MXPA06004635A - Novel nitrophenyl mustard and nitrophenylaziridine alcohols and their corresponding phosphates and their use as targeted cytotoxic agents - Google Patents

Novel nitrophenyl mustard and nitrophenylaziridine alcohols and their corresponding phosphates and their use as targeted cytotoxic agents

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Publication number
MXPA06004635A
MXPA06004635A MXPA/A/2006/004635A MXPA06004635A MXPA06004635A MX PA06004635 A MXPA06004635 A MX PA06004635A MX PA06004635 A MXPA06004635 A MX PA06004635A MX PA06004635 A MXPA06004635 A MX PA06004635A
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Mexico
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amino
ethyl
compound
bis
bromoethyl
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MXPA/A/2006/004635A
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Spanish (es)
Inventor
Alexander Denny William
Robert Wilson William
J Atwell Graham
Yang Shangjin
Vorn Patterson Adam
Ann Helsby Nuala
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Atwell Graham John
Auckland Uniservices Limited
Alexander Denny William
Ann Helsby Nuala
Vorn Patterson Adam
Robert Wilson William
Yang Shangjin
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Application filed by Atwell Graham John, Auckland Uniservices Limited, Alexander Denny William, Ann Helsby Nuala, Vorn Patterson Adam, Robert Wilson William, Yang Shangjin filed Critical Atwell Graham John
Publication of MXPA06004635A publication Critical patent/MXPA06004635A/en

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Abstract

The present invention relates to novel nitrophenyl mustard and nitrophenylaziridine alcohols, to their corresponding phosphates, to their use as targeted cytotoxic agents;as bioreductive drugs in hypoxic tumours, and to their use in cell ablation, including gene-directed enzyme-prodrug therapy (GDEPT) and antibody-directed enzyme -prodrug therapy (ADEPT), in conjunction with nitroreductase enzymes.

Description

NEW ALCOHOLS OF NITROPHENYLAZIRIDINE AND MUSTARD NITROPHENYL AND ITS CORRESPONDING PHOSPHATES AND THEIR USE AS CITOTOXIC AGENTS SELECTED AS A PURPOSE FIELD OF THE INVENTION The present invention relates to novel nitrophenylaziridine alcohols and nitrophenyl mustard, to their corresponding phosphates, to their use as targeted cytotoxic agents; as bio-reductive drugs in hypoxic tumors, and their use in cell ablation, including prodrug therapy and gene-directed enzyme (GDEPT) and prodrug therapy and antibody-directed enzyme (ADEPT), in conjunction with nitroreductase enzymes. BACKGROUND OF THE INVENTION The use of selective tumor prodrugs (Relatively inactive compounds that can be selectively converted to more active compounds in vivo) is a valuable concept in cancer therapy (see, for example Denny, Eur. J. Med. Chem. (2001) 36, 577) . For example, a prodrug can be converted to an anti-tumor agent under the influence of an enzyme that can bind to a monoclonal antibody that will bind to an antigen associated with tumor. The combination of this prodrug with a conjugate of monoclonal antibody and enzyme represents * a very powerful clinical agent. This approach to REF. : 172416 Cancer therapy, often referred to as "antibody-directed enzyme prodrug therapy" (ADEPT), is described in WO 88/07378. An additional therapeutic approach called "virus directed enzyme prodrug therapy" (VDEPT) has been proposed as a method for treating tumor cells in patients using prodrugs. The tumor cells are targeted with a viral vector having a gene encoding an enzyme capable of activating a prodrug. The gene can be regulated transcriptionally by a tissue-specific promoter or by enhancer sequences. The viral vector enters the tumor cell and expresses the enzyme, so that a prodrug is converted to an active drug within the tumor cells (Huber et al., Proc. Nati. Acad. Sci. USA (1991) 88 , 8039). Alternatively, non-viral methods have been used for the distribution of genes. These methods include co-precipitation with calcium phosphate, 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" (prodrug therapy and gene-directed enzyme) is used to include both viral and non-viral delivery systems (Denny et al US 6,310,237). The 4-nitroaromatic compounds are reduced by Flavoprotein enzymes, both mammalian and bacterial, that effect the gradual addition of up to six electrons. The main enzymatic metabolite is usually the reduced 4-electron species (hydroxylamine). Several nitrophenyl mustards and nitrophenylaziridines have been reported as prodrugs for use in gene directed enzyme prodrug (GDEPT) therapy in conjunction with nitroreductase enzymes. In particular, CD 1954 [5- (aziridin-1-yl) -2, -dinitrobenzamide; (1) [shown below] which is a substrate for the aerobic nitroreductase NTR (nfsB gene product) isolated from E. coli B (Boland et al., Biochem Pharmacol, 1991, 41, 867-875; Anlezark et al. , Biochem Pharmacol, 1992, 44, 2289-2295; Parkinson et al., J. Med. Chem. 2000, 43, 3624). This compound has been used as a prodrug in both ADEPT applications (Knox et al., Biochem. Pharmacol., 1995, 49, 1641-1647) as GDEPT (Bridgewater et al., Eur. J. Cancer, 1995, 31A, 2362-2370; Bailey et al., Gene Ther., 1996, 3, 1143-1150; Bailey and Hart, Gene Ther., 1997, 4, 80-81; Green et al., Cancer Gene Ther., 1997, 4, 229-238), including a clinical trial (Chung-Faye et al., Clin Cancer Res., 2001, 7, 2662-2668). Similarly, dinitrophenyl mustard SN 23862 (2) is also a substrate for NTR, and shows selective toxicity towards the cell lines that express the enzyme It is activated by nitro group reduction (Palmer et al., J. Med. Chem., 1995, 38, 1229, Kestell et al., Cancer Chemother, Pharmacol., 2000, 46, 365-374). The 4-S02Me derivative (3) was also a substrate (Atwell et al., Anti-Cancer Drug Des., 1996, 11, 553), as were the regioisomers (4) and (5) (Friedlos et al. , J. Med. Chem., 1997, 40, 1270).
However, compounds of this type were not very effective as bioreductive prodrugs when these compounds were activated in hypoxic tumor tissue by endogenous reductase enzymes, showing power ratios of 2-5 times under hypoxic conditions in relation to oxic connections in the line wild-type AA8 cell, using a clonogenic assay (Palmer et al., J. Med. Chem. 1996, 39, 2518-2528). Some phosphate analogs of mustards have been described, for the purpose of solubilizing the compounds. The best known is estramustine phosphate (Estracyt; 6), which has been shown to bind to tubulin-binding domains in several microtubule-associated proteins (Moraga et al., Biochim, Biophys. Acta, 1992, 1121, 97- 103), and that shown to be active in advanced breast cancer (Keren-Rosenberg et al., Semin. Oncol., 1997, 24 (Suppl 3), 26-29), but it has not been shown to be activated by NTR or hypoxia. Another study has also shown that estramustine phosphate is a radiation sensitizer (Kim et al., Int. J. Radiat, Oncol. Biol. Phys., 1994, 29, 555-557). The analogue of phenol mustard phosphate 7 is a carboxypeptidase substrate that is not activated under hypoxic conditions, (Matsui et al., Japanese Patent 07082280 A2, 1995), and solubilized mustard 8 has been described as a phosphatase inhibitor but it has not been shown to activate under hypoxic conditions (Workman, Chem. -Biol., Interact., 1978, 20, 103-112).
It is an object of the present invention to provide a specific class of nitrophenyl mustards and aziridines, which have short chain alcohols, and their corresponding phosphates for use as selected cytotoxic agents. as a target or bioreductive prodrugs or to provide at least the public with a useful alternative.
DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides new phosphate compounds of Formula I wherein: X represents at any available position of the ring -CONH-, -S02NH-, -0-, -CH2-, -NHCO- or -NHS02-; R represents a C? _6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2) 2 or -N (CH2CHMe) 2, wherein each is independently selected from halogen or -0S02Me. Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; and pharmaceutically acceptable salts and derivatives thereof. In a preferred embodiment, the phosphate compound of the formula (I) is selected from a represented compound by the formulas (la), (Ib) or (le) (le) where Y can represent and wherein n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each is independently selected from halogen or -OS02Me and pharmaceutically acceptable salts and derivatives thereof. Preferably, the phosphate compound of Formula (I) is selected from the following: 2- [[2- [Bis (2-bromoethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl diacid phosphate; 3- [[5- [Bis (2-chloroethyl) amino] -2, 4- diacid phosphate dinitrobenzoyl] amino] ropyl; 3- [[5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzoyl] amino] -propyl phosphate; 2- [[2- [Bis (2-chloroethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl diacid phosphate; 2- [(2-Chloroethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl-methanesulfonate; 2- (. {2- [Bis (2-bromopropyl) amino] -3,5-dinitrobenzoyl} amino) ethyl phosphate; 2- [(2-Bromoethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl-methanesulfonate; 2- [[2- [Bis (2-iodoethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl diacid phosphate; 2- [(2-Yodoethyl) -2,4-dinitro-6- ( { [2- (phosphonooxy) ethyl] amino} carbonyl) -anilino] ethyl-methanesulfonate; 2- [(2-Chloroethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) ropyl] amino] -carbonyl] anilino] ethyl-methanesulfonate; 3- (. {3- [Bis (2-bromoethyl) amino] -2,6-dinitrobenzoyl} amino) propyl phosphate; 2- [(2-Bromoethyl) -2,4-dinitro-3- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl- methanesulfonate; 2- [(2-Bromoethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) propyl] amino] -carbonyl] anilino] ethyl-methanesulfonate; and 2- [(2-Yodoethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) propyl] amino] -carbonyl] anilino] ethyl-methanesulfonate. In a second aspect, the present invention provides alcohol compounds of Formula (II): wherein: X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS02-; Y represents in any available position of the ring, -N-aziridinyl, -N (CH2CH2W) 2 or -N (CH2CHMeW) 2, wherein each is independently selected from halogen or -OSC ^ Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; R represents a C? -6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives thereof; with the proviso that when Z represents N02 and Y represents N (CH2CH2CI) 2, X and R together can not represent -CONHCH2 (CHOH) CH2- and also with the proviso that the following compounds are excluded In a preferred embodiment, the alcohol compound of the formula (II) is selected from a compound represented by the formulas (lia), (Ilb) or (lie) (llb) (Hc) where Y can represent and wherein n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each W is independently selected from halogen or -OS02Me and pharmaceutically acceptable salts and derivatives thereof with the proviso that when Z represents N02 and Y represents N (CH2CH2CI) 2, X and R together can not represent -CONHCH2 ( CHOH) CH2- and also with the condition that are excluded The following compounds Preferably, the compound of the Formula (II) is selected from the following: N- (2-Hydroxyethyl) -5- [bis (2-bromoethyl) amino] -2, 4- dinitrobenzamide; N- (4-Hydroxybutyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide; N- (5-Hydroxypentyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide; N- (6-Hydroxyhexyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide; 5- [Bis (2-bromoethyl) amino] N- (2-hydroxyethyl) -4- (methylsulfonyl) -2-nitrobenzamide; 2 - [(2-Bromoethyl) -5 - [[(3-hydroxypropyl) amino] carbonyl] -2,4-dinitroanilino] ethyl-methanesulfonate; 5- [Bis (2-iodoethyl) amino] -N- (2-hydroxyethyl) -2,4-dinitrobenzamide; 2- [Bis (2-Chloroethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (2-hydroxyethyl) -3,5-initrobenza ida; 2- [Bis (2-chloroethyl) amino] -N- (3-hydroxypropyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (3-hydroxypropyl) -3,5-dinitrobenzamide; 2- [Bis (2-chloroethyl) amino] -N- (4-hydroxybutyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (4-hydroxybutyl) -3,5- dinitrobenzamide; 2- [Bis (2-chloroethyl) amino] -N- (5-hydroxypentyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (5-hydroxypentyl) -3,5-dinitrobenzamide; 2- [Bis (2-chloroethyl) amino] -N- (6-hydroxyhexyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (6-hydroxyhexyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromopropyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide; 2- ((2-Bromoethyl) -2- { [(2-hydroxypropyl) amino] carbonyl} -4-6-dinitroanilino) ethyl-methanesulfonate; 2- ((2-Bromoethyl) -2- { [(2-hydroxyethyl) amino] carbonyl} -4-6-dinitroanilino) ethyl-methanesulfonate; 2- ((2-Chloroethyl) -2- { [(2-hydroxyethyl) amino] carbonyl, 4,6-dinitroanilino) ethyl-methanesulfonate; 2- [Bis (2-iodoethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide; 2- ((2-Yodoethyl) -2- { [(2-hydroxyethyl) amino] carbonyl, 4,6-dinitroanilino) ethyl-methanesulfonate; 3- [Bis (2-bromoethyl) amino] -N- (2-hydroxyethyl) -2,6- dinitrobenzamide; 2- ((2-Bromoethyl) -3- { [(2-hydroxyethyl) amino] carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; 3- [Bis (2-bromoethyl) amino] N- (3-hydroxypropyl) -2,6-dinitrobenzamide; 2- ((2-bromoethyl) -3- { [(3-hydroxypropyl) amino] carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; 3- [Bis (2-bromoethyl) amino] -N- (4-hydroxybutyl) -2,6-dinitrobenzamide; 2- ((2-Bromoethyl) -3- { [(4-hydroxybutyl) amino] carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; 2- ((2-Chloroethyl) -3- { [(3-hydroxypropyl) amino] carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; and 2- ((2-Yodoethyl) -3- { [(3-hydroxypropyl) amino] carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate. In a third aspect of the invention, there is provided a method for preparing the phosphates represented by the general formula (I); wherein: X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS02-; R represents a C? _6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; Y represents, in any available position of the ring, -N-aziridinyl, -N (CH2CH2) 2, wherein each is independently selected from halogen or -0S02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; and pharmaceutically acceptable salts and derivatives thereof; the method including the step of (i) phosphorylating a compound of Formula (II) N02 AND (ID wherein X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHC0- or -NHS02-; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2) 2, or -N (CH2CHMe) 2, wherein each is independently selected from halogen or -OS02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S0Me; and R represents a C6-lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof. In a fourth aspect, there is provided a method for preparing a compound of Formulas (lia), (Ilb) or (líe). (llb) (Hc) where Y can represent and wherein n represents 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein Wi is halogen and W2 is -0S02Me and pharmaceutically acceptable salts and derivatives thereof; the method including the step of reacting a compound of the formulas (lia '), (Ilb') or (lie ') optionally with heating (lie ") where Y can represent wherein W'i and W'2 are each halogen; with an effective amount of silver methanesulfonate (AgOMs) in a solvent to give a compound of the formulas (lia), (Ilb) or (lie) defined above. It will be appreciated that in the method defined immediately above where W'i and W2 are either iodine and / or bromine, the iodine and / or bromine can be partially or completely substituted with -0S02Me. In the situation where either or both of '? and W2 represent chlorine, chlorine is inert and can not be substituted with -OS02Me. Preferably, the solvent is selected from MeCN or another polar non-protic solvent. In a fifth aspect, a method for preparing a compound of the formulas (la), (Ib) or (le) is provided. (le) where Y can represent 0 and wherein n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each is independently selected from halogen or -0S02Me and pharmaceutically acceptable salts and derivatives thereof the method including the step of phosphorylating a compound represented by the formulas (Ha), (Ilb) or (He) (Uc) where Y represents and where n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each is independently selected from halogen or -0S02Me and pharmaceutically acceptable salts and derivatives thereof. In a sixth aspect, there is provided a compound of the formula (I), formula (la), (Ib) or (le) or formula (Ha), (Ilb) or (He) obtained by any of the preparative methods defined above . In a seventh aspect, the present invention provides a method for use as suitable prodrugs for (i) GDEPT (prodrug therapy to gene directed enzyme) or ADEPT (prodrug therapy to enzyme directed by antibody) in conjunction with at least one nitroreductase enzyme; or (ii) Use as one or more hypoxia-selective cytotoxins, which includes the step of administering a compound of Formula (I) as defined above or a compound of Formula (II). where X represents in any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS02-; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2) 2, or -N (CH2CHMeW) 2, wherein each is independently selected from halogen or -OS02Me; Z represents at any available position of the ring, -N02 / -halogen, -CN, -CF3 or -S02Me; R represents a C? _6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives thereof; or a mixture thereof in a therapeutically effective amount to tumor cells in a subject. Preferably, the nitroreductase enzyme is encoded by the nfsB gene of either E. coli or by orthologous genes in the Clostridia species. Preferably, the method includes the additional step of irradiating the tumor cells. In an eighth aspect, the present invention provides a method for use as suitable prodrugs for GDEPT (prodrug therapy to gene directed enzyme) or ADEPET (prodrug therapy to antibody directed enzyme) in conjunction with at least one enzyme of Nitroreductase, as an anticancer agent that includes the step of administering a compound of Formula (I) as defined above or a compound of Formula (II). wherein X represents at any available position of the ring -CONH-, -S02NH-, -0-, -CH2-, -NHCO- or -NHS02-; -, Q Y represents in any available position of the ring, -N-aziridinyl, -N (CH2CH2W) 2 or -N (CH2CHMe) 2, wherein each W is independently selected from halogen or -0S02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; R represents a C? _6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives of l ° s themselves; or a mixture thereof in a therapeutically effective amount to target tumor cells in a subject. Preferably, the nitroreductase enzyme is encoded by the nfsB gene of E. coli or by orthologous genes in the Clostridia species.
Preferably, the method includes the additional step of irradiating the tumor cells. It will be appreciated that with ADEPT it may be necessary to provide a reducing co-factor, because these can not be present in significant concentrations outside the cells. It is contemplated that a synthetic cofactor may be used to stimulate pro-drug activation by the preferences of an intracellular enzyme. The same issue does not arise with GDEPT because there are several intracellular reducing co-factors such as the NADH and NADPH preferences in significant concentrations. In a ninth aspect, the present invention provides a method for providing anticancer treatment, wherein a compound of Formula (i) is co-administered or defined above in an amount to a subject. Preferably, the amount of the method is between about 20% to 100% of the maximum tolerated dose of the subject. Preferably, the method includes the additional step of applying radiation or chemotherapeutic agents to the tumor cells. In a tenth aspect of the present invention, there is provided a method of cellular ablation using at least one nitroreductase enzyme, wherein the method includes the step of administering a compound of Formula (I) as defined above or a compound of the Formula (ID wherein X represents at any available position of the ring -CONH-, -S02NH-, -0-, -CH2-, -NHCO- or -NHS02-; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2) 2 or -N (CH2CHMeW) 2, wherein each W is independently selected from halogen or -0S02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; R represents a C? -6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives thereof, or a mixture thereof in an amount effective to remove the cells, wherein the cells express at least one nitroreductase enzyme. Preferably, the nitroreductase enzyme is encoded by the nfsB gene in E. coli or by orthologous genes and in the Clostridia species. Preferably, the cells that are Select as targets for ablation are tumor cells in the tissue in a tissue. Preferably, the cell ablation method using at least one nitroreductase enzyme is distributed either by ADEPT or GDEPT technology. In an eleventh aspect of the present invention, there is provided a pharmaceutical composition that includes a therapeutically effective amount of a compound of the Formula (I) or a compound of Formula (II). wherein: X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS02-; Y represents in any available position of the ring, -N-aziridinyl, -N (CH2CH2) 2 or -N (CH2CHMe) 2, wherein each is independently selected from halogen or -0S02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; R represents a C6-lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives of the same, or a mixture thereof, and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabilizer. The pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabilizer should preferably be non-toxic and should not interfere with the efficiency of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, such as cutaneous, subcutaneous or intravenous. It will be appreciated that these factors can easily be determined by one skilled in the art without undue experimentation. The pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. A tablet can comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oil. Physiological saline, dextrose or other solution of saccharides or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such as gelatin. For intravenous, cutaneous or subcutaneous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has a suitable pH, adequate isotonicity and stability.
Those skilled in the art will be able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride injection, Ringer's injection, Lactated Ringer's injection, preservatives, stabilizers, buffers, antioxidants and / or other additives which may include As required. In a twelfth aspect of the present invention, there is provided, the use in the manufacture of a medicament in an effective amount of a compound of the Formula (I) as defined above or a compound of the Formula (II) wherein X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS02-; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2W) 2 or -N (CH2CHMe) 2, wherein each W is independently selected from halogen or -OS02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; R represents a C? _6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives thereof, or mixtures thereof, for use in (i) GDEPT or (ii) as a hypoxia-selective cytotoxin, the target cancer cells in a subject in need thereof. In a thirteenth aspect of the present invention there is provided, the use in the manufacture of a medicament of an effective amount of a compound of the Formula (I) as defined above or a compound of the Formula (II) wherein X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS0-; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2) 2 or -N (CH2CHMeW) 2, wherein each W is independently selected from halogen or -OS02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; R represents a C? _ Lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives thereof, or mixtures thereof for use in cell ablation therapy to target cancer cells in a subject in need thereof. While the compounds of the present invention will typically be used to target tumor cells or tumor tissues in human subjects, they can be used to target tumor cells or tumor tissues in other warm-blooded animals such as other primates, animals of farm such as cattle, and sports animals and pets such as horses, dogs and cats. As used in the specification from beginning to end, the term "therapeutically effective amount" is to be understood as an amount of a compound of Formula (I) or Formula (II) as defined above or a compound of any of the compounds Ia-Ic, or Halle as defined above or a mixture thereof which is sufficient to show benefit to a subject with cancer cells. The actual amount, speed and time course of administration will depend on the nature and severity of the disease being treated. The prescription of treatment is within the responsibility of general practitioners and other medical doctors.
As used from beginning to end in the specification, the term "halogen" includes chlorine, bromine or iodine. It is to be understood that the compounds of the invention as defined above can be administered alone or in combination with other treatments, especially radiotherapy and cytotoxic chemotherapeutic drugs, either simultaneously or sequentially depending on the condition to be treated. As used throughout the specification, pharmaceutically acceptable derivatives and salts thereof include salts derived from acid formed from hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic, isethionic and the like and salts derived from base formed from sodium and potassium carbonate, sodium and potassium hydroxide, ammonia, triethylamine, triethanolamine and the like. As used from beginning to end in the specification, the term "cell ablation" is to be understood as the annihilation of cells, which have been engineered to express an enzyme, such as a nitroreductase, by administration of a prodrug that is activated by that enzyme. . As a result, cell ablation can be used to selectively remove specified target cells or target tissue specified through the specific enzymatic expression of a nitroreductase, for example, which are specifically expressed by the tissue and which can then be employed to activate a prodrug in an active metabolite to remove the targeted tissue or target cells. (Gusterson et al., Endocrine Related Cancer, 1997, 4, 67-74.) The term "substantially minimal surrounding effect" is to be understood as meaning that the annihilation of attached cells not targeted is minimal because there is little or no diffusion between targeted cells and non-targeted cells of the activated metabolite that arises from the enzymatic activation of a compound of Formula (I) or Formula (II) as defined above or a compound of any of the compounds Ia-Ic, or Ha-Hc as defined above or a mixture thereof. The pharmaceutically acceptable salts of the formula (I) include the basic compounds or acids of the formula (I) which form pharmaceutically acceptable salts with both organic and inorganic acids and / or organic or inorganic bases. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic, isethionic, and the like. Examples of suitable bases for salt formation are sodium and potassium carbonate, sodium and potassium hydroxide, ammonia, triethylamine, triethanolamine, and the like. Additional aspects of the present invention will become apparent from the following description given by way of example only and with reference to the attached synthesis reaction schemes. Examples of the compounds of Formula (I) wherein X is -CONH- can be prepared by the processes described in Reaction Scheme 1, where Z is as defined above for Formula (I). Reaction Scheme 1 compounds of the formula (I) H m iv The following Tables la and 2a set forth physical data for compounds within General Formula (I) and (II), representative of them, and capable of being prepared by the processes of the invention. 4 (Uc) ede to represent Table the representative examples of alcohols of origin A-methyl mustard Notes References for known compounds 1. Khan AH, Ross CJ. Tumor-growth inhibitory nitrophenylaziridines and related compounds. Structure-activity relations. II. Chem. -Biol. Int., 1971, 4, 11-22. 2. NZ Patent No.240785 3. Co-pending NZ Application No. 521851 4. Wilson WR, Pullen SM, Hogg A, Helsby NA, Hicks KO, Denny WA. Quantitation of bystander effects in nitroreductase suicide gene therapy using three-dimensional cell cultures. Cancer Res., 2002, 62, 1425-1432. The compounds of Table 1 can be prepared by the general methods set forth in Reaction Schemes 2a-2k, and exemplified in Examples 1-20 below. (the) (Ib) (Ic) where Y can represent 25 Table Ib Examples of phosphates of the Formulas Ia-Ic ilfa-ME The compounds of Table Ib can be prepared by the general methods set forth in the Reaction Scheme, and exemplified in Examples 26-39 below.
Reaction scheme 2a 1: Q = CI lla-2: R = (CH2) 2OH, Q = Ci 2: Q = Br Ha-3: R = (CH2) 3OH, Q = Cl lla-7: R = (CH2) 2OH, Q = Br (i) SOCI2 or oxalyl bromide, then RNH, Ila-8: R = (CH2) 3OH, Q = Br lla-9: R = (CH2) 4OH, Q = Br Ha-10: R = (CH2) sOH , Q = Br Ha-11; R = (CH2) 6OH, Q = Br Reaction Scheme 2b (i) SOCl2 / eOH; (ii) diethanolamine; (iii) MsCI / py, then LiBr; (iv) KOH; (v) (COCi cat. DMF, then 2-aminoethanol Reaction Scheme 2c - 8: R4 = OMs. r- 10: R3 = OMs L- ^ IIa-13: R4 = Br, OMs IV - Ha-14: R3 = I (i) S0Cl2 / cat DMF or (COCI) 2 / cat DMF, then 2-amipoetapol (ii) ) S0C (2 / cat DMF or (COBr) 2 / cat DMF, then 3-amylo-1-propanol (iii) UBr (1.4 equiv) / DMF, (iv) Nal (excess) / MeCN.
Reaction Scheme 2d (i) SOCI2 / cát DMF, then RNH2; (I) HN (CH2CH2Cl) 2; (iii) LiBr / 3-Mebuapopa Reaction Scheme 2e (i) diisopropanolamine; (ii) MsCl / EtaN; (iii) 1N HCI / THFr (iv) ü'Br (excess). Reaction Scheme 2f I- 22: 2 = 0H i- 24: R2 = 0Ms OMs ^ llM ^ zOMs.Br (i) 3,4-dMdro-2 £ f-pyran, -TsOH; (ii) diethanolamine; (in) MsCl / Et3N; (iv) lNHCl / THF; (v) LiBr (1.2eq).
Reaction Scheme 2g llb-7 IIb-12 25 Reaction Scheme 2h (i) LÍCI (1,2 eq) 00 Nal or LIBr (1.2 eq) then MeSOaAg (1.5 eq) Reaction Scheme 2i (i) Nal (1.5 eq) Reaction Scheme 2j 26: R = (CH2) 2 OH Hc-12: R = (CH2) 2 OH Hc-7: R = (CH2) 2 OH 27: R = (CH2) 3 OH Hc-13: R = (CH2) 3 OH Hc-8: R = (CH 2) 3 OH 28: R = (CH 2) 4 OH llc-14: R = (CH 2) 4 OH llc-9: R = (CH 2) 4 OH Reaction Scheme 2k Reaction Scheme 3 I I-nE (i) EtaNP (? tBu) 2 or i-Pr2NP (0lBu) 2/1 H-tetrazole, then mCPBA or H202 (ii) TFA / CH2Ci2 In Reaction Scheme 3, X, Y, Z and r are as specified for formula (I) and (II) above.
EXAMPLES The invention and the best mode for practicing it are illustrated by the following examples 1-25 (alcohols) and examples 26-39 (phosphates).
Example 1 (Reaction Scheme 2a). N- (3-Hydroxypropyl) -5- [bis (2-chloroethyl) amino] -2,4-dinitrobenzamide (Ha-3). A suspension of 5- [bis (2-chloroethyl) amino] -2,4-dinitrobenzoic acid [Palmer et al., J. Med. Chem., 1994, 37, 2175] (1) (2.50 g, 7.1 mmol) in S0C12 (20 mL) containing DMF (2 drops) was heated under reflux for 1 hour, then concentrated to dryness under reduced pressure and re-evaporated with benzene. The resulting crude benzoyl chloride was diluted with Me2CO (50 mL) and the cooled solution (-5 ° C) was treated with a cold solution of 3-amino-1-propanol (1.09 g, 14.5 mmol) in water (25 mL ). The reaction mixture was stirred at room temperature for 5 minutes, then diluted with water (25 mL), concentrated to half volume, and extracted with CH2C12 (2x). The organic extract was washed with 0.1 N HCl and water was then treated to give a solid which was chromatographed on silica gel, eluting with EtOAc to give Ha-3 (2.37 g, 82%); p.f. (EtOAc / i-Pr20) 90-91 ° C; NMR XH [(CD3) 2SO] d 8.63 (t, J = 5.6 Hz, 1 H, CONH), 8.53 (s, .l H, H-3), 7.42 (s, 1 H, H-6), 4.46 (t , J = 5.1 Hz, 1 H, OH), 3.82 (t, J = 5.9 Hz, 4 H, N (CH2CH2C1) 2), 3.68 (t, J = 5.9 Hz, 4 H, N (CH2CH2C1) 2), 3.49 (q, J = 6.0 Hz, 2 H, CH2OH), 3.29 (q, partially obscured, J = 5.9 Hz, 2 H, CONHCH2), 1.68 (pent, J = 6.7 Hz, 2 H, CH2CH2CH2). Anal. (C? 4 H 18 Cl 2 N 406) C, H, N, Cl.
Example 2 (Reaction Scheme 2a). N- (3-hydroxypropyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide (IIa-8). A suspension of 5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzoic acid powder (2) (1.10 g, 2.49 mmol) in benzene (170 mL) was treated at 20 ° C with oxalyl bromide (1.10 mL, 11. 7 mmol) and DMF (2 drops). The mixture was stirred at 20 ° C for 2 hours, then concentrated under reduced pressure, and re-evaporated to dryness in the presence of benzene under high vacuum. The resulting acid bromide was dissolved in Me2CO (20 mL) and the solution was treated at -5 ° C with a cold solution of 3-amino-1-propanol (0.39 g, 5.19 mmol) in water (10 mL). The mixture was stirred at room temperature for 5 minutes, then diluted with water and extracted with EtOAc (2x). The organic extract was treated and the resulting residue was chromatographed on silica gel, eluting with EtOAc to give Ha-8 (1.06 g, 85%): m.p. (EtOAc / i-Pr20) 85-86 ° C; NMR XH [(CD3) 2S0] d 8.64 (t, J = 5.6 Hz, 1 H, CONH), 8.53 (s, 1 H, H-3), 7.41 (s, 1 H, H-6), 3.77- 3.64 (m, 8 H, N (CH 2 CH 2 Cl) 2), 4.46 (br s, 1 H, OH), 3.49 (t, J = 6.3 Hz, 2 H, CH20H), 3.33-3.25 (m, partially obscured, 2 H, C0NHCH2), 1.68 (pent, J = 6. 72 Hz, 2 H, CH2CH2CH2). Anal. (C? 4H18Br2N406) C, H, N, Br.
Example 3 (Reaction Scheme 2a). N- (2-hydroxyethyl) -5- [bis- (2-bromoethyl) amino] -2,4-dinitrobenzamide (Ha-7). Similar reaction of acid bromide of 2 with 2-aminoethanol from Ha-7 (0.78 g, 46%): p.f. (MeOH / EtOAc / pet ether.) 151-152 ° C; X H NMR [(CD3) 2 SO] d 8.73 (t, J = 5.7 Hz, 1 H, CONH), 8.53 (s, 1 H, H-) 3), 7.43 (s, 1 H, H-6), 4.76 (t, J = 5.6 Hz, 1 H, OH), 3.77-3.64 (m, 8 H, N (CH2CH2Br) 2), 3.53 '(q , J = 6.0 Hz, 2 H, CH2OH), 3.31 (q, partially obscured, J = 6.1 Hz, 2 H, CONHCH2). Anal. (C13H? 6Br2N4Os) C, H, N, Br.
Example 4 (Reaction Scheme 2a). N- (4-hydroxybutyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide (Ha-9) Similar reaction of acid bromide of 2 with 4-amino-1-butanol in cold Me 2 CO, followed by chromatography on silica gel and elution with EtOAc da Ha-9 (69%) as a yellow solid: m.p. (EtOAc / iPr20) 123-124 ° C; NMR [(CD3) 2SO] d 8.62 (t, J = 5.6 Hz, 1 H), 8.53 (s, 1 H), 7.39 (s, 1 H), 4.39 (t, J = 5.1 Hz, 1 H) , 3.78-3.64 (m, 8 H), 3.47-3.40 (m, 2 H), 3.27-3.20 (m, 2 H), 1.61-1.44 (m, 4 H). Anal. (C15H20Br2N4Os) C, H, N, Br.
Example 5 (Reaction Scheme 2a). N- (5-hydroxypentyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide (Ha-10).
Similar reaction of the acid bromide of 2 with 5-amino-1-pentanol in cold Me 2 CO, followed by chromatography on silica gel and elution with EtOAc gives 10 (66%) as a yellow foam; NMR XH [(CD3) 2S0] d 8.62 (t, J = 5.6 Hz, 1 H), 8.53 (s, 1 H), 7.38 (s, 1 H), 4.34 (t, J = 5.1 Hz, 1 H) , 3.79-3.64 (m, 8 H), 3.44-3.37 (, 2 H), 3.26-3.18 (, 2 H), 1.59-1.29 (m, 4 H). HRMS (FAB) Calculated for C? GH2379Br2N406 [M + H +] m / z 524. 9984, found 524,964.
Example 6 (Reaction Scheme 2a). N- (6-hydroxyhexyl) -5- [5-bis (2-bromoethyl) amino] -2,4-dinitrobenzamide (Ha) -11). Similar reaction of acid bromide of 2 with 6-amino-1-hexanol in cold Me 2 CO, followed by chromatography on silica gel and elution with EtOAc da Ha-11 (72%) as a yellow foam, XE-NMR [(CD3)] 2SO] d 8.61 (t, J = 5.6 Hz, 1 H), 8.53 (s, 1 H), 7.38 (s, 1 H), 4.31 (t, J = 5.2 Hz, 1 H), 3.79-3.64 (m, 8 H), 3.43-3.36 (m, 2 H), 3.27- 3.19 (m, 2 H), 1.58-1.26 (m, 4 H). HRMS (FAB) Calculated for C17H2579Br2N406 [M + H +] m / z 539. 0141, found 539.0137.
Example 7 (Reaction Scheme 2b). 5- [bis (2-bromoethyl) amino] -N- (2-hydroxyethyl) -4- (methylsulfonyl) -2-nitrobenzamide (Ha-7s). 5-Fluoro-4- (methylsulfonyl) -2-nitrobenzoic acid was heated [Atwell et al., ACDD, 1996, 11, 553] (3) in excess SOCl2 / catalytic DMF to provide the acid chloride, which was reacted with dry MeOH to give methyl 5-fluoro-4- (methylsulfonyl) -2-nitrobenzoate (4): m.p. (EtOAc / hexane) 134-135 ° C; NMR XH [(CD3) 2SO] d 8.49 (d, J = 5.9 Hz, 1 H), 8.14 (d, J = 9.3 Hz, 1 H), 3.92 (s, 3 H), 3.46 (s, 3 H). Anal. (C9H8FN06S) C, H, N. A mixture of 4 (1.48 g, 5.34 mmol) in diethanolamine (1.40 g, 13.3 mmol) in DMA (6 mL) was stirred at ° C for 1 hour, and then diluted with EtOAc (60 mL). The solution was washed with brine (2x) and concentrated under reduced pressure. The residue was purified by chromatography on silica gel, eluting with EtOAc / MeOH, followed by recrystallization from EtOAc / iPr20 to give 5- [bis (2-hydroxyethyl) amino [4- (methylsulfonyl) -2-nitrobenzoate methyl (5) (1.41 g, 73%): mp 99-100 ° C; NMR [(CD3) 2SO] d 8.56 (s, 1 H), 7.73 (s, 1 H), 4.62 (t, J = 4.9 Hz, 2 H), 3.89 (s, 3 H), 3.59-3.49 ( m, 8 H), 3.45 (s, 3 H). Anal. (C13H18N208S) C, H, N. A solution of 5 (1.48 g, 4.08 mmol) in dry pyridine (15 mL) was treated dropwise at 0 ° C with MsCl (0.80 mL, 10.3 mmol). The reaction was stirred at 0 ° C for 2 hours, then poured into 10% aqueous NaBr. The resulting crude dimesylate was collected, washed well with water, dried, dissolved in DMF (15 mL) and stirred with NaBr (21.6 g, 25 mmol) at 70 ° C for 1.5 hours. The cooled reaction was poured into water and the resulting solid was purified by chromatography, on silica gel, eluting with CH2C12, then recrystallization from CH2Cl2 / iPr20 to give 5- [bis (2-bromoethyl) amino] -4- ( Methylsulfonyl) -2-nitrobenzoate methyl (6) (1.47 g, 74%): mp 161-162 ° C; NMR XH [(CD3) 2SO] d 8.58 (s, 1 H), 7.94 (s, 1 H), 3.90 (s, 3 H), 3.82 (t, J = 7.0 Hz, 4 H), 3.63 (t, J = 6 9 Hz, 4 H), 3 .48 (s, 3 H). Anal (C? 3H16Br2N2OsS) C, H, N. A solution of 6 (1.00 g, 2.05 mmol) in dioxane / MeOH (1: 1, 20 mL), was treated at 10 ° C with 4 N aqueous KOH (5 mL) and stirred at 10 ° C for 45 minutes. The mixture was acidified to pH 2 with 1 N aqueous HBr, concentrated to a small volume under reduced pressure, and then diluted with saturated aqueous NaBr (20 mL). The resulting semi-solid was isolated and recrystallized twice from MeOH / H2 to give 5- [bis- (2-bromoethyl) amino] -4- (methylsulfonyl) -2-nitrobenzoic acid (7) (0.70 g, 72% ): pf 174-176 ° C; NMR XH [(CD3) 2SO] d 8.50 (s, 1 H), 7.88 (s, 1 H), 3.79 (t, J = 7.0 Hz, 4 H), 3.62 (t, J = 7.0 Hz, 4 H), 3.48 (s, 3 H). Anal.
(C12H14Br2N2OeS) C, H, N. A finely-divided suspension of 7 was treated. (260 mmg, 0.55 mmol) in dry benzene (50 mL) with (COBr) 2 (2.13 mL, 0.20 mmol) and catalytic DMF. The mixture was stirred for 2 hours, then concentrated to dryness under reduced pressure and re-evaporated with benzene under high vacuum. The resulting crude acid bromide was dissolved in Me2CO (10 mL) and treated at -5 ° C with a cold solution of 2-aminoethanol. (101 mg, 1.65 mmol) in water (5 mL). The mixture was stirred at 0 ° C for 5 minutes, then acidified to pH 4 with aqueous HBr 1.
N, and concentrated under reduced pressure. The residue was chromatographed on silica gel, eluting with EtOAc to give Ha-7s (222 mg, 78%): m.p. (? tOAc / iPr20) 126-127 ° C; NMR XH [(CD3) 2SO] d 8.75 (t, J = 5.6 Hz, 1 H), 8.51 (s, 1 h), 7.68 (s, 1 H), 4.79 (t, J = 5.4 Hz, 1 H), 3.76 (t, J = 7.1 Hz, 4 H), 3.62 (t, J = 7.0 Hz, 4 H), 3.54 (q, J = 5.9 Hz, 2 H), 3.48 (s, 3 H), 3.31 (after exchange of D20, t, J = 6.0 Hz, 2 H). HRMS (FAB) Calculated for C? 4H2079Br2N306S (MH +) m / z 515.9440; found 515.9425.
Example 8 (Reaction Scheme 2c). 2- [(2-bromoethyl) -5 - [[(3-hydroxypropyl) amino [carbonyl] -2,4-dinitroanilino] -ethylmethanesulfonate (Ha-13) and 5- [bis (2-iodoethyl) amino] -N - (2-hydroxyethyl) -2,4-dinitrobenzamide (Ha-14). 5- (Bis. {2- 2- [(Methylsulfonyl) oxy] ethyl} amino) -2,4-dinitrobenzoic acid was heated [A method for preparing this compound is described in co-pending NZ application No. 521851] (9) under reflux in excess S0C12 (60 mL) and catalytic DMF for 1 hour. Evaporation under reduced pressure, followed by azeotrope formation in benzene, gives the crude acid chloride. This is dissolved in dry Me2CO and treated at 0 ° C with 3-amino-1-propanol at 0 ° C for 5 minutes. The mixture was acidified to pH 2-3 with 0.2 N HCl, concentrated to half volume, and then solid NaBr was added, followed by extraction with EtOAc (2x). Evaporation, and chromatography of the residue on silica gel, eluting with EtOAc / MeOH (9: 1), gives 2- (5- { [3 (3-hydroxypropyl) amino] carbonyl.}. [(methylsulfonyl) oxy] ethyl} -2,4-dinitroanilino) ethylmethanesulfonate (8) (68%) as a yellow gum; NMR XH [(CD3) 2S0] d 8.54 (t, J = 5.7 Hz, 1 H), 8.53 (S, 1 H), 7.45 (s, 1 H), 4.43 (t, J = 5.1 Hz, 1 H) , 4.33 (t, J = 5.2 Hz, 4 H), 3.69 (t, J = 5.2 Hz, 4 H), 3.5 7 (q, J = . 9 Hz, 2 H), 3.26 (after exchange of D0, t, J = 7.0 Hz, 2 H), 3.12 (s, 6 H), 1.66 (pent, J = 6.7 Hz, 2 H). HRMS (FAB) Calculated for C16H2sN40? 2S (MH +) m / z 529. 0910; found 529. 0904. A solution of 8 in DMF was treated with LiBr (1.4 equivalent) and treated as above, and the product was chromatographed on silica gel. Elution with EtOAc gave a small amount of dibromo mustard, while elution with EtOAc / MeOH (19: 1) gave Ha-13 (31%) as a yellow gum: XH NMR [(CD3) 2SO] d 8.60 (t, J = 5.6 Hz, 1 H), 8.54 (s, 1 H), 7.44 (s, 1 H), 4.45 (t, J = 5.2 Hz, 1 H), 4.33 (t, J = 5.1 Hz, 2 H), 3.74 (t, J = 5.2 Hz, 2 H), 3.72-3.66 (m, 4 H), 3.49 (q, J = 5.9 Hz, 2 H), 3.27 (after change of D20, t, J = 7.0 Hz, 2 H), 3.14 (s, 3 H), 1.68 (pent, J = 6.7 Hz, 2 H). HRMS (FAB) Calculated for C15H2279BrN409S (MH +) m / z 515. 0270; found 515.0283. The similar treatment of the acid chloride of 9 (activation with (COCl) 2 / DMF) with 2-aminoethanol gave 2- (5. {[[(2-hydroxyethyl) amino] carbonyl] { 2- 2- [ (methylsulfonyl) oxy] -ethyl.} -2,4-dinitroanilino) ethylmethanesulfonate (10). A stirred mixture of 10 (1.42 g, 2.76 mmol) and Nal (3.3 g, 22 mmol) in dry MeCN (45 mL) was heated under reflux for 1 hour, then concentrated under reduced pressure. The residue was partitioned between EtOAc and water, and the organic layer was washed with water and evaporated The residue was chromatographed on silica gel, eluting with CH2Cl2 / EtOAc (1: 4), followed by recrystallization from MeOH / EtOAc / i-Pr20 to give IIa-14 (2.9 g, 81%): m.p. 142-143 ° C; NMR [(CD3) 2SO] d 8.73 (t, J = 5.7 Hz, 1 H), 8.53 (s, 1 H), 7.38 (s, 1 H), 4.76 (t, J = 5.5 Hz, 1 H) , 3.68 (t, J = 6.9 Hz, 4 H), 3.57-3.49 (m, 2 H), 3.39 (t, J = 6.9 Hz, 4 H), 3.34-3.26 (m, partially obscured, 2 H). Anal. (C? 3H16I2N4Os) C, H, N.
Example 9 2- (aziridin-1-yl) -N- (6-hiroxyhexyl) -3,5-dinitrobenzamide (IIA-1). A solution of 2-chloro-N- (6-hydroxyhexyl) -3,5-dinitrobenzamide (16) [for preparation see Example 14 below] (118 mg, 0.34 mmol) and Et3N (200 mg) in EtOAc (200 mL) it was treated with aziridine (100 mg) at room temperature for 3 hours. The mixture was diluted with EtOAc and washed three times with water, then dried, concentrated under reduced pressure to about 20 mL, the yellow solid was collected and gave 101 mg of product (84%); RMN ^? [(CD3) 2SO] d 8.74 (d, J = 2.8 Hz, 1 H), 8.63 (m, 1 H), 8.29 (d, J = 2.8 Hz, 1 H), 4.31 (m, 1 H), 3.39 (m, 2 H), 3.25 (, 2 H), 2.37 (s, 4 H), 1.56 (m, 2 H), 1.43 (m, 2 H), 1.33 (m, 4 H). Anal. (C? 5H2oN4Oe) C, H, N.
Example 10 (Reaction Scheme 2d) 2- [bis (2-chloroethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide (Hb- 2) and 2- [bis (2-bromoethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide (Hb-7). 2-Chloro-3,5-dinitrobenzoic acid (1) (18 g, 81 mmol) was treated with S0C12 (250 mL) containing a drop of DMF and heated under reflux for 6 hours. Evaporation of the reagent followed by azeotrope formation with toluene gave the crude acid chloride, which was dissolved in THF (200 mL) and a solution made of 25 mL of 2-aminoethanol in THF (400 mL) was added dropwise and it was cooled with a dry ice-acetone bath. After stirring for 20 minutes, the reaction mixture was acidified to pH 4-5 with 1 N HCl, the majority of the solvent was evaporated, and the residue was partitioned between water (250 mL) and EtOAc (300 mL). The aqueous phase was extracted with EtOAc, and the combined organic phases were washed with saturated NaHCO 3, 1 N HCl and brine respectively, then concentrated to give 2-chloro-N- (2-hydroxyethyl) -3,5-dinitrobenzamide (2 21.34 g (91 %) as a white solid: m.p. (EtOAc) 159-160 ° C; NMR XH [(CD3) 2S0] d 8.99 (d, J = 2.6 Hz, 1 H, H-4), 8.86 (m, 1 H, CONH), 8.56 (d, J = 2.6 Hz, 1 H, H-6), 4.83 (, 1 H, OH), 3.54 (m, 4 H). Anal. (C9H8CIN306) C, H, N. A solution of 12 (1.52 g, 5.3 mmol) and Et3N (4 mL) in p-dioxane (60 mL) was treated with N, N-bis (2-chloroethyl) amine hydrochloride (3.0 g, 16.5 mmol) at 50 ° C for 24 hours. The mixture was poured into water and extracted with EtOAc to give the crude product, which was chromatographed on silica gel. The elution with EtOAc / petroleum ether (4: 1) and the concentration of the product eluted under reduced pressure gave an oily residue which was dissolved in a minimum amount of EtOAc. Petroleum ether was added and slowly to incipient cloudiness, and the solution was allowed to stand overnight to precipitate 2- [bis (2-chloroethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide (Hb-). 2) (2.07 g, 100%) as yellow crystals: pf (EtOAc / petroleum ether) 109-111 ° C; NMR [(CD3) 2 SO] d 8.73 (d, J = 2.6 Hz, 1 H, H-4), 8.72 (m, 1 H, CONH), 8.34 (d, J = 2.6 Hz, 1 H, H- 6), 4.83 (m, 1 H, OH), 3.72 (m, 4 H, 2 x CH 2 Cl), 3.55 (m, 2 H), 3.42 (m, 4 H, 2 x CH 2 N), 3.34 (m, 2 H); 13 C NMR 165.3, 145.8, 145.3, 141.0, 136.3, 127.5, 122.1, 59.1, 54.1, 42.1, 41.5. HRMS (FAB) [MH +] Calculated for C13H? 735Cl2N4Os m / z 395.0525. Found; 395.0525. A solution of Hb-2 (1.20 g, 3.0 mmol) and LiBr (5.0 g, 58 mmol) in 3-methyl-2-butanone (20 mL) was heated under reflux for 6 hours, then cooled and poured into water. Extraction with EtOAc gave a crude product (<95% pure), which was re-treated with LiBr (5.0 g, 58 mmol) in 3-methyl-2-butanone for an additional 4 hours, then treated and gel chromatographed. of silica, eluting with EtOAc / petroleum ether (from 1: 1 to 1: 0) to give Hb-7 (1.39 g, 95%): mp (? tOAc / petroleum ether) 105-108 ° C XH NMR [(CD3) 2SO] d 8.74 (d, J = 2.7 Hz, 1 H, H-4), 8.73 (m, 1 H, CONH), 8.34 (d, J = 2.7 Hz, 1 H, H-6), 4.83 (m, 1 H, OH), 3.59-3.29 (m, 12 H); 13 C NMR 165.3, 145.4, 145.3, 141.1, 136.5, 127.4, 122.1, 59.3, 53.9, 42.1, 30.0. HRMS (FAB) Calculated for C? 3 H1779Br2N4O? [M + H +] m / z 482.9515. Found; 482.9492. Anal. (C 13 H 16 Br 2 N 406) H, N, Br, C: found 32.9; calculated A * o.
Example 11 (Reaction Scheme 2d) 2- [bis (2-chloroethyl) amino] -N- (3-hydroxypropyl) -3,5-dinitrobenzamide (Hb-3) and 2- [bis (2-bromoethyl) aino] -N- (3-hiroxypropyl) -3,5-dinitrobenzamide (Hb-8). Reaction of the acid chloride of 11 (17 g) with 3-aminopropanol (7.5 g) in Me 2 CO (120 mL) at 0 ° C as described above, gave 2-chloro-N- (3-hydroxypropyl) -3, 5-dinitrobenzamide (13) (5.06 g, 26%): mp (EtOAc / petroleum ether) 120-121 ° C; NMR [(CD3) 2 SO] d 8.99 (d, J = 2.6 Hz, 1 H, H-4), 8.79 (m, 1 H, CONH), 8.51 (d, J = 2.6 Hz, 1 H, H- 6), 4.50 (m, 1 H, OH), 3.49 (m, 2 H), 3.32 (m, 2 H), 1.70 (m, 2 H). Anal. (C? 0H10ClN3O6) C, H, N. A solution of 13 (1.39 g, 4.58 mmol) and Et3N (4 mL) in p-dioxane (60 mL) was treated with N, N-bis (2-chloroethyl) hydrochloride. ) amine (2.9 g, 16.0 mmol) at 50 ° C for 24 h.Treatment as described above gave Hb-3 (1.84 g, 100%): m.p. (EtOAc / petroleum ether) 89-91 ° C; NMR XH [(CD3) 2SO] d 8.74 (d, J = 2.7 Hz, 1 H, H-4), 8.71 (m, 1 H, CONH), 8.30 (d, J = 2.7 Hz, 1 H, H-6) , 4.52 (m, 1 H, OH), 3. 71 (m, 4 H, 2 x CH 2 Cl), 3.50 (m, 2 H), 3.42 (m, 4 H, 2 x CH 2 N), 3.32 (m, 2 H), 1.71 (m, 2 H); 13 C NMR 165.1, 145.7, 145.5, 141.0, 136.4, 127.3, 122.1, 58.4, 54.1, 41.5, 36.7, 31.8. HRMS (FAB) Calculated for C 14 H 19 35 C 12 N 406 [M + H +] m / z 409.0682. Found; 409.0678. Treatment of Hb-3 with LiBr in 3-methyl-2-butanone twice, as described above, gave I Ib- 8 (74% yield): p.f. (EtOAc / petroleum ether) 89-94 ° C; NMR [(CD3) 2SO] d 8.74 (d, J = 2.7 Hz, 1 H, H-4), 8.72 (m, 1 H, CONH), 8.30 (d, J = 2.7 Hz, H, H-6) ), 3.77-3.44 (m, 12 H), 1.70 (, 2 H); 13 C NMR 165.1, 145.5, 145.3, 141.2, 136.5, 127.3, 122.1, 58.4, 54.0, 36.7, 31.8, 29.9. HRMS (FAB) Calculated for C14H? 979Br2N0ß [M + H +] m / z 496.9671. Found; 496.9658.
Example 12 (Reaction Scheme 2d). 2- [bis (2-chloroethyl) amino] -N- (4-hydroxybutyl) -3,5-dinitrobenzamide (Hb-4) and 2- [bis) (2-bromoethyl) amino] -N- (4-hydroxybutyl) -3,5-dinitrobenzamide (IIb-9). The acid chloride reaction of 11 (2.65 g, 10 mmol) with 4-aminobutanol (1.9 g) as before, followed by acidification to pH 4.-5 with 1 N HCl and evaporation of most of the solvent gives a residue. This was partitioned between water (50 mL) and EtOAc (100 mL). The aqueous phase was extracted with EtOAc, and the combined organic phase was washed with saturated NaHCO3, 1 N HCl and brine, respectively, then concentrated to give 2-chloro-N- (4-hydroxybutyl) -3,5-dinitrobenzamide (14) 11.11 g (35%): m.p. (EtOAc) 121-124 ° C; NMR [(CD3) 2 SO] d 8.98 (d, J = 2.7 Hz, 1H), 8.79 (m, 1 H), 8.52 (d, J = 2.7 Hz, HH), 4.43 (m, 1 H), 3.43. (m, 2H), 3.26 (m, 2 H), 1.54 (m, 4 H); 13 C NMR 162.6, 148.4, 145.9, 140.4, 128.2, 125.8, 120.4, 60.2, 39.1, 29.8, 25.3. Anal. (CnH12CIN3Os) C, H, N. A solution of 14 (0.75 g, 2.3 mmol) and Et3N (2 mL) in p-dioxane (30 mL) was treated with N, N-bis (2-chloroethyl) amine hydrochloride. (1.5 g, 8.0 mmol) at 50 ° C for 24 hours. The mixture was poured into water and extracted with EtOAc to give the crude product which was chromatographed on silica gel. Elution with EtOAc / petroleum ether (4: 1) gave Hb-4 (0.99 G, 100%) as a yellow foam NMR XH [(CD3) 2S0] d 8.71 (d, J = 2.8 Hz, 1 H), 8.69 (m, 1 H), 8.27 (d, J = 2.8 Hz, 1 H), 4.37 (m, 1 H), 3.70 (m, 4 H), 3.38 (m, 6 H), 3.25 (, 2 H) ), 1.56 (m, 2 H), 1.47 (m, 2 H); 13 C NMR 165.0, 145.7, 145.5, 141.0, 136.4, 127.2, 122.0, 60.2, 54.2, 41.5, 39.2, 29.8, 25.2. HRMS (FAB) Calculated for C15H2135Cl2N4Oe [M + H +] m / z 423.0838. Found; 423.0847. A solution of Hb-4 (0.96 g, 3.04 mmol) and LiBr (5 g) in 3-methyl-2-butanone (15 mL) was heated under reflux for 6 hours, then cooled and poured into water. Extraction with EtOAc gave a crude product (<95% pure), which was re-treated with LiBr (5 g) in 3-methyl-2-butanone for 4 hours. additional hours, then treated and chromatographed on silica gel, eluting with EtOAc / Petroleum ether (from 1: 1 to 3: 1) to give Hb-9 (1.01 g, 87%) as a yellow foam; NMR [(CD3) 2SO] d 8.74 (d, J = 2.8 Hz, 1 H), 8.72 (m, 1 H), 8.28 (d, J = 2.8 Hz, 1 H), 3.60-3.26 (m, 12 H), 1.58 (m, 2 H), 1.49 (m, 2 H); 13 C NMR 165.0, 145.6, 145.2, 141.2, 136.5, 127.2, 122.0, 60.2, 54.1, 39.2, 29.9, 29.8, 25.2. HRMS (FAB) Calculated for C15H2? 79Br2N4? 6 [M + H +] m / z 510.9828. Found; 510.9832.
Example 13 (Reaction Scheme 2d). 2- [Bis (2-chloroethyl) amino] -N- (5-hydroxypentyl) -3,5-dinitrobenzamide (Hb-5) and 2- [bis (2-bromoethyl) amino] -N- (5-hydroxypentyl) -3,5- dinitrobenzamide (Hb-10). Similar reaction of the acid of ll with 5-aminopentanol as before gave 2-chloro-N- (5-hydroxy-pentyl) -3,5-dinitrobenzamide (15), 1.3 g (39%), m.p.
(EtOAc) 105 = 108 ° C; NMR XH [(CD3) 2 SO] d 8.98 (d, J = 2.7 Hz, ÍH), 8.79 (m, 1 H), 8.50 (d, J = 2.7 Hz, 1 H), 4.35 (m, 1 H), 3. 39 (m, 2 H), 3.26 (m, 2 H), 1.54 (m, 2 H), 1.44 (m, 2 H), ° 1.36 (m, 2 H); 13 C NMR 162.7, 148.4, 145.9, 140.4, 128.2, 125.8, 120.4, 60.5, 39.1, 32.0, 28.4, 22.8. Anal. (C12H14C1N306) C, H, N. A solution of 15 (0.63 g, 2.3 mmol) and Et3N (2 mL) in p-dioxane (30 mL) was treated with N, N-bis (2-5 chloroethyl) hydrochloride amine (1.5 g, 8.0 mmol) at 50 ° C for 24 hours.
The mixture was poured into water and extracted with EtOAc to give the crude product which was chromatographed on silica gel. Elution with EtOAc / petroleum ether (4: 1) gave Hb-5 (0.82 g, 100%) as a yellow foam; NMR [(CD3) 2SO] d 8.73 (d, J = 2.8 Hz, 1 H), 8.69 (m, 1 H), 8.28 (d, J = 2.8 Hz, 1 H), 4.32 (m, 1 H) , 3.70 (m, 4 H), 3.40 (m, 6 H), 3.25 (m, 2 H), 1.55 (m, 2 H), 1.47 (m, 2 H), 1.37 (m, 2 H); 13 C NMR 165.0, 145.7, 145.5, 141.0, 136.4, 127.2, 122.0, 60.5, 54.2, 41.5, 39. 3, 32.0, 28.3, 22.9. HRMS (FAB) Calculated for CleH2335Cl2N4Os [M + H +] m / z 437.0995. Found; 437.0991. The similar reaction of Hb-5 (1.3 g) with LiBr gave Hb-10 (1.35 g, 86%) as a yellow foam; NMR [(CD3) 2SO] d 8.74 (d, J = 2.8 Hz, 1 H), 8.71 (m, 1 H), 8.28 (d, J = 2.8 Hz, 1 H), 3.60-3.26 (m, 12 H), 1.55 (m, 2 H), 1.48 (m, 2 H), 1.37 (m, 2 H); NMR X3C d 165.0, 145.6, 145.2, 141.2, 136.5, 127.2, 122.0, 60.5, 54.1, 39.3, 32.0, 29.8, 28. 4, 22.9. HRMS (FAB) Calculated for C16H2379Br2N406 [M + H +] m / z 524.9984. Found; 524,999.
Example 14 (Reaction Scheme 2d). 2- [Bis (2-chloroethyl) amino] -N- (6-hydroxyhexyl) -3,5-dinitrobenzamide (Hb-6) and 2- [bis (2-bromoethyl) amino] -N- (6-hydroxyhexyl) -3,5-dinitrobenzamide (Hb-11). The similar reaction of the acid chloride of 11 with 6-aminohexanol as above gave 2-chloro-N- (6-hydroxyhexyl) -3,5-dinitrobenzamide (16), 0.9 g (26%), m.p.
(EtOAc) 88-91 ° C; NMR [(CD3) 2SO] d 8.98 (d, J = 2.7 Hz, HH), 8.78 (m, 1 H), 8.49 (d, J = 2.7 Hz, 1 H), 4.32 (m, 1 H), 3.39 (m, 2H), 3.26 (m, 2 H), 1.54 (m, 2 H), 1.44 (m, 2 H), 1.34 (m, 4 H); NMR X3C d 162.7, 148.4, 145.9, 140.4, 128.2, 125.8, 120.4, 60.5, 39.1, 32.3, 28.6, 26.2, 25.1. Anal. (C13H? 6ClN3Oe) C, H, N. A solution of 16 (0.67 g, 2.5 mmol) and Et3N (2 mL) in p-dioxane (30 mL) was treated with N, N-bis (2-chloroethyl) hydrochloride ) amine (1.5 g, 8.0 mmol) at 50 ° C for 24 hours. The mixture was poured into water and extracted with EtOAc to give the crude product which was chromatographed on silica gel, elution with EtOAc / petroleum ether (4: 1) gave Hb-6 (0.87 g, 100%) as a yellow foam; NMR [(CD3) 2 SO] d 8.73 (d, J = 2.8 Hz, 1 H), 8.70 (m, 1 H), 8.28 (d, J = 2.8 Hz, 1 H), 4.31 (m, 1 H) , 3.70 (m, 4 H), 3.38 (m, 6 H), 3.25 (m, 2 H), 1.54 (m, 2 H), 1.40 (m, 2 H), 1.32 (m, 4 H); NMR X3C d 165.0, 145.7, 145.6, 141.0, 136.4, 127.2, 122.0, 60.5, 54.2, 41.5, 39.2, 32.3, 28.5, 26.3, 25.1. HRMS (FAB) Calculated for Cl? 7H2535Cl2N4Os [M + H +] m / z 451.1151. Found; 451.1154. The similar reaction of Hb-6 (0.97 g) with LiBr gave Hb-11 (0.96 g, 81%) as a yellow foam; NMR XH [(CD3) 2SO] d 8.74 (d, J = 2.8 Hz, 1 H), 8.70 (m, 1 H), 8.28. (d, J = 2.8 Hz, 1 H), 3.60-3.26 (m, 12 H), 1.54 (m, 2 H), 1. 43 (m, 2 H), 1.32 (m, 4 H); NMR X3C d 165.0, 145.6, 145.2, 141.2, 136.5, 127.2, 122.0, 60.6, 54.1, 39.2, 32.4, 29.9, 28. 5, 26.3, 25.1. HRMS (FAB) Calculated for C? 7H2579Br2N406 [M + H +] m / z 539.0141. Found; 539.0135.
Example 15 (Reaction Scheme 2e). 2- [Bis (2-bromopropyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide (Hb-7a). The reaction of 2-chloro-3,5-dinitro-N- [2- (tetrahydro-2H-pyran-2-yloxy) ethyl] benzamide (17) (1.02 g) [For preparation method see NZ request copending No .521851] with diisopropanolamine (0.8 g) as before gave 2- [bis (2-hydroxypropanyl) amino] -3,5-dinitro-N- [2- (tetrahydro-2H-pyran-2-yloxy) ethyl] benzamide ( 18) (1.29 g, 100%): as a yellow foam; NMR XH [(CD3) 2S0] d 9.22 (br, HH), 8.66 (d, J = 2.8 Hz, HH), 8.29 (d, J = 2.8 Hz, HH), 4.99 (m, 1 H), 4.85 ( br, 1 H), 4.62 (br, 1 H), 3.94 (m, 2 H), 3.77 (m, 2 H), 3.53 (m, 4 H), 3.26 (m, 2 H), 1.48 (m, 10 H), 0.98 (m, 6 H); NMR X3C d 166.5, 147.8, 142.4, 138.2, 132.6, 128.8, 123. 8, 98.1, 64.8, 63.5, 61.5, 60.1, 30.1, 25.0, 20.5, 20.2, 19 .1. HRMS (FAB) Calculated for C20H31N4O9 [M + H +] m / z 471.2091.
Found; 471.2089. Reaction of 18 with MsCl as before gave 1-methyl-2- [. { 2- [(methylsulfonyl) oxy] propyl} -2,4-dinitro-6- ( { [2- (tetrahydro-2H-pyran-2-yloxy) ethyl] amino} carbonyl) anilino] ethyl methanesulfonate (19) (2.52 g, 100%): as a yellow foam; which was used directly for the next step. A solution of 19 (2.52 g, 4.03 mmol) in THF (150 mL) was treated with 1 N HCl (100 mL), and the solution was stirred at 20 ° C for 1 hour, then diluted with water (100 mL), neutralized with saturated NaHCO 3 and extracted with EtOAc (3x80 mL ). The combined organic phases were washed with brine, dried, the solvent was evaporated, and the residue was purified by chromatography on silica gel, eluting with EtOAc / MeOH (100: 1), to give 2- (2-. {[[ (2-hydroxyethyl) amino] carbonyl.} { 2 - [(methylsulfonyl) oxy] propyl} -4-6-dinitroanilino) -1-methylethyl methanesulfonate (20) (0.80 g, 37%): as a yellow foam; NMR [(CD3) 2SO] d 8.94 (m, 1 H), 8.72 (m, 1 H), 8.35 (m, 1 H), 4.92 (m, 2 H), 3.56 (m, 2 H), 3.30 (m, 6 H), 3.16 (s, 6 H), 1.32 (m, 6 H); NMR X3C d 165.9, 145.8, 143.4, 139.4, 133.6, 128.0, 123.1, 76.3, 59.2, 57.3, 42.2, 37.7, 18.6. HRMS (FAB) Calculated for C17H27N4O? 2S2 [M + H +] m / z 543.1067. Found; 543.1074. Treatment of 20 (0.52 g, 0.96 mmol) with LiBr (0.5 g, 5.8 mmol) in EtOAc (50 mL) at 60 ° C for 3 hours, and chromatography of the product on silica gel, eluting with EtOAc / petroleum ether (from 2: 1 to 1: 0) gave Hb-7a (0.31 g, 62%): as a yellow solid: mp (EtOAc / petroleum ether) 127-130 ° C; XR NMR [(CD3) 2SO] d 8.91 (m, 1H, CONH), 8.70 (d, J = 2.8 Hz, 1H, H-4), 8.32 (d, J = 2.8 Hz, ÍH, H-6), 4.80 (m, 1 H), 4.42 (m, 2 H), 3.55 (m, 4 H), 1.62 (m, 6 H); NMR X3C d 165.8, 144.8, 143.5, 139.6, 133.6, 128.0, 122.9, 60.6, 59.2, 47.9, 42.2, 23.4. Anal. (C15H2oBr2N4Os) C, H, N.
Example 16 (Reaction Scheme 2f). 2- ((2-Bromoethyl) -2- { [(2-hydroxypropyl) amino] carbonyl} -4-6-dinitroanilino) ethyl methanesulfonate (Hb-13). A solution of 13 (1.22 g, 4.0 mmol) in 50 mL of CH2C12 was cooled in an ice bath, and 3,4-dihydro-2H-pyran (1.0 mL) and p-toluenesulfonic acid (0.1 g) were added. The reaction mixture was stirred for 2 hours, then concentrated under reduced pressure. Chromatography of the residue on silica gel, eluting with EtOAc / petroleum ether (from 1: 2 to 2: 1), gave 2-chloro-3,5-dinitro-N- [2- (tetrahydro-2H-pyran 2-yloxy) propyl] benzamide (21) (1.45 g, 94%): as a pale yellow oil; NMR XH [(CD3) 2S0] d 8.99 (d, J = 2.7 Hz, 1 H, H-4), 8.81 (m, 1 H, CONH), 8.51 (d, J = 2.7 Hz, 1 H, H-6), 4.57 (m, 1 H), 3.72 (, 2 H), 3.46-3.25 (m, 4 H), 1.82-1.44 (m 8 H). NMR X3C d 162.7, 148.4, 145.9, 140.3, 128.2, 125.8, 120.5, 98.0, 64.2, 61.3, 36.5, 30.2, 28.9, 24.9, 19.1. HRMS (FAB) Calculated for C? SH1935CHN3? 7 [M + H +] m / z 388.0912. Found; 388.0915. The reaction of 21 (1.45 g, 3.75 mmol) with diethanolamine (1.67 g) as before gave 2- [bis (2-hydroxyethyl) amino] -3,5-dinitro-N- [2- (tetrahydro-2H-pyran- 2-yloxy) propyl] benzamide (22) (1.62 g, 95%): as a yellow foam; NMR XH [(CD3) 2SO] d 8.96 (m, HH, CONH), 8.66 (d, J = 2.8 Hz, HH, H-4), 8.31 (d, J = 2.8 Hz, HH, H-6), 4.95 (m, 2H), 4.56 (m, ÍH), 3.79-3.16 (m, 14H), 1.80-1.45 (m, 8 H); NMR X3C d 166.2, 148.1, 143.6, 139.3, 133.8, 128.9, 123.8, 98.5, 64.8, 61.7, 58.5, 54.6, 37.3, 30.6, 29.2, 25.4, 19.6. HRMS (FAB) Calculated for C? 9H29N406 [M + H +] m / z 457.1935. Found; 457.1939. The reaction of 22 (1.62 g, 3.55 mmol) with MsCl (2 mL) as before gave 2- [. { 2- [(methylsulfonyl) oxy] ethyl} 4,6-dinitro-6- ( { [2- (tetrahydro-2H-pyran-2-yloxy) propyl] -amino.} Carbonyl) anilino] ethyl methanesulfonate (23) (2.17 g, 100%) : like a yellow foam; NMR [(CD3) 2 SO] d 8.71 (d, J = 2.8 Hz, HH), 8.71 (m, HH), 8.31 (d, J = 2.8 Hz, 1H), 4.26 (m, 4 H), 3.71- 3.37 (m, 10 H), 3.13 (s, 6 H), 3.10 (m, 2 H), 1.82-1.43 (m, 8 H); X3C NMR 165.1, 146.3, 145.4, 140.9, 135.9, 127.4, 122.2, 98.0, 67.2, 64.3, 51.4, 45.7, 36.5, 30.2, 28.7, 24.9, 19.1, 8.5. HRMS (FAB) Calculated for C 21 H 33 N 4 O 3 S 2 [M + H +] m / z 613.1486. Found; 613.1481. A solution of 23 (2.95 g, 3.55 mmol) in THF (120 mL) was treated with 1 N HCl (80 mL), and the solution was stirred at 20 ° C for 1 hour, then diluted with water (100 mL) , neutralized with saturated NaHCO3, and extracted with EtOAc (3x80 mL). The combined organic phases were washed with brine and dried, the solvent was evaporated, and the residue was purified by chromatography on silica gel, eluting with EtOAc / MeOH (100: 1), to give 2- (2-. [(3-hydroxypropyl) amino] carbonyl, {.2- [(methylsulfonyl) oxy] ethyl} - 4,6-dinitroanilino) ethyl methanesulfonate (24) (1.4 g, 75%): as a yellow solid: m.p. (EtOAc / petroleum ether) 130-133 ° C; NMR [(CD3) 2 SO] d 8.74 (d, J = 2.8 Hz, 1H), 8.72 (m, HH), 8.32 (d, J = 2.8 Hz, 1H), 4.29 (m, 4 H), 3.47 ( m, 8 H), 3.14 (s, 6 H), 1.71 (m, 2 H); NMR X3C d 165.2, 146.3, 145.3, 140.8, 135.9, 127.5, 122.3, 67.3, 58.4, 51.4, 36.8, 36.5, 31.7. Anal. (C? GH24N4012S2) C, H, N. The treatment of 24 (0.25 g, 0.45 mmol) with LiBr (53 mg, 0.61 mmol) in EtOAc (50 mL) at 60 ° C for 3 hours, and chromatography of the product on silica gel, eluting with EtOAc / petroleum ether (from 2: 1 to 1: 0) gave Hb-13 (0.16 g, 66%): as a yellow foam; NMR XH [(CD3) 2SO] d 8.74 (d, J = 2.8 Hz, ÍH), 8.73 (m, 1H), 8.31 (d, J = 2.8 Hz, ÍH), 4.28 (m, 2 H), 3.65-3.44 (m, 10 H), 3.13 (s, 3 H), 1.70 (m, 2 H); NMR X3C d 165.1, 145.7, 145.4, 141.0, 136.2, 127.3, 122.1, 67.5, 58.4, 51.1, 36.7, 36.5, 31.7, 29.6. HRMS (FAB) Calculated for d5H2279BrN409S [M + H +] m / z 513.0291. Found; 513.0281.
Example 17 (Reaction Scheme 2g). 2- ((2-bromoethyl) -2- { [(2-hydroxyethyl) amino] carbonyl} -4,6-dinitroanilino) ethyl methanesulfonate (Hb-12). The solid llb-7 '(300 mg, 0.62 mmol) and silver methanesulfonate (130 mg, 0.65 mmol) in dry MeCN (15 mL) were heated under reflux for 3 hours, then cooled and filtered. The solid AgBr was washed with EtOAc to give a 98% yield of AgBr. He The solvent was removed under reduced pressure and the residue was separated by chromatography on silica gel, eluting with EtOAc / petroleum ether (from 1: 1 to 1: 0), to give successively: - starting material (Hb-7) ( 28 mg, 9%). - Hb-12 (123 mg, 38%) as a yellow foam; NMR [(CD3) 2SO] d 8.77 (m, 1 H, CONH), 8.74 (d, J = 2.7 Hz, 1 H, H-4), 8.36 (d, J = 2.7 Hz, 1 H, H- 6), 4.28 (m, 2 H, CH2OMs), 3.58 (m, 4 H), 3.44 (m, 4 H), 3.14 (s, 3 H, OS02CH3); X3C NMR d 165.3, 145.8, 145.2, 140.9, 135.1, 127.5, 122.2, 67.5, 59.2, 54.2, 51.0, 42.1, 36.4, 29.7; HRMS m / z required for C14H2079BrN409S 499.01344; Found 499.01324. The column was eluted finally with EtOAc / MeOH (9: 1) to give 2- (2. {[[(2-hydroxyethyl) amino] carbonyl]. {2 - [(methylsulfonyl) oxy] ethyl}. -4, 6-dinitroanilino) ethyl-methanesulfonate 25 (159 mg, 53%) as a yellow solid: mp 128-132 ° C (EtOAc / petroleum ether); X H NMR [(CD3) 2 SO] d 8.78 (m, 1 H, CONH), 8.74 (d, J = 2.7 Hz, 1 H, H-4), 8.36 (d, J = 2.7 Hz, 1 H, H- 6), 4.29 (m, 4 H, 2xCH2OMs), 3.56 (m, 2 H), 3.45 (m, 6 H), 3.14 (s, 6 H, 2xOS02CH3); NMR X3C d 165.4, 146.3, 145.1, 140.6, 135.8, 127.6, 122.3, 67.3, 59.2, 51.3, 42.1, 36.4; HRMS: C15H23N40X2S2 required m / z 515.0754. Found: 515.0744.
Example 18 (Reaction Scheme 2h). 2- ((2-Chloroethyl) -2- { [(2-hydroxyethyl) amino] carbonyl} -4-6-dinitroanilino) ethyl methanesulfonate (Hb-2m). A solution of 25 (5.3 g, 10.3 mmol) in DMF (100 mL) was treated with LiCl (524 mg, 12.4 mmol) at 60 ° C for 2 hours, and then cooled, poured into dilute HCl and extracted with EtOAc (3x150 mL). Treatment and chromatography of the product on silica gel, eluting with EtOAc / petroleum ether from 1: 1 to 1: 0, gave Hb-2 (2.4 g, 59%), and then Hb-2m (1.94 g, 41% ) as a yellow solid; NMR XH [(CD3) 2SO] d 8.77 (m, HH, CONH), 8.74 (d, J = 2.7 Hz, HH, H-4), 8.36 (d, J = 2.7 Hz, 1H, H-6), 4.28 (m, 2H, -CH20-Ms), 3.58 (m, 4H), 3.44 (m, 4H), 3.14 (s, 3 H, - OS02CH3); X3C NMR d 165.3, 145.8, 145.2, 140.9, 135.1, 127.5, 122.2, 67.5, 59.2, 54.2, 51.0, 42.1, 36.4, 29.7. Hb-2 was prepared by an alternative method as follows: A solution of Hb-2 (12.50 g, 31.6 mmol) in 3-methyl-2-butanone (150 mL) was treated at 25 ° C with Nal (5.69 g, 38.0 mmol) and the mixture was stirred at 70 ° C for 2 hours and then concentrated under reduced pressure. The residue was partitioned between water (250 mL) and EtOAc (250 mL) and the organic layer was washed with water, dried (NaS0) and then concentrated under reduced pressure. The resulting oil (15.23 g) was dissolved in CH 3 CN (80 mL), treated with silver methanesulfonate (9.63 g, 47.4 mmol) and the mixture was stirred at 25 ° C for 1 hour and then concentrated under reduced pressure. The residue was extracted with EtOAc (200 mL), filtered, the solids were washed with EtOAc (100 mL) and the EtOAc solution was evaporated and the mixture was evaporated.
Oily was separated by chromatography on silica gel as before and gave starting material (3.61 g, 29%), Hb-2m (4.55 g, 32%) and 25 (4.98 g, 31%). When Nal was replaced by LiBr, the reaction gave a similar result.
Example 19 (Reaction Scheme 2i). 2- [Bis (2-iodoethyl) amino] N- (2-hydroxyethyl) -3,5-dinitrobenzamide (Hb-14) and 2- ((2-iodoethyl) -2- { [(2-hydroxyethyl) amino] carbonyl, 4, 6-dinitroanilino) ethyl methanesulfonate (Hb-15). Treatment of 25 (6.7 g, 13.0 mmol) with Nal (2.9 g, 20 mmol) in EtOAc (200 mL) at 60 ° C for 3 hours, and chromatography of the product on silica gel, eluting with EtOAc / petroleum ether (from 2: 1 to 1: 0) gave Hb-14 (3.3 g, 44%) as a yellow solid: m.p. (EtOAc / petroleum ether) 129-131 ° C; NMR [(CD3) 2 SO] d 8.72 (d, J = 2.8 Hz, 1 H, H-4), 8.70 (m, 1 H, CONH), 8.32 (d, J = 2.8 Hz, 1 H, H-6), 4.80 (m, 1 H), 3.55 (m, 2 H), 3.43 (, 4 H), 3.31 (m, 6 H); 13 C NMR 165.3, 145.2, 144.7, 141.0, 136. 3, 127.3, 122.0, 59.3, 54.7, 42.1, 2.94. Anal (C13H16N4I2Oe) C, H, N. Finally the eluted products gave Hb-15 (1.35 g, 19%) as a yellow foam; NMR XH [(CD3) 2S0] d 8.74 (d, J = 2.8 Hz, HH, H-4), 8.74 (m, HH, CONH), 8.34 (d, J = 2.8 Hz, 1 H, H-6) , 4.28 (m, 2 H), 3.56 (m, 2 H), 3.43 (m, 2 H), 3.31 (m, 6 H), 3.13 (s, 3 H); NMR X3C d 165.3, 145.5, 145.2, 140.8, 136.1, 127.4, 122.1, 67.5, 59.2, 55.4, 50.6, 42. 1, 36.5, 2.6. HRMS (FAB) Calculated for C14H20IN409S [M + H +] m / z 546.9996. Found; 546,997.
Example 20 (Reaction Scheme 2j). 3- [Bis (2-bromoethyl) amino] -N- (2-hydroxyethyl) -2,6-dinitrobenzamide (IIc-7) and 2- ((2-bromoethyl) -3-. {[[(2-hydroxyethyl)] ) amino] -carbonyl.} -2,4-dinitroanilino) ethyl methanesulfonate (Hc-12). The treatment of 2- (3. {[[(2-hydroxyethyl) amino] -carbonyl.}. {2- [(Methylsulfonyl) oxy} ethyl} -2,4-dinitroanilino) -ethyl-methanesulfonate (26) [for preparation method see NZ application No. 521851] (310 mg, 0.6 mmol) in EtOAc (50 mL) with LiBr (78 mg, 0.9 mmol), followed by chromatography on silica gel and elution with EtOAc / petroleum ether (from 1: 1 to 1: 0) gave Hc-7 (70 mg, 25%) as a foam; NMR XH [(CD3) 2S0] d 8.80 (m, 1H, CONH), 8.24 (d, J = 9.4 Hz, 1H), 7.63 (d, J = 9.4 Hz, 1H), 4.66 (m, 1 H), 3.70 (m, 4 H), 3.60 (m, 4 H), 3.45 (m, 2 H), 3.22 (m, 2 H); NMR X3C d 161.4, 145.8, 140.2, 137.5, 129.2, 127.6, 122.6, 59.0, 52.6, 41.7, 30.0 .. HRMS (FAB) Calculated for C? 3 H1779Br2N406 [M + H +] m / z 482.9515. Found; 482.9508. Further elution with EtOAc / MeOH (50: 2) gave Hc-12 (118 mg, 39%): m.p. 94-97 ° C; NMR [(CD3) 2SO] d 8.80 (m, 1H, CONH), 8.25 (d, J = 9.4 Hz, ÍH), 7.64 (d, J = 9.4 Hz, HH), 4.67 (m, 1 H), 4.27 (m, 2 H), 3.63 (m, 4 H), 3.57 (m, 2 H), 3.45 (m, 2 H), 3.26 (m, 2 H), 3.15 (s, 3 H); RM3 X3C d 161. 4, 146.2, 140.5, 137.7, 129.2, 127.5, 122.9, 66.8, 59.0, 50.0, 41.7, 36.6, 29.9. Anal. (C14H? 9BrN409S) C, H, N.
Example 21 (Reaction Scheme 2j). 3- [Bis (2-bromoethyl) amino] -N- (3-hydroxypropyl) -2,6-dinitrobenzamide (Hc-8) and 2- ((2-bromoethyl) -3- { [(3-hydroxypropyl) amino] -carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate (Hc-13). The treatment of 2- (3. {[[(3-hydroxypropyl) amino] carbonyl] -2- [(methylsulfonyl) oxy] ethyl} -2,4-dinitroanilino) ethyl-methanesulfonate (27) ) [for preparation preparation method see Co-pending application NZ No. 521851] (716 mg, 1.36 mmol) in EtOAc (200 mL) with LiBr (175 mg, 2.0 mmol), followed by chromatography on silica gel and elution with EtOAc / petroleum ether (from 1: 1 to 1: 0) gave Hc-8 (289 mg, 42%) as a yellow solid; p.f. (EtOAc / petroleum ether) 142-144 ° C; NMR [(CD3) 2 SO] d 8.75 (t, J = 5.8 Hz, 1 H, CONH), 8.23 (d, J = 9.4 Hz, 1 H, H-5), 7.62 (d, J = 9.4 Hz, 1 H, H-6), 4.47 (m, 1 H, CHOH), 3.68 (m, 4 H), 3.57 (m, 4 H), 3.43 (m, 2 H), 3.20 (m, 2 H), 1.60 (m, 2 H); NMR 13, d 161.2, 146.9, 140.2, 137.5, 129. 4, 127.7, 122.6, 58.3, 52.6, 36.4, 31.6, 30.1. HRMS (FAB) Calculated for C? H? 979Br2N4Os [M + H +] m / z 496.9671. Found: 496.9667. Further elution with EtOAc / MeOH (50: 2) gave H-13 (270 g, 39%): mp 115-117 ° C; NMR XH [(CD3) 2S0] d 8.75 (t, J = 5.8 Hz, 1 H, CONH), 8.24 (d, J = 9.4 Hz, 1 H, H-5), 7. 64 (d, J = 9.4 Hz, 1 H, H-6), 4.43 (m, 1 H, CHOH), 4.27 (m, 2 H, CH2OMs), 3.66 (m, 4 H, 2xCH2N), 3.59 (m , 2 H), 3.44 (m, 2 H), 3.22 (m, 2 H), 3.15 (s, 3 H, CH3S03), 1.60 (m, 2 H); NMR X3C d 161.1, 146.2, 140.5, 137.7, 129.2, 127.6, 122.9, 66.8, 58.2, 52.9, 50.0, 36.6, 36.4, 31.6, 30.0. Anal. (C? SH2? BrN409S) C, H, N.
Example 22 (Reaction Scheme 2j). 3- [Bis (2-bromoethyl) amino] -N- (4-hydroxybutyl) -2,6-dinitrobenzamide (IIc-9) and 2- ((2-bromoethyl) -3-. {[[(4-hydroxybutyl)] ) amino] -carbonyl.} -2,4-dinitroanilino) ethyl methanesulfonate (Hc-14). The treatment of 2- (3. {[[(4-hydroxybutyl) amino] -carbonyl.} { 2 - [(methylsulfonyl) oxy] ethyl} -2,4-dinitroanilino) -ethyl-methanesulfonate (28) [for preparation method see NZ application No. 521851] (500 mg, 0.92 mmol) in EtOAc (100 mL) with LiBr (110 mg, 1.4 mmol), followed by chromatography on silica gel and elution with EtOAc / petroleum ether (from 1: 1 to 1: 0) gave Hc-9 (100 mg, 21%) as a foam; NMR XH [(CD3) 2SO] d 8.73 (m, 1H, CONH), 8.25 (d, J = 9.4 Hz, HH), 7.63 (d, J = 9.4 Hz, HH), 4.38 (m, 1 H), 3.69 ( m, 4 H), 3.57 (m, 4 H), 3.40 (m, 2 H), 3.14 (m, 2 H), 1.47 (m, 4 H); NMR X3C d 161.0, 145.8, 140.2, 137.6, 129.3, 127.6, 122.6, 60.2, 52.6, 30.0, 29.6, 24.8. HRMS (FAB) Calculated for C? 5H2o79Br2N4Os [M + H +] m / z 510.9828. Found; 510.9819. Further elution with EtOAc / MeOH (50: 2) gave He- 14 (117 mg, 30%): p.f. 114-117 ° C; NMR [(CD3) 2SO] d 8.74 (m, 1 H, CONH), 8.25 (d, J = 9.4 Hz, 1 H), 7.65 (d, J = 9.4 Hz, 1 H), 4.37 (m, 1) H), 4.27 (m, 2 H), 3.65 (m, 4 H), 3.57 (m, 2 H), 3.35 (m, 2 H), 3.16 (m, 2 H), > 3.15 (s, 3 H), 1.47 (m, 4 H); NMR X3C d 160.0, 146.1, 140.6, 137.8, 129.2, 127.5, 122.9, 66.8, 60.2, 52.9, 50.0, 36.6, 29.9, 29.6, 24.9. Anal. (C16H23BrN409S) C, H, N.
Example 23 (Reaction Scheme 2k). 2- (3 - ([(3-hydroxypropyl) amino] carbonyl, {.2- [(methylsulfonyl) oxy] ethyl} - 2,4-dinitroanilino) ethyl methanesulfonate (27) and 2- (( 2-bromoethyl) -3- { [(3-hydroxypropyl) amino] carbonyl} -2,4-dinitroanilino) ethyl methanesulfonate (Hc-13). Solid Hc-8 was added (2.15 g, 4.3 mmol) was added to a warm solution of silver methanesulfonate (0.992 g, 4.9 mmol) in dry MeCN (40 mL) .The mixture was heated under reflux for 3 hours, then cooled and filtered.The solvent was removed under reduced pressure and the residue was separated by chromatography on silica gel, eluting with EtOAc / petroleum ether (from 1: 1 to 1: 0), to successively give IIc-13 (0.5 g, "25%), Hc-8 (0.3 g, 14%) and 27 (0.4 g, 18%).
Example 24 (Reaction Scheme 2k). 2- ((2-Chloroethyl) -3- { [(3-hydroxypropyl) amino] carbonyl} -2,4-dinitroanilino) ethyl methanesulfonate (Hc-6). A solution of 27 (9.0 g, 17.0 mmol) in DMF (110 mL) was treated with LiCl (860 mg, 20.4 mmol) at 60 ° C for 2 hours, then cooled, poured into dilute HCl, and extracted with EtOAc (3x150 mL). Treatment and chromatography of the product on silica gel, eluting with EtOAc / petroleum ether from 1: 1 to 1: 0, gave Hc-6 (4.0 g, 50%) as yellow crystals: m.p. 104-109 ° C; NMR [(CD3) 2 SO] d 8.75 (t, J = 5.8 Hz, HH, CONH), 8.24 (d, J = 9.4 Hz, 1 H, 'H-5), 7.64 (d, J = 9.4 Hz, 1H, H-6), 4.44 (m, 1 H, CHOH), 4.26 (m, 2H), 3.72 (, 2H), 3.65 (m, 2H), 3.59 (m, 2H), 3.43 (m, 2H) , 3.20 (m, 2H), 3.15 (s, 3H), 1.60 (m, 2H); NMR X3C d 161.1, 146.4, 140.5, 137.7, 129.2, 127.6, 122.9, 66.8, 58.2, 52.9, 50.1, 41.4, 36.6, 36.4, 31.6. Anal. (C15H2? ClN409S) C, H, N.
Example 25 (Reaction Scheme 2k). 2- ((2-Yodoethyl) -3- { [(3-hydroxypropyl) amino] carbonyl} -2,4-dinitroanilino) ethyl methanesulfonate (Hc-15). A solution of 27 (5.28 g, 10.0 mmol) in EtOAc (250 mL) was treated with Nal (1.8 g, 12.0 mmol) at 60 ° C for 2 hours, and the product was chromatographed on silica gel, eluting with EtOAc / petroleum ether from 1: 2 to 1: 0, to give Hc-15 (2.29 g, 41%) as yellow crystals: mp 100-103 ° C; H NMR [(CD3) 2S0] d 10.05 (s, 1H), 7.40 (d, J = 11.5 Hz, 1 H), 7.09 (s, 1 H), 6.70 (d, J = 11.5 Hz, 1 H), 2.50 (m, 2 H), 2.21 (m, 2 H), 2.03 (s, 3 H), 1.52 (m, 4 H); NMR X3C d 161.1, 145.8, 140.5, 137.7, 129.2, 127.6, 122.9, 66. 8, 58.2, 53.9, 49.9, 41.4, 36.6, 36.4, 31.6. Anal. (C15H21IN409S) C, H, N.
Preparation of Phosphates (Reaction Scheme 3) Example 26 Diacid phosphate of 2- [[2- [bis (2-bromoethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl (Ib-7P). A solution of the alcohol IIb-7 (2.58 g, 5.33 mmol) and di-diethylphosphoramidite; tert -butyl (93%, 2.0 mL, 6.9 mmol) in dry DMF (20 mL) under N2 was treated with lH-tetrazole (3% by weight in CH3CN, 55 mL, 18.7 mmol) and stirred at 20 ° C for 1.5 hours The reaction mixture was then cooled to -50 ° C and 3-chloroperoxybenzoic acid solution (55%, 2.68 g, 8.54 mmol) was rapidly added such that the temperature was maintained below -5 ° C. The reaction mixture was warmed to room temperature and diluted with CH2C12 (150 mL). The solution was washed with 5% aqueous Na2S205 (2 50 mL), 10% aqueous NaHCO3 (2 x 50 mL), water (2 x 50 mL), dried, concentrated under reduced pressure below 30 ° C. and the residue was stirred with i-Pr20 / hexane and refrigerated. The resulting solid was purified by chromatography on silica gel, eluting with CH2Cl2 / EtOAc, followed by recrystallization from CH2Cl2 / hexane (below 40 ° C) to give di-tert-butyl phosphate 2 - [[2- [ bis (2-bromoethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl (Ib-7E) (2.59 g, 72%) as an unstable yellow solid: mp 99-101 ° C (decomposition); NMR XH [(CD3) 2S0] d 8. 93 (t, J = 5 .6 Hz, 1 H), 8.76 (d, J = 2.8 Hz, 1 H), 8.33 (d, J = 2.8 Hz, 1 H), 4.01 (g, J = 6.1 Hz, 2 H), 3.62- 3.42 (m, 10 H), 1.43 (s, 18 H). HRMS (FAB) calculated for C2? H3479Br2N409P (MH +) m / z 675.0430 found 675.0398; calculated for C2? H3479Br81BrN409P (MH +) m / z 677.0410, found 677.0397; calculated for C2? H348XBr2N409P (MH) m / z 679.0389, found 679.0398. Anal. (C21H33Br2N409P). A solution of Ib-7E (2.80 g, 4.14 mmol) and TFA (15 mL) in dry CH2C12 (15 mL) was stirred at 20 ° C for 1 hour, then concentrated under reduced pressure. The residual TFA was removed azeotropically with CH3CN (2 x) and the resulting residue was dissolved in EtOAc. The addition of excess hexane precipitated a semi-solid which was dried under high vacuum at 20 ° C to give Ib-7P (98%) as a yellow foam. NMR xH [(CD3) 2 SO] d 8.93 (t, J = 5.6 Hz, 1 H), 8.75 (d, J = 2.8 Hz, 1 H), 8.36 (d, J = 2.8 Hz, 1 H), 3.97 (q, J = 6.3 Hz, 2 H), 3.62-3.43 (m, 10 H). HRMS (FAB) calculated for C? 3 H1879Br2N409P (MH +) m / z 562.9178, found 562.9171; calculated for C13H1879Br8XBrN409P (MH +) m / z 564.9158, found 564.9152; calculated for C? 3H188XBr2N09P. (MH +) m / z 566.9137, found 566. 9121. Treatment of the diacid Ib-7P with NaHCO 3 (2.0 equivalent) gave the disodium salt.
Example 27 3- [[5- [Bis (2-chloroethyl) amino] -2,4-dinitrobenzoyl] amino] propyl (I-3P) diacid phosphate. Phosphorylation similar to Ha-3, followed by chromatography of the product on silica gel and elution with CH2Cl2 / EtOAc (2: 3), gave di-tert-butyl-3- [[5- [bis (2-chloroethyl)] phosphate amino] -2,4-dinitrobenzoyl] mino] propyl (Ia-3E) (76%) as a yellow solid: mp (EtOAc / i-Pr2? / Hexane) 120-121 ° C (decomposition); NMR XH [(CD3) 2 SO] d 8.70 (t, J = 5.6 Hz, 1 H), 8.55 (s, 1 H), 7.45 (s, 1 H), 3.96 (q, J = 6.7 Hz, 2 H), 3. 82 (t, J = 5.8 Hz, 4 H), 3.69 (t, J = 5.8 Hz, 4 H), 3.34 (after exchange of D20, t, J = 6.8 Hz, 2 H), 1.86 0 (pent, J = 6.6 Hz, 2 H), 1.42 (s, 18 H). Anal. (C22H35C12N409P) C, H, N. Similar treatment of ester Ia-3E with TFA gave Ia-3P diacid (99%) as a yellow hygroscopic solid. NMR [(CD3) 2 SO] d 8.71 (t, J = 5.6 Hz, 1 H), 8.54 (s, 1 H), 7.45 (s, 1 H), 3.92 (q, J = 6.7 Hz, 2 H ), 3.82 (t, J = 5.8 Hz, 4 H), 3.69 (t, J = 5.8 Hz, 4 H), 3.31 (q, J = 6.5 Hz, 2 H), 1.84 (pent, J = 6.6 Hz, 2 H). HRMS (FAB) Calculated for C 14 H 20 35 Cl 2 N 4 O 9 P [M + H] + m / z 489.0345; found 489.0344. Calculated for C 14 H 20 35 Cl 37 ClN 4 O 9 P [M + H] + m / z 491.0316; ° found 491.0317. Calculated for C14H2037Cl2N4O9P [M + H] + m / z 493. 0286; found 493.0312. Treatment of the I-3P diacid with NaHCO 3 (2: 0 equivalent) gave the disodium salt.
EXAMPLE 28 Diacid phosphate of 3- [[5- [Bis (2-bromoethyl) amino] -5,2,4-dinitrobenzoyl] amino] propyl (Ia-8P). Phosphorylation similar to Ha-8, followed by chromatography of the product on silica gel and elution with CH2Cl2 / EtOAc (1: 1), gave the di-tert-butyl-3- [[5- [bis (2-bromoethyl ) amino] -2,4-dinitrobenzoyl] amino] propyl (Ia-8E) (66%) as a yellow solid: mp (EtOAc / i-Pr20 / hexane) 110-111 ° C (dec.). NMR xH ((CD3) 2SO) d 8.70 (t, J = 5.6 Hz, 1 H), 8.55 (s, 1 H), 7.44 (s, 1 H), 3.96 (q, J = 6.7 Hz, 2 H) , 3.79-3.63 (m, 84 H), 3.35 (after exchange of D20, t, J = 6.8 Hz, 2 H), 1.86 (pent, J = 6.6 Hz, 2 H), 1.42 (s, 18 H) . Anal. (C22H35Br2N409P) C, H, N. Similar treatment of Ia-8E ester with TFA gave the diacid Ia-8P (99%) as a yellow hygroscopic solid. NMR [(CD3) 2SO] d 8.71 (t, J = 5.6 Hz, 1 H), 8.55 (s, 1 H), 7.43 (s, 1 H), 3.93 (q, J = 6.7 Hz, 2 H) , 3.79-3.63 (m, 8 H), 3.31 (q, J = 6.5 Hz, 2 H), 1.85 (pent, J = 6.6 Hz, 2 H). HRMS (FAB) calculated for C14H2079Br2N4O9P (MH +) m / z 576. 9335, found 576. 9314; calculated for C14H2079Br8XBrN4O9P (MH +) m / z 578 9314, found 578. 9305; calculated for C14H2o8XBr2N409P (MH +) m / z 580. 9294, found 580. 9297 Treatment of the diacid Ia-8P with NaHCO 3 (2.0 equivalent) gave the disodium salt.
Example 29 Diacid phosphate of 2- [[2- [Bis (2-chloroethyl) aminoj-3,5-dinitrobenzoyl] amino] ethyl [(lb-2P)]. Similar phosphorylation of Hb-2, followed by chromatography of the product in silica gel and elution with CH2Cl2 /? t0Ac (13: 7), gave di-tert-butyl-2- [[2- [bis (2-chloroethyl) mino] -3,5-dinitrobenzoyl] amino] phosphate] ethyl (lb-2E) (72%) as a yellow solid: mp (EtOAc / i-Pr20 / hexane) 107-108 ° C (dec.); NMR XH [(CD3) 2SO] 6 8.90 (t, J = 5.6 Hz, 1 H), 8.75 (d, J = 2.8 Hz, 1 H), 8.33 (d, J = 2.8 Hz, 1 H), 4.01 (q, J = 6.1 Hz, 2 H), 3.72 (t, J = 6.8 Hz , 4 H), 3.53 (q, J = 5.5 Hz, 2 H), 3.43 (t, J = 6.8 Hz, 4 H), 1.43 (s, 18 H). Anal. (C21H33C12N409P) C, H, N, P. CRL 11363. Similar treatment of the ester Ib-2E with TFA gave the diacid Ib-2P (98%) as a yellow foam. NMR XH [(CD3) 2SO] d 8.89 (t, J = 5.6 Hz, 1 H), 8.74 (d, J = 2.8 Hz, 1 H), 8.36 (d, J = 2.8 Hz, 1 H), 3.98 (q, J = 6.2 Hz, 2 H), 3. 72 (t, J = 6.7 Hz, 4 H), 3.51 (q, J = 5.6 Hz, 2 H), 3.43 (t, J = 6.7 Hz, 4 H). HRMS (FAB) Calculated for C? 3H1835Cl2N209P [M + H] + m / z 475.0189; found 475.0189. Calc'd for C13H? 835 Cl37ClN209P [M + H] + m / z 477.0159; found 477.0167. Calculated for C 13 H 835 Cl 2 N 209 P [M + H] + m / z 479.0130; found 479.0160. Treatment of the diacid Ib-2P with NaHCO 3 (1.0 equivalent.) Gave the monosodium salt.
EXAMPLE 30 2- [(2-Chloroethyl) -2,4-dinitro-6- [[2- (phosphonooxy) -ethyl] amino] -carbonyl] anilino] ethylmethanesulfonate (Ib-2mP). Similar phosphorylation of Hb-2m, followed by chromatography of the product on silica gel and elution with EtOAc, gave 2- [(2- chloroethyl) -2- (6-tert-butoxy-8,8-dimethyl-6-oxido-5,7-dioxa-2-aza-6-phosphason-1-anoyl) -4,6-dinitroanilino] ethylmethanesulfonate (Ib -2mE) (80%) as a yellow foam. NMR XH [(CD3) 2S0] d 8.94 (t, J = 5.6 Hz, 1 H), 8.75 (d, J = 2.8 Hz, 1 H), 8.34 (d, J = 2.8 Hz, 1 H), 4.28 ( t, J = 5.4 Hz, 2 H), 4.02 (q, j = 6.2 Hz, 2 H), 3.74-3.43 (m, 8 H); 3.13 (s, 3 H), 1.43 (s, 18 H). NMR X3C d 265.6, 146.2, 145.3, 140.8, 135.6, 127.5, 122.4, 81.7, 67.5, 64.2, 54.3, 51.3, 41.4, 36.5, 29.5. The similar treatment of Ib-2mE ester with TFA gave the diacid Ib-2mP (68%) as a yellow solid. P.f.
(EtOAc / CH2Cl2): 132-134 ° C; X H NMR [(CD3) 2 SO] d 8.92 (t, J = 5.6 Hz, 1 H), 8.74 (d, J = 2.8 Hz, 1 H), 8.37 (d, J = 2.8 Hz, 1 H), 4.29 (t, J = 5.4 Hz, 2 H), 3.98 (q, J = 6.0 Hz, 2 H), 3.58-3.40 (after the exchange of D20, m, 8 H), 3.13 (s, 2 X3C NMR 165.5, 146.1, 145.3, 140.8, 135.7, 127.6, 122.3, 67.5, 63.3, 63.2, 54.3, 51.3, 41.3, .36.5 Anal. (C14H20C1N4012PS) C, H, N.
EXAMPLE 31 Diacid phosphate of 2- (. {2- [Bis (2-bromopropyl) -amino] -3,5-dinitrobenzoyl}. Amino) ethyl (Ib-7aP). Similar phosphorylation of alcohol IIb-7a (0.67 g, 1.3 mmol) with di-tert-butyl diethylphosphoramidite (93%, 489 mg, 2.0 mmol), followed by flash column chromatography on silica gel, eluting with EtOAc / ether of oil (1: 1) gave Ib-7aE as a yellow solid (0.74 g, 81%): m.p. (EtOAc / petroleum ether) 121-123 ° C; NMR [(CD3) 2SO] d 9.09 (m, 1 H), 8.73 (m, 1 H), 8.32 (m, 1 H), 4.44 (m, 2 H), 4.00 (m, 2 H), 3.39 (m, 2 H), 3.60 (m, 4 H), 1.62 (m, 6 H), 1.44 (s, 18 H). NMR X3C d 165.9, 144.8, 143.6, 139.6, 133.2, 128.0, 123. 1, 81.6, 64.0, 60.4, 39.9, 29.4, 23.5. Anal.
(C23H37Br2N409P) C, H, N. The similar treatment of Ib-7aE (100 mg) with TFA (6 mL), followed by crystallization of CH2Cl2 / EtOAc, gave Ib-7aP as a yellow solid (70 mg, 85%) : pf 157-161 ° C; NMR XH [(CD3) 2S0] d 9.07 (m, 1 H), 8.72 (m, 1 H), 8.36 (m, 1 H), 4.43 (m, 2 H), 4.00 (m, 2 H), 3.52 (m , 6 H), 1.62 (m, 6 H). 13 C NMR 165.9, 144.8, 143.6, 139.7, 133.4, 128.1, 123.1, 63.2, 60.4, 47.9, 39.9, 23.5. Anal. (C15H21Br2N409P) C, H, N.
Example 32 2- [(2-Bromoethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) -ethyl] amino] -carbonyl] anilino] ethylmethanesulfonate (lb-12P). Similar phosphorylation of Ha-12, followed by chromatography of the product on silica gel and elution with EtOAc, gave 2 - [(2-bromoethyl) -2- (6-tert-butoxy-8,8-dimethyl-6-oxide -5,7-dioxa-2-aza-6-phosphason-l-anoyl) -4,6-dinitroanilino] ethyl methanesulfonate (lb-12E) (66%) as a yellow foam. NMR XH [(CD3) 2SO] d 8.94 (t, J = 5.6 Hz, 1 H), 8.75 (d, J = 2.8 Hz, 1 H), 8.34 (d, J = 2.8 Hz, 1 H), 4.28 ( t, J = 5.4 Hz, 2 H), 4.02 (q, j = 6.2 Hz, 2 H), 3.62-3.43 (m, 8 H), 3.13 (s, 3 H), 1.43 (s, 18 H). HRMS (FAB) calculated for C22H3779BrN4O? 2PS [M + H] + m / z 693.1029; found 693.1010. The similar treatment of ester Ib-12? with TFA gave the diacid Ib-12P (98%) as a yellow foam. X H NMR [(CD3) 2 SO] d 8.92 (t, J = 5.6 Hz, 1 H), 8.74 (d, J = 2.8 Hz, 1 H), 8.37 (d, J = 2.8 Hz, 1 H), 4.28 (t, J = 5.4 Hz, 2 H), 3.98 (q, J = 6.0 Hz, 2 H), 3.58-3.40 (after the exchange of D20, m, 8 H), 3.13 (s, 2 H). HRMS (FAB) calculated for C? 4H2? 79BrN4012PS [M + H] + m / z 578.9798; found 578.9784; 0 calculated for C? 4H218XBr81BrN4012PS [M + H] + m / z 580.9777; found 580.9784. Treatment of the diacid Ib-12P with NaHCO 3 (1.0 equivalent) gave the monosodium salt.
EXAMPLE 33 Diacid phosphate of 2- [[2- [Bis (2-iodoethyl) amino] -5,3,5-dinitrobenzoyl] amino] ethyl (Ib-14P). Similar phosphorylation of Ib-14, followed by chromatography of the product on silica gel and elution with CH2Cl2 / EtOAc (3: 1), gave di-tert-butyl-2- [[2- [bis (2- iodoethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl (Ib-14E) (67%) as a yellow solid: mp (CH2Cl2 / i-Pr20 / hexane) 108-110 ° C (decomposition); NMR XH [(CD3) 2SO] d 8.91 (t, J = 5.6 Hz, 1 H), 8.74 (d, J = 2.8 Hz, 1 H), 8.30 (d, J = 2.8 Hz, 1 H), 4. 01 (q, J = 6.3 Hz, 2 H), 3.53 (q, J = 5.7 Hz, 2 H), 3.45 (t, J 7.8 Hz, 4 H), 3.24 (after exchange D20, t, J = 5 7.6 Hz, 4 H), 1.44 (s, 18 H). Anal. (C21H33I2N409P), C, H, N, P. The similar treatment of the ester Ib-14E with TFA gave the diacid Ib-14P (97%) as a yellow foam. NMR XH [(CD3) 2SO] d 8.90 (t, J = 5.6 Hz, 1 H), 8.73 (d, J = 2.8 Hz, 1 H), 8.34 (d, J = 2.8 Hz, 1 H), 3.98 (q, J = 6.4 Hz, 2 H), 3. 49 (after exchange of D20 t, J = 5.6 Hz, 2 H), 3. 45 (t, J = 7.8 Hz, 4 H), 3.29 (t, J = 7.7 Hz, 4 H). HRMS (FAB) Calculated for C? 3H18I2N409 [M + H] + m / z 658.3911; found 658.3907. Treatment of the diacid Ib-14P with NaHCO 3 (2.0 equivalent.) Gave the disodium salt.
EXAMPLE 34 2- [(2-Yodoethyl) -2,4-dinitro-6- ( { [2- (phosphonooxy) -ethyl] amino} carbonyl) -anilino] ethyl-methanesulfonate (Ib-15P). Similar phosphorylation of alcohol Hb-15 (1.68 g, 3.1 mmol) with di-tert-butyl diethylphosphoramidite (93%, 1.15 g, 4.5 mmol), followed by flash column chromatography on silica gel, eluting with EtOAc / ether of petroleum (1: 1), and crystallization from EtOAc / petroleum ether, gave Ib-15E as a yellow solid (2.23 g, 97%): mp (EtOAc / petroleum ether) 109-111 ° C; NMR [(CD3) 2 SO] d 8.98 (m, 1 H), 8.76 (d, J = 2.8 Hz, 1 H), 8.33 (d, J = 2.8 Hz, 1 H), 4.27 (m, 2 H) , 4.00 (m, 2 H), 3.53 (m, 2 H), 3.46 (m, 4 H), 3.14 (s, 3 H), 1.43 (s, 18 H). NMR X3C d 165.5, 145.6, 145.2, 140.8, 135.6, 127.4, 122.4, 81.7, 67.5, 64.2, 55.4, 50.7, 39.9, 36.5, 29.3, 2.6. Anal. (C22H3SIN4O? 2PS), C, H, N.
The similar treatment of Ib-15E (405 mg) with TFA (6 mL) and crystallization of the product from CH2Cl2 / petroleum ether gave the diacid Ib-15P as a solid yellow (306 mg, 89%): m.p. 147-150 ° C; NMR [(CD3) 2SO] d 8.93 (m, 1 H), 8.74 (d, J = 2.8 Hz, 1 H), 8.36 (d, J = 2.8 Hz, 1 H), 4.27 (m, 2 H) , 4.00 (m, 2 H), 3.46 (m, 6 H), 3.31 (m, 2 H), 3.12 (s, 3 H). NMR X3C d 165.5, 145.6, 145.2, 140.8, 135.7, 127.6, 122.3, 67.6, 63.3, 55.5, 50.7, 39.9, 36.5, 2.7. Anal. (C? 4H20IN4O9PS), C, H, N.
EXAMPLE 35 2- [(2-Chloroethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) -propyl] amino] -carbonyl] anilino] ethyl-methanesulfonate (Ic-6P). Similar phosphorylation of He-6, followed by chromatography of the product on silica gel and elution with EtOAc / petroleum ether (from 1: 2 to 1: 1), gave 2- [(2-chloroethyl) -3- (7 -tert-butoxy-9, 9-dimethyl-7-oxido-6,8-dioxa-2-aza-7-phosphahex-l-anoyl) -2,4-dinitroanilino] ethyl-methanesulfonate (Ic-6E) (98 %) as a yellow solid: pf (EtOAc / petroleum ether) 98-102 ° C. NMR [(CD3) 2SO] d 8.83 (t, J = 5.6 Hz, 1 H), 8.26 (d, J = 9.4 Hz, 1 H), 7.65 (d, J = 9.4 Hz, 1 H), 4.29 ( t, J = 5.3 Hz, 2 H), 3.92 (q, J = 6.7 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.62-3.55 (m, 2 H), 3.23 (q, J = 6.5 Hz, 2 H), 3.15 (s, 3 H), 1.79 (pent, J = 6.7 Hz, 2 H), 1.42 (s, 18 H). NMR X3C d 161.3, 146.4, 140.4, 137.6, 129.1, 127.6, 123.0, 81.2, 66.8, 64.1, 64.0, 52.9, 50.1, 41.4, 36.6, 35.9, 29.3. Anal.
(C23H38CIN4O? 2PS) C, H, N. Similar treatment of the ester Ic-6E with TFA gave the diacid Ic-6P (84%) as a yellow solid: m.p. (EtOAc / CH2Cl2) 98-102 ° C; X H NMR [(CD3) 2 SO] d 8.84 (t, J = 5.7 Hz, 1 H), 8.26 (d, J = 9.4 Hz, 1 H), 7.65 (d, J = 9.4 Hz, 1 H), 4.28 ( t, J = 5.3 Hz, 2 H), 3.88 (q, J = 6.8 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.53 (after the exchange of D20, t, J = 6.0 Hz, 2 H), 3.23 (q, J = 6.6 Hz, 2 H), 3.15 (s, 3 H), 1.76 (pent, J = 6.7 Hz, 2 H). Anal. (C? 5H22CIN4012PS) C, H, N.
Example 36 Diacid phosphate of (. {3- [Bis (2-bromoethyl) amino] -2,6-dinitrobenzoyl}. Amino) propyl (Ic-8P). Similar phosphorylation of alcohol Hc-8 (1.41 g, 2.83 mmol) with di-tert-butyl diethylphosphoramidite (93%, 1.25 g, 5.0 mmol), followed by flash column chromatography on silica gel, eluting with EtOAc / ether of oil (1: 1), gave Ic-8E as a yellow solid (1.77 g, 91%): m.p.
(EtOAc / petroleum ether) 112-114 ° C; X H NMR [(CD3) 2 SO] d 8.86 (m, 1 H), 8.24 (d, J = 9.4 Hz, 1 H), 7.63 (d, J = 9.4 Hz, 1 H), 3.92 (m, 2 H) , 3.70 (m, 4 H), 3.60 (m, 4 H), 3.22 (m, 2 H), 1.78 (m, 2 H), 1.41 (s, 18 H). NMR X3C d 161.4, 145.9, 139.9, 137.3, 129.2, 127.8, 122.5, 81.3, 64.1, 52.5, 35.9, 30.1, 29.4. 29.1. Anal. (C22H35Br2N409P), C, H, N. Similar treatment of Ic-8E (900 mg) with TFA (10 mL) gave the diacid Ic-8P as a yellow foam (754 mg, 100%): XH NMR [(CD3) 2S0] d 8.83 (m, 1 H), 8.24 (d, J = 9.4 Hz, 1 H ), 7.63 (d, J = 9.4 Hz, 1 H), 3.86 (m, 2 H), 3.73 (m, 4 H), 3.60 (m, 4 H), 3.22 (m, 2 H), 1.76 (m , 2 H). NMR X3C d 161.3, 145.9, 140.1, 137.4, 129.2, 127.6, 122.5, 62.9, 52.5, 36.0, 30.0, 29.3. HRMS (FAB) calculated for C? 4H2079Br2N4O9P. [M + H] + m / z 576.9335, found 576.9326.
Example 37 2- [(2-Bromoethyl) -2,4-dinitro-3- [[[2- (phosphonooxy) -ethyl] amino] -carbonyl] anilino] ethyl-methanesulfonate (Ic-12P). Similar phosphorylation of IIc-12, followed by chromatography of the product on silica gel and elution with EtOAc / petroleum ether (from 1: 2 to 1: 0), gave (Ic-12E) (99%) as a yellow solid : pf (EtOAc / petroleum ether) 82-86 ° C (decomposition). NMR xH [(CD3) 2S0] d 9.00 (t, J = 5.6 Hz, 1 H), 8.26 (d, J = 9.4 Hz, 1 H), 7.65 (d, J = 9.4 Hz, 1 H), 4.28 ( t, J = 5.3 Hz, 2 H), 3.92 (q, J = 6.7 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.62-3.55 (m, 2 H), 3.23 (q, J = 6.5 Hz, 2 H), 3.15 (s, 3 H), 1.42 (s, 18 H). Anal. (C22H36BrN4O? 2PS) C, H, N. Similar treatment of ether Ic-12E with TFA gave the diacid Ic-12P (100%) as a yellow solid: m.p.
(EtOAc / CH2Cl2) 93-97 ° C; NMR XH [(CD3) 2S0] d 8.99 (t, J = 5.7 Hz, 1 H), 8.26 (d, J = 9.4 Hz, 1 H), 7.65 (d, J = 9.4 Hz, 1 H), 4.28 (t, J = 5.3 Hz, 2 H), 3.88 (q, J = 6.8 Hz, 2 H), 3. 72-3.62 (m, 4 H), 3.53 (after exchange of D20, t, J = 6.0 Hz, 2 H), 3.23 (q, J = 6.6 Hz, 2 H), 3.15 (s, 3 H). Anal. (C14H20BrN4012PS) C, H, N.
EXAMPLE 38 2- [(2-Bromoethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) -propyl] amino] -carbonyl] anilino] ethyl-methanesulfonate (Ic-13P). Similar phosphorylation of IIc-13, followed by chromatography of the product on silica gel and elution with CH2Cl2 / EtOAc (1: 3), gave 2- [(2-bromoethyl) -3- (7-tert-butoxy-9 , 9-dimethyl-7-oxido-6,8-dioxa-2-aza-7-phosphahex-1-anoyl) -2,4-dinitroanilino] ethyl-methanesulfonate (Ic-13E) (70%) as a yellow solid : pf (CH2Cl2 / i-Pr20) 95-96 ° C (decomposition) NMR XH [(CD3) 2 SO] d 8.83 (t, J = 5.6 Hz, 1 H), 8.26 (d, J = 9.4 Hz, 1 H), 7.65 (d, J = 9.4 Hz, 1 H), 4.28 (t, J = 5.3 Hz, 2 H), 3.92 (q, J = 6.7 Hz , 2 H), 3.72- 3.62 (m, 4 H), 3.62-3.55 (m, 2 H), 3.23 (q, J = 6.5 Hz, 2 H), 3.15 (s, 3 H), 1.79 (pent, J = 6.7 Hz, 2 H), 1.42 (s, 18 H). Anal. (C23H38BrN4012PS) C, H, N, P. Similar treatment of the ester Ic-13E with TFA gave the diacid Ic-13P (98%) as a yellow hygroscopic solid. NMR [(CD3) 2 SO] d 8.84 (t, J = 5.7 Hz, 1 H), 8. 26 (d, J = 9.4 Hz, 1 H), 7.65 (d, J = 9.4 Hz, 1 H), 4.28 (t, J = 5.3 Hz, 2 H), 3.88 (q, J = 6.8 Hz, 2 H), 3.72-3.62 (m, 4 H), 3.53 (after the exchange of D20, t, J = 6.0 Hz, 2 H), 3.23 (q, J = 6.6 Hz, 2 H), 3.15 (s, 3 H), 1.76 (pent, J = 6.7 Hz, 2 H). HRMS (FAB) calculated for C? 5H2379BrN4O? 2PS [M + H] + m / z 592.9954; found 592,956. Treatment of the diacid Ic-13P with NaHCO 3 (1: 0 equivalent) gave the monosodium salt.
Example 39 2- [(2-Yodoethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) -propyl] amino] -carbonyl] anilino] ethyl-methanesulfonate (Ic-15P).
Similar phosphorylation of Hc-15, followed by chromatography of the product on silica gel and elution with CH2Cl2 / EtOAc (1: 3), gave 2- [(2-iodoethyl) -3- (7-tert-butoxy-9, 9-dimethyl-7-oxido-6,8-dioxa-2-aza-7-phosphahex-l-anoyl) -2,4-dinitroanilino] ethyl-methanesulfonate (Ic-15E) (58%) as a yellow solid: pf (EtOAc / iPr20) 90-100 ° C. NMR XH [(CD3) 2S0] d 8.86 (t, J = 5.6 Hz, 1 H), 8.25 (d, J = 9.4 Hz, 1 H), 7.63 (d, J = 9.5 Hz, 1 H), 4.27 (t, J = 5.2 Hz, 2 H), 3. 91 (q, J = 6.7 Hz, 2 H), 3.67 (t, J = 5.2 Hz, 2 H), 3.60 (t, J = 7.1 Hz, 2 H), 3.26-3.17 (after exchange of D20, partially obscured, 2 H), 3.23 (q, J = 6.5 Hz, 2 H), 3.15 (s, 3 H), 1.78 (pent, J = 6.6 Hz, 2 H). Anal. (C23H38IN4012PS) C, H, N, P. Similar treatment of the ester Ic-15E with TFA gave the diacid Ic-15P (97%) as a yellow hygroscopic solid: m.p. (CH3CN / Et0Ac) 84-86 ° C. NMR XH [(CD3) 2SO] d 8. 90 (t, J = 5.6 Hz, 1 H), 8.24 (d, J = 9.4 Hz, 1 H), 7.57 (d, J = 9.5 Hz, 1 H), 4.25 (t, J = 5.2 Hz, 2 H), 3.81 (after exchange of D20, q, J = 6.7 Hz, 2 H), 3.62 (after exchange of D20, t, J = 5.2 Hz, 2 H), 3.56 (t, J = 7.1 Hz, 2 H), 3.26 (t, J = 6.9 Hz, 2 H), 3.20 (q, J = 6.5 Hz, 2 H), 3.09 (s, 3 H), 1.73 (pent, J = 6.6 Hz, 2 H).
HRMS (FAB) calculated for C15H22IN012PS (MH +) m / z 640.9816.
Found; 640.9795. Anal. (C15H22IN4012PS) C, H.
Table 2 Combustion analysis data for new compounds of Tables la and Ib Representative alcohols of Formula (I) (listed in Table la) show selective cytotoxicity towards lines of human cancer cells transfected with either the E. coli nitroreductase cDNA (NTR) (Table 3, columns 2 and 3), or cytochrome P450-reductase (P450R) human under hypoxic conditions (Table 3, columns 4 and 5). In this table, sensitivity ratios are displayed to indicate the degree of selectivity for either NTR expression (column 3 or hypoxia (column 5).) However, overexpression of P450R for hypoxic selectivity is not required. of IC50 are derived from cell proliferation experiments, after 4 hours of exposure to the drug under a gas phase of either 20% oxygen or 0% oxygen (anoxia, achieved using an anaerobic chamber). under aerobic conditions for an additional 5 days, and cell density was estimated using the sulforhodamine B assay, to determine the concentration of the prodrug required to inhibit growth at 50% control.
Table 3 Selective cytotoxicities of representative examples of the alcohols of Table Ia The activity of the phosphates as hypoxic cytoxins is demonstrated by the data in Figure 1 for the representative Example (Ib-7P). This employs an in vivo excision assay with the Rif-1 tumor, where they are sterilized oxic tumor cells using 15 Gy of radiation, and the cytotoxicity of an agent against the remaining hypoxic cells can be quantified. Unexpectedly, the Ib-7P phosphate activity is found to exceed that of its source alcohol (Hb-7) at its respective maximum tolerated doses (Ib-7P = 750 μmol / kg, Hb-7 = 1000 μmol / kg ). This experiment demonstrates that the Ib-7P phosphate is more active against hypoxic cells than the reference hypoxic cytotoxin, tirapazamine, and that it is more active against hypoxic cells than the oxic cells (ie, when it is given after irradiation then when it is given without irradiation). In this way, Ib-7P acts as a cytotoxin selective to hypoxia in vivo. Although less active against tumorous tumor cells, this activity is significant showing that the compound also has utility as an inhibitory agent (without radiation). The remarkable activity of the phosphates of the Formula (I) against hypoxic cells in human tumor xenografts is illustrated by the data in Table 4. In these experiments, human cervical carcinoma SiHa cells were cultured subcutaneously in nude CD-1 (immunodeficient) mice. Compounds were administered at doses corresponding to 75% to 20% of the maximum tolerated dose (MTD), 5 minutes after a full body dose of ionizing radiation (cobalt radium-gamma-60) sufficient to sterilize the oxic cells (15 Gy). The tumors were excised 18 hours later, dissociated with a cocktail of proteases, and cell survival was determined using a clonogenic assay. The logarithms of cell annihilation were calculated from the difference in clonogenes numbers per gram of tumor tissue between treated and control tumors. All the phosphates tested showed great effects against hypoxic cells at 75% MTD (Table 4, column 4). This was selective for hypoxia as demonstrated by less cellular annihilation in the absence of radiation. However, cell annihilation by the compounds alone was significant in all cases (Table 4, column 5) demonstrating that the compounds also have antitumor activity as individual agents. The activity against hypoxic cells was also demonstrated for the same compounds at doses corresponding to only 20% of the MTD (Table 4, column 7). The reference hypoxic cytotoxin, tirapazamine, and reference nitrogen mustards (melphalan, chloranbucil, and cyclophosphamide) lacked activity at 20% of their respective BAT. Table 4 Phosphate activity of Formula (I) against hypoxic and hypoxic cells in human tumor xenografts SiHa in nude mice. The compounds were administered as i.p. individual in saline solution.
In relation to controls, in the same experiment, treated with vehicle (saline solution) only. Logarithmic annihilation = Logio (clonogenes / g of tumor for control tumors) -logio (clonogenes / g of tumor for tumor for treated tumors). b Regarding the mean for radiation only, in the same experiment. Logarithmic annihilation = Logio (clonogenes / g of tumor for solar radiation) - logio (clonogenes / g of tumor for tumors treated with more ccomposed radiation). c None detected.
A representative example of the phosphates of Formula (I) as NTR activated cytotoxins is provided in Figure 2. In the in vivo growth retardation assay of WiDr, xenografts containing mixtures of WiDrwt and WiDr15'1'11 at 300mm3 were cultured and treated with a single dose of prodrug to their MTD. Tumor growth is monitored during the period and the animals are euthanized with medium tumor volume >; 1600mm3. The data is presented as time to death. Unexpectedly, phosphate activity (Ib-7P) is observed to exceed that of its source alcohol (Hb-7), when administered at its respective maximum tolerated doses (750 μmol / kg). Ib-7P is superior to Hb-7 with respect to (i) time to first treatment failure (77 days versus 17 days) and (ii) complete survival (40% versus 6%). Figures 3a-3d show pharmacokinetics of the phosphate esters Ib-2mP, Ib-7P, Ib-12P and Ic-12P after administration to female CD-1 nude mice by intraperitoneal injection at a dose corresponding to 75% of the maximum tolerated dose. The monosodium salts of the compounds were dissolved in buffered saline solution of phosphate, pH 7.4, with the addition of one equivalent of sodium bicarbonate. Blood samples were obtained in series by small bleedings from the tail vein, and 10 μl of plasma from each was prepared. The proteins were precipitated by the addition of 3 volumes of methanol, and the concentrations of the phosphate esters and the corresponding alcohols were determined by HPLC using either UV detection or mass spectrometry. The data show that the phosphate esters are efficiently converted to the corresponding alcohols in the mice. Alcohols are the species activated by nitroreduction in hypoxic cells or expressing NTR. Where the foregoing description refers to reagents, or integers having known equivalents thereof, then these equivalents are incorporated herein as if they were presented individually. While the invention has been described with reference to certain embodiments and examples, it will be appreciated that modifications and further variations to the embodiments and examples may be made without departing from the scope of the invention. BRIEF DESCRIPTION OF THE FIGURES The figure shows an in vivo activity of Hb-7 and its corresponding pre-prodrug of phosphate Ib-7P, with relationship to the known hypoxic cytotoxin, tirapazamine. Figure Ib shows the cleavage test method: 300-500 mg of xenograft Rif-1 - »15 Gy - Dosage of individual prodrug @ MTD -» 18 hr - > excision - »disagregar -» plate 102-105 cells - > 12 days - »counted colonies. Figures 2a and 2b show an in vivo activity of Hb-7 and its corresponding phosphate pre-prodrug Ib-7P, against human colon carcinoma xenografts cultured by inoculating mixtures of Wi Dr cells, 90% NTR-ve and NTR + go to 10%. Figures 3a-3d are graphs of the plasmatic concentration versus time of the compound phosphate esters Ib-2mP, Ib-27P, Ib-12P and Ic-12P after administration to female CD-1 nude mice by intraperitoneal injection at a dose that corresponds to 75% of the maximum tolerated dose. The monosodium salts of the compounds were dissolved in phosphate buffered saline, pH 7.4, with the addition of one equivalent of sodium bicarbonate. Blood samples were obtained in series by small bleedings from the tail vein, and 10 μl of plasma from each was prepared. The proteins were precipitated by the addition of 3 volumes of methanol, and the concentrations of the phosphate esters and the corresponding alcohols were determined by HPLC using either UV detection or mass spectrometry.
It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.

Claims (64)

  1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Phosphate compound of the Formula (I) characterized in that X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS02-; R represents a Cx-lower salt optionally substituted with one or more groups including hydroxy, amino and N-oxides. thereof or dialkylamino and N-oxides thereof; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2W) 2 or -N (CH2CHMeW) 2, wherein each W is independently selected from halogen or -OS02Me. Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; and pharmaceutically acceptable salts and derivatives thereof.
  2. 2. Phosphate compound of the formula (I) according to claim 1, characterized in that it is selected from a compound represented by the formulas (la), (Ib) or (le) (le) where Y can represent and wherein n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each W is independently selected from halogen or -0S02Me and pharmaceutically acceptable salts and derivatives thereof.
  3. 3. Phosphate compound of the formula (I) according to claim 1 or claim 2, characterized in that it is selected from: 2- [[2- [Bis (2-bromoethyl) amino] -3 diacid phosphate, 5-dinitrobenzoyl] amino] ethyl; 3- [[5- [Bis (2-chloroethyl) amino] -2,4- dinitrobenzoyl] amino] propyl; 3- [[5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzoyl] amino] propyl phosphate; 2- [[2- [Bis (2-chloroethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl diacid phosphate; 2- [(2-Chloroethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl methanesulfonate; 2- (. {2- [Bis (2-bromopropyl) amino] -3,5-dinitrobenzoyl} amino) ethyl phosphate; 2- [(2-Bromoethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl methanesulfonate; 2- [[2- [Bis (2-iodoethyl) amino] -3,5-dinitrobenzoyl] amino] ethyl diacid phosphate; 2- [(2-Yodoethyl) -2,4-dinitro-6- ( { [2- (phosphonooxy) ethyl] amino.} Carbonyl) -anilino] ethyl methanesulfonate, - 2- [(2-chloroethyl) - 2,4-dinitro-3- [[[3- (phosphonooxy) propyl] amino] -carbonyl] anilino] ethylmethanesulfonate; 3- (. {3- [Bis (2-bromoethyl) amino] -2,6-dinitrobenzoyl} amino) propyl phosphate; 2- [(2-Bromoethyl) -2,4-dinitro-3- [[[2- (phosphonooxy) ethyl] amino] -carbonyl] anilino] ethyl- methanesulfonate; 2- [(2-Bromoethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) propyl] amino] -carbonyl] anilino] ethyl methanesulfonate; and 2- [(2-Yodoethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) propyl] amino] -carbonyl] anilino] ethylmethanesulfonate.
  4. 4. Method for preparing a phosphate represented by the general formula (I); wherein: X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or ~ NHS02 ~; R represents a C? -6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2W) 2, wherein each W is independently selected from halogen or -0S02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me, -and pharmaceutically acceptable salts and derivatives of the same; the method is characterized in that it includes the step of (i) phosphorylating a compound of Formula (II) wherein X represents at any available position of the ring -CONH-, -S02NH-, -O-, -CH2-, -NHCO- or -NHS02-; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2W) 2, or -N (CH2CHMeW) 2, wherein each W is independently selected from halogen or -OS02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; and R represents a C? _6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof.
  5. 5. Method for preparing a compound of the formulas (la), (Ib) or (le) (le) where Y can represent and wherein n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each W is independently selected from halogen or -OS02Me and pharmaceutically acceptable salts and derivatives thereof the method is characterized in that it includes the step of phosphorylating a compound represented by the formulas (Ha), (Hb) or (He) (He) where Y represents and wherein n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each W is independently selected from halogen or -0SO2Me and pharmaceutically acceptable salts and derivatives thereof.
  6. 6. Compound of formula (I), characterized in that it is obtained by the method defined in claim 4.
  7. 7. Compound of formulas (la), (Ib) or (le), characterized in that it is obtained by the method defined in claim 5.
  8. 8. Anticancer treatment method, characterized in that it includes the step of administering an amount of a compound of the formula (I) as defined in any of claims 1 to 3 to a subject.
  9. 9. A method for killing hypoxic cells in a tumor, characterized in that it includes the step of administering an amount of a compound of the formula (I) as defined in any of claims 1 to 3 to a subject with a tumor.
  10. Method according to claim 8 or claim 9, characterized in that it includes the additional step of applying irradiation to one or more chemotherapeutic agents to a subject.
  11. 11. Method of compliance with any of claims 8 to 10, characterized in that the subject is a human.
  12. Method according to any of claims 8 to 11, characterized in that the amount administered is between approximately 20% to 100% of the maximum tolerated dose of the subject.
  13. 13. Method of cellular ablation using at least one nitroreductase enzyme, characterized in that it includes the step of using a compound of the formula (I) as defined above in any of claims 1 to 3 in an amount effective to remove the cells that express at least one nitroreductase enzyme.
  14. 14. Cellular ablation method using at least one nitroreductase enzyme, characterized in that it includes the step of using a compound of the formula (I) as defined above in any of claims 1 to 3 in an effective amount to a subject to be excised the cells that express at least one nitroreductase enzyme.
  15. 15. Method according to claim 15 or claim 14, characterized in that at least one nitroreductase enzyme is encoded by the nfsB gene of either E. coli or by orthologous genes in the Clostridia species.
  16. 16. Method according to claim 14 or claim 15, characterized in that the cells expressing at least one nitroreductase enzyme are tumor cells in tissue in a subject.
  17. Method according to any of claims 14 to 16, characterized in that the cellular ablation is achieved through GDEPT (prodrug therapy and gene-directed enzyme).
  18. 18. Method according to any of claims 14 to 17, characterized in that the cellular ablation is achieved through ADEPT (prodrug therapy and antibody directed enzyme).
  19. 19. Method according to any of claims 14 to 18, characterized in that the cells They are from a mammal.
  20. 20. Method according to any of claims 14 to 19, characterized in that the amount administered is between about 20% to 100% of the maximum tolerated dose of the subject.
  21. 21. Method according to any of claims 14 to 20, characterized in that it includes the additional step of applying irradiation to one or more chemotherapeutic agents to a subject.
  22. 22. Pharmaceutical composition, characterized in that it includes a therapeutically effective amount of a compound of the formula (I) as defined in any of claims 1 to 3 and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabilizer.
  23. 23. The use in the manufacture of a medicament of an effective amount of a compound of the formula (I) as defined in any of claims 1 to 3 for treating cancer in a subject.
  24. 24. The use according to claim 23, wherein the medicament is further adapted for use in cellular ablation together with at least one nitroreductose enzyme that includes GDEPT (prodrug therapy and gene directed enzyme) or ADEPT (therapy of prodrug and enzyme directed by antibody).
  25. 25. The use according to claim 24, wherein at least one nitroreductase enzyme is encoded by the nfsB gene of either E. coli or by orthologous genes in the Clostridia species.
  26. 26. The use according to any of claims 23 to 25, wherein the medicament is adapted for a mammalian subject.
  27. 27. Alcohol compound of Formula (II) characterized in that: X represents at any available position of the ring -CONH-, -S02NH-, -0-, -CH2-, -NHCO- or -NHS02-; Y represents at any available position of the ring, -N-aziridinyl, -N (CH2CH2W) 2 or -N (CH2CHMeW) 2, wherein each W is independently selected from halogen or -0S02Me; Z represents at any available position of the ring, -N02, -halogen, -CN, -CF3 or -S02Me; R represents a C? _6 lower alkyl optionally substituted with one or more groups including hydroxy, amino and N-oxides thereof or dialkylamino and N-oxides thereof; and pharmaceutically acceptable salts and derivatives thereof; with the proviso that when Z represents N02 and Y represents N (CH2CH2CI) 2, X and R together can not represent -CONHCH (CHOH) CH2- and also with the additional condition that the following compounds Exclude
  28. 28. Alcohol compound of Formula (II) of according to claim 27, characterized in that it is selected from a compound represented by the formulas (Ha), (Ilb) or (He) where Y can represent and wherein n represents from 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein each W is independently selected from halogen or -0S02Me and pharmaceutically acceptable salts and derivatives thereof with the proviso that when Z represents N02 and Y represents N (CH2CH2CI) 2, X and R together can not represent - C0NHCH2 ( CHOH) CH2- and also with the additional condition that the following compounds
  29. 29. An alcohol compound of the Formula (II) characterized in that it is selected from a compound of the Formula (Hb) or (He) as defined in claim 28.
  30. 30. Alcohol compound of the Formula (II) according to claim 28 or claim 29, characterized in that it is selected from: N- (2-Hydroxyethyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide; N- (4-Hydroxybutyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide; N- (5-Hydroxypentyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide; N- (6-Hydroxyhexyl) -5- [bis (2-bromoethyl) amino] -2,4-dinitrobenzamide; 5- [Bis (2-bromoethyl) amino] N- (2-hydroxyethyl) -4- (methylsulfonyl) -2-nitrobenzamide; 2 - [(2-Bromoethyl) -5 - [[(3-hydroxypropyl) amino] -carbonyl] -2,4-dinitroanilino] ethyl-methanesulfonate; 5- [Bis (2-iodoethyl) amino] -N- (2-hydroxyethyl) -2,4-dinitrobenzamide; 2- [Bis (2-Chloroethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide; 2- [Bis (2-chloroethyl) amino] -N- (3-hydroxypropyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (3-hydroxypropyl) -3,5-dinitrobenzamide; 2- [Bis (2-chloroethyl) amino] -N- (4-hydroxybutyl) -3,5- dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (4-hydroxybutyl) -3,5-dinitrobenzamide; 2- [Bis (2-chloroethyl) amino] -N- (5-hydroxypentyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (5-hydroxypentyl) -3,5-dinitr? Benzamide; 2- [Bis (2-chloroethyl) amino] -N- (6-hydroxyhexyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromoethyl) amino] -N- (6-hydroxyhexyl) -3,5-dinitrobenzamide; 2- [Bis (2-bromopropyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide; 2- ((2-Bromoethyl) -2- { [(2-hydroxypropyl) amino] -carbonyl} -4-6-dinitroanilino) ethyl-methanesulfonate; 2- ((2-Bromoethyl) -2- { [(2-hydroxyethyl) amino] -carbonyl} -4-6-dinitroanilino) ethyl-methanesulfonate; 2- ((2-Chloroethyl) -2- { [(2-hydroxyethyl) amino] -carbonyl} -4-6-dinitroanilino) ethyl-methanesulfonate; 2- [Bis (2-iodoethyl) amino] -N- (2-hydroxyethyl) -3,5-dinitrobenzamide; 2- ((2-Yodoethyl) -2- { [(2-hydroxyethyl) amino] -carbonyl} -4-6-dinitroanilino) ethyl-methanesulfonate; 3- [Bis (2-bromoethyl) amino] -N- (2-hydroxyethyl) -2,6-dinitrobenzamide; 2- ((2-Bromoethyl) -3- { [(2-hydroxyethyl) amino] -carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; 3- [Bis (2-bromoethyl) amino] N- (3-hydroxypropyl) -2,6-dinitrobenzamide; 2- ((2-bromoethyl) -3- { [(3-hydroxypropyl) amino] -carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; 3- [Bis (2-bromoethyl) amino] -N- (4-hydroxybutyl) -2,6-dinitrobenzamide; 2- ((2-Bromoethyl) -3- { [(4-hydroxybutyl) amino] -carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; 2- ((2-Chloroethyl) -3- { [(3-hydroxypropyl) amino] -carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate; and 2- ((2-Yodoethyl) -3- { [(3-Hydroxypropyl) amino] -carbonyl} -2,4-dinitroanilino) ethyl-methanesulfonate.
  31. 31. Method for preparing a compound of the formulas (Ha), (Ilb) or (líe) (He) where Y can represent and wherein n represents 1 to 6, Z represents -N02, -halogen, -CN, -CF3 or -S02Me; and wherein Wi is halogen and W2 is -0S02Me and pharmaceutically acceptable salts and derivatives thereof, - the method is characterized in that it includes the step of reacting a compound of the formulas (Ha '), (Hb') or (He ') optionally with heating ("c ') where Y can represent wherein W'i and W'2 are each halogen; with an effective amount of silver methanesulfonate (AgOMs) in a solvent to give a compound of the formulas (Ha), (Hb) or (He) defined above.
  32. 32. Method according to claim 31, characterized in that the solvent is selected from MeCN or another polar non-protic solvent.
  33. 33. Compound of the formulas (Ha), (Hb) or (He), characterized in that it is obtained by the method defined in claim 31 or claim 33.
  34. 34. Anticancer treatment method, characterized in that it includes the step of administering an amount of a compound of the formula (II) as defined in claim 27 to a subject.
  35. 35. Method for killing hypoxic cells in a tumor, characterized in that it includes the step of administering an amount of a compound of the formula (II) as defined in claim 27 to a subject with the tumor.
  36. 36. Method according to claim 34 or claim 35, characterized in that it includes the additional step of applying irradiation to one or more chemotherapeutic agents or to a subject.
  37. 37. Method according to any of claims 34 to 36, characterized in that the subject is a human.
  38. 38. Cellular ablation method using at least one nitroreductase enzyme, characterized in that it includes the step of using a compound of formula (II) as defined in claim 27 in an amount effective to remove cells expressing at least one nitroreductase enzyme.
  39. 39. Method of cell ablation using at least one nitroreductase enzyme, characterized in that it includes the step of administering a compound of the formula (II) as defined in claim 27 in an amount effective to a subject to excise the expressing cells to the minus one nitroreductase enzyme.
  40. 40. Method according to claim 39, characterized in that at least one nitroreductase enzyme is encoded by the nfsB gene of either E. coli or by orthologous genes in the Clostridia species.
  41. 41. Method according to claim 39 or claim 40, characterized in that the cells expressing at least one nitroreductase enzyme are tumor cells in tissue in a subject.
  42. 42. Method according to any of claims 39 to 41, characterized in that the cellular ablation is achieved through GDEPT (prodrug therapy and gene-directed enzyme).
  43. 43. Method according to any of claims 39 to 41, characterized in that the cellular ablation is achieved through ADEPT (prodrug therapy and 2 enzyme directed by antibody).
  44. 44. Method according to any of claims 39 to 43, characterized in that the cells are mammalian.
  45. 45. Method according to any of claims 39 to 44, characterized in that it includes the additional step of applying irradiation to one or more chemotherapeutic agents to a subject.
  46. 46. Pharmaceutical composition, characterized in that it includes a therapeutically effective amount of a compound of the formula (II) as defined in claim 27 and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabilizer.
  47. 47. Use in the manufacture of a medicament of an effective amount of a compound of the formula (II) as defined in claim 27 as an anticancer agent in a subject.
  48. 48. Use according to claim 47, wherein the medicament is adapted for use cell ablation is achieved through GDEPT (prodrug therapy and gene directed enzyme) or ADEPT (prodrug therapy and antibody directed enzyme) .
  49. 49. Use according to claim 48, wherein at least one nitroreductase enzyme is encoded by the nfsB gene of either E. coli or by orthologous genes in the Clostridia species.
  50. 50. Use according to any of claims 47 to 49, wherein the medicament is adapted for a mammalian sujto.
  51. 51. A compound, characterized in that it is selected from: 2- [(2-Bromoethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) -ethyl] -amino] -carbonyl] anilino] ethyl-methanesulfonate; 2- [(2-Bromoethyl) -2,4-dinitro-3- [[[2- (phosphonooxy) -ethyl] amino] -carbonyl] anilino] ethyl-methanesulfonate, 2- [(2-Bromoethyl) -2 , 4-dinitro-3- [[[3- (phosphonooxy) -propyl] amino] -carbonyl] anilino] ethyl-methanesulfonate; and 2- [(2-Yodoethyl) -2,4-dinitro-3- [[[3- (phosphonooxy) -propyl] amino] -carbonyl] anilino] ethyl-methanesulfonate.
  52. 52. The compound, characterized in that it is: 2- [(2-Bromoethyl) -2,4-dinitro-6- [[[2- (phosphonooxy) -ethyl] amino] -carbonyl] anilino] ethyl-methanesulfonate.
  53. 53. A pharmaceutical composition, characterized in that it includes a therapeutically effective amount of a compound as claimed in claims 51 or claims 52 and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabilizer.
  54. 54. Use in the manufacture of a medicament of an effective amount of a compound of Formula (I) as claimed in claim 51 or claim 52 to treat cancer in a kid.
  55. 55. Use as claimed in claim 54, wherein the subject has a tumor.
  56. 56. Use as claimed in claim 54 or claim 55, wherein the subject is a human.
  57. 57. Cellular ablation method using at least one nitroreductase enzyme, characterized in that it includes the step of using a compound as defined above in claim 51 or claim 52 in an amount effective to remove cells expressing at least one nitroreductase enzyme , with the proviso that when the cells are human cells they are ex vivo cells.
  58. 58. Use in the manufacture of a medicament in an effective amount of a compound of Formula (I) as defined above in claim 51 or claim 52 for removing cells in a subject, wherein the cells express at least one enzyme of nitroreductase.
  59. 59. Use according to claim 58, wherein at least one nitroreductase enzyme is encoded by the nfsB gene of either E. coli or orthologous genes in the Clostridia species.
  60. 60 Use according to claim 58 or claim 59, wherein the cells expressing at least one nitroreductase enzyme are tumor cells.
  61. 61. Use according to any of claims 50 to 58 in GDEPT (prodrug therapy and gene directed enzyme).
  62. 62. Use according to any of claims 58 to 60 in ADEPT (prodrug therapy and antibody directed enzyme).
  63. 63. Use according to any of claims 58 to 62, wherein the cells are mammalian.
  64. 64. Use according to any of claims 58 to 63, wherein the medicament provides between about 20% to 100% of the maximum tolerated dose of a compound of Formula I to the subject.
MXPA/A/2006/004635A 2003-10-31 2006-04-26 Novel nitrophenyl mustard and nitrophenylaziridine alcohols and their corresponding phosphates and their use as targeted cytotoxic agents MXPA06004635A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ529249 2003-10-31
NZ535618 2004-09-28

Publications (1)

Publication Number Publication Date
MXPA06004635A true MXPA06004635A (en) 2006-12-13

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