said alkyl or alkoxy being optionally 5 substituted with one or more of the following groups: hydroxy (OH), ether (OR), amino (NH 2 ), mono-substituted amino (NRxH), di-substituted amino (NRxRx 2 ), cyclic C 1 5 alkylamino, imidazolyl, C,- 6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRx), tetrazole, carboxy (COOH), carboxylate (COORx,), sulphoxy (S(=O) 2 0H), sulphonate (S(=0) 2 0R), suphonyl (S(=O) 2 Rj), sulphixy (S(=O)OH), sulphinate (S(=O)ORx), sulphinyl 10 (S(=O)Rx), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORx) 2 ), where Rx, R~' and R 2 are selected from a C, 6 alkyl group, a C 3 - 20 heterocyclyl group or a C 5 - 20 aryl group; a is 0,1,2,3 or 4; Y represents H or C,6 alkyl; 1, 2 or 3 of the members Z of the 5-membered aromatic ring are independently selected from -O-,-S-,-N = or -NR-, where R is H or C,6 alkyl optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORR), 15 amino (NH 2 ), mono-substituted amino (NRRH), di-substituted amino (NRRRR2), cyclic C,. 5 alkylamino, imidazolyl, alkylpiperazinyl, morpholino, thiol (SH), thioether (SRR), tetrazole, carboxy (COOH), carboxylate (COORR), sulphoxy (S(=O) 2 0H), sulphonate (S(=O) 2 0RR), sulphonyl (S(=O) 2 RR), sulphixy (S(=O)OH), sulphinate (S(=O)ORR), sulphinyl (S(=O)RR), phosphonooxy (OP(=O)(OH) 2 ) and phosphate (OP(=O)(ORR) 2 ), where RR, RR 1 and RR 2 are 20 selected from a C-6 alkyl group, a C 3 - 20 heterocyclyl group or a C 5 - 2 0 aryl group, the other ring atoms being C; n is 0 or 1; and E represents a moiety such that EH is an amine; (ii) measuring the rates of fragmentation of the compounds to release EH when the nitro group is reduced and selecting a compound having a desired rate of decomposition; and (iii) providing the amine to be protected with a protecting group corresponding to that in the 25 selected compound.
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