NZ515044A - N-protected amines and their use as prodrugs - Google Patents

N-protected amines and their use as prodrugs

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Publication number
NZ515044A
NZ515044A NZ515044A NZ51504400A NZ515044A NZ 515044 A NZ515044 A NZ 515044A NZ 515044 A NZ515044 A NZ 515044A NZ 51504400 A NZ51504400 A NZ 51504400A NZ 515044 A NZ515044 A NZ 515044A
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New Zealand
Prior art keywords
alkyl
compound according
substituted
amino
etoac
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NZ515044A
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William Alexander Denny
Michael Patrick Hay
William Robert Wilson
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Auckland Uniservices Ltd
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Publication of NZ515044A publication Critical patent/NZ515044A/en

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    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • 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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    • C07D231/10—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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    • 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
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Abstract

A compound of formula (II), wherein X, Y, Z, E and n are as described within the specification. This compound has uses in the treatment of neoplastic disease.

Description

New Zealand Paient Spedficaiion for Paient Number 515044 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 groups which are susceptible to reduction, leading to loss of the protecting group and liberation of the amine. The amine is desirably biologically active, the activity being substantially suppressed by N-protection. Thus it 10 may be an amine-based cytotoxin, e.g actinomycin D, an anthracycline, an aniline mustard or an enediyne.
Thus suitable protected amines may be valuable as anticancer agents, and/or may be useful as prodrugs for antibody-directed enzyme prodrug therapy (ADEPT) or gene-directed enzyme prodrug therapy (GDEPT), in conjunction with nitroreductase enzymes. 15 BACKGROUND TO THE INVENTION The use of prodrugs (relatively inactive compounds which can be converted into more active compounds in vivo) represents a clinically very valuable concept, 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 W088/07378.
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 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], Cf^NHR O^NHR 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, 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.
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 (op) of the substituent, as shown in Table 1. The maximum half-lives (M^1/2) 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 (tm) of species R is calculated from the equation ln([R]</[R],) = f(ln2//1/2). 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.
X(E = N02) Y (E = NHOH) Table 1 Half-lives for fragmentation (MtI/2) and percent of amine released (t0) for 15 substituted 4-hydroxylaminobenzyl carbamates (derived from the corresponding 4-nitrobenzyl carbamates by radiolytic reduction).
D A °p Mt1/7 (min) M%) 2-NO? H 0.78 88 18 3-NO, H 0.71 65 22 3-CO,Me H 0.37 44 3-OMe H 0.12 17 37 H H 0.0 16 40 2-OMe H -0.27 12 48 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 formula (II) II wherein: X represents H, C,.6 alkyl or C,.6 alkoxy, said alky] or alkoxy being optionally 25 substituted with one or more of the following groups: hydroxy (OH), ether (ORx), amino (NH2), mono-substituted amino (NRXH), di-substituted amino (NRjR^2), cyclic C,_5 alkylamino, imidazolyl, C,.6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRJ, ^ R 0 I ^ -5 tetrazole, carboxy (COOH), carboxylate (COORJ, sulphoxy (S(=0)20H), sulphonate (S(=0)20Rx), sulphonyl (S(=0)2Rx), sulphixy (S(=0)0H), sulphinate (S(=0)0Rx), sulphinyl (S(=0)Rx), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0Rx)2), where R^, R^1 and Rx2 are selected from a C,.6 alkyl group, a C3.20 heterocyclyl group or a C5.20 aryl group, 5 preferably a C|_6 alkyl 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 -S-,-N= or -NR-(where R is H or CN6 alkyl optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORr), amino (NH2), mono-substituted amino (NRrH), di-substituted amino (NRr'Rr2), C,.5 cyclic amino, imidazolyl, alkylpiperazinyl, morpholino, thiol (SH), 10 thioether (SRr), tetrazole, carboxy (COOH), carboxylate (COORr), sulphoxy (S(=0)20H), sulphonate (S(=0)20Rr), sulphonyl (S(=0)2Rr), sulphixy (S(=0)0H), sulphinate (S(=0)0Rr), sulphinyl (S(-O)Rr), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0Rr)2), where Rr, Rr' and Rr2 are selected from a C,.6 alkyl group, a C3.20 heterocyclyl group or a C5.20 aryl group, preferably a C,_6 alkyl group), the other ring atoms 15 being C; n is 0 or 1; and ; E is selected from formulae (III-XIII), wherein R, represents H or C,_6 alkyl, being optionally substituted with one or more of the following groups: one or more of the following groups: hydroxy (OH), ether (ORE), amino (NH2), mono-substituted amino (NReH), di-substituted amino (NRe'Re2), cyclic C,.5 alkylamino, imidazolyl, C,.6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRe), tetrazole, carboxy (COOH), 20 carboxylate (COORE), sulphoxy (S(=0)20H), sulphonate (S(=0)20RE), sulphonyl (S(=0)2RE), sulphixy (S(=0)0H), sulphinate (S(=0)0RE), sulphinyl (S(=0)RE), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0RE)2), where RE, RE' and RE2 are selected from a C,_6 alkyl group, a C3.20 heterocyclyl group or a C5.20 aryl group, more preferably from a C,.6 alkyl group; R2 represents H, C,.6 alkyl, C,.6 alkoxy, OH, halogen, 25 N02, NH2, NHMe, NMe2, S02Me, CF3, CN, CONH2 or CONHMe; each R3 is independently selected from CI, Br, I and OMS; and R4 is selected from -C(=0)Me and -C(=0)CH20H; Q represents substituted indole, substituted 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: i ^ "NR-i R3" "R3 R3 an) (iv) (v) (VD OH O HN (Vila) (Vllb) R1 (vm) c (IX)) o (*) vv+iere L= (XI) O OH (XII) HO R|N (XII!) (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 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 10 actuation by the appropriate enzyme, and thus kill tumour cells further away from the site of actuation (the "bystander" effect).
In a second aspect, the present invention relates to a compound represented by the general formula (II) as shown above, wherein X, Y, Z, E, a and n are as defined above.
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 (II) may be basic or acidic and may thus form pharmaceutically acceptable salts with both organic and inorganic acids and bases. These are 20 included within the scope of the second aspect.
In a first type of preferred embodiment, X represents C,^ alkyl or C,6 alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy, ether (ORJ, amino, alkylamino (NR^H), dialkylamino (NR^'R^2), cyclic C,.5 alkylamino, imidazolyl, C,.6 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRJ, tetrazole 25 and -C02X' where X' is selected from the possibilities listed for X and R^, R^1 and R^2 are selected from C,_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 -0-,-S-,-N= or -NR-, (where R is H or lower alkyl optionally substituted with one or more of the following groups: hydroxy, ether (ORr), amino, alkylamino (NRRH), dialkylamino (NRr'Rr2), cyclic C,.5 30 alkylamino, imidazolyl, C,_6 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRr), tetrazole and -C02R' where R' is selected from the possibilities listed for R and Rr, Rr' and Rr2 are selected from C,_6 alkyl); and E represents R, represents H or C,_6 alkyl, being optionally substituted with one or more of the following groups hydroxy, ether (ORE), amino, alkylamino (NREH), dialkylamino (NRE'RE2), cyclic C,.5 alkylamino, imidazolyl, C,.6 alkylpiperazinyl, morpholino, thiol, alkylthioether (SRe), tetrazole and -C02X' where X' is selected from the possibilities listed for X and Re, Re' and RE2 are selected from C,_6 alkyl.
In a second preferred type of embodiment, the compound is of formula (II) with one Z 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 N02 at the 3 position. In this type 10 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 third preferred type of embodiment, the compound is of formula (II), a is 0, and Z is S. It is further preferred that the S is in the 2 position in the ring, and the N02 is attached to the 3 position. In this type of embodiment, Y is preferably selected from H or Me. 15 In a fourth 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 C02Et. In this type of embodiment, Y is preferably selected from H or Me.
In a third aspect the invention provides a compound according to the second aspect for 20 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 activating means for simultaneous or separate administration, the activating means typically 25 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 XVII where X, n, Z and Y are as defined for the second aspect and T is OH or an activated alcohol functionality (such as -O.CO.L where L is a leaving group such as CI) suitable for reaction 30 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 6 1 T n XVII (XVII) in protecting an amine. This may include activation of an alcohol XVII where T is OH) with a reagent such as phosgene, diphosgene or triphosgene or a chloroformate, e.g. 4-nitrophenylchloroformate or pentafluorophenylchloroformate, optionally in conjunction with HOBT(l -hydroxybenzotriazole). of the general formula (II); examples of the methods are outlined in Schemes 25-42 The following compounds are used in these routes: Amine 1 [G.J. Atwell, W.R. Wilson, W.A. Denny, Bioorg. Med. Chem. Lett., 1997, 7,1483] ci In a further aspect, the present invention relates to a method of preparing compounds NH.
Amine 9; OTBDMS Thus (Scheme 25), (2-nitro-l//-imidazol-5-yl)methanol (115) is obtained from the known ethyl 2-nitro-l//-imidazol-5-ylcarboxylate (113) [B. Cavalleri, R. Ballotta, G.C Lancini. J. Heterocyclic Chem. 1972, 9, 979.] by basic hydrolysis to the acid 114 and 515 G :' 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 /V,/V-bis-(2-chloroethyl)amine to give carbamate 117.
Scheme 25 no2 no2 no2 no2 no2 jx (0 (') J\ (»')' (K/) L N NMe >-N NMe vN NMe >N NMe >> N NMe C02Me C02H ^OH ^ O O \= O 113 114 115 116 ' 117 O, - N N \ Reagents: (i) NaOH, aq. MeOH; (ii) CDI, NaBhU, THF; (ii) N02Ph0C0CI, pyridhe, THF; (iv)HN(CH2CH2CI)2, "ci pyridine. N02 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 10 standard conditions.
Scheme 26 NO, (i) OR TBDMS 118 R = H ,, 119R = OTBDMS^(i) (ii) OTB DM S 120 no2 N^NMe Reagents: (i) TBDMSCI, imidazole (ii) Pd/C, H2, EtOH; (ii) 116, pyridine; (iv) TBAF, THF; (v) (a) MsCI, pyridine, (b) LCI, DMF.
O 121 R = OTBDMS 122 R = OH > (iv) 123 R = Q J(v) N—\ In another example (Scheme 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 (or pyridine, or another organic base) gave an intermediate isocyanate which was coupled with alcohol 115 using catalytic dibutyltin diacetate to give carbamate 124.
Scheme 27 N02 X... « .
NMe • OH N 115 U VNO? Reagents: (i) 1, triphosgene, Et3N, DCM, _ Me then 115, nBu2Sn(0Ac)2.
\ /""" ^ A ^ | OMe — OMe In another example (Scheme 28), reaction of the carbonate 116 with doxorubicin (13) 10 and triethylamine in DMF gave the carbamate 125.
Scheme 28 (') 116 Reagents: (0 13, Et3N, DMF O OH 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 15 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 in DMF gave the carbamate 129.
Scheme 29 116 (i) N02 N^ NMe L O HN^o or 126 r = tbdms "~\ (j) 127 r = h >w 128 C02PhN02 / (ii) (iv) N' O i! OH , OH OH 0 Me P \ HN- : Y T V OMeO OH O Me'; i°/ / NH HO / A -—, o o ) O2N—< l| 129 Reagents: (i) 9, HOBT. Et3N, mol. sieves, THF; (ii) HCI, aq. MeOH; (iii) 4-N02Ph0C0CI, THF; (vi) 13, Et3N, 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. 5 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. 132 no2 Reagents: (i) 03, DCM, MeOH; (ii) NaBH4, EtOH; (iii) 1, triphosgene, Et3N, DCM, then 131, nBu2Sn(OAc)2.
In another example (Scheme 31), treatment of (N-methyl-5-nitro-1 //-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 7V,A/-bis(2- chloroethyl)amine to give the carbamate 135.
Scheme 31 ,oh NO, 133 (0 a:>,a N NMe 134 NO, a NO, (•) O^N.
.X Reagents: (i) N02Ph0C0CI, pyridine,THF; (i) HN(CH2CH2CI)2, pyridine. 135 NO 2 a Similarly (Scheme 32), activation of 133 with diphosgene and subsequent reaction 5 with A^//'-bis(2-chloroethyl)-l ,4-benzenediamine hydrochloride (136) gave the carbamate 137.
Scheme 32 O R OH ^ . N^NMe ° «>2 (i) ^N02 133 137 Reagents: (i) diphosgene, Et3N, 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 10 gave an intermediate isocyanate which was coupled with alcohol 133 using catalytic dibutyltin diacetate to give carbamate 138.
Scheme 33 no2 X Ny NMe OH (i) 115 Reagents: (i) 1, triphosgene, Et3N, DCM, then 115, nBu2Sn(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 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 5 triethylamine in DMF gave the carbamate 142.
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.
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.
Reagents: (i) 9, HOBT, Et3N, mol. sieves, THF; (ii) HCI, aq. MeOH; 4-N02Ph0C0CI, THF; (vi) 13, Et3N, DMF.
Scheme 35 J5_ Me i N^N'"\,.OH N02 143 OH N^N' (■0. (%) j -OR N^N' NO, NO, 144 R = H Nj m 146 145 R = TBDMS r (v) -OTBDMS Reagents: (i) NaOMe, PhCHO, DMSO; (i) TBDMSTf, pyridine, DCM; (ii) 03, DCM, MeOH; (vi) NaBH4, EtOH; (v) 1, triphosgene, Et3N, DCM, then 146, nBujSnfOAc^; (vi) HCI aq. MeOH.
CI rf y !: I V OMe HN- Y OMe OMe HN, .0 f N—i 147 R = TBDMS 148 R = H N02 -OR ). (vi) 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 5 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 give carbamate 150. ci In another example (Scheme 37), reaction of the 5-nitrofuran-l -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 15 V/vrl-bis(2-hydroxyethyl)-l,4-benzenediamine (57) to give the carbamate diol 153. The diol 153 was converted to the dichloride 154 using standard methods.
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 A/1 -bis(2-hydroxyethyl)-1,4-benzenediamine (57) 5 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-l-methanol (151) [J.M.
Berry, C.Y. Watson, W.J. Whish, and M.D. Threadgill, J. Chem. Soc. Perkin Trans. I, \991, 1147], 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. Watson, W. J. D. Whish, and M. D. Threadgill, J. Chem. Soc. Perkin Trans. 1,1997,1147.] 15 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. Aust. J. Chem. 1979, 32, 2647] using catalytic dibutyltin diacetate to give carbamate 160.
Scheme 37 Reagents: (i) NC^PhOCOCl, pyridine, THF, (i) 57, pyridine, (ii) (a) MsCI, pyridine, (b) LiCI, DMF. 153 X = O, R = OH -s 154 X=0, R = a > (si) 158 X = S, R = OH 159 X = S, R = CI > Scheme 38 OH (i) 151 X = O 156 X = S Reagents: (i) 1, triphosgene, Et3N, DCM, then alcohol, nBu2Sn(OAc)2. a OMe OMe 155 x= o 160 x = s x no2 In another example (Scheme 39), l-methyl-5-nitro-l//-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 triethylamine gave an intermediate isocyanate which was coupled with alcohol 162 using catalytic dibutyltin diacetate to give carbamate 163.
Scheme 39 Me ,Nn V no2 co2h 161 (i) Me <VN0* —oh 162 (ii) Reagents: (i) BH3.DMS, THF; (ii) 1, triphosgene, Et3N, DCM, then 162, nBu2Sn(OAc)2.
OMe OMe HtV° Ki Y /=N 163 °^yNMe o2n In another example (Scheme 40), ethyl 4-formyl-5-nitro-lif-pyrrole-2-carboxylate (164) [P. Fornari, M. Farnier, C. Foumier, 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 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 5 gave an intermediate isocyanate which was coupled with alcohol 168 using catalytic dibutyltin diacetate to give carbamate 169.
Scheme 40 02N^/^\^C02Et U7 <®> OHC7 UN 164R = H "s 166R = C02Et~\ I 165 R = Me A 168 R = H ? ( ( ) °-^^.NMe o2n Reagents: (i) Me2S04, K2C03, DMSO; (ii) NaBH,), EtOH; (ii) 1, trphosgene, EtaN, DCM, then alcohol, nBu2Sn(OAc)2; 169 R - (K/)NaOH,aq. EtOH; (v)Cu,quinolhe.
Similarly (Scheme 41), ethyl 5-formyl-4-nitro-l//-pyrrole-2-carboxylate (170) [P. 10 Fornari, M. Farnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-291] was alkylated with dimethyl sulfate to give pyrrole 171. Reduction of 171 with sodium borohydride gave the 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 15 carbamate 173.
In another example (Scheme 41), hydrolysis of ester 172 followed by decarboxylation 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 20 diacetate to give carbamate 175. 19- ^ ^ p v\ 5 CI ^ Scheme 41 OHC.. 0,N 170 R = H 171 R = Me > (0 HO (i) Me I H \.-R (Si) 02N 172R = C02Et\ 174 R = H A (w). (v) Reagents: (i) Me2S04, K2C03, DMSO; (ii) NaBH4, EtOH; (ii) 1, trphosgene, Et3N, DCM, then alcohol, nBii2Sn(OAc^; (K/)NaOH, aq. EtOH; (v)Cu, quinoline.
CI OM e HN' OMe OMe HN..O R r. MeN—' O. 0,N 173 R = COoEt 175 R = H In another example (Scheme 42), 1 -methyl-5-nitro- l//-pyrrole-2-carbaldehyde (176) [ P. Fournari, 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 isocyanate which was coupled with alcohol 177 using catalytic dibutyltin diacetate to give carbamate 178.
Scheme 42 Me Me OH 2 NVVCH0 (i) > WVJJL, XOMe 176 177 Reagents: (i) NaBH4, EtOH; (ii) 1, trphosgene, Et3N, DCM, then 177,nBu2Sn(OAc)2.
OMe OMe In a further preferred aspect, the present invention relates to the use of a compound of 10 (II) as defined in the second aspect of the invention, in conjunction with a 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 (II) 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 15 lung tumours, including small cell lung carcinoma.
The invention also provides a pharmaceutical composition comprising a compound of ^ ^ 0 ll the formula (II) as defined in the second aspect of the invention together with a pharmaceutically acceptable earner or diluent.
Detailed Description of the Invention GDEPT 5 - 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 10 for the transformation of hepatoma, breast, colon or skin cells. Culver et al. (Science (1992) 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). 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. 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 p-globin 5' untranslated region up to the initiation ATG is supplied to direct efficient translation of the enzyme.
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-la 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.
- Nitroreductase Compounds of the formula (II) can be activated by reduction of one (or more) of the available nitro groups by nitroreductase.
Englehardt et al. (Nature Genetics (1993) 4; 27-34) describe the use of adenovirus Other vector systems including vectors based on the Molony murine leukaemia virus Suitable promoters which may be used in vectors described above, include MLV, Preferably, the enzyme is a non-mammalian nitroreductase enzyme, such as a bacterial nitroreductase. An E.coli nitroreductase as disclosed in W093/08288 is particularly preferred. 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 5 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 made may be any which still leaves the enzyme with the ability to reduce the nitro group in formula II but alters other properties of the enzyme, for example its rate of reaction or 10 selectivity.
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 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, 20 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 25 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.
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 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 5 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 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. 10 Compounds of the present invention may also be tested in combination with additional anticancer 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 15 cells in a patient with neoplastic disease which comprises administering to a patient in need of treatment a compound of formula (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 20 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. 25 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 30 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 (II) is minimised.
In ADEPT the degree of localisation of the enzyme/agent conjugate (in terms of the ratio of localized to freely circulating active conjugate) can be further enhanced using the clearance and/or inactivation systems described in W089/10140. This involves, usually 5 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 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, 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 15 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 20 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/m2 (e.g. 20,000 enzyme units/m2) and a suitable dose of prodrug may be from about 0.1 to 200 mg/Kg, preferably about from 10 to 25 100 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 30 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 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 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. 10 Alternatively the vectors may be packaged into liposomes. The liposomes may be targeted to 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 15 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. 20 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 25 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 30 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 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.
Administration of prodrug While it is possible for a compound of formula (II) to be administered alone it is 10 preferable to present it as a pharmaceutical formulation. Suitable formulations comprise the 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 15 the positively charged lipid (N[ 1 -(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), those comprising dioleoylphosphatidylethanolamine (DOPE), and those comprising 3p[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Choi).
Formulations suitable for parenteral or intramuscular administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, 20 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, for example 25 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 30 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 nonaqueous 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 delivery and injection, typically parenteral or intramuscular injection or intratumoral injection.
The exact dosage regime will, of course, need to be determined by individual 5 clinicians for individual patients and this, in turn, will be controlled by the exact nature of the compound of formula (II) but some general guidance can be given. Typical dosage ranges generally will be those described above which may be administered in single or multiple doses. Other doses may be used according to the condition of the patient and other factors at the discretion of the physician.
The following Examples illustrate the invention.
General procedures Analyses were carried out in the Microchemical Laboratory, University of Otago, Dunedin, 15 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 13C spectra. Spectra were obtained in CDC13 unless otherwise specified, and are referenced to Me4Si. Chemical shifts and coupling constants were recorded in units of ppm and Hz, 20 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 25 in organic solvents were dried with anhydrous Na^O,,. 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 F254) with visualisation of components by UV light (254 nm) or exposure to I2. Column chromatography was carried out on silica gel, (Merck 230-400 mesh). All compounds designated for biological testing were analyzed at 30 >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 NaH2P04,0.75 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 dichloromethane; DIEA refers to diisopropylethylamine, DMF refers to dry dimethyl formamide; DMSO refers to dimethylsulphoxide; ether refers to diethyl ether; EtOAc refers to 5 ethyl acetate; EtOH refers to ethanol; iPr20 refers to diisopropyl ether; light petroleum refers to petroleum ether, boiling range 40-60 °C; MeOH refers to methanol; THF refers to tetrahydrofiiran dried over sodium benzophenone ketyl. All solvents were freshly distilled.
Example 1A. Preparation of (l-methyl-2-nitro-l//-imidazoI-5-yl)methyl bis(2-10 chloroethyl)carbamate (117). l-MethyW-nitro-l/^-imidazole-S-carboxylic acid (114). Sodium hydroxide solution (1 M, 125 ml, 125 mmol) was added slowly to a stirred suspension of ethyl l-methyl-2-nitro-l//-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 °C 15 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 °C (lit. (B. Cavalleri, R. Ballotta, V. Arioli, G.C. Lancini, J. Med. Chem. 1973,16, 557) (EtOAc) 161-163 °C); 'H NMR [(CD3)2SO] 5 13.60 (br s, 1 H, C02H), 7.37 (s, 1 H, H 4), 20 4.20 (s, 3 H, NCH3); 13C NMR [(CD3)2SO] 5 160.3, 147.2, 133.7, 127.0, 35.0. (l-methyl-2-nitro-l//-iniidazol-5-yl)methanol (115). A solution of CDI (7.0 g, 43.1 mmol) and 114 was stirred at 20 °C for 30 min and then added to a stirred solution of NaBH4(4.07 g, 108 mmol) in EtOH (10 mL) and the mixture stirred at 20 °C for 1 h. 5 M 25 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 °C); 'H NMR [(CD3)2SO] 57.12 (s, 1 H, H 4), 5.49 (br s, 1 H, OH), 4.55 (s, 2 H, CH20), 3.92 (s, 3 H, NCH3); 13C NMR 30 [(CD3)2SO] 5 145.6, 138.6, 126.5, 52.9, 34.0. (l-Methyl^-nitro-lif-imidazol-S-ytymethyl 4-nitrophenyl carbonate (116). A solution of 4-nitrophenylchloroformate (0.67 g, 3.34 mmol) in THF (5 mL) was added to a stirred solution of alcohol 115 (0.50 g, 3.18 mmol) and pyridine (283 [A,, 3.50 mmol) in THF (50 mL) at 20 °C under N2. The solution was stirred at 20 °C for 16 h, the solvent evaporated and the residue dissolved in EtOAc (lOOmL). The solution was washed with water (2 x 50 5 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 white solid, mp (EtOAc) 156.5-157.5 °C; 1R N 1771, 1537, 1359 cm1; 'H NMR [(CD3)2SO] 5 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, CH20), 4.00 (s, 3 H, NCH3); 13C NMR 10 [(CD3)2S0] 5 155.1, 151.4, 146.3, 145.2, 131.5, 129.6, 125.4 (2), 122.5 (2), 59.4, 34.3; Anal. (C12Hl0N4O7) C, H. N calc. 17.4, found 16.7%. (l-Methyl-2-nitro-l//-imidazoI-5-y])methyl bis(2-chloroethyI)carbamate (117). A solution of carbonate 116 (0.68 g, 2.11 mmol) in pyridine (3 mL) was added to a solution 15 of bis-(2-chloroethyl)amine hydrochloride (0.75 g, 4.22 mmol) in pyridine (30 mL) under N2. The solution was stirred at 20 °C 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 20 white crystals, mp (EtOAc) 100-101 °C; IR N 1703, 1489, 1344 cm1; 'H NMR 5 7.23 (s, 1 H, H 4), 5.21 (s, 2 H, CH20), 4.05 (s, 3 H, NCH3), 3.58-3.70 (m, 8 H, 2 x CH2N, 2 x CH2C1); nC NMR 5 154.8, 144.5, 132.1, 129.7, 56.2, 50.8 (2), 41.6 (2), 34.3; Anal. (C10H)4Cl2N4O4) C, H, N.
Example IB. Preparation of (l-methyI-2-nitro-l/7-imidazol-5-yl)methyl 4-[bis(2-chloroethyl)amino]phenyIcarbamate (123).
AyV-Bis(2-{(ter/-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 N,N-bis(2-hydroxyethyl)-4-nitroaniline (118) (3.0 g, 13.26 mmol) and imidazole (3.79 g, 55.7 30 mmol) in DMF (50 mL) and the solution stirred at 20 °C 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 ""J (~\ "? T *■) 1 ■*, (;, I A a • ' 1 KM •£ 4 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 °C; IR N 1597, 1520, 1300, 1202, 1107 cm"1; '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 CH2N), 3.63 (dd, J = 5.9, 5.7 Hz, 4 H, 2 x CH20), 0.86 (s, 18 H, 2 x SiC(CH3)3), 0.01 (s, 12 H, 2 x Si(CH3)2); 13C NMR 5 153.0, 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) calc. for C22H42N204Si2 (M+) m/z 454.2683, found 454.2668; Anal. (C22H42N204Si2) C, H, N.
J/V1^V1-Bis(2-{[^er?-butyl(dimethyI)siIyl]oxy}ethyl)-l,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 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 15 directly without further purification or characterization. (l-Methyl-2-nitro-l//-imidazol-5-yI)methyl 4-[bis(2-{[tert- butyl(dimethyl)silyl]oxy}ethyI)amino]phenylcarbamate (121). A solution of carbonate 116 (0.87 g, 2.68 mmol), 120 (3.39 mmol), and pyridine (217 yuL, 2.68 mmol) in THF (50 20 mL) was stirred at 20 °C 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 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 °C; IRN 3258,1721,1539,1257, 1103 cm"1; 'H NMR [(CD3)2SO] 5 7.23 (s, 1 H, H 4'), 7.15 (br d,J= 8.9 Hz, 2 H, H 3, H 5), 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, CH20), 4.06 (s, 3 H, NCH3), 3.73 (dd, 7 = 6.5, 6.3 Hz, 4 H, 2 x CH20), 3.47 (dd, J= 6.5, 6.3 Hz, 4 H, 2 x CH2N), 0.88 (s, 18 H, 2 x Si(C(CH3)3), 0.02 (s, 12 H, 2 x Si(CH3)2); 13C NMR [(CD3)2SO] 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, 30 26.0 (6), 18.2 (2), -5.4 (4); Anal. (C28H49N506Si2) C, H, N. (l-Methyl-2-nitro-l//-imidazol-5-yI)methyI 4-[bis(2- hydroxyethyI)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 °C. The solution was stirred for 30 minutes and the solvent evaporated. The residue was purified by chromatography, eluting with a gradient (0-10%) of MeOH/EtOAc, to give 122 5 (0.59 g, 88%) as a white solid, mp (MeOH) 171-174 °C; IRN 3445, 3329, 3266, 1717, 1609, 1549, 1491, 1375, 1248 cm '; 'H NMR [(CD3)2SO] 6 9.37 (s, 1 H, OCONH), 7.28 (s, 1 H, H 4'), 7.19 (brd,J= 9.1 Hz, 2 H, H 3, H 5), 6.61 (d, .7=9.1 Hz, 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, NCH3), 3.51 (dd, J= 6.4, 5.9 Hz, 4 H, 2 x CH20), 3.34 (dd, J = 6.2, 5.9 Hz, 4 H, 2 x CH2N); 13C NMR [(CD3)2SO] 5 10 152.8, 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 C16H22N506 (MH+) 380.1570, found 380.1579; Anal. (C16H21N506) C, H, N. (l-Methyl-2-nitro-l//-imidazol-5-yl)methyl 4-[bis(2-15 chloroethyl)amino]phenylcarbamate (123). Methanesulfonyl chloride (191 fuL, 2.47 mmol) was added dropwise to a stirred solution of diol 122 (312 mg, 0.82 mmol) in pyridine (10 mL) at 5 °C and the solution stirred at 20 °C for 1 h. 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, 20 and the solvent evaporated. The residue was dissolved in DMF (10 mL), LiCl (210 mg, 4.9 mmol) added and the mixture stirred at 80 °C 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% EtOAc/light 25 petroleum, to give 123 (227 mg, 66%) as a white solid, mp (MeOH) 156-157 °C; IRN 3408, 3246, 1725, 1531, 1354, 1221 cm '; 'H NMR [(CD3)2SO] 5 9.49 (s, 1 H, OCONH), 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, CH20), 3.96 (s, 3 H, NCH3), 3.64-3.70 (m, 8 H, 2 x CH2N, 2 x CH2C1); ,3C NMR [(CD3)2SO] 5 152.8, 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. 30 Anal. (CI6H,9C12N504) C, H, N, CI.
Example 1C. Preparartion of (l-methyl-2-nitro-l//-imidazol-5-yl)methyl 1- 51 (chIoromethyl)-3-[(5,6,7-trimethoxy-li/-indol-l-yl)carbonyI]-2,3-dihydro-l//-benzo[e]indol-5-ylcarbamate (124). A solution of triphosgene (14.5 mg, 49 /umol) 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 ^mol) and 5 Et3N (39 mL, 280 jumol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of alcohol 115 ( 24 mg, 154 ^mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C 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 °C; 'H NMR 8 9.49 (br s, 1 H, indole-10 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,H4'),5.31 (d,J= 13.6 Hz, 1 H, CH20), 5.25 (d,J= 13.6 Hz, 1 H, CH20), 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, OCH3), 4.01 (br s, 3 H, NCH,), 3.94-3.98 (m, 4 H, CH2C1, 15 OCH3), 3.92 (s, 3 H, OCH3), 3.47 (dd, J- 10.8, 10.8 Hz, 1 H, CH2C1); ,3C NMR 5 160.4 (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 (C 3'), 97.6 (C 4'), 61.5 (OCH3), 61.2 (OCH3), 56.3 (OCH3), 55.8 (CH20), 54.9 (C 2), 45.8 20 (CH2C1), 43.4 (C 1), 34.3 (NCH3); MS (FAB+) m/z 649 (MH+, 2%); HRMS (FAB+) calc. for C31H3035ClN6O8 (MH+) m/z 649.1814, found 649.1767; calc. for C31H3037ClN6O8 (MH+) m/z 651.1784, found 651.1819; Anal (C3IH29C1N608.'/2H20) C, H, N.
Alternative preparation of (l-methyl-2-nitro-l//-imidazol-5-yl)methyl 1-25 (chIoromethyl)-3-[(5,6,7-trimethoxy-lJfiT-indoI-l-yI)carbonyl]-2,3-dihydro-l//-benzo[e]indol-5-y!carbamate (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) and pyridine (9 juL, 0.11 mmol) in DCM (2 mL) at 20 °C. The mixture was stirred at 20 °C for 2 h, the solvent evaporated and the residue dissolved in THF (5 mL). A solution of 30 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 °C for 16 h. The mixture was partitioned between EtOAc (50 mL) and sat. aq. KHC03 solution, 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.
Example ID. Preparation of (l-methyl^-nitro-l/^-imidazoI-S-yOmethyl doxorubicin carbamate (125). A solution of (l-methyl-2-nitro-l//-imidazol-5-yl)methyl 4-nitrophenyl carbonate (116) (33 mg, 104 yumol) in DMF (2 mL) was added dropwise to a stirred 10 solution of doxorubicin (13) (46 mg, 86 //mol) and Et3N (15 jj.L, 104 //mol) in DMF (5 mL) at 20 °C 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 °C; 'H NMR [(CD3)2SO] 5 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, 15 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 = 2.6 Hz, 1 H,H 1'), 5.07 (s, 2 H,CH20), 4.86-4.91 (m, 2 H, H 7, 14-OH), 4.73 (d,J= 5.9 Hz, 1H, 4-OH), 4.58 (d, J = 5.9 Hz, 2 H, H 14), 4.13-4.17 (m, 1 H,H5'),3.96 (s,3H,4-OCH3), 3.88 (s, 3 H, NCH3), 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), 20 2.17 (dd, J= 14.0, 5.4 Hz, 1 H, H 8), 1.84 (dt, J= 12.8, 3.5 Hz, 1 H,H2'), 1.57(dd,J = 12.8, 3.8 Hz, 1 H, H 2'), 1.12 (d, J= 6.4 Hz, 3 H, H 6');13C NMR [(CD3)2SO] 5 213.7 (C 13), 186.3 (C 5), 186.2 (C 12), 160.7 (C4), 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"), 119.8 (C 4a), 119.6 (C 1), 118.9 (C 3), 110.6 (C 5a), 110.5 (C 11a), 100.3 (C 1'), 74.9 (C 25 9), 69.8 (C 7), 67.8 (C 4'), 66.6 (C 5'), 63.7 (C 14), 56.5 (4-OCH3), 54.7 (CH20), 47.3 (C 3'), 38.4 (C 8), 34.1 (NCH3), 32.0 (C 10), 29.7 (C 2'), 17.0 (C 6'); MS (FAB+) m/z 727 (MH+, 0.2%); HRMS (FAB+) calc. for C33H34N4015 (MH+) m/z 727.2099, found 727.2075; Anal (C33H34N4015.'/2H20) C, H, N.
Example IE. Preparation of 4-({[(l-methyl-2-nitro-l//-imidazol-5-yl)methoxy]carbonyl}amino)benzyl doxorubicin carbamate (129). (l-Methyl-2-nitro-l//-imidazol-5-yl)methyl 4-{{\tert- i ""1 v, l ■"* ^ 1 ar# ^ -33 butyl(dimethyl)silyl]oxy}methyl)phenylcarbamate (126). Et3N (0.26 mL, 1.86 mmol) was added to a stirred suspension of (l-methyl-2-nitro-l//-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 5 mmol), and 4 A molecular sieves (500 mg) in THF (80 mL) and the mixture stirred at 20 °C 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 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%) 10 as a white solid, mp (EtOAc/light petroleum) 131-132 °C; 'H NMR 5 7.33 (brd, 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, OCONH), 5.22 (s, 2 H, CH20), 4.69 (s, 2 H, CH20), 4.05 (s, 3 H, NCH3), 0.93 (s, 9 H, SiC(CH3)3), 0.09 (s, 6 H, Si(CH3)2); ,3C NMR 5 152.3 (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), 15 55.4 (CH20), 34.3 (NCH3), 25.9 (SiC(CH3)3), 18.4 (SiC(CH3)3), -5.3 (Si(CH3)2); Anal. (C19H28N405Si) C, H, N. (1 -Methyl-2-nitro-l//-imidazol-5-yl)methyI 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 20 mmol) in MeOH (10 mL) and stirred at 20 °C 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 with a gradient (50-100%) EtOAc/light petroleum, to give 127 (247 mg, 87%) as a pale yellow solid, mp (EtOAc) 180-181 °C; 'H NMR [(CD3)2SO] 5 25 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, CH20), 3.97 (s, 3 H, NCH3); 13C NMR [(CD3)2SO] 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 (CH20), 55.0 (CH20), 34.2 (NCH3); Anal. (Cl3H14N405) C, H, N. 4-({[(l-methyl-2-nitro-l//-imidazol-5-yI)methoxy]carbonyl}amino)benzyI 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 °C for 96 h. The 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 50% EtOAc/light petroleum, to give 128 (62 mg, 18%) as a white solid; 'H NMR [(CD3)2SO] 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 H, H 2", H 6"), 7.51 (d, J = 8.6 Hz, 2 H, H 3, H 5), 7.40 (d, J= 8.6 Hz, 2 H, H 2, H 6), 7.31 (s, 1 H, H 4'), 5.33 (s, 2 H, CH20), 5.24 (s, 2 H, CH20), 3.98 (s, 3 H, NCH3); 13C NMR 10 [(CD3)2SO] 5 155.2 (0C02), 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 (CH20), 34.2 (NCH3); MS (FAB+) m/z 472 (MH+,1%), 443 (0.5); HRMS (FAB+) calc. for C20HlgN5O9 (MH+) m/z 472.1105, found 472.1106. 4-({[(l-Methyl-2-nitro-lJHr-imidazol-5-yl)methoxy]carbonyl}amino)benzyl doxorubicin carbamate (129). A solution of carbonate 128 (81 mg, 172 //mol) in DMF (2 mL) was added dropwise to a stirred solution of doxorubicin (45 mg, 86 //mol) and Et3N (15 //L, 103 //mol) in DMF (5 mL) at 20 °C and the solution stirred for 24 h. The solvent was 20 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 °C; 'H NMR [(CD3)2SO] 5 13.99 (s, 1 H, 6-OH), 13.24 (s, 1 H, 11-OH), 9.82 (s, 1 H, OCONH), 7.84-7.89 (m, 2 H, H 1, H 2), 7.60-7.63 (m, 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, CH20), 5.21 (d, J= 2.9 Hz, 1 H, H 1'), 4.89-4.91 (m, 1 H, 9-OH), 4.87 (s, 2 H, CH20), 4.84 (dd, J =6.3, 5.8 Hz, 1 H, 14-OH), 4.69 (d,J=5.7Hz, 1 H, 4-OH), 4.58 (d,J= 6.0 Hz, 2 H, H 14), 4.13-4.17 (m, 1 H, H 5'), 3.97 (s, 3 H, OCH3), 3.95 (s, 3 H, NCH3), 3.68-3.75 (m, 1 H, H 3'), 3.43-3.46 (m, 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, 30 H 8), 1.84(dt,y= 12.9, 3.6 Hz, 1 H,H2'), 1.47 (dd, J= 12.9, 3.8 Hz, 1 H, H 2'), 1.13 (d, J = 6.5 Hz, 3H,H6'); ,3CNMR [(CD3)2SO] 5 213.7 (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 (C4a), 119.6 (C 1), 118.9 (C 3), 118.0 (C3",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 (CH20), 56.5 (OCH3), 55.1 (CH20), 47.0 (C 3'), 36.5 (C 8), 34.1 5 (NCH3), 32.0 (C 10), 29.8 (C 2'), 16.9 (C 6'); MS (FAB+) m/z 876 (MH+, 0.2%); Anal. (C41H41N50I7.H20)C,H,N.
Example IF. Preparation of (l-methyM-nitro-l/Z-imidazoI-S-ytymethyl 1-(chloromethyI)-3-[(5,6,7-trimethoxy-l//-indoI-l-yl)carbonyI]-2,3-dihydro-l/f-10 benzo[e]indoI-5-y!carbamate (132). (l-MethyI-4-nitro-l/f-imidazol-5-yl)methanol (131). Ozone was bubled through a solution of l-methyl-4-nitro-5-[(£}-2-phenylethenyl]-l//-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) in DCM/MeOH (1:1, 120 mL) at -78 °C until a blue colour persisted. The solution was 15 warmed to -40 °C with a nitrogen purge to remove excess ozone. A solution of NaBH4 (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 was evaporated and the residue triturated with hot acetone (60 mL). The mixture was 20 filtered and the solution concentrated to give 131 (523 mg, 78%) as a tan powder, mp (acetone) 135-137 °C; 'H NMR 5 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, CH20), 3.75 (s, 3 H, NCH3); I3C NMR 5 143.5 (C 4), 136.8 (C 2), 133.2 (C 5), 51.4 (CH20), 32.5 (NCH3); Anal. (C5H7N303) C, H, N. (l-Methyl-4-nitro-l//-imidazol-5-yl)methyl l-(chloromethyl)-3-|(5,6,7-trimethoxy-l/7-indoI-l-yl)carbonyl]-2,3-dihydro-l/7-benzo[e]indol-5-ylcarbamate (132). A solution of triphosgene (13 mg, 43 //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] (51 mg, 109 /imol) and Et3N (34 /uL, 246 //mol) in DCM (10 mL) and stirred at 20 30 °C for 2 h. A solution of (l-methyl-4-nitro-l//-imidazol-5-yl)methanol 131 (23 mg, 147 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C for 24 h. The solvent was evaporated and the residue purified by ^ r r ; p- h !h 51 $ v J chromatography, eluting with EtOAc, to give 132 (40 mg, 56%) as a white powder, mp (EtOAc/light petroleum) 229-231 °C; '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,H9),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 5 (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, OCH3, CH2C1), 3.95 (s, 3 H, OCH3), 3.92 (s, 3 H, OCH3), 3.83 (br s,3H,NCH3), 3.44 (dd,J= 10.9, 10.7 Hz, 1 H, CH2C1); 13C 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"), 10 133.4 (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 (OCH3), 61.1 (OCH3), 56.3 (OCH3), 54.9 (C 2), 54.4 (CH20), 45.8 (CH2C1), 43.4 (C 1), 33.2 (NCH3); MS (FAB+) m/z 651 (MH+, 1%), 649 (MH+, 2); HRMS (FAB+) calc. for C31H3035ClN6O8 (MH+) m/z 649.1814, found 649.1818; calc. for 15 C3iH3037C1N6O8 (MH+) m/z 651.1784, found 651.1805; Anal. (C31H29C1N608) C, H, N.
Example 1G. Preparation of (l-methyl-5-nitro-l/7-imidazol-2-yI)methyl bis(2-ch!oroethyl)carbamate (135). (l-Methyl-5-nitro-l//-imidazoI-2-yl)methanoI (133). A mixture of l-methyl-5-nitro-l//-20 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 °C, EtOH (50 mL) was added, and the suspension was filtered. The filtrate was concentrated and the residue was purified by chromatography on alumina, eluting with a gradient of MeOH/CHCl3 (0-10%), to give 25 133 (0.71 g, 57%) as a white solid, mp (CHC13) 116-118 °C (lit. [C. Rufer, H. J. Kessler, E. Schroder. J. Med. Chem. 1971,14, 94] mp 111 °C); 'H NMR [(CD3)2SO] 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, CH20), 3.99 (s, 3 H, NCH3); ,3C NMR [(CD3)2S0] 8 151.8, 138.8, 130.9, 56.1, 32.9. (l-Methyl-5-nitro-l//-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 515 0 A 4 THF (50 mL) at 20 °C under N2. The mixture was stirred at 20 °C for 16 h, then partitioned between Et0Ac/H20. The organic fraction was washed with saturated aqueous NaHC03 (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 [(CD3)2SO] 5 8.33 (ddd, J= 9.1, 3.2, 2.2 5 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 (s, 2 H, CH20), 3.97 (s, 3 H, NCH3); ,3C [(CD3)2SO] 8 155.0, 151.3, 145.9, 145.2, 139.5, 131.6, 125.3 (2), 122.4 (2), 61.7, 33.5. (l-Methyl-5-nitro-l//-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 solution of N,jV-bis-(2-chloroethyl)amine hydrochloride (1.5 g, 8.4 mmol) in pyridine (30 mL) at 0 °C. The solution was stirred at 20 °C 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 15 chromatography, eluting with 50% EtOAc/light petroleum, to give 135 (2.0 g, 95%) as an oil; 'H NMR 8 7.99 (s, 1 H, H 4') 5.27 (s, 2 H, CH20), 4.03 (s, 3 H, NCH3), 3.60-3.71 (m, 8 H, 2 x CH2N, 2 x CH2C1); 13C NMR 8 154.8,146.9,139.6,132.2, 58.7, 51.1 (2), 41.8 (2), 39.7; HRMS calc. for C10H14Cl2N4O4(M+) m/z 324.1392; found 324.1381.
Example 1H. Preparation of (l-methyI-5-nitro-l//-imidazol-2-yl)methyl 4-[bis(2-chloroethyl)amino]pheny!carbamate (137). Diphosgene (85mL, 0.7 mmol) was added dropwise to a stirred solution of (l-methyl-5-nitro-l//-imidazol-2-yl)methanol (133) (0.2 g, I.27 mmol) and Et3N (98 mL, 0.7 mmol) in THF (10 mL) at 5 °C. The suspension was stirred at 5 °C for 30 min and a mixture of/Vl,vV'-bis(2-chloroethyl)-l,4-benzenediamine hydrochloride (136) (J. L. Everett, W. C. J. Ross. J. Chem. Soc. 1949, 1972] (0.38 g, 1.40 mmol) and Et3N (195 mL, 1.40 mmol) in THF (4 mL) was added dropwise to the above suspension. The mixture was stirred at 20 °C for 4 h, the solvent evaporated and the residue purified by chromatography, eluting with 50% EtOAc/light petroleum, to give 137 (0.19 g, 36%), mp (CHCl3/pet. ether) 164-164.5 °C; 1R n 3250, 3185, 3127, 1723, 1603, 1547, 30 1516, 1381 cm1; 'H NMR ([(CD3)2SO] 5 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, CH20), 3.95 (s, 3 H, NCH3), 3.65-3.72 (m, 8 H, 2 x CH2N, 2 x CH2C1); ,3C NMR [(CD3)2SO] 8 152.7, 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; MS (DEI) m/z 415 (M+, 1%), 366 (2), 316 (2), 258 (20), 211 (30), 209 (100); HRMS (DEI) m/z calc. for C16H1935C12N504 (M+) 415.0814, found 415.0808; calc. for C,6H!935C137C1N504 (M+) 417.0785, found 417.0781; calc. for CI6HI937C12N504 (M+) 419.0755, found 419.0769; 5 Anal. (CI6H19C12N504) C, H, N.
Example II. Preparation of (l-methyI-5-nitro-l//-imidazoI-2-yI)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indoI-2-yl)carbonyl]-2,3-dihydro-l//-benzo[e]indoI-5-yIcarbamate (138). A solution of triphosgene (12 mg, 41 //mol) in DCM 10 (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 //mol) and Et3N (32 //L, 231 (Umol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of (l-methyl-5-nitro-l//-imidazol-2-yl)methanol (133) (20 mg, 127 ,umol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C for 24 h. The solvent was 15 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 °C; 'H NMR 5 9.47 (s, 1 H, 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, 7= 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 20 (d, J= 13.4 Hz, 1 H, CH20), 5.34 (d, J = 13.4 Hz, 1 H, CH20), 4.79 (dd,7= 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, OCH3), 4.02 (br s, 3 H, NCH3), 3.95 (s, 3 H, OCH3), 3.92-3.94 (m, 4 H, OCH3, CH2C1), 3.45 (dd, J = 10.9,10.7 Hz, 1 H, CH2C1); 13C NMR 5 160.4 (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"), 25 129.7 (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'), 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 (OCH3), 61.2 (OCH3), 58.4 (CH20), 56.3 (OCH3), 54.9 (C 2), 45.8 (CH2C1), 43.4 (C 1), 33.8 (NCH3); MS (FAB+) m/z 649 (MH+, 3%), 651 (1.5); HRMS (FAB+) calc. for C31H3035C1N6O8(mH+) m/z 649.1814, found 649.1797; calc. for C3IH3037ClN6O8 (MH+) m/z 30 651.1784, found 651.1802; Anal. (C31H29C1N608) C, H, N.
Example 1 J. Preparation of 4-({[(l-methyl-5-nitro-l//-imidazol-2- yl)methoxy]carbonyl}amino)benzyI doxorubicin carbamate (142). 1-Methy I-5-nitro-lfMmidazol-2-yI 4-({ [tert- butyl(dimethyl)silyI]oxy}methyl)phenylcarbamate (139). Et3N (1.10 mL, 7.87 mmol) was added to a stirred suspension of (l-methyl-5-nitro-l//-imidazol-2-yl)methyl 4-5 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 °C 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 10 (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 °C; 'H NMR 8 7.99 (s, 1 H, H 4'), 7.32 (br d, J= 8.1 Hz, 2 H, H 3, H 5), 7.27 (d, J= 8.1 Hz, 2 H, H 2, H 6), 6.96 (br s, 1 H, OCONH), 5.30 (s, 2 H, CH20), 4.69 (s, 2 H, CH20), 4.05 (s, 3 H, NCH3), 0.93 (s, 9 H, 15 SiC(CH3)3), 0.09 (s, 6 H, Si(CH3)2); 13C NMR 8 152.3 (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 (CH20), 58.0 (CH20), 33.7 (NCH3), 25.9 (SiC(CH3)3), 18.4 (SiC(CH3)3), -5.3 (Si(CH3)2); Anal. (C19H28N405Si) C, H, N. (l-Methyl-5-nitro-l//-imidazol-2-yl)methyl 4-(hydroxymethyl)phenyIcarbamate (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 °C 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 crystallized 25 from EtOAc/light petroleum, to give 140 (0.86g, 47%) as a white solid, mp (EtOAc/light petroleum) 181-183 °C; 'H NMR [(CD3)2SO] 8 9.85 (br s, 1 H, OCONH), 8.09 (s, 1 H, H 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, CH20), 4.42 (s, 2 H, CH20), 3.96 (s, 3 H, NCH3), 3.79 (br s, 1 H, OH); 13C NMR [(CD3)2SO] 8 152.5 (OCONH), 147.8 (C 5'), 139.3 (C 2'), 137.4 (C 1), 136.8 (C 4), 131.7 30 (C 2, C 6), 127.0 (C 3, C 5), 118.0 (C 4), 62.4 (CH20), 57.5 (CH20), 33.4 (NCH3); Anal. (C13H!4N405) C, H, N. 515 C 4-({[(l-MethyI-5-nitro-li/-imidazol-2-yl)methoxy]carbonyl}amino)benzyI 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, 2.37 mmol) and Et3N (0.66 mL, 4.73 mmol) in THF (40 mL) and the solution stirred at 20 5 °C 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 [(CD3)2SO] 5 10.02 (s, 1 H, OCONH), 8.31 (ddd J= 9.1, 3.3, 2.1 Hz, 2 H, H 3, H 5), 8.08 (s, 1 H, H 4"), 7.57 (ddd, 7=9.1, 3.3, 2.1 Hz, 2 H, H 2, H 6), 7.51 (d, J= 8.5 Hz, 2 H, H 3', 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, CH20), 3.97 10 (s, 3 H, NCH3); MS (FAB+) m/z 472 (MH+,1.5%); HRMS (FAB+) calc. for C20H18N5O9 (MH+) m/z 472.1105, found 472.1108. 4-({[(l-Methyl-5-nitro-l//-imidazol-2-yl)methoxy]carbonyl}amino)benzyl doxorubicin carbamate (142). A solution of carbonate 141 (61 mg, 129 yumol) in DMF (2 mL) was 15 added dropwise to a stirred solution of doxorubicin (13) (45 mg, 86 yumol) and Et3N (18 /xL, 129 yumol) in DMF (5 mL) at 20 °C 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 °C; 'H NMR [(CD3)2SO] 8 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'"), 20 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, CH20), 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= 6.0 Hz, 2 H, H 14), 4.12-4.18 (m, 1 H, H 5'), 3.97 (s, 3 H, OCH3), 3.95 (s, 3 H, NCH3), 25 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,H2'), 1.47 (dd,J= 12.9,4.0 Hz, 1 H,H2'), 1.12 (d, J = 6.5 Hz, 3 H, H 6'); l3C NMR [(CD3)2SO] 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 30 (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", C 5"), 110.7 (C 5a), 110.5 (C 11a), 100.2 (CI'), 74.9 (C 9), 69.8 (C 7), 67.9 (C 4'), 66.6 (C '), 64.8 (C 14), 63.6 (CH20), 57.6 (CH20), 56.5 (OCH3), 47.0 (C 3'), 36.5 (C 8), 33.4 (NCHj), 32.0 (C 10), 29.7 (C 2'), 16.9 (C 6'); MS (FAB+) m/z 876 (MH\ 0.6%); HRMS (FAB+) calc. for C41H42N50,7(MH+) m/z 876.2576, found 876.2573; Anal. (C41H41N50I7.H20)C,H,N.
Example IK. Preparation of [l-(2-hydroxyethyl)-5-nitro-l//-imidazoI-2-yI]methyl 1-(chloromethyl)-3-|(5,6,7-trimethoxy-l//-indol-2-yl)carbonyl]-2,3-dihydro-l//-benzo[e)indol-5-ylcarbamate (148). 2-{5-Nitro-2-[(£')-2-phenylethenyl]-l//-imidazoI-l-yI}ethanol (144). A solution of Na 10 (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 °C. 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% 15 EtOAc/light petroleum, to give 144 (4.0 g, 26%) as a yellow powder, mp 155 °C (lit mp 156-157 °C [ W. J. Ross, W. B. Jamieson, and M. C. McCowen, J. Med. Chem. 1972,15, 1035-1039]); 'H NMR 5 8.06 (s, 1 H, H 4'), 7.83 (d, J= 15.8 Hz, 1 H, CH=), 7.52-7.58 (m, 2 H, H 2", H 6"), 7.33-7.38 (m, 3 H, H 3", H 4", H 5"), 7.05 (d, J= 15.8 Hz, 1 H, CH=), 4.64 (dd, 5.1, 5.0 Hz, 2 H, H 1), 4.07 (dd, 5.1, 5.0 Hz, 2 H, H 2), 2.42 (br s, 1 H, 20 OH); l3C 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). ter/-Butyl(dimethyl)siIyl 2-{5-nitro-2-[(£)-2-phenylethenyl]-l//-imidazol-l-yl}ethyl ether (145). TBDMS triflate (2.7 mL, 11.75 mmol) was added dropwise to a stirred 25 solution of alcohol 144 (2.77 g, 10.7 mmol) and pyridine (1.3 mL, 16.0 mmol) in DCM (100 mL) at -5 °C and the solution stirred at 20 °C for 16 h. The reaction was quenched with MeOH (5 mL) and poured in sat. aq. KHC03 (100 mL). The mixture was extracted with DCM (3 x 50 mL), the combined organic fraction dried and the solvent evaporated. The residue was purified by chromatography, eluting with 20% EtOAc/light petroleum, to 30 give 145 (4.00 g, 100%) as a yellow solid, mp (EtOAc/light petroleum) 99-100.5 °C; 'H NMR 5 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 (m, 3 H, H 3", H 4", H 5"), 7.06 (d,J= 15.8 Hz, 1 H, CH=), 4.62 (dd,J = n\ ii ;■/h i) y ^ H .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(CH3)3), 0.10 (s, 6 H, Si(CH3)2); l3C NMR 5 151.2 (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(CH3)3), 18.1 (SiC(CH3)3), -5.8 (Si(CH3)2); Anal. (C19H27N303Si) C, H, N. [l-(2-{[ter/-Butyl(dimethyl)silyl]oxy}ethyl)-5-nitro-l//-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 °C until a blue colour persisted. The solution was warmed to -40 °C with a N2 purge to remove excess ozone. A solution of NaBH4 (132 mg, 3.48 mmol) in 10 EtOH (10 mL) was added dropwise over 15 min and the mixture stirred for 30 min. The 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 15 petroleum, to give 146 (0.92 g, 88%) as a white solid, mp 104-105 °C; "H NMR 5 7.97 (s, 1 H, H 4), 4.79 (s, 2 H, CH20), 4.62 (t, J = 4.8 Hz, 2 H, CH20), 3.97 (t, J= 4.8 Hz, 2 H, CH2N), 3.80 (br s, 1 H, OH), 0.81 (s, 9 H, SiC(CH3)3), 0.10 (s, 6 H, Si(CH3)2); 13C NMR 5 157.2 (C 2), 138.8 (C 5), 132.3 (C 4), 62.0 (CH20), 57.2 (CH20), 48.3 (CH2N), 25.7 (SiC(CH3)3), 18.2 (SiC(CH3)3), -5.8 (Si(CH3)2); Anal. (Cl2H23N304Si) C, H, N. [l-(2-{[tert-Butyl(dimethyI)silyl]oxy}ethyI)-5-nitro-l//-imidazoI-2-yl]methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indoI-2-yl)carbonyl]-2,3-dihydro-l/f-benzo[e]indol-5-ylcarbamate (147). A solution of triphosgene (25 mg, 84 //mol) in DCM (3 mL) was added dropwise to a stirred solution of amine 1 [G. J. Atwell, W. R. Wilson, W. 25 A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (112 mg, 240 //mol) and Et3N (67 /uL, 481 Mmol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of alcohol 146 (78 mg, 264 //mol) in DCM (3 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C 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 30 oil, 'H NMR 5 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, 7=2.2 Hz, 1 H, H 3'), 5WQ 6.88 (s, 1 H, H 4'), 5.44 (d, 7 = 13.5 Hz, 1 H, CH20), 5.39 (d, 7 = 13.5 Hz, 1 H, CH20), 4.81 (dd, 7 = 10.7, 1.5 Hz, 1 H, H 2), 4.65-4.74 (in, 3 H, H 2, CH2N), 4.16-4.22 (m, 1 H, H 1), 4.10 (s, 3 H, OCH3), 3.95-3.99 (m, 4 H, CH2C1, OCH3), 3.89-3.93 (m, 5 H, OCH3, CH2N), 3.48 (t, 7 = 10.9 Hz, 1 H, CH2C1)), 0.81 (s, 9 H, SiC(CH3)3), -0.08 (s, 6 H, 5 Si(CH3)2); MS (FAB+) m/z 795 (MH+, 12%), 793 (25); HRMS (FAB+) calc. for C38H4635ClN609Si(MH+) m/z 793.2784, found 793.2762; calc. for C38H4637ClN609Si (MH+) m/z 795.2755, found 795.2751. [l-(2-hydroxyethyl)-5-nitro-l//-imidazol-2-yl]methyl l-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-2-yl)carbonyl]-2,3-dihydro-l//-benzo[e]indol-5-ylcarbamate (148). 1M HCI (0.23 mL, 230 //mol) was added to a stirred solution of silyl ether 147 (91 mg, 115 yumol) in MeOH (5 mL) and the solution stirred at 20 °C for 4 h. The solvent was evaporated and the residue partitioned between EtOAc (40 mL) and water (40 mL). The 15 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 (0-10%MeOH/40%EtOAc/DCM), to give 148 as a white solid, mp (EtOAc/light petroleum) 148-150 (dec.); 'H NMR 5 [(CD3)2SO] 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, 7 = 8.5 Hz, 1 H, H 6), 7.97 (d, 7= 8.3 Hz, 1 20 H, H 9), 7.58 (ddd, 7= 8.3, 7.2, 0.8 Hz, 1 H, H 8), 7.46 (ddd, 7= 8.5, 7.2, 0.8 Hz, 1 H, H 7), 7.09 (d, 7 = 2.0 Hz, 1 H, H 3'), 6.97 ( s, 1 H, H 4'), 5.37 (s, 2 H, CH20), 5.12 (t, 7= 5.4 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, CH20), 4.32-4.38 (m, 1 H,H 1), 4.06 (dd, 7= 11.1,3.2 Hz, 1 H, CH2C1), 3.91-3.95 (m,4H, OCH3, CH2C1), 3.83 (s, 3 H, OCH3), 3.81 (s, 3 H, OCH3), 3.70-3.75 (m, 2 H, CH2N); 13C NMR 5 25 [(CD3)2SO] 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'), 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 (OCH3), 60.9 (OCH3), 59.7 (CH20), 58.1 (CH20), 55.9 (OCH3), 54.9 (C 2), 48.2 (CH2N), 47.8 (CH2C1), 41.1 (C 1); MS 30 (FAB+) m/z 681 (MH+, 5%), 679 (MH+, 12%); HRMS (FAB+) calc. for C32H3235C1N609 (MH+) m/z 679.1919, found 679.1797; calc. for C32H3237C1N609 (MH+) m/z 681.1890, found 681.1892; Anal. (C32H31C1N609) C, H, N. il 44 Example 1L. Preparation of (l-methy]-5-nitro-l//-imidazol-4-yl)methyI 1-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-l-yl)carbonyl]-2,3-dihydro-l/f-benzo[<?]indol-5-yJcarbamate (150). A solution of triphosgene (21 mg, 70 //mol) in DCM 5 (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 //mol) and Et3N (55 //L, 400 //mol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of (l-methyl-5-nitro-l//-imidazol-4-yl)methanol 149 [ D. C. Baker, S.R. Putt, H. D. H. Showalter, 7. Heterocyclic Chem., 1983, 20, 629-634.] (37 mg, 240 //mol) in DCM (3 mL) was added, 10 followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C 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 °C; 'H NMR [(CD3)2SO] 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, 7 = 8.4 Hz, 1 H, H 9), 7.57 (ddd, J= 8.4, 7.2, 0.7 15 Hz, 1 H, H 8), 7.45 (ddd, 7 = 8.5, 7.2, 0.7 Hz, 1 H, H 7), 7.09 (d, 7 = 2.0 Hz, 1 H, H 3'), 6.97 ( s, 1 H, H 4'), 5.40 (s, 2 H, CH20), 4.79 (dd, 7= 10.8, 1.4 Hz, 1 H, H 2), 4.52 (dd, 7 = 11.0, 1.9 Hz, 1 H, H 2), 4.31-4.36 (m, 1 H,H 1), 4.07 (dd, 7= 11.1, 3.0 Hz, 1 H, CH2C1), 3.89-3.95 (m, 7 H, OCH3, CH2C1, NCH3), 3.83 (s, 3 H, OCH3), 3.81 (s, 3 H, OCH3); 13C NMR [(CD3)2SO] 8 160.1 (CO), 154.3 (OCONH), 154.2 (C 5"), 149.1 (C 5'), 141.4 (C 2"), 20 141.2 (C 3a), 139.9 (C 6'), 139.0 (C 7'), 135.1 (C4"), 134.4 (C 5), 130.8 (C 9a), 129.4 (C 2'), 127.0 (C 8), 125.5 (C 5a), 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 (OCH3), 60.9 (OCH3), 59.6 (CH20), 55.9 (OCH3), 54.8 (C 2), 47.5 (CH2C1), 41.1 (C 1), 35.1 (NCH3); MS (FAB+) m/z 651 (MH+, 2%), 649 (MH+, 8); HRMS (FAB+) calc. for C31H3035ClN6O8 (MH+) m/z 25 649.1814, found 649.1802; calc. for C31H3037ClN6O8 (MH+) m/z 651.1784, found 651.1761; Anal. (C31H29C1N608) C, H, N.
Example 2A. Preparation of (5-nitro-2-furyl)methyl 4-[bis(2-chloroethyI)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 solution of (5-nitrofuran-2-yl)methanol (151) [J. M. Berry, C. Y. Watson, W. J. D. Whish, 9 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 °C under N2. The mixture was stirred at 20 °C for 16 h, then partitioned between EtOAc (100 mL) and H20 (100 mL). The organic layer was washed with saturated aqueous NaHC03 (50 mL), dried, and the solvent 5 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; IRN 1775, 1526, 1352, 1215 cm1; 'HNMR [(CD3)2SO] 5 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, 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, CH20); ,3C [(CD3)2SO] 10 5 155.0, 154.1, 151.8, 151.5, 145.2, 125.4(2), 122.5(2), 115.1, 113.3, 61.5; Anal. (C12H8N208)C,H,N. (5-Nitro-2-furyl)methyI 4-[bis(2-hydroxyethyl)amino]phenyIcarbamate (153). A solution of carbonate 152 (1.00 g, 3.24 mmol), TV1 ,jV'-bis(2-hydroxyethyl)-1,4-15 benzenediamine (57) (3.24 mmol), and pyridine (260 /j.L, 3.24 mmol) in THF (80 mL) was stirred at 20 °C 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 [(CD3)2SO] 5 9.44 (s, 1 H, OCONH), 7.68 (d, J= 3.7 Hz, 1 H, H 4'), 7.20 (br d, J= 9.1 Hz, 2 H, H2, H 6), 6.93 (d, J= 3.7 Hz, 1 H, H 3'), 6.62 (d, J= 9.1 20 Hz, 2 H, H 3, H 5), 5.19 (s, 2 H, CHaO), 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 (m, 4 H, 2 x CH2N); 13C NMR [(CD3)2SO] 5 154.0,152.7, 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 C16Hl9N307 (M+) m/z 365.1223, found 365.1218. (5-Nitro-2-furyI)methyl 4-[bis(2-chloroethyI)amino]phenylcarbamate (154). Methane-sulphonyl chloride (460 //L, 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 °C and the solution stirred at 20 °C for 2 h. The solvent was evaporated and the residue partitioned between DCM (100 mL) and water 30 (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), LiCl (0.51 g, 12.0 mmol) added and the mixture stirred at 80 °C for 3 h. The solvent was 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, CH20), 3.63-3.72 (m, 8 H, 2 x CH2N, 2 x CH2C1); I3CNMR (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 C16H1735C12N305(M+) m/z 10 401.0545, found 401.0546; calc. for C16HI735C137C1N305(M+) m/z 403.0516, found 403:0521; calc. for C16H1737C12N305 (M+) m/z 405.0486, found 405.0498.
Example 2B. Preparation of (5-nitro-2-furyl)methyl l-(chIoromethyI)-3-[(5,6,7-trimethoxy-l//-indoI-l-yl)carbonyI]-2,3-dihydro-l//-benzo[e]indoI-5-ylcarbamate 15 (155). A solution of triphosgene (14 mg, 48 //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] (57 mg, 122 //mol) and Et3N (38 //L, 275 //mol) in DCM (10 mL) and stirred at 20 °C 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 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 ( 2 drops) and the solution stirred at 20 °C 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 °C; '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, 8.5 Hz, 1 H, H 6), 7.98 25 (d, J= 8.3 Hz, 1 H, H 9), 7.72 (d, 7= 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,H7),7.09 (d,J=2.1 Hz, 1 H,H3'), 6.98-7.00 (m, 2 H, H 4', H 3"), 5.30 (s, 2 H, CH20), 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, CH2C1), 3.91-3.96 (m, 4 H, OCH3, CH2C1), 3.83 (s, 3 H, OCH3), 3.80 (s, 3 H, OCH3); ,3C 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 54 ^ a m S ;) 0 3"), 113.6 (C 4"), 113.1 (C 4), 106.2 (C 3'), 98.0 (C 4'), 61.0 (OCH3), 60.9 (OCH3), 57.7 (CH20), 55.9 (OCH3), 54.8 (C 2), 47.5 (CH2C1), 41.1 (C 1); MS (FAB+) m/z 635 (MH+, 6%), 637 (MH+, 3); HRMS (FAB+) calcd for C31H2835C1N409 (MH+) m/z 635.1545, found 635.1552; calcd for C31H2837C1N409 (MH+) m/z 637.1515, found 637.1514; Anal.
(C3IH27C1N409) Example 3A. Preparation of (5-nitro-2-thienyI)methyl 4-[bis(2-chloroethyl)amino]phenyIcarbamate (159). 4-Nitrophenyl (5-nitro-2-thienyl)methyl carbonate (157). 4-Nitrophenyl chloroformate 10 (2.58 g, 12.8 mmol) in dry THF (20 mL) was added slowly to a stirred solution of (5-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 °C under N2. The mixture was stirred at 20 °C for 16 h, then partitioned between EtOAc (100 mL) and H20 (100 mL). The organic layer was washed with saturated aqueous NaHC03 15 (50 mL), and the solvent evaporated to give 157 (1.86g, 49%), mp (EtOAc/light petroleum) 121-122 °C; IRN 1763, 1522, 1345, 1231 cm1; 'HNMR [(CD3)2SO] 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); 13C NMR [(CD3)2SO] 8 155.0, 151.6, 148.2, 145.2, 144.8, 129.5, 129.0, 125.4(2), 122.6(2), 64.4; Anal.
(C12H8N207S) C, H, N. (5-Nitro-2-thienyI)methyl 4-[bis(2-hydroxyethyI)amino]phenylcarbamate (158). A solution of 157 (0.75 g, 2.3 mmol), A^IJV'-bis(2-hydroxyethyl)-l,4-benzenediamine (57) (2.5 mmol), and pyridine (206 /uL, 2.5 mmol) in THF (50 mL) was stirred at 20 °C for 16 25 h. The solvent was evaporated and the residue purified by chromatography, eluting with EtOAc to give 158 (0.56 g, 64%), mp (EtOAc/light petroleum) 139-140.5 °C; IR N 3360, 3208, 1730, 1530, 1337, 1215 cm1; 'H NMR [(CD3)2SO] 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 31), 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, CH20), 4.72 (t, J= 5.5 Hz, 2 H, 2 * OH), 30 3.49-3.56 (m, 4 H, 2 x CH20), 3.36 (t, J = 6.2 Hz, 4 H, 2 x CH2N); I3C NMR [(CD3)2SO] 5 153.0,150.8, 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,6H]9N306S) C, H; N, calc 11.0, found 10.5%. (5-Nitro-2-thienvl)methyI 4-(bis(2-chloroethyl)aminojphenylcarbamate (159).
Methane-sulphonyl chloride (260 /iL, 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 °C 5 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 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 °C for 3 h. The solvent was evaporated and the residue partitioned between EtOAc (100 mL) and water (100 mL). 10 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 159 (0.35 g, 69%) as pale green needles, mp (EtOAc/light petroleum) 99-100 °C; IRN 3353, 1723, 1547, 1530, 1339, 1219 cm1; 'H NMR [(CD3)2SO] 5 9.58 (br s, 1 H, OCONH), 8.04 (d, J = 4.2 Hz, 1 H, H 4'), 7.28-7.30 (m, 3 H, 15 H 3', H 3, H 5), 6.71 (d, J= 9.1 Hz, 2 H, H 2, H 6), 5.34 (s, 2 H, CH20), 3.65-3.72 (m, 8 H, 2 x CH2N, 2 x CH2C1); 13C NMR [(CD3)2SO] 8 153.0, 150.8, 148.2, 142.4, 129.5, 128.6, 127.5, 120.4 (2), 112.3 (2), 60.1, 52.2 (2), 41.1 (2); Anal. (CI6H19C12N504) C, H, N, CI.
Example 3B. Preparation of (5-nitro-2-thienyI)methyl l-(chloromethyl)-3-[(5,6,7-20 trimethoxy-l//-indol-l-yI)carbonyl]-2,3-dihydro-l/7-benzo[e]indol-5-ylcarbamate (160). A solution of triphosgene (15 mg, 51 /umol) 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] (61 mg, 131 ptmol) and Et3N (41 ptL, 294 ju,mol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of (5-nitro-2-thienyl)methanol (156) [P. J. 25 Narcombe, R. K. Norris. Aust. J. Chem. 1979, 32, 2647] (28 mg, 176 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C for 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 °C; 'H NMR [(CD3)2SO] 8 11.48 (br s, 1 H, indole-NH), 9.34 (br s, 1 H, 30 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 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, CH20), 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 (m, 1 H, H 1), 4.07 (dd, J = 11.0, 3.0 Hz, 1 H, CH2C1), 3.92-3.96 (m, 4 H, CH2C1, OCH3), 3.92 (m, 3 H, OCH3), 3.81 (s, 3 H, OCH3); l3C NMR f(CD3)2SO] 5 160.3 (CO), 154.4 (OCONH), 151.1 (C 5"), 149.3 (C 5'), 148.2 (C 2"), 141.6 (C 3a), 140.0 (C 5 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 (C4), 106.4 (C 3'), 98.1 (C 4'), 61.2 (OCH3), 61.0 (OCH3), 60.8 (CH20), 56.0 (OCH3), 55.0 (C 2), 47.7 (CH2C1), 41.2 (C 1); MS (FAB+) m/z 653 (MH+, 4%), 651 (MH+, 8); HRMS (FAB+) calc. for C31H2835ClN4OgS (MH+) m/z 651.1316, found 10 651.1311; calc. for C31H2837ClN4OgS (MH+) m/z 653.1287, found 653.1307; Anal. (C31H27C1N408S)C,H,N.
Example 4. Preparation of (l-methyl-5-nitro-l/f-pyrazol-4-yl)methyI 1-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indoI-l-yl)carbonyl]-2,3-dihydro-l//-15 benzo[e]indol-5-ylcarbamate (163). (l-methyl-5-nitro-1//-pyrazol-4-yl)methanol (162). Borane dimethylsulfide (2 M solution in THF, 4.2 mL, 8.4 mmol) was added to a solution of l-methyl-5-nitro-l//-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 N2, and the mixture stirred at reflux temperature 20 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 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 °C. 'H NMR 5 7.58 (s, 1 H, H 3), 25 4.82 (d, 3.4 Hz, 2 H, CH20), 4.25 (s, 3 H, NCH3), 2.39 (br s, 1 H, OH); Anal. (C5H7N303) C, H, N. (l-Methyl-5-nitro-l#-pyrazol-4-yI)methyl l-(chloromethyl)-3-((5,6,7-trimethoxy-l//-indol-l-yl)carbonyl]-2,3-dihydro-l//-benzo[e]indol-5-ylcarbamate (163). A solution of 30 triphosgene (14.3 mg, 48 //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] (57 mg, 122 //mol) and Et3N (38 //L, 275 //mol) in DCM (10 mL) and stirred at 20 & ^ 'l 4 i 'fj y l£ Hfr °C for 2 h. A solution of alcohol 162 (26 mg, 165 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C 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) 201-5 202 °C; 'H NMR [(CD3)2SO] 5 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= 1.6Hz, 1 H,H 3'), 6.98 (s, 1 H,H4'),5.33 (s,2H, CH20), 4.80 (dd, J= 11.0, 9.4 Hz, 1 H, H 2), 4.53 (dd, J= 11.0, 1.8 Hz, 1 H,H2), 4.32-4.38 (m, 1 H,H 1), 4.17 (s, 3 H, NCH3), 4.07 (dd, J= 11.0, 3.1 Hz, 1 H, CH2C1), 3.91-3.96 (m, 4 H, OCH3, CH2C1), 3.82 (s, 3 H, OCH3), 3.80 (s, 3 H, OCH3); 13C NMR [(CD3)2SO] 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), 123.3 (C 6), 123.1 (C 3a'), 122.0 (C 9b), 117.4 (C 4"), 113.0 (C 4), 106.2 (C3'), 98.0 (C 15 4'), 61.0 (OCH3), 60.9 (OCH3), 57.2 (CH20), 55.9 (OCH3), 54.8 (C 2), 47.5 (CH2C1), 41.1 (C 1), 40.8 (NCH3); MS (FAB+) m/z 650 (MH+, 2%), 648 (MH+, 5); HRMS (FAB+) calc. for C31H3035ClN6O8 (MH+) m/z 649.1814, found 649.1803; calc. for C3IH3037ClN6O8 (MH+) m/z 651.1784, found 651.1796; Anal. (C31H29C1N608) C, H, N.
Example 5A. Preparation of ethyl 4-({[({l-(chIoromethyl)-3-[(5,6,7-trimethoxy-lfl-indol-l-yl)carbonyl]-2,3-dihydro-l//-benzo[e]indol-5-yl}amino)carbonyl]oxy}methyl)-l-methyl-5-nitro-l//-pyrrole-2-carboxylate (167).
Ethyl 4-formyl-l-methyl-5-nitro-l//-pyrrole-2-carboxylate (165). Dimethyl sulfate (0.31 mL, 3.2 mmol) was added to a mixture of ethyl 4-formyl-5-nitro-l//-pyrrole-2-25 carboxylate (164) [P. Fornari, M. Farnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-291] (0.57 g, 2.7 mmol) and K2C03 (0.56 g, 4.0 mmol) in DMSO (4 mL) and the brown suspension was stirred at 20 °C for 1 h. The mixture was diluted with water (50 mL), acidified with HCI (2 N), and extracted with EtOAc (2 x 50 mL). The combined extract was dried, the solvent evaporated. The residue was chromatograped, eluting with 30 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, CH2), 4.33 (s, 3 H, NCH3), 1.39 (t, J= 7.1 Hz, 3 H, CH3); Anal. (c9h10n2o5) c, h, n.
Ethyl 4-(hydroxymethyI)-l-methyl-5-nitro-l#-pyrrole-2-carboxylate (166). NaBH4 (0.33 g, 8.7 mmol) was added in portions to a solution of aldehyde 165 (3.96 g, 17.5 mmol) 5 in EtOH (100 mL) and the mixture was stirred at 20 °C for 20 min. Water (5 mL) was 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 10 s, 2 H, CH20), 4.36 (q, J= 7.1 Hz, 2 H, CH2), 4.31 (s, 3 H, NCH3), 2.49 (br s, 1 H, OH), 1.38 (t, J= 7.1 Hz, 3 H, CH3); Anal. (C9H12N205) C, H, N. The mother liquor was evaporated and purified by chromatography 10% EtOAc/light petroleum to give more 166 (1.46 g, 37%).
Ethyl 4-({[({l-(chloromethyl)-3-[(5,6,7-trimethoxy-l#-indol-l-yl)carbonyl]-2,3-dihydro-l//-benzo[e]indol-5-yl}amino)carbonyl]oxy}inethyl)-l-methyl-5-nitro-l/7-pyrrole-2-carboxylate (167). A solution of triphosgene (13.8 mg, 46 //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] (55 mg, 118 //mol) and Et3N (37 //L, 20 265 //mol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of ethyl 4- (hydroxymethyl)-l-methyl-5-nitro-l//-pyrrole-2-carboxylate (166) (36 mg, 159 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C 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 25 (EtOAc/light petroleum) 248-250 °C; 'H NMR [(CD3)2SO] 5 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, 7=2.0 Hz, 1 H,H3"'), 7.06 (brs, 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"), 4.35-30 4.40 (m, 1 H, H 1"), 4.29 (q, J= 7.1 Hz, 2 H, H 1'), 4.19 (s, 3 H, NCH3), 4.07 (dd, J= 11.0, 3.0 Hz, 1 H, CH2C1), 3.92-3.96 (m, 4 H, CH2C1, OCH3), 3.83 (m, 3 H, OCH3), 3.81 (s, 3 H, OCH3), 1.31 (t, J = 7.1 Hz, 3 H, H 2'); ,3CNMR [(CD3)2SO] 5 160.2 (CO), 159.4 (C02), 154.3 (OCONH), 149.1 (C5'"), 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 (C2'"), 127.1 (C 8"), 125.6 (C 5a"), 125.5 (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 (OCH3), 60.9 (OCHj), 59.6 (CH20), 55.9 (OCH3), 54.9 (C 2"), 47.5 (CH2C1), 41.1 (C 1"), 35.2 (NCH3), 14.0 (C 2'); MS (FAB+) m/z 722 (MH+, 0.3%), 720 (MH+, 0.6); HRMS (FAB+) calc. for C35H3535ClN5O10 (MH+) m/z 720.2073, found 720.2059; calc. for C35H3537ClN5Ol0 (MH+) m/z 722.2043, found 722.2031; Anal. (C35H34ClN5O10) C, H, N.
Example 5B. Preparation of (l-methyI-2-nitro-l//-pyrrol-3-yl)methyl 1- (chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-l-yl)carbonyl]-2,3-dihydro-l//-benzo|e]indol-5-ylcarbamate (169). (l-methyl-2-nitro-l//-pyrrol-3-yl)methanoI (168). A solution of NaOH (2.7 g, 68 mmol) 15 in water (15 mL) was added to a solution of ester 166 (1.02 g, 4.47 mmol) in EtOH (30 mL), and the mixture was stirred at 20 °C for 1 h. The EtOH was evaporated, and the aqueous phase washed with EtOAc (20 mL) and then acidified (HCI). 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)-l-methyl-5-nitropyrrole-2-carboxylic 20 acid (0.84 g, 96%) as a red-brown solid.
The acid was suspended in quinoline (6 mL) with Cu powder (0.44 g) and the mixture was heated at 170-180 °C 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 25 as a pink solid (0.45 g, 64%), mp (benzene) 79-80.5 °C; ]H NMR 8 6.78 (d,J= 2.8 Hz, 1 H, H 5), 6.27 (d, J = 2.5 Hz, 1 H, H 4), 4.80 (d, y = 6.8 Hz, 2 H, CH20), 4.00 (s, 3 H, NCH3), 2.75 (t, J= 6.8 Hz, 1 H, OH); I3C NMR 8 131.6 (C 3), 129.1 (C 4), 109.0 (C 5), 58.8 (CH20), 38.6 (NCH3); Anal. (C6H8N203) C, H, N. (l-Methyl-2-nitro-l//-pyrrol-3-yl)methyI l-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indoI-l-yl)carbonyI]-2,3-dihydro-l//-benzo[^]indol-5-ylcarbamate (169). A solution of triphosgene (13.3 mg, 45 //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] (53 mg, 114 nmol) and Et3N (36 /jlL, 256 //mol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of (l-methyl-2-nitro-l//-pyrrol-3-yl)methanol (168) (24 mg, 153 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution 5 stirred at 20 °C 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 [(CD3)2SO] 5 11.46 (br s, 1 H, 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, 10 1 H, H 7), 7.33 (d, 7=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, CH20), 4.80 (dd, 7 = 11.0, 9.3 Hz, 1 H, H 2), 4.52 (dd, J= 11.0, 1.9 Hz, 1 H, H 2), 4.31-4.37 (m, 1 H,H 1), 4.07 (dd, J = 11.1, 3.0 Hz, 1 H, CH2C1), 3.96 (s, 3 H, NCH3), 3.91-3.94 (m, 4 H, CH2C1, OCH3), 3.82 (s, 3 H, OCH3), 3.80 (s, 3 H, OCH3); l3CNMR [(CD3)2SO] 5 160.2 (CO), 154.4 (OCONH), 149.1 (C 5'), 141.5 15 (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 3a'), 122.0 (C 9b), 113.0 (C 4), 108.1 (C 5"), 106.1 (C3'), 98.0 (C 4'), 61.0 (OCH3), 60.9 (OCH3), 60.2 (CH20), 55.9 (OCH3), 54.8 (C 2), 47.5 (CH2C1), 41.1 (C 1), 37.9 (NCH3); MS (FAB+) m/z 650 (MH+, 1.5%), 648 (MH+, 3.5); HRMS (FAB+) calc. for C32H3135C1N508 20 (MH+) m/z 648.1861, found 648.1844; calc. for C32H3137ClN5Og (MH+) m/z 650.1832, found 650.1826; Anal. (C32H30ClN5O8) C, H, N.
Example 5C. Preparation of ethyl 5-({[({l-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-l-yI)carbonyI]-2,3-dihydro-l//-benzo[e]indol-5-yl}amino)carbonyI]oxy}methyl)-25 l-methyI-4-nitro-li/-pyrrole-2-carboxyIate (173).
Ethyl 5-formyl-l-methyl-4-nitro-l/Z-pyrrole-2-carboxyIate (171). Dimethyl sulfate (0.31 mL, 3.2 mmol) was added to a mixture of ethyl 5-formyl-4-nitro-l//-pyrrole-2-carboxylate (170) [P. Fomari, M. Farnier, C. Fournier, Bull. Soc. Chim. Fr. 1972, 283-291] (0.57 g, 2.7 mmol) and K2C03 (0.56 g, 4.0 mmol) in DMSO (4 mL) and the brown 30 suspension was stirred at 20 °C for 1 h. The mixture was diluted with water (50 mL), acidified with HCI (2 N), and extracted with EtOAc (2 x 50 mL). The combined extract was dried, the solvent evaporated. The residue was chromatograped, eluting with !• L-A P £ %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, CH2), 4.33 (s, 3 H, NCH3), 1.40 (t, J = 7.2 Hz, 3 H, CH3); Anal. (C9H10N2O5) C, H, N.
Ethyl 5-(hydroxymethyl)-l-methyl-4-nitro-l//-pyrrole-2-carboxylate (172). NaBH4 (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 °C for 20 min. Water (5 mL) was slowly added, the EtOH was evaporated, and the residue was diluted with brine and 10 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 °C; 'H NMR 5 7.48 (s, 1 H, H 3), 4.97 (d, J = 6.8 Hz, 2 H, CH20), 4.32 (q, J - 7.1 Hz, 2 H, CH2), 4.06 (s, 3 H, NCH3), 2.72 (t, J= 7.1 Hz, 1 H, OH), 1.37 (t, J= 7.1 Hz, 3 H, CH3); Anal. (C9H12N205) C, H, N.
Ethyl 5-({[({l-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-l-yI)carbonyl]-2,3-dihydro-l//-benzo[^]indol-5-yl}amino)carbonyl]oxy}methyl)-l-methyl-4-nitro-l//-pyrrole-2-carboxylate (173). A solution of triphosgene (13.5 mg, 46 //mol) in DCM (2 20 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 Et3N (36 //L, 260 //mol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of ethyl 5-(hydroxymethyl)-l-methyl-4-nitro-l//-pyrrole-2-carboxylate (172) (36 mg, 156 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 25 °C 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 °C; 'H NMR [(CD3)2SO] 5 11.46 (s, 1 H, indole-NH), 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 30 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, CHzO), 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 = 1.1 Hz, 2 H, H 1'), 4.00-4.08 (m, 4 H, CH2C1, NCH3), 3.91-3.95 (m, 4 H, CH2C1, OCH3), 3.83 (m, 3 H, OCH3), 3.80 (s, 3 H, OCH3), 1.31 (t, J = 7.1 Hz, 3 H, H 2'); 13CNMR [(CD3)2SO] 5 160.1 (CO), 159.3 (C02), 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"), 111.2 (C 3), 106.2 (C 3'"), 98.0 (C 4'"), 61.0 (OCH3), 60.9 (OCH3), 60.8 (CH20), 55.9 (OCH3), 54.8 (C 2"), 54.6 (C 1'), 47.5 (CH2C1), 41.1 (C 1"), 33.6 (NCH3) 13.9 (C 2'); MS (FAB+) m/z 722 (MH+, 2.5%), 720 (MH+, 6); HRMS (FAB+) calc. for C35H3535ClN5O10 (MH+) m/z 720.2073, found 720.2045; calc. for C35H3537C1N50IO (MH+) m/z 722.2043, found 722.2039; Anal. (C35H34ClN5O10) C, H, N.
Example 5D. Preparation of (l-methyl-3-nitro-l^T-pyrroI-2-yl)methyl 1-(chloromethyI)-3-[(5,6,7-trimethoxy-li/-indol-l-yl)carbonyl]-2,3-dihydro-l//-benzo[e]indol-5-ylcarbamate (175). 2-(Hydroxymethyl)-l-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 °C 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 was adjusted to 2 with 1 M HCI. The aqueous mixture was extracted with EtOAc (3 ' 50 20 mL), the combined organic extract was dried and the solvent evaporated to give crude 4-(hydroxymethyl)-l-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 heated at 180-190 °C for 50 min under N2. The cooled mixture was diluted with EtOAc (50 25 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 °C (benzene); 'H NMR 5 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, J= 7.2 Hz, 2 H, CH20), 3.73 (s, 3 H, NCH3), 2.83 (t, J = 7.2 Hz, 1 H, OH); ,3C NMR 5 30 134.9 (C 3), 133.5 (C 2), 122.0 (C 4), 105.7 (C 5), 53.8 (CH20), 34.8 (NCH3); Anal. (C6H8N203)C,H,N. (l-Methyl-3-nitro-l//-pyrrol-2-yl)methyl l-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-l-yl)carbonyl]-2,3-dihydro-l/7-benzo[e]indol-5-ylcarbamate (175). A solution of triphosgene (15 mg, 50 //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, 5 1483] (59 mg, 127 //mol) and Et3N (40 //L, 284 //mol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of alcohol 174 (27 mg, 171 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C 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 °C; 10 'H NMR 5 9.42 (s, 1 H, indole-NH), 8.94 (s, 1 H, OCONH), 7.87 (d,J= 8.4 Hz, 1 H, H 6), 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, 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, CH20), 4.81 (dd,7= 10.7,91.7 Hz, 1 H, H 2), 4.67 (dd, 10.7,8.6 Hz, 1 H, H 2), 4.15-4.20 (m, 1 15 H, H 1), 4.10 (s, 3 H, OCH3), 3.95-3.99 (m, 4 H, OCH3, CH2C1), 3.92 (s, 3 H, OCH3), 3.80 (br s, 3 H, NCH3), 3.48 (dd, J = 11.0, 10.7 Hz, 1 H, CH2C1); 13C NMR 8 160.4 (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 5a), 123.6 (C 3a'), 123.1 (C 9), 122.6 (C 6), 122.2 (C4"), 121.6 (C 9b), 113.0 (C4), 106.5 (C 20 3'), 106.1 (C 5"), 97.7 (C 4'), 61.5 (OCH3), 61.1 (OCH3), 56.3 (OCH3), 55.3 (CH20), 54.9 (C 2), 45.8 (CH2C1), 43.4 (C 1), 35.3 (NCH3); MS (FAB+) m/z 650 (MH+, 0.6%), 648 (MH+, 1.5); HRMS (FAB+) calc. for C32H3135C1N508 (MH+) m/z 648.1861, found 648.1850; calc. for C32H3I37C1N508 (MH+) m/z 650.1832, found 650.1841; Anal. (C32H30ClN5O8) C, H, N.
Example 5E. Preparation of (l-methyl-5-nitro-l//-pyrrol-2-yl)methyl 1-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-l-yl)carbonyl]-2,3-dihydro-l//-benzo[e]indol-5-ylcarbamate (178). (l-Methyl-5-nitro-l//-pyrrol-2-yl)methanol (177). NaBH4 (0.19 g, 5.03 mmol) was 30 added to a stirred solution of l-methyl-5-nitro-l//-pyrrole-2-carbaldehyde (176) [ P. Fournari, Bull. Soc. Chun. Fr. 1963, 488-491] (0.78 g, 5.07 mmol) in MeOH (40 mL) at room temperature under N2. 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 K2C03. The mixture was extracted with EtOAc (3 ' 50 mL), the combined organic fraction dried, and the solvent evaporated to give 177 (0.77 g, 97%)as a white solid, mp (EtOAc/light petroleum) 76-77 °C; *H NMR 5 7.16 (d, J = 4.3 Hz, 1 H, H 4), 6.17 (d, J = 5 4.3 Hz, 1 H, H 3), 4.68 (s, 2 H, CH2), 4.02 (s, 3 H, CH3); Anal. (C6H8N203) C, H, N. (l-Methyl-5-nitro-l//-pyrroI-2-yI)methyl l-(chloromethyl)-3-[(5,6,7-trimethoxy-l//-indol-l-yl)carbonyI]-2,3-dihydro-liy-benzo[e]indol-5-ylcarbamate (178). A solution of triphosgene (16 mg, 52 jumol) in DCM (2 mL) was added dropwise to a stirred solution of 10 amine 1 [G. J. Atwell, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett., 1997, 7, 1483] (62 mg, 133 //mol) and Et3N (42 //L, 299 //mol) in DCM (10 mL) and stirred at 20 °C for 2 h. A solution of alcohol 177 (28 mg, 179 //mol) in DCM (2 mL) was added, followed by nBu2Sn(OAc)2 (2 drops) and the solution stirred at 20 °C for 24 h. The solvent was evaporated and the residue purified by chromatography, eluting with 20% 15 EtOAc/DCM, to give 178 (54 mg, 63%) as a white solid, mp (EtOAc) 212-214 °C; 'H NMR [(CD3)2SO] 5 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,/=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, 20 CH20), 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), 4.32-4.37 (m, 1 H,H 1), 4.07 (dd,J= 11.1, 3.1 Hz, 1 H, CH2C1), 3.91-3.96 (m, 7 H, CH2C1, NCH3, OCH3), 3.83 (s, 3 H, OCH3), 3.81 (s, 3 H, OCH3); ,3C NMR [(CD3)2SO] 5 160.2 (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 5a), 125.2 25 (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"), 113.0 (C 4), 110.6 (C 4"), 106.2 (C 3'), 98.0 (C 4'), 61.0 (OCH3), 60.9 (OCH3), 57.6 (CH20), 55.9 (OCH3), 54.8 (C 2), 47.5 (CH2C1), 41.1 (C 1), 33.9 (NCH3); MS (FAB+) m/z 650 (MH+, 1%), 648 (MH+, 2); HRMS (FAB+) calc. for C32H3135C1N508 (MH+) m/z 648.1861, found 648.1852; calc. for C32H3137C1N508 (MH+) m/z 650.1832, found 650.1836; 30 Anal. (C32H30ClN5O8) C, H, N.
Elemental analysis data r-TT3 L* fj 4 1- f! f—ru <S.v No Formula Calculated (%) Found (%) IA J17 CmH,4Cl7N404 C, 36.9; H. 4.3; N, 17.2; CI, 21.8 C, 37.4; H, 4.1; N, 17.2; CI, 21.8 IB 123 C.AH.^WO, C, 46.2; H, 4.6; N, 16.8; CI, 17.0 C, 46.3; H, 4.8; N, 17.1; CI, 17.1 1C 124 C„H?,ClNr,0s-'/2H70 C, 56.6; H, 4.6; N, 12.8 C, 56.6; H, 4.4; N, 12.5 ID 125 CnH^NAv'/d^O C, 53.9; H, 4.8; N, 7.6 C, 53.7; H, 4.8; N, 7.3 IE 129 C41H4tN,0„.H,0 C, 55.1; H, 4.85; N, 7.8 C, 54.7; H, 4.9; N, 7.5 IF 132 C„H,qC1N„Os C, 57.4; H, 4.5; N, 12.95 C, 57.7; H, 4.5; N, 12.9 1G 135 C,„HI4C1,N404(M+) 324.1392 324.1381 1H 137 c1#h„cj,n,o4 C, 46.2; H, 4.6; H, 16.8 C, 46.1; H, 4.6; N, 16.7 11 138 c„h„cin110„ C, 57.4; H, 4.5; N, 12.95 C, 57.5; H, 4.6; N, 12.9 IJ 142 C4,H4,Ns0n.H,0 C, 55.1; H, 4.85; N, 7.8 C, 55.2; H, 4.9; N, 7.9 IK 148 C, 56.6; H, 4.6; N, 12.4 C, 56.5; H, 49; N, 12.1 1L 150 C„H„ClNfiO» C, 57.4; H, 4.5; N, 12.95 C, 57.2; H, 4.5; N, 12.9 2A 154 ClnHl73iCl,N10,(M+) 401.0545 401.0546 2B 155 C^H^CIKA C, 58.6; H, 4.3; N, 8.8 C, 58.5; H, 4.3; N, 8.9 3A 159 c14h„ci,n,o4 C, 45.9; H, 4.1; N, 10.1; CI, 17.0 C, 46.2; H, 4.0; N, 9.9; CI, 17.2 3B 160 C^H^CIHAS C, 57.2; H, 4.2; N, 8.6 C, 57.4; H, 4.0; N, 8.4 4 163 C„H„ClN(iOR C, 57.4; H, 4.5; N, 12.95 C, 57.5; H, 4.6; N, 13.1 5A 167 C„H«ClN,0,n C, 58.4; H, 4.8; N, 9.7 C, 58.2; H, 4.9; N, 9.7 5B 169 Ci2HwClN,Os C, 59.3; H, 4.7; N, 10.8 C, 59.4; H, 4.8; N, 10.8 5C 173 C„H14C1N,0,„ C, 58.4; H, 4.8; N, 9.7 C, 58.6; H, 4.9; N, 9.8 5D 175 CwH1(iC1N,Os C, 59.3; H, 4.7; N, 10.8 C, 59.1; H, 4.7; N, 10.9 5E 178 c„h,0cin,o8 C, 59.3; H, 4.7; N, 10.8 C, 59.4; H, 4.8; N, 10.7 Example 10 Biological activity Selected compounds were evaluated for cytotoxicity (measured as IC50 values in 30 /iM 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 carcinoma (EMT6-V) is wild-type, while the EN2A is the NR+ transfectant. Ratios (NR-35 /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 NR- IC50 ratios NR- Ratio NR- Ratio ICsn(^mol) IQnOumol) IC,«(/imol) IB 123 2.3 28 1.7 6.7 - - 1C 124 0.075 21 0.075 40 - - ID 125 3.74 14.5 3.59 .3 0.46 . 6.4 IE 129 >1.5 >12 >1.5 >5.6 0.24 4.95 11 138 0.15 50 0.23 99 0.078 71 IJ 142 >3 >9.0 2.72 1.64 0.23 >1.22

Claims (1)

  1. -59- IK 148 0.23 30 0.38 <38 0.067 <7 1L 150 0.061 5.6 0.024 3.4 0.063 6.5 3B 160 0.01 7.0 0.018 13.5 0.004 16.5 4 163 0.032 30 0.011 13.5 0.039 87 5A 167 0.097 9.6 0.094 13.2 0.028 11.6 ■&zs» y fa i w* i;L <s;60;CLAIMS;1. A compound of formula (II): wherein:;ii;X represents H, Ci-6 alkyl or Ci-6 alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORx), amino (NH2), mono-substituted amino (NRXH), di-substituted amino (NRx'Rx2), cyclic C1.5 alkylamino, imidazolyl, Ci-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRX), tetrazole, carboxy (COOH), carboxylate (COORx), sulphoxy (S(=0)20H), sulphonate (S(=0)20RX), sulphonyl (S(=0)2RX), sulphixy (S(=0)0H), sulphinate (S(=0)0Rx), sulphinyl (S(=0)Rx), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0RX)2), where Rx, RXJ and Rx2 are selected from a Ci.6 alkyl group, a C3.2o heterocyclyl group or a C5-20 aryl group; a is 0,1,2,3 or 4; Y represents H or Ci-6 alkyl; 1, 2 or 3 of the members Z of the 5-membered aromatic ring are independently selected from -S-,-N= or -NR-, where R is H or Ci-6 alkyl optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORr), amino (NH2), mono-substituted amino (NRrH), di-substituted amino (NRr'Rr2), cyclic C1-5 alkylamino, imidazolyl, alkylpiperazinyl, morpholino, thiol (SH), thioether (SRr), tetrazole, carboxy (COOH), carboxylate (COORr), sulphoxy (S(=0)20H), sulphonate (S(=0)20Rr), sulphonyl (S(=0)2Rr), sulphixy (S(=0)0H), sulphinate (S(=0)0Rr), sulphinyl (S(=0)Rr), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0Rr)2), where Rr, Rr1 and Rr2 are selected from a Ci-6 alkyl group, a C3.2o heterocyclyl group or a Cs.20 aryl group, the other ring atoms being C; n is 0 or 1; and;E is selected from formulae (III-XIII); wherein, R] represents H or C)_6 alkyl, being optionally substituted with one or more of the following groups: one or more of the following groups: hydroxy (OH), ether (ORe), amino (NH2), mono-substituted amino (NReH), di-substituted amino (NRe'Re2), cyclic C1.5 alkylamino, imidazolyl, Ci-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRe), tetrazole, carboxy (COOH), carboxylate (COORe), sulphoxy (S(=0)20H), sulphonate (S(=0)20RE), sulphonyl (S(=0)2Re), sulphixy (S(=0)0H), sulphinate (S(=0)0RE), sulphinyl (S(=0)RE), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0Re)2), where Re, Re' and RE2 are selected from a Ci-6 alkyl group, a C3.2o heterocyclyl group or a C5.20 aryl group; R2 represents H, Ci-6 alkyl, Ci-6 alkoxy, OH, halogen, NO2, NH2, NHMe, NMe2, S02Me, CF3, CN, CONH2 or CONHMe; each R3 is independently selected from CI, Br, I and OMS; and R4 is selected from -C(=0)Me and -C(=0)CH20H; Q represents substituted indole, substituted 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.;63;2. A compound according to claim 1, wherein Rx, Rx', Rx2, Rr, Rr' and Rr2 are independently Ci-6 alkyl groups.;3. A compound according to either claims 1 or claim 2, wherein the compound is of formula (II) with two Z, one Z being -N= and the other Z being -NR-.;4. A compound according to claim 3, wherein R is either Me or Et.;5. A compound according to claim 4, wherein R is Et substituted with hydroxy.;6. A compound according to any one of claims 3 to 5, wherein a is 0.;7. A compound according to any one of claims 3 to 6, wherein the -N= and -NR- are not adjacent in the heterocyclic ring.;8. A compound according to any one of claims 3 to 7, wherein E is of formula V.;9. A compound according to any one of claims 3 to 7, wherein E is of formula XIII;10. A compound according to claim 9, wherein n = 1.;11. A compound according to claim 1, wherein the compound is of formula (II), a is 0, and Z is S.;12. A compound according to claim 1, wherein the compound is of formula (II), Z is NR, and a is either 0 or 1.;13. A compound according to claim 12, wherein Z is NMe.;14. A compound according to either claim 12 or 13, wherein a is 1 and X is CC^Et.;15. A compound according to any one of the preceding claims for pharmaceutical use.;64;16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier or diluent.;17. 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 14.;18. 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 14.;19. A compound according to any one of claims 1 to 14, a composition according to claim 18, or a system according to claims 17 or 18 for use in a method of medical treatment.;20. The use of a compound according to any one of claims 1 to 14 for the manufacture of a composition for use in the treatment of a hyper-proliferative disease.;21. A method of providing an amine with a protecting group comprising:;(i) providing a plurality of different compounds selected from compounds of formula (II);ytE;o ii wherein:;X represents H, C]_6 alkyl or Ci_6 alkoxy, said alkyl or alkoxy being optionally substituted with one or more of the following groups: hydroxy (OH), ether (ORx), amino (NH2), mono-;substituted amino (NRXH), di-substituted amino (NR^R*2), cyclic C1.5 alkylamino, imidazolyl, C|_6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRX), tetrazole, carboxy (COOH), carboxylate (COORx), sulphoxy (S(=0)20H), sulphonate (S(=0)20Rx), sulphonyl (S(=0)2Rx), sulphixy (S(=0)0H), sulphinate (S(=0)0Rx), sulphinyl (S(=0)Rx), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0Rx)2), where Rx, Rx' and Rx2 are selected from a alkyl group, a C3-2o heterocyclyl group or a C5-2o aryl group; a is 0,1,2,3 or 4; Y represents H or C1-6 alkyl; 1, 2 or 3 of the members Z of the 5-membered aromatic ring are independently selected from -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 (NH2), mono-substituted amino (NRrH), di-substituted amino (NRr'Rr2), cyclic C1-5 alkylamino, imidazolyl, alkylpiperazinyl, morpholino, thiol (SH), thioether (SRr), tetrazole, carboxy (COOH), carboxylate (COORr), sulphoxy (S(=0)20H), sulphonate (S(=0)20Rr), sulphonyl (S(=0)2Rr), sulphixy (S(=0)0H), sulphinate (S(=0)0Rr), sulphinyl (S(=0)Rr), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0Rr)2), where Rr, Rr1 and Rr2 are selected from a Ci-6 alkyl group, a C3.20 heterocyclyl group or a C5.2o aryl group, the other ring atoms being C; n is 0 or 1; and E is selected from formulae (IH-XIII); wherein, Ri represents H or Ci_6 alkyl, being optionally substituted with one or more of the following groups: one or more of the following groups: hydroxy (OH), ether (ORe), amino (NH2), mono-substituted amino (NREH), di-substituted amino (NRe'Re2), cyclic C1.5 alkylamino, imidazolyl, C1-6 alkylpiperazinyl, morpholino, thiol (SH), thioether (SRe), tetrazole, carboxy (COOH), carboxylate (COORe), sulphoxy (S(=0)20H), sulphonate (S(=0)20RE), sulphonyl (S(=0)2RE), sulphixy (S(=0)0H), sulphinate (S(=0)0RE), sulphinyl (S(=0)RE), phosphonooxy (0P(=0)(0H)2) and phosphate (0P(=0)(0RE)2), where Re, Re1 and RE2 are selected from a C1-6 alkyl group, a C3-2o heterocyclyl group or a C5.2o aryl group; R2 represents H, Ci^ alkyl, Ci-6 alkoxy, OH, halogen, N02, NH2, NHMe, NMe2, S02Me, CF3, CN, CONH2 or CONHMe; each R3 is independently selected from CI, Br, I and OMS; and R4 is selected from -C(=0)Me and -C(=0)CH20H; Q represents substituted indole, substituted benzofuran or substituted cinnamoyl; in (IX) and (X), each n is independently 66 from 2-4, and each m is independently from 2-4, and p = 0 or 1 r3' r3 r3 (III) (IV) (v) (Vila) OH O HN OH O HN (Vllb) R1 (VI) Nvs (vni) (1^0) N- (X) vtoe l= (XI) NH, O OH OMe O OH O (XII) Me—'T~-~o-^7 HO'^N ^ (XIII) 67 (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.
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