Indian Patents
Title of Invention

NOVEL NITROPHENYL MUSTARD AND NITROPHENYLAZIRIDINE ALCOHOLS AND THEIR CORRESPONDING PHOSPHATES AND THEIR USE AS TARGETED CYTOTOXIC AGENT
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.
Full Text 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.
Background to the invention
The use of tumour-selective 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 may be converted into an anti-tumour agent under the influence of
an enzyme that is linkable to a monoclonal antibody that will bind to a tumour associated
antigen. The combination of such a prodrug with such an enzyme monoclonal/antibody
conjugate represents a very powerful clinical agent. This approach to cancer therapy, often
referred to as "antibody directed enzyme/prodrug therapy" (ADEPT), is disclosed in
W088/07378.
A further therapeutic approach termed "virus-directed enzyme prodrug therapy" (VDEPT)
has been proposed as a method for treating tumour cells in patients using prodrugs.
Tumour cells are targeted with a viral vector carrying a gene encoding an enzyme capable
of activating a prodrug. The gene may be transcriptionally regulated by tissue specific
promoter or enhancer sequences. The viral vector enters tumour cells and expresses the
enzyme, in order that a prodrug is converted to an active drug within the tumour cells
(Huber et al., Proc. Natl. Acad. Sci. USA (1991) 88, 8039). Alternatively, non-viral
methods for the delivery of genes have been used. Such methods include calcium
phosphate co-precipitation, microinjection, liposomes, direct DNA uptake, and receptor-
mediated DNA transfer. These are reviewed in Morgan & French, Annu. Rev. Biochem.,

1993, 62; 191. The term "GDEPT" (gene-directed enzyme prodrug therapy) is used to
include both viral and non-viral delivery systems (Denny et al US 6,310,237).
4-Nitroaromatic compounds are reduced by both mammalian and bacterial flavoprotein
enzymes, which effect stepwise addition of up to six electrons. The major enzymatic
metabolite is usually the 4-electron reduced species (hydroxylamine).
A number of nitrophenyl mustards and nitrophenylaziridines have been reported as
prodrugs for use in gene-directed enzyme-prodrug therapy (GDEPT) in conjunction
with nitroreductase enzymes. In particular, CB 1954 [5-(aziridin-l-yl)-2,4-
dinitrobenzamide; (1) [shown below] is reported to be 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 (Knox et al., Biochem. Pharmacol., 1995,49,
1641-1647) and GDEPT (Bridge water .et al.,. Eur. J. Cancer, 1995,31 A, 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) applications, including a
clinical trial (Chung-Faye et al., Clin. Cancer Res., 2001, 7, 2662-2668).
Similarly, the dinitrophenyl mustard SN 23862 (2) is also a substrate for NTR, and shows
selective toxicity towards 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-SO2Me 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 tumour tissue by endogenous reductase enzymes,
showing potency ratios of 2-5 fold under hypoxic conditions relative to oxic conditions in the
wild-type AA8 cell line, using a clonogenic assay (Palmer et al., J. Med. Chem. 1996,39,
2518-2528).
Some phosphate analogues of mustards have been described, for the purpose of solubilising
the compounds. The best known is estramustine phosphate (Estracyt; 6), which has been
shown to bind to tubulin binding domains on various microtubule-associated proteins
(Moraga et ah, Biochim. Biophys. Acta, 1992, 1121, 97-103), and which has been shown
to be active in advanced breast cancer (Keren-Rosenberg et al., Semin. Oncol., 1997, 24
(Suppl. 3), 26-29), but has not been shown to be activated by NTR or hypoxia. Another
study has also shown estramustine phosphate to be a radiation sensitizer (Kim et al., Int. J.
Radiat. Oncol. Biol. Phys., 1994,29, 555-557). The phenol mustard phosphate analogue 7
is a carboxypeptidase substrate that is not activated under hypoxic conditions, (Matsui et
al., Japanese Patent 07082280 A2,1995), and the solubilised mustard 8 has been
described as a phosphatase inhibitor but has not been shown to be activated 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, bearing short-chain alcohols, and their corresponding phophates for use as
targeted cytotoxic agents or bioreductive prodrugs or to at least provide the public with a _
useful alternative.
Summary and detailed description
In a first aspect, the present invention provides novel phosphate compounds of Formula 1

wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2., -NHCO- or
-NHSO2-;

R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2 or
-N(CH2CHMeW)2, where each W is independently selected from halogen or -OSO2Me.
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
and pharmaceutically acceptable salts and derivatives thereof.
In a preferred embodiment, the phosphate compound of Formula (I) is selected from a
compound represented by formulae (Ia), (Ib) or (Ic)

and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where each W is independently selected from halogen or -OSO2Me

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 dihydrogen phosphate;
3-[[5-[Bis(2-chloroethyl)amino]-2,4-dinitrobenzoyl]amino]propyl dihydrogen phosphate;
3-[[5-[Bis(2-bromoethyl)amino]-2,4-dinitrobenzoyl]amino]propyl dihydrogen phosphate;
2-[[2-[Bis(2-chloroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogen 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 dihydrogen 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 dihydrogen phosphate;
2-[(2-Iodoethyl)-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]ethyl methanesulfonate;
3-({3-[Bis(2-bromoethyl)amino]-2,6-dinitrobenzoyl}amino)propyl dihydrogen 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-Iodoethyl)-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 ring position -CONH-, -SO2NH-, -O-, -CH2., -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2, or
-N(CH2CH MeW)2 where each W is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;

are excluded.
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom; and
pharmaceutically acceptable salts and derivatives thereof, with the proviso that

In a preferred embodiment, the alcohol compound of Formula (II) is selected from a
compound represented by formulae (IIa), (IIb) or (IIc)

and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where each W is independently selected from halogen or -OSO2Me
and pharmaceutically acceptable salts and derivatives thereof with the proviso that


Preferably the compound of Formula (II) is selected from the following:
N-(3 -Hydroxypropyl)-5 - [bis(2-chloroethyl)amino] -2,4-dinitrobenzamide;
N-(3-Hydroxypropyl)-5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzamide;
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-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-Iodoethyl)-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-hy_droxybutyl)-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-Iodoethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroanilino)ethyl
methanesulfonate.
In a third aspect of the invention there is provided a method of preparing the phosphates
represented by the general formula (I);

wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or
-NHSO2-;
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom;

Y represents at any available ring position -N-aziridinyl or -N(CH2CH2W)2, where each W
is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
and pharmaceutically acceptable salts and derivatives thereof;
the method including the step of
(i) phosphorylating a compound of formula (II)

wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2, or -N(CH2CH
MeW)2 where each W is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me; and
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom.
In a fourth aspect there is provided a method of preparing a compound of formulae (IIa),
(IIb) or (IIc)


and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where W1 is halogen and W2 is -OSO2Me
and pharmaceutically acceptable salts and derivatives thereof;
the method including the step of
reacting a compound of formulae (IIa), (IIb') or (IIc) optionally with heating


with an effective amount of silver methanesulfonate (AgOMs) in a solvent to give a
compound of formulae (IIa), (IIb) or (IIc) defined above.
It is to be appreciated that in the method defined immediately above where W'1 and W'2
are either iodine and/or bromine that the iodine and/or bromine can be partially or
completely substituted with -OSO2Me. In the situation where either or both of W'1 and
W'2 represent chlorine, the chlorine is inert and cannot be can be substituted with - fj
OSO2Me.
Preferably the solvent is selected from MeCN or other polar non-protic solvent.
In a fifth aspect there is provided a method of preparing a compound of formulae (Ia), (Ib)
or(Ic)


and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where each W is independently selected from halogen or -OSO2Me
and pharmaceutically acceptable salts and derivatives thereof
the method including the step of
phosphorylating a compound represented by formulae (IIa), (IIb) or (IIc)


and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where each W is independently selected from halogen or -OSO2Me
and pharmaceutically acceptable salts and derivatives.
In a sixth aspect there is provided a compound of formula (I), formula (Ia), (Ib) or (Ic) or
formula (IIa), (IIb) or (IIc) obtained by any one of the preparative methods defined above.
In a seventh aspect, the present invention provides a method for the use as prodrugs
suitable for'
(i) GDEPT (gene-directed enzyme-prodrug therapy) or ADEPT (anti-body directed
enzyme prodrug therapy) in conjunction with at least one nitroreductase
enzyme; or
(ii) Use as one or more hypoxia-selective cytotoxins,
including the step of administering a compound of Formula (I) as defined above or a
compound of Formula (II)


wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2., -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2, or -N(CH2CH
MeW)2 where each W is independently selected from halogen or -OSO2Me; r
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom; and
pharmaceutically acceptable salts and derivatives thereof;
or a mixture thereof in a therapeutically effective amount to tumour cells in a subject.
Preferably, the nitroreductase enzyme is encoded for by the nfsB gene of either E.
coli or by orthologous genes in Clostridia species.
Preferably the method includes the further step of irradiating the tumour cells. '
In an eighth aspect, the present invention provides a method for the use as prodrugs
suitable for GDEPT (gene-directed enzyme-prodrug therapy) or ADEPT (antibody-
directed enzyme prodrug therapy) in conjunction with at least one nitroreductase enzyme,
as an anticancer agent including the step of administering a compound of Formula (I) as
defined above or a compound of Formula (II)


wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2 or -N(CH2CH
MeW)2, where each W is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom; and
pharmaceutically acceptable salts and derivatives thereof;
or a mixture thereof in a therapeutically effective amount to target tumour cells in a
subject.
Preferably the nitroreductase enzyme is encoded for by the nfsB gene of E .coli or by
orthologous genes in Clostridia species
Preferably the method includes the further step of irradiating the tumour cells.
It is to be appreciated that with ADEPT it may be necessary to supply a reducing co-
factor, because these may not be present in significant concentrations outside cells. It
is envisaged that a synthetic co-factor could be used to stimulate activation of the
pro-drug by the likes of an intracellular enzyme. The same issue does not arise with
GDEPT because there are several intracellular reducing co-factors such as the likes
of NADH and NADPH in significant concentrations.

In a ninth aspect of the present invention, there is provided a method of cell ablation
utilising at least one nitroreductase; enzyme, wherein the method includes the step of
administering a compound of Formula (I) as defined a above or a compound of Formula
(H)

wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2 or -N(CH2CH
MeW)2, where each W is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
R represents a lower Cl-6 alkyl optionally substituted with one or more groups
including hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides
therefrom; and pharmaceutically acceptable salts and derivatives thereof,
or a mixture thereof in an effective amount to ablate cells, wherein said cells express
at least one nitroreductase enzyme.
Preferably the nitroreductase enzyme is encoded for by the nfsB gene in E. coli or by
orthologous genes in Clostridia species.
Preferably, the cells that are targeted for ablation are tumor cells in tissue in a subject.

Preferably, the method of cell ablation utilising at least one nitroreductase enzyme is
delivered by either ADEPT or GDEPT technology.
Preferably, the cell ablation provides a substantially minimal bystander effect.
In a tenth aspect, the present invention provides a method of providing anticancer
therapy, wherein a compound of Formula (I) as defined above is administered in a
therapeutically effective amount to tumour cells in a subject.
Preferably the therapeutically effective amount of said method is between about 20%
to 100% of the maximum tolerated dose of said subject.
Preferably, the method includes the further step of irradiating the tumour cells.
In an eleventh aspect of the present invention there is provided a pharmaceutical
composition including a therapeutically effective amount of a compound of Formula (I) or
a compound of Formula (II)

wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2 or -N(CH2CH
MeW)2, where each W is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;

R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom; and
pharmaceutically acceptable salts and derivatives thereof,
or a mixture thereof, and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer
or stabiliser.
The pharmaceutically acceptable excipient, adjuvant, carrier, buffer or stabiliser should
preferably be non-toxic and should not intertere with the efficacy 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 is to be appreciated that these factors could be readily determined by
someone skilled in the art without undue experimentation.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or
liquid form. A tablet may comprise a solid carrier or an adjuvent. Liquid pharmaceutical
compositions generally comprise a liquid carrier such as water, petroleum, animal or
vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other
saccharide solution 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, isotonicity and stability. Those of relevent skill in the art are well able to prepare
suitable solutions using, for example, isotonic vehicles such as Sodium Chloride injection,
Ringer's injection, Lactated Ringer's injection. Preservatives, stabilisers, buffers
antioxidants and/or other additives may be included as required.

In a twelfth aspect of the present invention there is provided, the use in the manufacture of
a medicament of an effective amount of a compound of Formula (I) as defined above or a
compound of Formula (II)

wherein:
X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2 or -N(CH2CH
MeW)2, where each W is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom; and
pharmaceutically acceptable salts and derivatives thereof, or mixtures thereof,
for use in (i)GDEPT or (ii) as a hypoxia selective cytotoxin, to 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 Formula (I) as defined above or
a compound of Formula (II)

wherein:

X represents at any available ring position -CONH-, -SO2NH-, -O-, -CH2-, -NHCO- or
-NHSO2-;
Y represents at any available ring position -N-aziridinyl, -N(CH2CH2W)2 or -N(CH2CH
MeW)2, where each W is independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom; 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 tumour
cells or tumour tissues in human subjects, they may be used to target tumour cells or
tissues in other warm blooded animal subjects such as other primates, farm animals
such as cattle, and sports animals and pets such as horses, dogs, and cats.
As used throughout the specification 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 one of compounds Ia-Ic, or IIa-IIc as defined
above or a mixture thereof that is sufficient to show benefit to a subject with cancer
cells. The actual amount, rate and time-course of administration, will depend on the
nature and severity of the disease being treated. Prescription of treatment is within
the responsibility of general practitioners and other medical doctors.
As used throughout the specification the term "halogen" includes chlorine, bromine
or iodine.

It is to be understood that the compounds of the invention as defined above may be
administered alone or in combination with other treatments, especially radiotherapy and
cytotoxic chemotherapeutic drugs, either simultaneously or sequentially dependent upon
the condition to be treated.
As used throughout the specification the pharmaceutically acceptable derivatives and salts
thereof include acid derived salts formed from are hydrochloric, sulfuric, phosphoric, acetic,
citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic,
isethionic acids and the like and base derived salts formed from sodium and potassium
carbonate, sodium and potassium hydroxide, ammonia, triethylamine, triethanolamine and
the like.

As used throughout the specification, the term cell ablation is to be understood as the killing
of cells, that 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 ablate specified target cells or tissue through specific enzymatic
expression of a nitroreductase for example, that is specifically expressed by the tissue and
which can then be employed to activate a prodrug into an active metabolite to ablate the
specified target cells or tissue. (Gusterson et al. Endocrine Related Cancer, 1997,4,67-74.)
The expression "substantially minimal bystander effect" is to be understood as meaning that
the killing of adjoining non-targeted cells is minimal because there is little or no diffusion
between the targeted and non-targeted cells of an 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 one of compounds Ia-Ic, or IIa-IIc as defined above or a mixture thereof.
Pharmaceutically acceptable salts of formula (I) include the basic or acidic compounds of
formula (I) that form pharmaceutically acceptable salts with both organic and inorganic
acids and/or organic and 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.
Further aspects of the present invention will become apparent from the following
description given by way of example only and with reference to the accompanying
synthetic schemes.
Examples of the compounds of Formula (I) where X is -CONH- can be prepared by the
processes described in Scheme 1, where Z is as defined above for Formula (I).

The following Tables 1 a and 2a set out physical data for compounds within the general
Formula (I) and (II), representative of it, and capable of being prepared by the processes of
the invention.





References for known compounds.
1. Khan AH, Ross WCJ. 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, H1cks 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 a can be prepared by the general methods set out in Schemes
2a-2k, and exemplified in Examples 1-20 below.













(i) Et2NP(OtBu)2 or i-Pr2NP(OtBu)2/1H-tetrazole, then mCPBA or H202
(ii) TFA/CH2Cl2
In Scheme 3, X, Y, Z, and R are as specified for formula (I) and (II) above.
Examples
The invention and the best mode for practising the same are illustrated by the following
Examples 1-25 (alcohols) and Examples 26-39 (phosphates).
Example 1 (Scheme 2a). N-(3-HydroxypropyI)-5-[bis(2-chloroethyl)amino]-2,4-
dinitrobenzamide (IIa-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 SOCl2 (20 mL) containing DMF (2 drops) was heated under reflux for 1 h, then
concentrated to dryness under reduced pressure and re-evaporated with benzene. The
resulting crude benzoyl chloride was dissolved in Me2CO (50 mL) and the cooled (-5 °C)
solution was treated with a cold solution of 3-amino-l-propanol (1.O9 g, 14.5 mmol) in
water (25 mL). The reaction mixture was shaken at room temperature for 5 min, then
diluted with water (25 mL), concentrated to half volume, and extracted with CH2Cl2 (2x).
The organic extract was washed with 0.1 N HCl and water then worked up to give a solid
which was chromatographed on silica gel, eluting with EtOAc to give IIa-3 (2.37 g, 82%):
mp (EtOAc/i-Pr2O) 90-91 °C; 1H NMR [(CD3)2SO] δ 8.63 (t, J= 5.6 Hz, 1 H, CONH),
8.53 (s, 1 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(CH2CH2Cl)2), 3.68 (t, J= 5.9 Hz, 4 H, N(CH2CH2Cl)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. (Cl4H18Cl2N4O6) C, H, N, CI.
Example 2 (Scheme 2a). N-(3-Hydroxypropyl)-5-[bis(2-bromoethyI)amino]-2,4-
dinitrobenzamide (IIa-8). A suspension of powdered 5-[bis(2-bromoethyl)arnino]-2,4-
dinitrobenzoic acid (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 h, 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 shaken at room
temperature for 5 min, then diluted with water and extracted with EtOAc (2x). The organic
extract was worked up and the resulting residue was chromatographed on silica gel, eluting
with EtOAc, to give IIa-8 (1.O6 g, 85%): mp (EtOAc/i-Pr2O) 85-86 °C; 1H NMR
[(CD3)2SO] δ 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(CH2CH2Cl)2), 4.46 (br s, 1 H, OH), 3.49 (t, J= 6.3 Hz, 2 H, CH2OH),
3.33-3.25 (m, partially obscured, 2 H, CONHCH2), 1.68 (pent, J= 6.72 Hz, 2 H,
CH2CH2CH2). Anal. (Cl4H18Br2O6) C, H, N, Br.
Example 3 (Scheme 2a). N-(2-HydroxyethyI)-5-[bis(2-bromoethyl)amino]-2,4-
dinitrobenzamide (IIa-7). Similar reaction of the acid bromide of 2 with 2-aminoethanol
gave IIa-7 (0.78 g, 46%): mp (MeOH/EtOAc/pet. ether) 151-152 °C; 1H NMR [(CD3)2SO] δ 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. (Cl3H16Br2N4O6) C, H, N, Br.
Example 4 (Scheme 2a). N-(4-Hydroxybutyl)-5-[bis(2-bromoethyl)amino]-2,4-
dinitrobenzamide (IIa -9). Similar reaction of the acid bromide of 2 with 4-amino-l-

butanol in cold Me2CO, followed by chromatography on silica gel and elution with EtOAc
gave IIa-9 (69%) as a yellow solid: mp (EtOAc/iPr2O) 123-124 °C; 1H NMR [(CD3)2SO] δ 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.
(Cl5H20Br2N4O6)C,H,N,Br.
Example 5 (Scheme 2a). N-(5-Hydroxypentyl)-5-[bis(2-bromoethyl)amino]-2,4-
dinitrobenzamide (IIa -10). Similar reaction of the acid bromide of 2 with 5-amino-l-
pentanol in cold Me2CO, followed by chromatography on silica gel and elution with
EtOAc gave IIa -10 (66%) as a yellow foam; lH NMR [(CD3)2SO] δ 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 (m,
2 H), 3.26-3.18 (m, 2 H), 1.59-1.29 (m, 4 H). HRMS (FAB) Calcd. for Cl6H2379Br2N4O6
[M+H+] m/z 524.9984, found 524.9964.
Example 6 (Scheme 2a). N-(6-HydroxyhexyI)-5-[bis(2-bromoethyI)ammo]-2,4-
dinitrobenzamide (IIa -11). Similar reaction of the acid bromide of 2 with 6-amino-l-
hexanol in cold Me2CO, followed by chromatography on silica gel and elution with EtOAc
gave IIa -11 (72%) as a yellow foam; 1H NMR [(CD3)2SO] δ 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) Calcd. for Cl7H2579Br2N4O6
[M+H+] m/z 539.0141, found 539.0137.
Example 7. (Scheme 2b). 5-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-4-
(methylsulfony])-2-nitrobenzamide (IIa -7s). 5-Fluoro-4-(methylsulfonyl)-2-
nitrobenzoic acid [Atwell et al., ACDD, 1996, 11, 553] (3) was heated 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): mp (EtOAc/hexane) 134-135
°C; 1H NMR [(CD3)2SO] δ 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. (C9H8FNO6S) C, H, N.

A mixture of 4 (1.48 g, 5.34 mmol) and diethanolamine (1.40 g, 13.3 mmol) in DMA (6
mL) was stirred at 30 °C for 1 h, 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/iPr2O, to give methyl 5-[bis(2-hydroxyethyl)amino]-4-(methylsulfonyl)-2-
nitrobenzoate (5) (1.41 g, 73%): mp 99-100 °C; 1H NMR [(CD3)2SO] δ 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.
(Cl3H18N2O8S)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 h, 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
h. The cooled mixture was poured into water and the resulting solid was purified by
chromatography, on silica gel, eluting with CH2Cl2, then recrystallisation from
CH2Cl2Pr2O to give methyl 5-[bis(2-bromoethyl)amino]-4-(methylsulfonyl)-2-
nitrobenzoate (6) (1.47 g, 74%): mp 161-162 °C; 1H NMR [(CD3)2SO] δ 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. (Cl3H16Br2N2O6S) C, H, N.
A solution of 6 (1.00 g, 2.05 mmol) in a dioxane/MeOH (1:1, 20 mL) was treated at 10 °C
with 4N aqueous KOH (5 mL), and stirred at 10 °C for 45 min. 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 crystallized twice from MeOH/H20 to give 5-[bis(2-bromoethyl)amino]-4-
(methylsulfonyl)-2-nitrobenzoic acid (7) (0.70 g, 72%): mp 174-176 °C; 1H NMR
[(CD3)2SO] δ 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. (Cl2H14Br2N2O6S) C, H, N.

A finely-divided suspension of 7 (260 mmg, 0.55 mmol) in dry benzene (50 mL) was
treated with (COBr)2 (2.13 mL, 0.20 mmol) and catalytic DMF. The mixture was stirred
for 2 h, 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 min, then acidified to pH 4 with 1 N
aqueous HBr, and concentrated under reduced pressure. The residue was chromatographed
on silica gel, eluting with EtOAc, to give IIa -7s (222 mg, 78%): mp (EtOAc/iPr2O) 126-
127 °C; 1H NMR [(CD3)2SO] δ 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 D20 exchange, t, J= 6.0 Hz, 2 H). HRMS (FAB) calcd. for
Cl4H2079Br2N3O6S (MH+) m/z 515.9440; found 515.9425.
Example 8 (Scheme 2c). 2[(2-Bromoethyl)-5-[[(3-hydroxypropyI)amino]carbonyl]-
2,4-dinitroanilino]ethyl methanesulfonate (IIa-13) and 5-[bis(2-iodoethyl)amino]-N-
(2-hydroxyethyl)-2,4-dinitrobenzamide (IIa-14). 5-(Bis{2-
[(methylsulfonyl)oxy]ethyl}amino)-2,4-dinitrobenzoic acid [A method of preparing this
compound is disclosed in co-pending NZ Application No. 521851 ] (9) was heated under
reflux in excess SOCl2 (60 mL) and catalytic DMF for 1 h. Evaporation under reduced
pressure, followed by azeotroping in benzene, gave the crude acid chloride. This was
dissolved in dry Me2CO and treated at 0 °C with 3-amino-l-propanol at 0 °C for 5 min.
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), gave give 2-
(5-{[(3-hydroxypropyl)amino]carbonyl}{2-[(methylsulfonyl)oxy]ethyl}-2,4-
dinitroanilino)ethyl methanesulfonate (8) (68%) as a yellow gum; 1H NMR [(CD3)2SO] δ
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= 5.9 Hz, 2 H), 3.26 (after D20 exchange,

t, J= 7.0 Hz, 2 H), 3.12 (s, 6 H), 1.66 (pent, J = 6.7 Hz, 2 H). HRMS (FAB) calcd. for
Cl6H25N4O12S (MH+) m/z 529.O910; found 529.O904.
A solution of 8 in DMF was treated with LiBr (1.4 equiv.), and worked up as above, and
the product was chromatographed on silica gel. Elution with EtOAc gave a small amount
of the dibromo mustard, while elution with EtOAc/MeOH (19:1) gave Ha -13 (31%) as a
yellow gum: 1H NMR [(CD3)2SO] δ 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 D20 exchange, t,J= 7.0 Hz, 2 H), 3.14 (s, 3 H),
1.68 (pent, J= 6.7 Hz, 2 H). HRMS (FAB) calcd. for Cl5H2279BrN4O9S (MH+) m/z
515.0270; found 515.0283.
Similar treatment of the acid chloride of 9 (activation with (COCl)2/DMF) with 2-
aminoethanol gave 2-(5-{[(2-hydroxyethyl)amino]carbonyl} {2-
[(methylsulfonyl)oxy]ethyl}-2,4-dinitroanilino)ethyl methanesulfonate (10). A stirred
mixture of 10 (1.42 g, 2.76 mmol) and Nal (3.3g, 22 mmol) in dry MeCN (45 mL) was
heated at reflux for 1 h, 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 silic gel, eluting with CH2Cl2/EtOAc
(1:4), followed by recrystallisation from MeOH/EtOAc/i-Pr2O to give IIa -14 (2.9 g,
81%): mp 142-143 °C; 1H NMR [(CD3)2SO] δ 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. (Cl3H16I2N4O6) C, H, N.
Example 9.2-(Aziridin-l-yI)-N-(6-hydroxyhexyI)-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) was
treated with aziridine (100 mg) at room temperature for 3 h. The mixture was diluted with
EtOAc and washed three times with water, after dry, concentrated under reduced pressure

until about 20 mL, the yellow solid was collected and gave 101 mg product (84%); 1H
NMR [(CD3)2SO] δ 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 (m, 2 H), 2.37 (s, 4 H), 1.56 (m, 2 H), 1.43 (m, 2 H),
1.33 (m, 4 H). Anal. (Cl5H20N4O6) C, H, N.
Example 10 (Scheme 2d). 2-[Bis(2-ChIoroethyI)amino]-N-(2-hydroxyethyl)-3,5-
dinitrobenzamide (IIb-2) and 2-[bis(2-bromoethyI)amino]-N-(2-hydroxyethyl)-3,5-
dinitrobenzamide (IIb-7). 2-Chloro-3,5-dinitrobenzoic acid (1) (18 g, 81 mmol) was
treated with SOCl2 (250 ml) containing one drop of DMF and heated under reflux for 6 h.
Evaporation of reagent followed by azeotroping with benzene gave the crude acid chloride,
which was dissolved in THF (200 mL) and added dropwise to a solution made of 25 mL of
2-aminoethanol in THF (400 mL) and cooled with dryice-acetone bath. After stirring for
20 min. the reaction mixture was acidified to pH 4-5 with 1 N HCl, most 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 sat. NaHCO3, 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: mp
(EtOAc) 159-160 °C; 1H NMR [(CD3)2SO] δ 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 (m, 1 H, OH), 3.54 (m, 4 H). Anal.
(C9H8ClN3O6)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 h. 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) and
concentration of the eluate under reduced pressure gave a oily residue that was dissolved
in minimum amount of EtOAc. Petroleum ether was added slowly until incipient
cloudiness, and the solution was stood overnight to precipitate 2-[bis(2-
chloroethyl)amino]-N-(2-hydroxyethyl)-3,5-dinitrobenzamide (IIb-2) (2.07 g, 100%) as

yellow crystals: mp (EtOAc/petroleum ether) 1O9-111°C; 1H NMR [(CD3)2SO] δ 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, 2xCH2Cl), 3.55 (m, 2 H), 3.42 (m, 4 H, 2xCH2N), 3.34 (m, 2 H); 13C
NMR δ 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+] Calcd. For Cl3H1735Cl2N4O6 m/z 395.0525. Found; 395.0525.
A solution of IIb-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 h, then cooled and poured into water. Extraction
with EtOAc gave a crude product ( mmol) in 3-methyl-2-butanone for a further 4 h, then worked up and chromatographed on
silica gel, eluting with EtOAc/petroleum ether (from 1:1 to 1:0), to give IIb-7 (1.39 g,
95%): mp (EtOAc/petroleum ether) 105-108 °C; 1H NMR [(CD3)2SO] δ 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); 13C NMR δ 165.3,145.4,145.3,141.1, 136.5,127.4,122.1, 59.3,
53.9,42.1, 30.0. HRMS (FAB) Calcd. For Cl3H1779Br2N4O6 [M+H+] m/z 482.9515. Found;
482.9492. Anal. (Cl3H16Br2N4O6) H, N, Br; C: found, 32.9; calculated 32.3%.
Example 11 (Scheme 2d). 2-[Bis(2-chIoroethyl)amino]-N-(3-hydroxypropyI)-3,5-
dinitrobenzamide (IIb-3) and 2-[bis(2-bromoethyl)amino]-N-(3-hydroxypropyI)-3,5-
dinitrobenzamide (IIb-8). Reaction of the acid chloride of 11 (17 g) with 3-
aminopropanol (7.5 g) in Me2CO (120 mL) at 0 °C as described above, gave 2-chloro-N-
(3-hydroxypropyl)-3,5-dinitrobenzamide (13) (5.O6 g, 26%): mp (EtOAc/petroleum ether)
120-121 °C; 1H NMR [(CD3)2SO] δ 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. (Cl0H10ClN3O6) 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)amine hydrochloride (2.9 g, 16.0 mmol) at 50 C for 24 h.
Workup as described above gave IIb-3 (1.84 g, 100%): mp (EtOAc/petroleum ether) 89-

91 °C; 1H NMR [(CD3)2SO] δ 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, 2xCH2Cl), 3.50 (m, 2 H), 3.42
(m, 4 H, 2xCH2N), 3.32 (m, 2 H), 1.71 (m, 2 H); 13C NMR δ 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) Calcd. For
Cl4H1935Cl2N4O6 [M+H+] m/z 4O9.O682. Found; 4O9.O678.
Treatment of IIb-3 with LiBr in 3-methyl-2-butanone twice, as described above, gave Ilb-
8 (74% yield): mp (EtOAc/petroleum ether) 89-94 °C; 1H NMR [(CD3)2SO] δ 8.74 (d, J=
2.7 Hz, 1 H, H-4), 8.72 (m, 1 H, CONH), 8.30 (d, J= 2.7 Hz, 1H, H-6), 3.77-3.44 (m, 12
H), 1.70(m,2H);13CNMR δ 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) Calcd. For Cl4H1979Br2N4O6 [M+H+J m/z 496.9671.
Found; 496.9658.
Example 12 (Scheme 2d). 2-[Bis(2-chloroethyI)ammo]-N-(4-hydroxybutyI)-3,5-
dinitrobenzamide (IIb-4) and 2-[bis(2-bromoethyI)amino]-N-(4-hydroxybutyl)-3,5-
dinitrobenzamide (IIb-9). Reaction, of the acid chloride of 11 (2.65 g, 10 mmol) with 4-
aminobutanol (1.9 g) as above, followed by acidification to pH 4-5 with 1 N HCl and
evaporation of most of the solvent gave 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 were washed with sat. NaHCO3, 1 N HCl and brine respectively,
then concentrated to give 2-chloro-N-(4-hydroxybutyl)-3,5-dinitrobenzamide (14) 1.11 g
(35%): mp (EtOAc) 121-124 °C; 1H NMR [(CD3)2SO] δ 8.98 (d, J = 2.7 Hz, 1H), 8.79 (m,
1 H), 8.52 (d, J- 2.7 Hz, 1H), 4.43 (m, 1 H), 3.43 (m, 2H), 3.26 (m, 2 H), 1.54 (m, 4 H);
13C NMR δ 162.6, 148.4,145.9,140.4, 128.2, 125.8, 120.4, 60.2, 39.1,29.8, 25.3. Anal.
(CnH12ClN3O6)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 h. The
mixture was poured into water and extracted with EtOAc gave the crude product which

was chromatographed on silica gel. Elution with EtOAc/petroleum ether (4:1) gave IIb-4
(0.99 g, 100%) as yellow foam; 1H NMR [(CD3)2SO] δ 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 (m, 2 H),
1.56 (m, 2 H), 1.47 (m, 2 H); 13C NMR δ 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) Calcd. For Cl5H2135Cl2N4O6 [M+H+] m/z
423.0838. Found; 423.0847.
A solution of IIb-4 (0.96 g, 3.04 mmol) and LiBr (5 g) in 3-methyl-2-butanone (15 mL)
was heated under reflux for 6 h, then cooled and poured into water. Extraction with EtOAc
gave a crude product ( butanone for a further 4 h, then worked up and chromatographed on silica gel, eluting with
EtOAc/petroleum ether (from 1:1 to 3:1) give IIb-9 (1.01 g, 87%) as a yellow foam; 1H
NMR [(CD3)2SO] δ 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); 13CNMR δ 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) Calcd. For
Cl5H2179Br2N4O6 [M+H+] m/z 510.9828. Found; 510.9832.
Example 13 (Scheme 2d). 2-[Bis(2-chloroethyl)amino]-N-(5-hydroxypentyl)-3,5-
dinitrobenzamide (IIb-5) and 2-[bis(2-bromoethyl)amino]-N-(5-hydroxypentyl)-3,5-
dinitrobenzamide (lIb-10). Similar reaction of the acid chloride of 11 with 5-
aminopentanol as above gave 2-chloro-N-(5-hydroxypentyl)-3,5-dinitrobenzamide (15),
1.3 g (39%), mp (EtOAc) 105-108 °C; 1H NMR [(CD3)2SO] δ 8.98 (d, J= 2.7 Hz, 1H),
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); 13C NMR δ 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. (Cl2H14CIN3O6) 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-chloroethyl)amine hydrochloride (1.5 g, 8.0 mmol) at 50 °C for 24 h. The
mixture was poured into water and extracted with EtOAc to gave the crude product which

was chromatographed on silica gel. Elution with EtOAc/petroleum ether (4:1) gave IIb-5
(0.82 g, 100%) as yellow foam; 1H NMR [(CD3)2SO] δ 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); 13CNMR δ 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) Calcd. For
Cl6H2335Cl2N4O6 [M+Ff] m/z 437.O995. Found; 437.O991.
Similar reaction of IIb-5 (1.3 g) with LiBr gave Hb-10 (1.35 g, 86%) as a yellow foam; 1H
NMR [(CD3)2SO] δ 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); 13C NMR δ 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)
Calcd. For Cl6H2379Br2N4O6 [M+H+] m/z 524.9984. Found; 524.9975.
Example 14 (Scheme 2d). 2-[Bis(2-chloroethyl)ammo]-N-(6-hydroxyhexyl)-3,5-
dinitrobenzamide (IIb-6) and 2-[bis(2-bromoethyl)amino]-N-(6-hydroxyhexyI)-3,5-
dinitrobenzamide (IIb-11). 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%), mp (EtOAc) 88-91 °C; 1H NMR [(CD3)2SO] δ 8.98 (d, J= 2.7 Hz, 1H), 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); 13C NMR δ 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. (Cl3H16ClN3O6) 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)amine hydrochloride (1.5 g, 8.0 mmol) at 50 °C for 24 h. The
mixture was poured into water and extracted with EtO Ac to gave the crude product which
was chromatographed on silica gel. Elution with EtO Ac/petroleum ether (4:1) gave IIb-6
(0.87 g, 100%) as yellow foam; 1H NMR [(CD3)2SO] δ 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); 13C NMR δ 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) Calcd. For
Cl7H2535Cl2N4O6 [M+H+] m/z 451.1151. Found; 451.1154.
Similar reaction of IIb-6 (0.97 g) with LiBr gave IIb-11 (0.96 g, 81%) as a yellow foam;
1H NMR [(CD3)2SO] δ 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); 13C NMR δ 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) Calcd. For Cl7H2579Br2N4O6 [M+H+] m/z 539.0141. Found; 539.0135.
Example 15 (Scheme 2e). 2-[Bis(2-bromopropyl)amino]-N-(2-hydroxyethyl)-3,5-
dinitrobenzamide (IIb-7a). Reaction of 2-chloro-3,5-dinitro-N-[2-(tetrahydro-2H-pyran-
2-yloxy)ethyl]benzamide (17) (1.02 g)[For method of preparation see co-pending NZ
Application No. 521851] with diisopropanolamine (0.8 g) as above 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; 1H NMR [(CD3)2SO] δ 9.22 (br, 1H), 8.66 (d, J=
2.8 Hz, 1H), 8.29 (d, J= 2.8 Hz, 1H), 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, 4H), 3.26 (m, 2 H), 1.48 (m, 10 H), 0.98 (m, 6 H); 13C NMR
δ 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) Calcd. For C20H31N4O9 [M+H+] m/z 471.2O91. Found;
471.2089.
Reaction of 18 with MsCl as above gave l-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 h, then diluted with water (100 mL),
neutralized with satd. 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-{[(2-hydroxyethyl)amino]carbonyl} {2-[(methylsulfonyl)oxy]propyl}-4,6-
dinitroanilino)-l-methylethyl methanesulfonate (20) (0.80 g, 37%): as a yellow foam;
1H NMR [(CD3)2SO] δ 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); 13C NMR δ 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) Calcd. For
Cl7H27N4O12S2 [M+H+] m/z 543.1O67. 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 h, and chromatography of the product on silica gel, eluting with EtOAc/petroleum
ether (from 2:1 to 1:0) gave IIb-7a (0.31 g, 62%): as yellow solid: mp (EtOAc/petroleum
ether) 127-130 °C; 1H NMR [(CD3)2SO] δ 8.91 (m, 1H, CONH), 8.70 (d, J= 2.8 Hz, 1H,
H-4), 8.32 (d, .J=2.8 Hz, 1H, H-6), 4.80 (m, 1 H), 4.42 (m, 2 H), 3.55 (m, 4 H), 1.62 (m, 6
H); 13C NMR δ 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. (Cl5H20Br2N4O6) C, H, N.
Example 16 (Scheme 2f). 2-((2-Bromoethyl)-2-{[(2-hydroxypropyl)amino]carbonyl}-
4,6-dinitroanilino)ethyl methanesulfonate (IIb-13). A solution of 13 (1.22 g, 4.0 mmol)
in 50 mL of CH2Cl2 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 h, then
concentrated under reduced pressure. Chromatography of the residue on silica gel, eluting
with EtO Ac/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; 1H NMR
[(CD3)2SO] δ 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 (m, 2 H), 3.46-3.25 (m, 4 H), 1.82-1.44 (m, 8 H). 13C NMR δ
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) Calcd. For Cl5H1935ClIN3O7 [M+H+] m/z 388.O912. Found; 388.O915.

Reaction of 21 (1.45 g, 3.75 mmol) with diethanolamine (1.67 g) as above 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; 1H NMR [(CD3)2SO] δ 8.96 (m, 1H, CONH), 8.66
(d, .J=2.8 Hz, lH,H-4), 8.31 (d, .J=2.8 Hz, 1H, H-6), 4.95 (m, 2H), 4.56 (m, 1H),3.79-
3.16 (m, 14H), 1.80-1.45 (m, 8 H); 13CNMR δ 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) Calcd. For
Cl9H29N4O6 [M+H+] m/z 457.1935. Found; 457.1939.
Reaction of 22 (1.62 g, 3.55 mmol) with MsCl (2 mL) as above 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%): as a yellow foam; 1H
NMR [(CD3)2SO] δ 8.71 (d, J= 2.8 Hz, 1H), 8.71 (m, 1H), 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); 13C NMR
δ 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) Calcd. For C21H33N4O13S2 [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 h, then diluted with water (100 mL), neutralized
with satd. 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-hydroxypropyI)amino]carbonyI}{2-[(methyIsulfonyl)oxy]ethyl}-4,6-
dinitroanilino)ethyl methanesulfonate (24) (1.4 g, 75%): as a yellow solid: mp
(EtOAc/petroleum ether) 130-133 °C; 1H NMR [(CD3)2SO] δ 8.74 (d, J= 2.8 Hz, 1H),
8.72 (m, 1H), 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); 13C NMR δ 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. (Cl6H24N4O12S2) C, H, N.

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 h, and chromatography of the product on silica gel, eluting with
EtOAc/petroleum ether (from 2:1 to 1:0) gave IIb-13 (0.16 g, 66%): as yellow foam; 1H
NMR [(CD3)2SO] δ 8.74 (d, J= 2.8 Hz, 1H), 8.73 (m, 1H), 8.31 (d, J= 2.8 Hz, 1H), 4.28
(m, 2 H), 3.65-3.44 (m, 10 H), 3.13 (s, 3 H), 1.70 (m, 2 H); I3C NMR δ 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)
Calcd. For Cl5H2279BrN4O9S[M+H+] m/z 513.0291.' Found; 513.0281.
Example 17 (Scheme 2g). 2-((2-bromoethyI)-2-{[(2-hydroxyethyI)amino]carbonyl}-
4,6-dinitroanilino)ethyl methanesulfonate (IIb-12). Solid IIb-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 h, then cooled and filtered. The solid AgBr was washed with EtOAc to
give a 98% yield of AgBr. The solvent was removed at 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 (IIb-7) (28 mg, 9%).
- IIb-12 (123 mg, 38%) as a yellow foam; 1H NMR [(CD3)2SO] δ 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, OSO2CH3); 13C NMR δ 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
Cl4H2079BrN4O9S 499.01344; Found 499.01324.
The column was finally eluted 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); lH NMR [(CD3)2SO] δ 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, 2xOSO2CH3); 13C NMR δ 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: Cl5H23N4O12S2 requires m/z 515.0754. Found: 515.0744.

Example 18 (Scheme 2h). 2-((2-Chloroethyl)-2-{[(2-hydroxyethyl)amino]carbonyl}-
4,6-dinitroanilino)ethyl methanesulfonate (IIb-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 h, and then
cooled, poured into dilute HCl and extracted with EtOAc (3x150 mL). Workup and
chromatography of the product on silica gel, eluting with EtO Ac/petroleum ether from 1:1
to 1:0, gave IIb-2 (2.4 g, 59%), and then IIb-2m (1.94 g, 41%) as yellow oil; 1H NMR
[(CD3)2SO] δ 8.77 (m, 1H, CONH), 8.74 (d, J= 2.7 Hz, 1H, 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, -OSO2CH3);
I3CNMR δ 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.
IIb-2m was prepared by an alternative method as following: A solution of IIb-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 h and then concentrated under reduced
pressure. The residue was partitioned between water (250 mL) and EtO Ac (250 mL) and
the separated organic layer was washed with water, dried (Na2SO4) and then concentrated
under reduced pressure. The resulting oil (15.23 g) was dissolved in CH3CN (80 mL),
treated with silver methanesulfonate (9.63 g, 47.4 mmol) and the mixture was stirred at 25
°C for 1 h 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 EtO Ac
solution was evaporated and the oily mixture was separated by chromatography on silica
gel as above and gave starting material (3.61 g, 29%), IIb-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 (Scheme 2i). 2-[Bis(2-iodoethyl)amino]-N-(2-hydroxyethyl)-3,5-
dinitrobenzamide (IIb-14) and 2-((2-iodoethyI)-2-{[(2-hydroxyethyl)amino]carbonyl}-
4,6-dinitroanilino)ethyl methanesulfonate (IIb-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 h, and chromatography of the
product on silica gel, eluting with EtO Ac/petroleum ether (from 2:1 to 1:0) gave IIb-14
(3.3 g, 44%) as a yellow solid: mp (EtOAc/petroleum ether) 129-131 °C; lH NMR

[(CD3)2SO] δ 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 (m, 4 H), 3.31 (m, 6 H); 13C NMR δ 165.3,
145.2, 144.7,141.0,136.3, 127.3,122.0, 59.3, 54.7,42.1,2.94. Anal (Cl3H16N4O6) C, H,
N.
Later eluates gave IIb-15 (1.35 g, 19%) as a yellow foam; 1H NMR [(CD3)2SO] δ 8.74 (d,
J= 2.8 Hz, 1H, H-4), 8.74 (m, 1H, 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); 13C NMR δ 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) Calcd. For
Cl4H20IN4O9S [M+H+] m/z 546.9996. Found; 546.9997.
Example 20 (Scheme 2j). 3-[Bis(2-bromoethyl)amino]-N-(2-hydroxyethyl)-2,6-
dinitrobenzamide (IIc-7) and 2-((2-bromoethyl)-3-{[(2-
hydroxyethyI)ammo]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate (IIc-12).
Treatment of 2-(3-{[(2-hydroxyethyI)amino]carbonyl} {2-[(methylsulfonyl)oxy]ethyl}-
2,4-dinitroanilino)ethyl methanesulfonate (26) [for method of preparation 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 IIc-7 (70 mg, 25%) as a foam; 1H NMR [(CD3)2SO] δ 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); 13C NMR δ 161.4, 145.8,140.2, 137.5,129.2,
127.6,122.6, 59.0, 52.6,41.7, 30.0.. HRMS (FAB) Calcd. For Cl3HI779Br2N4O6 [M+H+]
m/z 482.9515. Found; 482.9508.
Further elution with EtOAc/MeOH (50:2) gave IIc-12 (118 mg, 39%): mp. 94-97 °C; 1H
NMR [(CD3)2SO] δ 8.80 (m, 1H, CONH), 8.25 (d, J= 9.4 Hz, 1H), 7.64 (d, J= 9.4 Hz,
1H), 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); I3C NMR δ 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. (Cl4H19BrN4O9S) C, H, N.

Example 21 (Scheme 2j). 3-[Bis(2-bromoethyl)amino]-N-(3-hydroxypropyl)-2,6-
dinitrobenzamide (IIc-8) and 2-((2-bromoethyl)-3-{[(3-
hydroxypropyl)amino]carbonyl}-2,4-dinitroanaino)ethyl methanesulfonate(IIc-13).
Treatment of 2-(3-{[(3-hydroxypropyI)amino]carbonyl } {2-[(methylsulfonyl)oxy]ethyl}-
2,4-dinitroanilino)ethyl methanesulfonatee (27) [for method of preparation see co-
pending NZ Application 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 IIc-8 (289 mg, 42%) as a yellow solid; mp
(EtOAc/petroleum ether) 142-144 °C; 1H NMR [(CD3)2SO] δ 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); 13C
NMR δ 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) Calcd. For Cl4H1979Br2N4O6 [M+H+] m/z 496.9671. Found: 496.9667.
Further elution with EtOAc/MeOH (50:2) gave IIc-13 (270 mg, 39%): mp. 115-117 °C; 1H
NMR [(CD3)2SO] δ 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, CH3SO3), 1.60 (m, 2
H); 13C NMR δ 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. (Cl5H21BrN4O9S) C, H, N.
Example 22 (Scheme 2j). 3-[Bis(2-bromoethyl)amino]-N-(4-hydroxybutyI)-2,6-
dinitrobenzamide (IIc-9) and 2-((2-bromoethyl)-3-{[(4-
hydroxybutyl)amino]carbonyl}-2,4-dinitroanilino)ethyl methanesulfonate (IIc-14).
Treatment of 2-(3-{[(4-hydroxybutyl)amino]carbonyI}{2-[(methylsulfonyI)oxy]ethyl}-
2,4-dinitroanilino)ethyI methanesulfonate (28) [for method of preparation 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 IIc-9 (100 mg, 21%) as a foam; 1H NMR [(CD3)2SO] δ 8.73 (m,
1H, CONH), 8.25 (d, J= 9.4 Hz, 1H), 7.63 (d,J= 9.4 Hz, 1H), 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); 13C NMR δ 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) Calcd. For
Cl5H2079Br2N4O6 [M+H+] mJz 510.9828. Found; 510.9819.
Further elution with EtOAc/MeOH (50:2) gave IIc-14 (117 mg, 30%): mp. 114-117 °C; lH
NMR [(CD3)2SO] δ 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); 13C NMR δ 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. (Cl6H23BrN4O9S) C, H, N.
Example 23 (Scheme 2k). 2-(3-{[(3-hydroxypropyI)amino]carbonyl}{2-
[(methylsulfonyl)oxy]ethyl}-2,4-dinitroanilino)ethyl methanesulfonate (27) and 2-((2-
bromoethyI)-3-{[(3-hydroxypropyl)amino]carbonyl}-2,4-dinitroaniIino)ethyI
methanesulfonate (IIc-13). Solid IIc-8 (2.15 g, 4.3 mmol) was added to a hot solution of
silver methanesulfonate (0.992 g, 4.9 mmol) in dry MeCN (40 mL). The mixture was
heated under reflux for 3 h, then cooled and filtered. The solvent was removed at 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 IIc-13 (0.5 g, 25%), IIc-8
(0.3 g, 14%) and 27 (0.4 g, 18%).
Example 24 (Scheme 2k). 2-((2-chIoroethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-
2,4~dinitroanilino)ethyl methanesulfonate (IIc-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 h, then
cooled, poured into dilute HCl, and extracted with EtO Ac (3x150 mL). Workup and
chromatography of the product on silica gel, eluting with EtO Ac/petroleum ether from 1:1
to 1:0, gave IIc-6 (4.0 g, 50%) as yellow crystals: mp104-1O9°C ;1H NMR [(CD3)2SO] δ
8.75 (t, J= 5.8 Hz, 1H, 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 (m, 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); !3C NMR δ 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. (Cl5H21ClN4O9S)
C, H, N.
Example 25 (Scheme 2k). 2-((2-Iodoethyl)-3-{[(3-hydroxypropyl)amino]carbonyl}-
2,4-dinitroanilino)ethyl methanesulfonate (IIc-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 h, and
the product was chromatographed on silica gel, eluting with EtOAc/petroleum ether from
1:2 to 1:0, to give IIc-15 (2.29 g, 41%) as yellow crystals: mp 100-103°C; 1H NMR
[(CD3)2SO] δ 10.05 (s, 1H), 7.40 (d, J= 11.5 Hz, 1 H), 7.O9 (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); 13C NMR δ 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.
(Cl5H21IN4O9S) C, H, N.
Preparation of Phosphates (Scheme 3)
Example 26.2-[[2-[Bis(2-bromoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl
dihydrogen phosphate (Ib-7P). A solution of alcohol IIb-7 (2.58 g, 5.33 mmol) and di-
tert-butyl diethylphosphoramidite (93%, 2.0 mL, 6.9 mmol) in dry DMF (20 mL) under N2
was treated with lH-tetrazole (3 wt. % in CH3CN, 55 mL, 18.7 mmol) and stirred at 20 °C
for 1.5 h. The reaction mixture was then cooled to -50 °C and a solution of 3-
chloroperoxybenzoic acid (55%, 2.68 g, 8.54 mmol) was rapidly added such that the
temperature was kept below -5 °C. The reaction mixture was warmed to room
temperature and diluted with CH2Cl2 (150 mL). The solution was washed with 5%
aqueous Na2S205 (2 x 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 shaken with i-
Pr20/hexane and refrigerated. The resulting solid was purified by chromatography on
silica gel, eluting with CH2Cl2/EtOAc, followed by recrystallisation from CH2Cl2/hexane

(below 40 °C) to give di-tert-butyl 2-[[2-[bis(2-bromoethyl)amino]-3,5-
dinitrobenzoyl]amino]ethyl phosphate (Ib-7E)(2.59 g, 72%) as an unstable yellow solid:
mp 99-101 °C (dec); 1H NMR [(CD3)2SO] δ 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) calcd for C21H3479Br2N4O9P (MH+) m/z 675.0430 found 675.0398;
calcd for C21H3479Br81BrN4O9P (MH+) m/z 677.0410, found 677.0397; calcd for
C2iH3481Br2N4O9P (MH+) m/z 679.0389, found 679.0398. Anal. (C21H33Br2N4O9P).
A solution of Ib-7E (2.80 g, 4.14 mmol) and TFA (15 mL) in dry CH2Cl2 (15 mL) was
stirred at 20 °C for 1 h, then concentrated under reduced pressure. Residual TFA was
removed azeotropically with CH3CN (2 x) and the resulting residue was dissolved in
EtOAc. Addition of excess hexane precipitated a semisolid which was dried under high
vacuum at 20 °C to give Ib-7P (98%) as a yellow foam. 1H NMR [(CD3)2SO] δ S.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) calcd for Cl3H1879Br2N4O9P (MH+) m/z 562.9178,
found 562.9171; calcd for Cl3H1879Br81BrN4O9P (MH+) m/z 564.9158, found 564.9152;
calcd for Cl3H1881Br2N4O9P. (MH+) m/z 566.9137, found 566.9121. Treatment of diacid
Ib-7P with NaHCO3 (2.0 equiv.) gave the disodium salt.
Example 27.3-[ [5-[Bis(2-chloroethyl)amino]-2,4-dinitrobenzoyI] amino] propyl
dihydrogen phosphate (Ia-3P). Similar phosphorylation of IIa-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)amino]-2,4-dinitrobenzoyl]amino]propyl phosphate (Ia-
3E) (76%) as a yellow solid: mp (EtOAc/i-Pr20/hexane) 120-121°C (dec); 1H NMR
[(CD3)2SO] δ 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 D20 exchange, t, J- 6.8
Hz, 2 H), 1.86 (pent, J= 6.6 Hz, 2 H), 1.42 (s, 18 H). Anal. (C22H35Cl2N4O9P) C, H, N.

Similar treatment of ester Ia-3E with TFA gave diacid Ia-3P (99%) as a hygroscopic
yellow solid. 1H NMR [(CD3)2SO] δ 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) Calcd. for C14H2035Cl2N4O9P
[M+H]+ m/z 489.0345; found 489.0344. Calcd. for C14H2035Cl37ClN4O9P [M+H]+ m/z
491.0316; found 491.0317. Calcd. for C14H2037Cl2N4O9P [M+H]+ m/z 493.0286; found
493.0312. Treatment of diacid I-3P with NaHCO3 (2:0 equiv) gave the disodium salt.
Example 28.3- [ [5- [Bis(2-bromoethyl)amino] -2,4-dinitrobenzoy I] amino] propyl
dihydrogen phosphate (Ia-8P). Similar phosphorylation of IIa-8, followed by
chromatography of the product on silica gel and elution with CH2Cl2/EtOAc (1:1), gave di-
tert-butyl 3-[[5-[bis(2-bromoethyl)amino]-2,4-dinitrobenzoyl]amino]propyl phosphate (Ia-
8E) (66%) as a yellow solid: mp (EtOAc/i-Pr20/hexane) 110-111°C (dec). 1H NMR
((CD3)2SO) δ 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 D20 exchange, t, J= 6.8 Hz, 2 H), 1.86 (pent, J= 6.6
Hz, 2 H), 1.42 (s, 18 H). Anal. (C22H35Br2N4O9P) C, H, N.
Similar treatment of ester Ia-8E with TFA gave diacid Ia-8P (99%) as a hygroscopic
yellow solid. 1H NMR [(CD3)2SO] δ 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) calcd for C14H2079Br2N4O9P (MH+) m/z 576.9335, found
576.9314; calcd for C14H2079Br81BrN4O9P (MH+) m/z 578.9314, found 578.9305; calcd for
C14H2081Br2N4O9P (MH4) m/z 580.9294, found 580.9297. Treatment of diacid Ia-8P with
NaHCO3 (2.0 equiv.) gave the disodium salt.
Example 29.2-[[2-[Bis(2-chloroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl
dihydrogen phosphate (Ib-2P). Similar phosphorylation of IIb-2, followed by
chromatography of the product on silica gel and elution with CH2Cl2/EtOAc (13:7), gave
di-tert-butyl 2-[[2-[bis(2-chloroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl phosphate

(Ib-2E) (72%) as a yellow solid: mp (EtOAc/i-Pr20/hexane) 107-108 °C (dec); 1H NMR
[(CD3)2SO] δ 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. (C21H33Cl2N4O9P) C, H, N, P. CRL 11363.
Similar treatment of ester Ib-2E with TFA gave diacid Ib-2P (98%) as a yellow foam. 1H
NMR [(CD3)2SO] δ 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) Calcd. for C13H1835Cl2N2O9P [M+H]+ m/z 475.0189;
found 475.0189. Calcd. for C13H1835Cl37ClN2O9P [M+H]+ m/z 477.0159; found 477.0167.
Calcd. for C13H1835Cl2N2O9P [M+H]+ m/z 479.0130; found 479.0160. Treatment of diacid
Ib-2P with NaHCO3 (1.0 equiv.) gave the monosodium salt.
Example 30.2-[(2-Chloroethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl] amino]-
carbonyl]anilino]ethyl methanesulfonate (Ib-2mP). Similar phosphorylation of IIb-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-phosphanon-l-
anoyl)-4,6-dinitroanilino]ethyl methanesulfonate (Ib-2mE) (80%) as a yellow foam. 1H
NMR [(CD3)2SO] δ 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). 13C NMR δ 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.
Similar treatment of ester Ib-2mE with TFA gave diacid Ib-2mP (68%) as a yellow solid.
Mp (EtOAc/CH2Cl2): 132-134 °C; 1H NMR [(CD3)2SO] δ 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 D20 exchange, m, 8 H), 3.13 (s, 2 H). 13C NMR δ 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.
(C14H20ClN4O12PS)C,H,N.

Example 31.2-({2-[Bis(2-bromopropyl)amino]-3,5-dinitrobenzoyl}amino)ethyl
dihydrogen phosphate (Ib-7aP). Similar phosphorylation of alcohol IIb-7a (0.67 g, 1.3
mmol) with di-tert-buty\ diethylphosphoramidite (93%, 489 mg, 2.0 mmol), followed by
flash column chromatography on silica gel, eluting with EtOAc/petroleum ether (1:1) gave
Ib-7aE as a yellow solid (0.74 g, 81%): mp (EtOAc/petroleum ether) 121-123 °C; 1H
NMR [(CD3)2SO] δ 9.O9 (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). 13C NMR δ 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.
(C23H37Br2N4O9P)C,H,N.
Similar treatment of Ib-7aE (100 mg) with TFA (6 mL), followed by crystallization from
CH2Cl2/EtOAc, gave Ib-7aP as a yellow solid (70 mg, 85%): mp 157-161 °C; 1H NMR
[(CD3)2SO] δ 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). 13C NMR δ 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. (C15H21Br2N4O9P) C, H, N.
Example 32.2-[(2-BromoethyI)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-
carbonyl]anilino]ethyl methanesulfonate (Ib-12P). Similar phosphorylation of IIa-12,
followed by chromatography of the product on silica gel and elution with EtO Ac, gave 2-
[(2-bromoethyl)-2-(6-tert-butoxy-8,8-dimethyl-6-oxido-5,7-dioxa-2-aza-6-phosphanon-l-
anoyl)-4,6-dinitroanilino]ethyl methanesulfonate (Ib-12E) (66%) as a yellow foam. 1H
NMR [(CD3)2SO] δ 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,y = 6.2 Hz, 2 H), 3.62-3.43 (m, 8 H), 3.13 (s, 3 H),
1.43 (s, 18 H). HRMS (FAB) calcd for C22H3779BrN4O12PS [M+H]+ m/z 693.1029; found
693.1010.
Similar treatment of ester Ib-12E with TFA gave diacid Ib-12P (98%) as a yellow foam.
1H NMR [(CD3)2SO] δ 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 D20
exchange, m, 8 H), 3.13 (s, 2 H). HRMS (FAB) calcd for C14H2179BrN4O12PS [M+H]+ m/z
578.9798; found 578.9784; calcd for C14H2181Br81BrN4O12PS [M+H]+ m/z 580.9777;
found 580.9784. Treatment of diacid Ib-12P with NaHCO3 (1.0 equiv) gave the
monosodium salt.
Example 33.2-[[2-[Bis(2-iodoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl
dihydrogen phosphate (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 phosphate (Ib-
14E) (67%) as a yellow solid: mp (CH2Cl2/i-Pr2O/hexane) 108-110 °C (dec); 1H NMR
[(CD3)2SO] δ 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, J7.8 Hz, 4 H), 3.24 (after D20
exchange, t, J= 1.6 Hz, 4 H), 1.44 (s, 18 H). Anal. (C21H33I2N4O9P), C, H, N, P.
Similar treatment of ester Ib-14E with TFA gave diacid Ib-14P (97%) as a yellow foam.
1HNMR [(CD3)2SO] δ 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 D20 exchange t, J= 5.6 Hz, 2 H), 3.45 (t, J
= 7.8 Hz, 4 H), 3.29 (t, J= 1.1 Hz, 4 H). HRMS (FAB) Calcd. for C13H18I2N4O9 [M+H]+
m/z 658.3911; found 658.3907. Treatment of diacid Ib-14P with NaHCO3 (2.0 equiv.)
gave the disodium salt.
Example 34.2-[(2-Iodoethyl)-2,4-dinitro-6-({[2-
(phosphonooxy)ethyl]amino}carbonyI)-anilino]ethyl methanesulfonate (Ib-15P).
Similar phosphorylation of alcohol IIb-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/petroleum ether (1:1), and
crystallization from EtOAc/petroleum ether, gave Ib-15E as a yellow solid (2.23 g, 97%):
mp (EtOAc/petroleum ether) 1O9-111 °C; 1H NMR [(CD3)2SO] δ 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). 13C NMR δ 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. (C22H36IN4012PS), C, H,N.
Similar treatment of Ib-15E (405 mg) with TFA (6 mL) and crystallization of the product
from CH2Cl2/petroleum ether gave diacid Ib-15P as a yellow solid (3O6 mg, 89%): mp
147-150 °C; 1H NMR [(CD3)2SO] δ 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). 13C
NMR δ 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. (C14H20IN4O9PS), 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 IIc-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: mp (EtOAc/petroleum ether) 98-102
°C. 1H NMR [(CD3)2SO] δ 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). 13C NMR δ 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.(C23H38ClN4O12PS) C, H, N.
Similar treatment of ester Ic-6E with TFA gave diacid Ic-6P (84%) as a yellow solid: mp
(EtOAc/CH2Cl2) 98-102 °C; 1H NMR [(CD3)2SO] δ 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 D20 exchange, 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. (C15H22ClN4O12PS) C, H, N.

Example 36.3-({3-[Bis(2-bromoethyI)amino]-2,6-dinitrobenzoyl}amino)propyl
dihydrogen phosphate (Ic-8P). Similar phosphorylation of alcohol IIc-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/petroleum ether (1:1),
gave Ic-8E as a yellow solid (1.77 g, 91%): mp (EtOAc/petroleum ether) 112-114 °C; 1H
NMR [(CD3)2SO] δ 8.86 (m, 1 H), 8.24 (d, J= 9.4 Hz, 1 H), 7.63 (d, J= 9A 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).
13C NMR δ 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. (C22H35Br2O9P), C, H, N.
Similar treatment of Ic-8E (900 mg) with TFA (10 mL) gave diacid Ic-8P as a yellow
foam (754 mg, 100%): 1H NMR [(CD3)2SO] δ 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). 13C NMR δ 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) calcd for C14H2079Br2N4O9P. [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
EtO Ac/petroleum ether (from 1:2 to 1:0), gave (Ic-12E) (99%) as a yellow solid: mp
(EtOAc/petroleum ether) 82-86 °C (dec). 1H NMR [(CD3)2SO] δ 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. (C22H36BrN4O12PS) C, H, N.
Similar treatment of ester Ic-12E with TFA gave diacid Ic-12P (100%) as a yellow solid:
mp (EtOAc/CH2Cl2) 93-97 °C; 1H NMR [(CD3)2SO] δ 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 D20 exchange, t, J= 6.0 Hz, 2 H), 3.23 (q, J= 6.6 Hz,
2 H), 3.15 (s, 3 H). Anal. (C14H20BrN4O12PS) C, H, N.
Example 38.2-[(2-BromoethyI)-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-l-anoyl)-2,4-dinitroanilino]ethyl methanesulfonate (Ic-13E) (70%) as a
yellow solid: mp (CH2Cl2/i-Pr2O) 95_-96 °C (dec). 1H NMR [(CD3)2SO] δ 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. (C23H38BrN4O12PS) C, H, N, P.
Similar treatment of ester Ic-13E with TFA gave diacid Ic-13P (98%) as a hygroscopic
yellow solid. 1H NMR [(CD3)2SO] δ 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 D20 exchange, 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) calcd for C15H2379BrN4O12PS [M+H]+ m/z
592.9954; found 592.9956. Treatment of diacid Ic-13P with NaHCO3 (1:0 equiv) gave the
monosodium salt.
Example 39.2-[(2-Iodoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-
carbonyl]anilino] ethyl methanesulfonate (Ic-15P). Similar phosphorylation of IIc-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: mp (EtOAc/iPr20) 90-100 °C. 1H NMR [(CD3)2SO] δ 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 D20

exchange, m, 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. (C23H38IN4O12PS) C, H, N, P.
Similar treatment of ester Ic-15E with TFA gave diacid Ic-15P (97%) as a hygroscopic
yellow solid: mp (CH3CN/EtOAc) 84-86 °C. 1H NMR [(CD3)2SO] δ 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
D2O exchange, q, J= 6.7 Hz, 2 H), 3.62 (after D2O exchange, 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.O9 (s, 3 H), 1.73
(pent, J= 6.6 Hz, 2 H). HRMS(FAB) calcd for C15H22IN4O12PS (MH+) m/z 640.9816.
Found; 640.9795. Anal. (C15H22lN4O12PS) C, H.



Representative alcohols of Formula (I) (listed in Table 1 a) show selective cytotoxicity
towards human cancer cell lines transfected with either the E. coli nitroreductase cDNA
(NTR) (Table 3, columns 2 and 3), or human cytochrome P450 reductase (P450R) 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 is not required for hypoxic selectivity.
IC50 values are derived from cell proliferation experiments, following 4 hour drug
exposure under a gas phase of either 20% oxygen or 0% oxygen (anoxia, achieved using
an anaerobic chamber). Cells were grown under aerobic conditions for a further 5 days,
and cell density estimated using the sulphorhodamine B assay, to determine the
concentration of prodrug required to inhibit growth to 50% of control.



The activity of the phosphates as hypoxic cytotoxins 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 tumour, where the oxic tumour cells are sterilised using 15Gy of radiation, and the
cytotoxicity of an agent against the remaining hypoxic cells can be quantitated.
Unexpectedly, the activity of the phosphate Ib-7P is found to exceed that of its parent
alcohol (IIb-7) at their respective maximum tolerated doses (Ib-7P = 750 µmol/kg; IIb-7

= 1000 µmol/kg). This experiment demonstrates that the phosphate Ib-7P is more active
against hypoxic cells than the reference hypoxic cytotoxin tirapazamine, and that it is more
active against hypoxic cells than oxic cells (i.e. when given post irradiation than when
given without irradiation). Thus Ib-7P acts as a hypoxia-selective cytotoxin in vivo.
Although less active against oxic tumour cells, this activity is significant demonstrating
that the compound also has utility as a single agent (without radiation).



The notable activity of the phosphates of Formula (I) against hypoxic cells in human
tumour xenografts is illustratecd by the data of Table 4. In these experiments SiHa human
cervical carcinoma cells were grown subcutaneously in CD-1 nude (immunodeficient)
mice. The compounds were administered at doses corresponding to 75% or 20% of the
maximum tolerated dose (MTD), 5 minutes after a whole body dose of ionising radiation
(cobalt-60 gamma radiation) sufficient to sterilise the oxic cells (15 Gy). The tumours
were excised 18 hours later, dissociated with a cocktail of proteases, and cell survival was
determined using a clonogenic assay. The logarithms of cell kill were calculated from the
difference in the numbers of clonogens per gram tumour tissue between treated and control
tumours. All of the phosphates tested showed large effects against hypoxic cells at 75% of
the MTD (Table 4, column 4). This was selective for hypoxia as demonstrated by the
lesser cell kill in the absence of radiation. However, cell killing by the compounds alone
was significant in all cases (Table 4, column 5) demonstrating that the compounds also
have antitumour activity as single agents. 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 the reference
nitrogen mustards (melphalan, chlorambucil and cyclophosphamide) lacked activity at
20% of their respective MTDs.



1 I I L__ 1 1 ■ ■ ■
a Relative to controls, in the same experiment, treated with vehicle (saline) only. Log kill = log10(clonogens/g
tumour for control tumours) - log10(clonogens/g tumour for treated tumours).
b Relative to the mean for radiation only, in the same experiment. Log kill = log10(clonogens/g tumour for
radiation alone) - log10(clonogens/g tumour for tumours treated with radiation plus compound).
c None detected
A representative example of the phosphates of Formula (I) as NTR activated cytotoxins is
provided in Figure 2. In the WiDr in vivo growth delay assay, xenografts containing
mixtures of WiDrWT and WiDrNTR cells are grown to 300mm3 and treated with a single
dose of prodrug at its MTD. Tumour growth is monitored over time and animals are
euthanased when mean tumour volume > 1600mm3. Data is presented as time to death.
Unexpectedly, the activity of the phosphate (Ib-7P) is observed to exceed that of its parent
alcohol (IIb-7), when administered at their respective maximum tolerated doses (750
µmol/kg). Ib-7P is superior to IIb-7 with respect to (i) time to first treatment failure (77-
days vs. 17-days) and (ii) over-all survival (40% vs. 6%).





Pharmacokinetics of the phosphate esters Ib-2mP, Ib-7P, Ib-12P and Ic-12P following
administration to female CD-1 nude mice by intraperitoneal injection at a dose
corresponding to 75% of the maximum tolerated dose. Monosodium salts of the
compounds were dissolved in phosphate buffered saline, pH 7.4, with addition of one
equivalent of sodium bicarbonate. Serial blood samples were obtained by small tail vein
bleeds, and 10 ul of plasma was prepared from each. Proteins were precipitated by addition
of 3 volumes of methanol, and concentrations of the phosphate esters and corresponding
alcohols were determined by HPLC using either UV or mass spectrometry detection.


The data shows that the phosphate esters are converted efficiently to the corresponding
alcohols in mice. The alcohols are the species activated by nitroreduction in hypoxic, or
NTR-expressing, cells.
Wherein the foregoing description reference has been made to reagents, or integers having
known equivalents thereof, then those equivalents are herein incorporated as if
individually set forth.
While this invention has been described with reference to certain embodiments and
examples, it is to be appreciated that further modifications and variations may be made to
embodiments and examples without departing from the scope of the invention.

WE CLAIM:
1. A phosphate compound of Formula (I)

wherein:
X represents at any available ring position -CONH-;
R represents a lower Cl-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkyJamino and N-oxides therefrom;
Y represents -N-aziridinyl, -N(CH2CH2W)2 or -N(CH2CHMeW)2,
where each W is independently selected from halogen or -OSO2Me,
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
and pharmaceutieally acceptable salts and derivatives thereof.
2. A phosphate compound of Formula (I) as claimed in claim I which is selected from a
compound represented by formulae (Ia), (Ib) or (Ic)


and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where each W is independently selected from halogen or -OSO2Me;
and pharmaceutically acceptable salts and derivatives thereof.
3. The phosphate compound of Formula (I) as claimed in claim 1 or claim 2 which is
selected from:
2-[[2-[Bis(2-bromoethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogen phosphate;
3-[[5-[Bis(2-chloroethyl)amino]-2,4-dinitrobenzoyl]amino]propyl dihydrogen phosphate;
3-[[5-fBis(2-bromoethyl)amino]-2,4-dinitrobenzoyl]amino]propyl dihydrogen phosphate;
2-[[2-tBis(2-chIoroethyl)amino]-3,5-dinitrobenzoyl]amino]ethyl dihydrogen phosphate;
2-[(2-Chloroemyl)-2,4-dirnitro-6-[r[2-Cphosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl
methanesulfonate;
2-({2-[Bis(2-bromopropyl)amino]-3,5-dinitrobenzoyl}amino)ethyl dihydrogen phosphate;
2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl
methanesulfonate;
2-t[2-[Bis(2-iodoethyl)amino]-3,5-dinitrobenzoyl]aminoJethyl dihydrogen phosphate;
2-[(2-Iodoethyl)-2,4-dininx)-6-({[2-(phosphonooxy)ethyl]amino}carbonyl)-anilino]ethyl
methanesulfonate;

2-[(2-ChIoroethyI)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethyI
methanesulfonate;
3-( {3-[Bis(2-bromoethyl)amino]-2}6-dinitrobenzoyl }amino)propyl dihydrogen phosphate;
2-[(2-Bromoethyl)-2,4-dinitro-3-[[[2-(phosphonooxy)ethy]]amino]-carbonyl]anilino]elhyl
methanesulfonate;
2-[(2-Bromoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyI]amino]-carbonyl]anilino]ethyl
methanesulfonate; and
2-[(2-Iodoethyl)-2,4-dmitro-3-[[[3-(phosphonooxy)propyl]aminoJ-carbonyl]amlino]ethyl
methanesulfonate.
4. A method of preparing a phosphate represented by the general formula (I);

wherein:
X represents at any available ring position -CONH-;
R represents a lower C1-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom;
Y represents -N-aziridinyl or -N(CH2CH2W)2, where each W is independently selected from
halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me;
and pharmaceutically acceptable salts and derivatives thereof;
the method including the step of
(i) phosphorylating a compound of formula (II)


wherein:
X represents at any available ring position -CONH-;
Y represents -N-aziridinyl, -N(CH2CH2W)2, or -N(CH2CH MeW)2 where each W is
independently selected from halogen or -OSO2Me;
Z represents at any available ring position -NO2, -halogen, -CN, -CF3 or -SO2Me; and
R represents a lower C1-6 alkyl optionally substituted with one or more groups including
hydroxy, amino and N-oxides therefrom or dialkylamino and N-oxides therefrom.
5. A method of preparing a compound of formulae (Ia), (Ib) or (1c)


and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where each W is independently selected from halogen or -OSO2Me;
and pharmaceutically acceptable salts and derivatives thereof;
the method including the step of
phosphorylating a compound represented by formulae (IIa), (IIb) or (IIc)

and wherein
n represents 1 to 6
Z represents -NO2, -halogen, -CN, -CF3 or -SO2Me; and
where each W is independently selected from halogen or -OSO2Me
and pharmaceutically acceptable salts and derivatives thereof.
6. A compound of Formula (I) when obtained by the method defined in claim 4.
7. A compound of Formula (Ia), (Ib) or (Ic) when obtained by the method defined in claim 5.

8. A pharmaceutical composition including a therapeutically effective amount of a
compound of Formula (I) as defined in any one of claims 1 to 3 and a pharmaceuticaily
acceptable excipient, adjuvant, carrier, buffer or stabiliser.
9. A compound selected from:
2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)ethyl]aminoJ-carbonyl]anilmo]ethyl
methanesulfonate.
2-[(2-Bromoethyl)-2,4-dinitro-3-[[[2-(pbosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl
methanesulfonate.
2-[(2-Bromoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino]-carbonyl]anilino]ethyl
methanesulfonate; and
2-[(2-Iodoethyl)-2,4-dinitro-3-[[[3-(phosphonooxy)propyl]amino3-carbonyl]anilino]ethyl
methanesulfonate.
10. The compound
2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2^hosphonooxy)ethyl]amino]-carbonyl]anilino]ethyl
methanesulfonate.
11. A pharmaceutical composition including a therapeutically effective amount of a compound
as claimed in claim 10 and a pharmaceuticaily acceptable excipient, adjuvant, carrier, buffer or
stabiliser.


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.

Documents:

01473-kolnp-2006-abstract.pdf

01473-kolnp-2006-assignment.pdf

01473-kolnp-2006-claims.pdf

01473-kolnp-2006-correspondence others.pdf

01473-kolnp-2006-decription complete.pdf

01473-kolnp-2006-form 1.pdf

01473-kolnp-2006-form 2.pdf

01473-kolnp-2006-form 3.pdf

01473-kolnp-2006-form 5.pdf

01473-kolnp-2006-international publication.pdf

01473-kolnp-2006-international search report.pdf

01473-kolnp-2006-pct form.pdf

1473-KOLNP-2006-(02-09-2011)-CORRESPONDENCE.pdf

1473-kolnp-2006-(21-12-2011)-CORRESPONDENCE.pdf

1473-KOLNP-2006-ABSTRACT.pdf

1473-KOLNP-2006-CANCELLED DOCOMENT.pdf

1473-KOLNP-2006-CLAIMS.pdf

1473-KOLNP-2006-CORRESPONDENCE 1.6.pdf

1473-KOLNP-2006-CORRESPONDENCE OTHERS 1.1.pdf

1473-KOLNP-2006-CORRESPONDENCE OTHERS 1.2.pdf

1473-KOLNP-2006-CORRESPONDENCE OTHERS.pdf

1473-KOLNP-2006-CORRESPONDENCE-1.3.pdf

1473-KOLNP-2006-CORRESPONDENCE-1.4.pdf

1473-KOLNP-2006-CORRESPONDENCE-1.5.pdf

1473-KOLNP-2006-CORRESPONDENCE.pdf

1473-KOLNP-2006-DESCRIPTION COMPLETE.pdf

1473-KOLNP-2006-FORM 1.pdf

1473-KOLNP-2006-FORM 13.pdf

1473-KOLNP-2006-FORM 18.pdf

1473-KOLNP-2006-FORM 2.pdf

1473-KOLNP-2006-FORM 3.pdf

1473-KOLNP-2006-FORM 5.pdf

1473-KOLNP-2006-GRANTED-ABSTRACT.pdf

1473-KOLNP-2006-GRANTED-CLAIMS.pdf

1473-KOLNP-2006-GRANTED-DESCRIPTION (COMPLETE).pdf

1473-KOLNP-2006-GRANTED-FORM 1.pdf

1473-KOLNP-2006-GRANTED-FORM 2.pdf

1473-KOLNP-2006-GRANTED-SPECIFICATION.pdf

1473-KOLNP-2006-PA.pdf

1473-KOLNP-2006-REPLY TO EXAMINATION REPORT.pdf

1473-KOLNP-2006-TRANSLATED COPY OF PRIORITY DOCUMENT.pdf


« Previous Patent
Next Patent »
Patent Number 252229
Indian Patent Application Number 1473/KOLNP/2006
PG Journal Number 18/2012
Publication Date 04-May-2012
Grant Date 02-May-2012
Date of Filing 31-May-2006
Name of Patentee AUCKLAND UNISERVICES LIMITED
Applicant Address LEVEL 10, 70 SYMONDS STREET, AUCKLAND
Inventors:
# Inventor's Name Inventor's Address
1 DENNY, WILLIAM ALEXANDER 165 GOSSAMER DRIVE, PAKURANGA, AUCLAND 1706
2 YANG, SHANGJIN 19 HAYCOCK AVENUE, MOUNT ROSKILL, AUCKLAND
3 WILSON, WILLIAM ROBERT 199 SMITH ROAD, RD2 WAIUKU
4 HELSBY, NUALA ANN 212 WEST TAMAKI ROAD, GLENDOWIE, AUCKLAND
5 PATTERSON, ADAM, VORN 18 MANUKA ROAD, WAIHEKE, AUCKLAND 1240
6 HELSBY, Nuala Ann 212 West Tamaki Road, Glendowie, Aucland,
7 PATTERSON, Adam, Vorn 18 Manuka Road, Waiheke, Auckland 1240,
8 ATWELL, GRAHAM JOHN 192 GOWING DRIVE, MEADOWBANK, AUCLAND
PCT International Classification Number C07C 233/67
PCT International Application Number PCT/NZ2004/000275
PCT International Filing date 2004-10-29
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 529249 2003-10-31 New Zealand
2 535618 2004-09-28 New Zealand