NITROIMIDAZOLE DERIVATIVES
Technical Field of the Invention
The present invention relates to compounds having activity against mycobacteria.
Certain compounds of the invention may be used in the treatment of mycobacterial
5 infections. The invention also provides radiolabelled compounds that are useful for in
vivo imaging in the diagnosis of mycobacterial infections. Methods and intermediates
useful for the preparation of certain compounds of the invention are also provided. The
invention also provides methods for using the compounds of the invention in treatment
and diagnosis.
10 Description of Related Art
Pulmonary tuberculosis (TB) is an airborne infection caused by Mycobacterium
tuberculosis (MTB) that causes high mortality and morbidity, particularly in developing
countries (Dye et al JAMA 1999; 282(7): 677-686). A recent factsheet produced by the
World Health Organisation reported that the number of new cases of TB continues to
15 increase each year in South-East Asia, the Eastern Mediterranean and Africa
(ht1p://www.who.int/mediacentre/factsheets/fsl04/eri//print.html). The antitubercular
nitroimidazoles, including two classes of new bicyclic agents with either fused oxazole or
oxazine rings, are one of the most exciting recent developments in the field of
antituberculosis chemotherapy, and two candidates are already in human clinical trials
20 for the treatment of both drug-susceptible and drug-resistant disease (in this regard the
reader is referred to the website http://www.newtbdrugs.org/pipeline.php). Sasaki et al
(J Med Chem 2006; 49(26): 7854-7860) have reported a series of novel optically active
6-nitro-2, 3-dihydroimidazo [2,1-/?]- oxazoles having various phenoxymethyl groups and
a methyl group at the 2-position. A particular compound that is potent and orally active
25 was found that is a promising candidate (OPC-67683) for the treatment of tuberculosis,
which is currently in clinical trials:
-1-
The unique structure of the cell wall of mycobacteria, rich in waxy my colic acid, is the
target of action of OPC-67683, which inhibits methoxy-mycolic and keto-mycolic acid
synthesis but at significantly lower concentrations.
With the recent emergence of drug-resistant strains of MTB there is still scope for
further improved agents to treat an otherwise incurable disease.
Radiolabelled nitroimidazoles are well-known for hypoxia imaging. Examples include
18F-misonidazole ([18F]FMISO) and 99mTcO(PnAO)-l-2-nitroimidazole (known as BMS-
181321):
These and other radiolabelled nitroimidazoles have been described as being particularly
useful in the detection of myocardial hypoxia (Strauss et al J Nuc Cardiol 1995; 2: 437-
445).
Accurate and prompt diagnosis is important in order to control the infection and also to
ensure the appropriate therapy for infected patients. Currently, a definitive diagnosis of
TB requires culture of MTB from a sample taken from a patient. Patients with clear
signs and symptoms of pulmonary disease with a sputum smear-positive result present
no problems to diagnose. However, there can be difficulty culturing the slow-growing
MTB organism in the laboratory. Furthermore the emergence of HIV has resulted in a
decreased likelihood of sputum smear positivity and an increase in non-respiratory
disease, such that ease of diagnosis is more difficult in these cases (see reviews by Jeong
& Lee Am J Roent 2008; 191: 834-844; Davies & Pai Int J Tuberc Lung Dis 2008;
12(11): 1226-1234; and, Lange & Mori Respirology 2010; 15: 220-240).
In vivo imaging methods are known to be useful in the diagnosis of TB. Chest x-ray is a
widely-used in vivo imaging method for screening, diagnosis and treatment monitoring in
patients with known or suspected TB. Chest computed tomography (CT) is more
sensitive than conventional x-ray and may be applied to identify early parenchymal
lesions or mediastinal lymph node enlargements and to determine disease activity in
tuberculosis (Lee & Im AJR 1995; 164(6): 1361-1367).
Nuclear imaging methods have also been reported for diagnosis and treatment
monitoring of TB. The positron-emission tomography (PET) tracer 18Ffluorodeoxyglucose
([18F]FDG) has been proposed as useful in the diagnosis of disease
activity and therapy monitoring in patients with TB (Demura et al Eur J Nuc Med Mol
Imag 2009; 36: 632-639). Roohi et al (Radiochim Acta 2006; 94: 147-152) describe a
99mTc-labelled isoniazid derivative, which localised to tubercular lesions in rabbits and
enabled the lesions to be visualised 2 hours following administration of the 99mTc-labelled
derivative. However, this 99mTc-labelled derivative comprises a 99mTc-chelate at a
location believed to be the active pharmacophore, which is not ideal.
There is therefore scope for improved strategies in the treatment and diagnosis of TB.
Summary of the Invention
The present invention provides novel compounds useful in the treatment and diagnosis
of mycobacterial infections. Compounds of the present invention have enhanced
biological properties as compared to the related known compounds. The present
invention also provides a precursor compound useful in the synthesis of certain
compounds of the invention, and a method to obtain these compounds using said
precursor compound. Methods of treatment and diagnosis in which the compounds of
the invention find use are also provided.
WO 2011/151320 PCT/EP2011/058938
Detailed Description of the Invention
Compound
In one aspect, the present invention provides a compound of Formula I:
02N N
5 wherein:
R1 is absent or is CM alkyl;
R2 is a halogen isotope; and,
X is -O- or -NH-.
Unless otherwise specified, the term "alkyl" alone or in combination, means a straight-
10 chain or branched-chain alkyl radical containing preferably from 1 to 4 carbon atoms.
Examples of such radicals include, methyl, ethyl, and propyl.
The term "halogen isotope" refers to any radioactive or non-radioactive isotope of a
halogen (also referred to herein as "radioactive halogen" and "non-radioactive halogen",
respectively). The terms radioactive and non-radioactive take their commonly-known
15 meaning, i.e. "radioactive" refers to giving off, or capable of giving off, radiant energy in
the form of particles or rays, as alpha, beta, and gamma rays, by the spontaneous
disintegration of atomic nuclei. The term''non-radioactive'' means not radioactive. The
term "halogen" suitably refers to an atom selected from iodine, fluorine, chlorine and
bromine, preferably to iodine and fluorine and most preferably to iodine.
20 R1 is preferably CM alkyl, and is most preferably methyl.
X is preferably -0-.
In one preferred embodiment, R2 is a gamma-emitting radioactive halogen selected from
WO 2011/151320 PCT/EP2011/058938
I, I and Br. For this embodiment said gamma-emitting radioactive halogen is
preferably 123I.
In another preferred embodiment, R2 is a positron-emitting radioactive halogen selected
from 17F, 18F, 75Br, 76Br and 124I. For this embodiment, said positron-emitting radioactive
5 halogen is selected from 18F and 124I, and is most preferably 124I.
127T 79T In a further preferred embodiment, R is a non-radioactive halogen selected from I, Br,
", 19F. For this embodiment, sai
and 19F, and is most preferably 127I.
81Br, 19F. For this embodiment, said non-radioactive halogen is preferably selected from 127I
If a chiral centre or another form of an isomeric centre is present in a compound
10 according to the present invention, all forms of such isomer, including enantiomers and
diastereoisomers, are encompassed by the present invention. Compounds of the
invention containing a chiral centre may be used as racemic mixture or as an
enantiomerically-enriched mixture, or the racemic mixture may be separated using wellknown
techniques and an individual enantiomer maybe used alone. In a preferred
15 embodiment, an individual enantiomer is used alone. Preferably, individual enantiomer
of the compound as defined herein is of Formula la:
R"
, . o
°2N N n ^
(la)
wherein R11, R12 and X1 are as suitably and preferably defined herein for R1, R2 and X,
respectively.
20 Precursor Compound
In another aspect, the present invention provides a precursor compound for the preparation
of compound of Formula I wherein R2 is a radioactive halogen as defined above, wherein
said precursor compound is a compound of Formula II:
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WO 2011/151320 PCT/EP2011/058938
(II)
wherein:
R21
is as defined above for R1 of Formula I;
R22 is a non-radioactive iodine or bromine, an organometallic derivative such as a
5 trialkylstannane or a trialkylsilane, an organoboron compound such as a boronate ester
or an organotrifluoroborate, or is selected from amino, hydroxy, nitro, bromo, iodo, tri-
Ci-3-alkylammonium, quaternary ammonium, diazonium, iodonium, tosylate, mesylate
and triflate; and,
X2 is as defined above for X of Formula I.
10 A "precursor compound" comprises a non-radioactive derivative of a radiolabeUed
compound, designed so that chemical reaction with a convenient chemical form of the
detectable label occurs site-specifically; can be conducted in the minimum number of
steps (ideally a single step); and without the need for significant purification (ideally no
further purification), to give the desired radiolabeUed compound. In the context of the
15 present invention, the term "radiolabeUed compound" refers to the compound of
Formula I wherein R2 is a radioactive halogen. Such precursor compounds are synthetic
and can conveniently be obtained in good chemical purity. In order to facilitate sitespecific
reaction, the precursor compound of the invention may optionally comprise a
suitable protecting group.
20 By the term "protecting group" is meant a group which inhibits or suppresses
undesirable chemical reactions, but which is designed to be sufficiently reactive that it
may be cleaved from the functional group in question to obtain the desired product
under mild enough conditions that do not modify the rest of the molecule. Protecting
groups are well known to those skilled in the art and are described in 'Protective Groups
25 in Organic Synthesis', Theorodora W. Greene and Peter G. M. Wuts, (Fourth Edition,
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WO 2011/151320 PCT/EP2011/058938
John Wiley & Sons, 2007).
An "organometallic derivative" is an organic substituent containing a metal, especially a
wherein a metal atom is bonded directly to a carbon atom. In the context of the present
invention the term preferably relates to trialkylstannane and trialkylsilane substituents. The
5 term''trialkylstannane'' refers to the moiety-Sn-(alkyl)3, wherein each alkyl is the same and
wherein the term alkyl is as defined above, and is preferably a Ci_6 alkyl, most preferably
methyl or butyl, and most especially preferably butyl. The term "trialkylsilane" refers to the
moiety -Si-(alkyl)3 wherein the (alkyl)3 portion is as defined for trialkylstannane.
The term "organoboron compound" (also known as organoborane compound) refers to a
10 substituent that is an organic derivative of BH3. A "boronate ester" is a substituent derived
from an alkyl or aryl substituted boric acid containing a carbon-boron bond belonging to the
larger class of organoboranes, wherein the terms alky and aryl are as defined herein. An
"organotrifluoroborate" is a substituent derivaed from an organoboron compound that
contains an anion with the general formula [RBF3]~.
15 The term "amino" refers to the group -NH2.
The term "hydroxy 1" refers to the group -OH.
The term "nitro" refers to the group -NO2.
The term "bromo" refers to a bromine substituent.
The term "iodo" refers to an iodine substituent.
20 The term "quaternary ammonium" refers to the group -NR3 wherein each R is an alkyl or an
aryl, wherein the terms alkyl and aryl are as defined herein. Preferably, each R is an alkyl,
most preferably a Ci_3 alkyl.
The term "diazonium" refers to the -N=N group.
The term "iodonium" in the context of the present invention refers to the ion RI+ wherein R
25 is any organic residue. R is preferably an aryl wherein the term "aryl" refers to aromatic
rings or ring systems having 5 to 12 carbon atoms, preferably 5 to 6 carbon atoms, in the
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WO 2011/151320 PCT/EP2011/058938
ring system, e.g. phenyl or naphthyl.
The term "tosylate" refers to the group -0-S(02)-/?-toluene.
The term "mesylate" refers to the group -0-S(02)-methyl.
The term "triflate" refers to the group -O- S(02)-CF3.
5 The preferred embodiments provided above for R1 and X of Formula I apply equally to R2
and X2, respectively of Formula II.
In a preferred embodiment, the precursor compound of the invention is of Formula Ha:
R31
•N' ^ ^ J " * "
. - 4> O
°>N N (Ila)
wherein:
10 R31 is as defined above for R21 of Formula II;
R32 is as defined above for R22 of Formula II;
X3 is as defined above for X of Formula II.
Precursor compounds of the present invention may be obtained by following the
methods described by Nagarajan et al (1989 Eur J Med Chem; 24: 631-633) by reaction
15 of 2,4-dinitroimidazole (1) with a substituted oxirane (2) as illustrated in Scheme 1
below:
-8-
Scheme 1
11 10 1 A0
wherein R , R and X are as suitably and preferably defined herein. R is either an R
group, or is an R12 group protected by a suitable protecting group wherein the
protecting group is removed in step (ii) of Scheme 1 following reaction in step (i) of 1
5 and 2 to obtain the precursor compound of the invention following deprotection. R42
may alternatively be a chemical group, or a suitably protected version thereof, which
may be converted using known organic chemistry methods into an R12 group in step (ii)
following completion of step (i).
In an alternative, the precursor compounds of the invention may be obtained by
10 following the methods described by Sasaki et al (2006 J Med Chem; 49 (26):7854-
7860), wherein a 2-chloro-5-nitro imidazole starting material (3) is converted to the
corresponding epoxide (4) and then reacted with the desired phenol (for X1 = -NH-) or
phenylamine (for X1 = -NH-) (5) to obtain the precursor compound of the invention, as
illustrated below in Scheme 2:
-9-
11 10 A0 1
In Scheme 2, R , R , R" and X1 are as described above for Scheme 1.
The precursor compound of the invention is ideally provided in sterile, apyrogenic form.
The precursor compound can accordingly be used for the preparation of a
5 radiopharmaceutical composition comprising the compound of the invention wherein R2
is a radioactive halogen, together with a biocompatible carrier suitable for mammalian
administration, which forms another aspect of the invention as described in more detail
below.
The precursor compound is also suitable for inclusion as a component in a kit or a
10 cassette for the preparation of such a pharmaceutical composition. These aspects of the
invention are also discussed in greater detail below.
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WO 2011/151320 PCT/EP2011/058938
Method to Prepare Compounds
With routine adaption, the above-described methods to obtain precursor compounds of
the invention can also be applied to obtain a compound of Formula I wherein R2 is a
non-radioactive halogen isotope.
5 In another embodiment, the present invention relates to a method for the preparation of the
compound of the invention wherein said compound comprises a radioactive halogen, and
wherein said method comprises reaction of the precursor compound as defined herein with a
suitable source of said radioactive halogen. The suitable and preferred aspects of the
compound of Formula I and the precursor compound of Formula II as defined herein apply
10 equally to this aspect of the invention.
The term "a suitable source said radioactive halogen" means the radioactive halogen in a
chemical form that is reactive with a substituent of the precursor compound such that the
radioisotope becomes covalently attached to the precursor compound. The person
skilled in the art of in vivo imaging agents will be familiar with sources of radioactive
15 halogen that are suitable for application in the present invention. The reader is referred
to the "Handbook of Radiopharmaceuticals" for a detailed presentation of the field
(2003; Wiley: Welch and Redvanly, Eds).
The step of "reaction" of the precursor compound with the suitable source of a
radioactive halogen involves bringing the two reactants together under reaction
20 conditions suitable for formation of the desired compound in as high a radiochemical
yield (RCY) as possible. Synthetic routes for obtaining particular compounds of the
present invention are presented in the experimental section below.
Methods of introducing radioactive halogens are described by Bolton (2002 J
LabCompRadiopharm; 45: 485-528).
25 It is known in the art that to introduce a radioactive halogen (which can be either a
gamma-emitting radioactive halogen or a positron-emitting radioactive halogen) the
precursor suitably comprises the following reactive groups: a non-radioactive precursor
halogen atom such as an aryl iodide or bromide (to permit radioiodine exchange); an
activated aryl ring (e.g. phenol or aniline groups); an imidazole ring; an indole ring; an
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WO 2011/151320 PCT/EP2011/058938
organometallic compound (eg. trialkyltin or trialkylsilyl); or an organic compound such
as triazene or a good leaving group for nucleophilic substitution such as an iodonium
salt. Methods of introducing radioactive halogens are described by Bolton (2002 J
LabCompRadiopharm; 45: 485-528). Examples of suitable aryl groups to which
5 radioactive halogens, especially iodine can be attached are given below:
Both contain substituents which permit facile radioiodine substitution onto the aromatic
ring. Alternative substituents containing radioactive iodine can be synthesised by direct
iodination via radiohalogen exchange wherein radioiodide ion is the suitable source of
10 radioactive iodine, e.g.:
Where R2 is radioactive iodine, a preferred precursor compound of Formula II comprises
at R22 a derivative which either undergoes electrophilic iodination. Examples of this are
organometallic derivatives such as a trialkylstannane (e.g. trimethylstannyl or
15 tributylstannyl), or a trialkylsilane (e.g. trimethylsilyl) or an organoboron compound (e.g.
boronate esters or organotrifluoroborates).
For electrophilic radioiodination, R22 of the precursor compound of Formula II
preferably comprises: an activated organometallic precursor compound (e.g. trialkyltin,
trialkylsilyl or organoboron compound). Precursor compounds and methods of
20 introducing radioiodine into organic molecules are described by Bolton (2002 J Lab
Comp Radiopharm; 45: 485-528). Suitable boronate ester organoboron compounds and
their preparation are described by Kabalaka et al (2002 Nucl Med Biol; 29: 841-843 and
2003 Nucl Med Biol; 30: 369-373). Suitable organotrifluoroborates and their
preparation are described by Kabalaka et al (2004 Nucl Med Biol 2004; 31: 935-938).
25 Preferred precursor compounds of Formula II for radioiodination comprise at R22 an
organometallic precursor compound, most preferably a trialkyltin, and especially
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WO 2011/151320 PCT/EP2011/058938
tributyltin.
Radiobromination can be achieved by methods similar to those described above for
radioiodination. Kabalka and Varma have reviewed various methods for the synthesis of
radiohalogenated compounds, including radiobrominated compounds (1989
5 Tetrahedron; 45(21): 6601-21).
The methods used when the radioactive halogen is 18F are described in detail in Chapter
6 of the Handbook of Radiopharmaceuticals (2003; Wiley: Welch and Redvanly, Eds).
18F has a relatively short half-life and therefore special considerations are required in the
synthesis of compounds comprising 18F.
10 Labelling with 18F can be achieved by nucleophilic displacement of a leaving group from
a precursor compound. In this way, the precursor compound may be labelled in one step
by reaction with a suitable source of [18F]-fluoride ion (18F~), which is normally obtained
as an aqueous solution from the nuclear reaction 180(p,n)18F and which is made reactive
by the addition of a cationic counterion and the subsequent removal of water to form a
15 suitable source of 18F. The radio fluorine atom attaches via a direct covalent bond to the
aromatic ring. 18F-fluoride nucleophilic displacement from an aryl diazonium salt, aryl
nitro compound or an aryl quaternary ammonium salt are suitable routes. Preferably,
where it is desired to add radioactive fluorine, R22 of said precursor compound is a
leaving group selected from hydroxyl, nitro, bromo, iodo, tri-Ci_3-alkylammonium,
20 quaternary ammonium, diazonium, iodonium, tosylate, mesylate and triflate, and said
suitable source of radioactive halogen is 18F-fluoride (18F~).
In one embodiment, the method for the preparation is automated. A cassette useful in
this automated method forms a further aspect of the invention describe in more detail
below.
25 Kit and Cassette
In a yet further aspect, the present invention provides a kit for the preparation of a
compound of the invention wherein R2 is a radioactive halogen, said kit comprising a
precursor compound of the invention as defined herein, so that reaction with a sterile
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WO 2011/151320 PCT/EP2011/058938
source of a radioactive halogen gives the desired compound with the minimum number
of manipulations. Such considerations are particularly important where the radioisotope
has a relatively short half-life, and for ease of handling and hence reduced radiation dose
for the radiopharmacist. The precursor compound is preferably present in the kit in
5 lyophilized form, and the reaction medium for reconstitution of such kits is preferably a
biocompatible carrier. Suitable and preferred embodiments of the precursor compound
for the kit of the invention are as provided above for the precursor compound of the
invention.
A "biocompatible carrier" is a fluid, especially a liquid, in which the resultant
10 radiolabeled compound of the invention is suspended or dissolved, such that the
composition is physiologically tolerable, i.e. can be administered to the mammalian body
without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable
carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such
as saline (which may advantageously be balanced so that the final product for injection is
15 either isotonic or not hypotonic); an aqueous solution of one or more tonicity-adjusting
substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g.
glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or
other non-ionic polyol materials (e.g. polyethyleneglycols, propylene glycols and the
like). The biocompatible carrier may also comprise biocompatible organic solvents such
20 as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or
formulations. Preferably the biocompatible carrier comprises pyrogen-free water for
injection, or isotonic saline. The pH of the biocompatible carrier for intravenous
injection is suitably in the range 4.0 to 10.5.
In the kit of the invention, the precursor compound is preferably presented in a sealed
25 container which permits maintenance of sterile integrity and/or radioactive safety, plus
optionally an inert headspace gas (e.g. nitrogen or argon), whilst permitting addition and
withdrawal of solutions by syringe. A preferred sealed container is a septum-sealed vial,
wherein the gas-tight closure is crimped on with an overseal (typically of aluminium).
Such sealed containers have the additional advantage that the closure can withstand
30 vacuum if desired e.g. to change the headspace gas or degas solutions.
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The precursor compound for use in the kit may be employed under aseptic manufacture
conditions to give the desired sterile, non-pyrogenic material. The precursor compound
may alternatively be employed under non-sterile conditions, followed by terminal
sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment
5 (e.g. with ethylene oxide). Preferably, the precursor compound is provided in sterile,
non-pyrogenic form. Most preferably the sterile, non-pyrogenic precursor compound is
provided in the sealed container as described above.
Preferably, all components of the kit are disposable to minimise the possibilities of
contamination between runs and to ensure sterility and quality assurance.
10 [18F] -radiotracers in particular are now often conveniently prepared on an automated
radiosynthesis apparatus. There are several commercially-available examples of such
apparatus, including Tracerlab™ and Fastlab™ (GE Healthcare Ltd). Such apparatus
commonly comprises a "cassette", often disposable, in which the radiochemistry is
performed, which is fitted to the apparatus in order to perform a radiosynthesis. The
15 cassette normally includes fluid pathways, a reaction vessel, and ports for receiving
reagent vials as well as any solid-phase extraction cartridges used in post-radiosynthetic
clean up steps.
The present invention therefore provides in another aspect a cassette for the automated
synthesis of compound of Formula I comprising 18F, wherein said cassette comprises:
20 (i) a vessel containing a precursor compound comprising a leaving group
wherein said leaving group is as defined herein for the precursor
compound of the invention; and
(ii) means for eluting the vessel with a suitable source of 18F-fluoride (18F~).
The cassette may additionally comprise:
25 (iii) an ion-exchange cartridge for removal of excess 18F-fiuoride (18F~).
Pharmaceutical Composition
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WO 2011/151320 PCT/EP2011/058938
In another aspect, the present invention provides a pharmaceutical composition comprising
the compound of Formula I together with a biocompatible carrier in a form suitable for
mammalian administration.
When R2 of the compound of Formula I in said pharmaceutical composition is a radioactive
5 halogen, said pharmaceutical composition is a radiopharmaceutical composition and the
biocompatible carrier is as defined above in relation to the kit of the invention. The
radiopharmaceutical composition may be administered parenterally, i.e. by injection, and is
most preferably an aqueous solution. Such a composition may optionally contain further
ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g. cyclodextrins or
10 surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable
stabilisers or antioxidants (such as ascorbic acid, gentisic acid or/?ara-aminobenzoic acid).
Where the compound of the invention is provided as a radiopharmaceutical composition, the
method for preparation of said compound may further comprise the steps required to obtain
a radiopharmaceutical composition, e.g. removal of organic solvent, addition of a
15 biocompatible buffer and any optional further ingredients. For parenteral administration,
steps to ensure that the radiopharmaceutical composition is sterile and apyrogenic also need
to be taken.
The suitable and preferred embodiments described herein for the compound of Formula I
wherein R2 is a radioactive halogen apply equally to the radiopharmaceutical composition of
20 the invention.
Where the pharmaceutical composition comprises the compound of Formula I wherein R2 is
a non-radioactive halogen, the biocompatible carrier may be a solid or liquid
pharmaceutically acceptable nontoxic carrier. Such pharmaceutical carriers can be sterile
liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic
25 origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred carrier when the pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerols solutions are also be employed as liquid
carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include
starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, magnesium
30 carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium
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WO 2011/151320 PCT/EP2011/058938
chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. These
compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders,
sustained release formulations and the like. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" (18th Edition; E. W. Martin, Ed: 1990 Mack
5 Publishing). Such compositions will contain an effective therapeutic amount of the
compound together with a suitable amount of carrier so as to provide the form for proper
administration to the host. While intravenous injection is a very effective form of
administration, other modes can be employed, e.g. oral administration.
In Vivo Imaging and Diagnosis
10 In a further aspect, the present invention provides an in vivo imaging method comprising:
(a) administration of the compound of Formula I wherein R2 is a radioactive halogen;
(b) allowing said compound to bind to the cell wall of any mycobacteria present in said
subject;
(c) detecting by an appropriate in vivo imaging procedure signals emitted by the
15 radioactive halogen comprised in said compound;
(d) generating an image representative of the location and/or amount of said signals;
and,
(e) determining the distribution of mycobacteria in said subject wherein said distribution
is directly correlated with said signals emitted by said radioactive halogen.
20 The "administration" step is preferably carried out parenterally, and most preferably
intravenously. The intravenous route represents the most efficient way to deliver the
compound throughout the body of the subject, and also does not represent a substantial
physical intervention on the body of the subject. By the term "substantial" is meant an
intervention which requires professional medical expertise to be carried out, or which entails
25 a substantial health risk even when carried out with the required professional care and
expertise. The compound is preferably administered as the pharmaceutical composition of
the invention, as defined herein. The in vivo imaging method of the invention can also be
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understood as comprising the above-defined steps (b)-(e) carried out on a subject to whom
said compound has been pre-administered. In this embodiment, the compound is preferably
administered as the radiopharmaceutical composition of the invention.
Following the administering step and preceding the detecting step, the compound is allowed
5 to bind to mycobacteria within said subject. For example, when the subject is an intact
mammal, the compound will dynamically move through the mammal's body, coming into
contact with various tissues therein. Once the compound comes into contact with any
mycobacteria, the two entities bind such that clearance of the compound from tissue in
which mycobacteria are present takes longer than from tissue without any mycobacteria
10 present. A certain point in time will be reached when detection of compound specifically
bound to mycobacteria is enabled as a result of the ratio between compound bound to tissue
with mycobacteria versus that bound in tissue without any mycobacteria. This is the optimal
time for the detecting step to be carried out.
The "detecting" step of the method of the invention involves detection of signals emitted by
15 the radioactive halogen by means of a detector sensitive to said signals. This detection step
can also be understood as the acquisition of signal data. Single-photon emission
tomography (SPECT) and positron-emission tomography (PET) are suitable in vivo imaging
procedures for use in the method of the invention. When R2 is a gamma-emitting
radioactive halogen, SPECT is suitable, and when R2 is a positron-emitting radioactive
20 halogen, PET is suitable.
The "generating" step of the method of the invention is carried out by a computer which
applies a reconstruction algorithm to the acquired signal data to yield a dataset. This
dataset is then manipulated to generate images showing the location and/or amount of
signals emitted by the radioactive halogen which is comprised in the compound used in said
25 in vivo imaging method. The signals emitted directly correlate with the presence of
mycobacteria such that the "deteirnining" step can be made by evaluating the generated
image.
The "subject" of the invention can be any human or animal subject. Preferably the subject of
the invention is a mammal. Most preferably, said subject is an intact mammalian body in
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vivo. In an especially preferred embodiment, the subject of the invention is a human. The in
vivo imaging method may be used in subjects known or suspected to have a pathological
condition associated with a mycobacterial infection. Preferably, said method relates to the
in vivo imaging of a subject known or suspected to have tuberculosis caused by
5 Mycobacrerium tuberculosis, and therefore has utility in a method for the diagnosis of said
condition. Where a subject is known to have tuberculosis caused by Mycobacrerium
tuberculosis, the in vivo imaging method of the invention may be carried out repeatedly
during the course of a treatment regimen for said subject, said regimen comprising
administration of a drug to combat tuberculosis caused by Mycobacrerium tuberculosis.
10 The present invention additionally provides a method for diagnosis of a mycobacterial
infection in a subject wherein said method comprises the in vivo imaging method as defined
herein, together with a further step (vi) of attributing the distribution of mycobacteria to a
mycobacterial infection. The term "mycobacterial infection" is defined herein as an infection
caused by a mycobacterium. The method of diagnosis is preferably used to diagnose
15 tuberculosis caused by Mycobacterium tuberculosis.
In a yet further aspect, the present invention provides the radiopharmaceutical composition
as suitably and preferably defined herein for use in a method of in vivo imaging wherein said
method of in vivo imaging is as suitably and preferably defined herein.
The present invention also provides the radiopharmaceutical composition as suitably and
20 preferably defined herein for use in a method of diagnosis wherein said method of diagnosis
is as suitably and preferably defined herein.
Treatment
In a yet further aspect, the present invention provides a method for the treatment of a
mycobacterial infection comprising administration of the compound of Formula I wherein R2
25 is a non-radioactive halogen. Preferably, said compound is administered as a pharmaceutical
composition. A suitable pharmaceutical composition for a compound of Formula I wherein
R2 is a non-radioactive halogen is defined above. As for the methods of in vivo imaging and
diagnosis of the invention, said mycobacterial infection is preferably tuberculosis caused by
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Mycobacterium tuberculosis.
As presented in the experimental examples herein, the compound of Formula I of the
present invention wherein R2 is a non-radioactive halogen has good activity against
Mycobacterium tuberculosis and as such has properties which make it a potentially useful
5 treatment against Mycobacterium tuberculosis.
The suitable and preferred embodiments of R2 as a non-radioactive halogen as presented
above in connection with the compound of Formula I apply equally to the method of
treatment of the invention.
In one embodiment, the method of treatment may also comprise the combined
10 administration of the compound of the invention with other known treatments for
tuberculosis. Non-limiting examples of such other treatments including isoniazid,
rifampicin, pyrazinamide, and ethambutol.
Brief Description of the Examples
Example 1 describes the synthesis of the unlabelled prior art compound, (R)-2-Methyl-6-
15 nitro-2-(phenoxymethyl)-2,3-dihydroimidazo[2,1 -b]oxazole.
Example 2 describes the synthesis of an iodinated version of the prior art compound
prepared in Example 1, (R)-2-((4-Iodophenoxy)methyl)-2-methyl-6-nitro-2,3-
dihydroimidazo[2,l-b]oxazole, a compound of Formula I of the invention wherein R2 is
non-radioactive iodine.
20 Example 3 describes the in vitro screening methods used to evaluate the compounds
obtained in Examples 1 and 2.
Example 4 describes the synthesis of (R)-2-((4-fluorophenoxy)methyl)-2-methyl-6-nitro-
2,3-dihydroimidazo[2,l-b]oxazole, a compound of Formula I of the invention wherein
R2 is non-radioactive fluorine.
25 Example 5 describes the synthesis of (R)-2-methyl-6-nitro-2-((4-
(tributylstannyl)phenoxy)methyl)-2,3-dihydroimidazo[2,1 -b]oxazole, a precursor
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compound of the invention.
List of Abbreviations used in the Examples
ATP adenosine triphosphate
DCM dichloromethane
5 DMF dimethylformamide
HPLC high-performance liquid chromatography
IC50 half maximal inhibitory concentration
LCMS liquid chromatography mass spectrometry
LORA low-oxygen recovery assay
10 MABAmicroplate alamar blue assay
MIC minimum inhibitory concentration
RBF round-bottom flask
VERO "verda reno" meaning "green kidney" in Esperanto; used to refer to a line of
kidney epithelial cells extracted from an African green monkey
15 (Cercopithecus aethiops).
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Examples
Example 1: Synthesis of(R)-2-Methvl-6-nitro-2-(phenoxvmethyl)-2,3-
dihvdroimidazo[2,l-bloxazole (prior art compound)
5 (R)-2-chloro-l- (2-methyl-2, 3-epoxypropyl)-4-nitroimidazole was obtained by
conversion of commercially-available 2-chloro-5-nitro imidazole starting material to the
corresponding epoxide following the method described by Sasaki et al (2006 J Med
Chem; 49: 7854-7860). (R)-2-chloro-l- (2-methyl-2, 3-epoxypropyl)-4-nitroimidazole
(57.7 mg, 0.267 mmol), and phenol (20.12mg, 0.214 mmol) were placed in a 50ml RBF
10 and dissolved in 2ml of DMF. The reaction mixture was cooled to 0°C and then to it,
NaH (16.48 mg, 0.256 mmol) was added carefully. The temperature was then increased
to 50°C and the reaction mass was stirred for 24-36 hours. The reaction was checked
for completion using the HPLC/LCMS and the reaction mass was concentrated on a
rotary evaporator. The dried material was then taken for purification on a CombiFlash
15 (Teledyne Isco) chromatography system (using a DCM-methanol solvent system). The
purified material was then taken for recrystallization using a DCM-hexane solvent
system to yield a pale yellow powder as the product. Yield = 7.4 mg; Purity = 96%; *H
NMR (CDC13): 8 1.8(dd (J=3.0,9.0), 2H,CH2), 4.22 (d (J=9), 1H, CH2), 4.5 (d (J=9),
1H, CH2), 6.86 (d (J=9.0), 2H, ArH), 7.6 (t (J=6.0, 1H, ArH), 7.33 (d (J=6.0), 2H,
20 ArH), 7.6 (s, 1H, ArH); MS: m/z 276 (M+l, 100%).
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Example 2:Synthesis of (R)-2-((4-Iodophenoxv)methvl)-2-methyl-6-nitro-2,3-
dihvdroimidazo[2,l-bloxazole (iodinated derivative of the prior art compound of
Example 1)
5 The method as described in Example 1 was used except that p-iodo phenol (28.38 mg,
0.129 mmol) was used in place of phenol. Yield = 4.2 mg; Purity = 96%; 1H NMR
(CDC13): 8 1.8(dd (J=3.0,9.0), 2H,CH2), 4.22 (d (J=9), 1H, CH2), 4.5 (d (J=9), 1H,
CH2), 6.64 (d (j=9.0), 2H, ArH), 7.6 (m, 3H, ArH) ; MS: m/z 402 (M+l, 100%).
Example 3: Methods used to Screen Compounds In Vitro
10 3d) Methods for Determining Minimum Inhibition Concentration (MIC)
Screening was done to get MIC for M. tuberculosis using both the microplate alamar
blue assay (MABA) and low-oxygen recovery assay (LORA).
The initial screen was conducted against Mycobacterium tuberculosis strain H37Rv
(American Type Culture Collection number 27294) in BACTEC 12B medium (Becton-
15 Dickinson) using the MABA. Compounds were tested in ten 2-fold dilutions, typically
from 100 p,g/mL to 0.19 p,g/mL. The MIC90 is defined as the concentration effecting a
reduction in fluorescence of 90% relative to controls. This value is determined from the
dose-response curve using a curve-fitting program. Any MIC90 value of <10ug/mL was
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considered "active" for antitubercular activity.
3(H) Method for Determining IC50
A VERO cell cytotoxicity assay was carried out in parallel with the TB Dose Response
assay. After 72 hours exposure, viability was assessed using Promega's Cell Titer Glo
5 Luminescent Cell Viability Assay, a homogeneous method of determining the number of
viable cells in culture based on quantitation of the ATP present. Cytotoxicity was
determined from the dose-response curve as the IC50 using a curve-fitting program.
3 (Hi) Method for Determining Calculated clogP
Chemdraw Ultra 10.0 (Cambridge Soft Software) was used to determine calculated
10 clogP values.
3(iv) In Vitro Screening Results
Compound
Example 1
Example 2
MABA MIC (ng/ml)
1.329
<0.195
IC50(ng/ml)
>50
>50
Calcd clogP
2.4
3.63
The above screening data demonstrates that introduction of iodine has reduced the MIC,
by a factor of over 6 which means iodine introduction has surprisingly increased the
activity of the parent compound.
15 Example 4: Synthesis of(R)-2-((4-fluorophenoxv)methvl)-2-methyl-6-nitro-2,3-
dih ydroimidazof2, l-bjoxazole
(R)-2-chloro-l-((2-methyloxiran-2-yl)methyl)-4-nitro-lH-imidazole (50.0 mg, 0.230
mmol) was transferred to clean, dry RBF and to it, added anhydrous DMF (2.0 ml). To
this mass, added p-fluoro phenol (20.66mg, 0.184 mmol) and stirred under nitrogen for
10 minutes. The mixture was then cooled to 0°C and then added sodium hydride (60%)
(8.84mg, 0.221 mmol) portion wise. The contents of the flask were allowed to stir in
cold conditions for about 10 minutes and then heated to 50 C. The reaction showed
completion within 30 hours on the LC/MS. The contents of the flask were allowed to
cool to room temperature and then concentrated on the rotary evaporator. The resulting
mass as such was taken for purification on the CombiFlash system using DCM/Methanol
as the gradient system. The resulting solid was then recrystallized using a DCM/Hexane
system to yield 5mg (74.6%) of the product as a whitish solid.
LC-MS: m/z calcd for C13H12FN304, 293.08; found, 294 (M+H)+.
Example 5: Synthesis of (R)-2-methyl-6-nitw-2-((4-
(tributvlstannvl)phenoxv)methvl)-2,3-dihvdroimidazof2,l-bJoxazole
A mixture of (R)-2-((4-iodophenoxy)methyl)-2-methyl-6-nitro-2,3-dihydroimidazo[2,1 -
b]oxazole prepared according to Example 2 (25mg, 0.0623 mmol), bis- (tributyltin)
(54.25mg, 47 ju.1, 0.0935 mmol) and tetrakis triphenylphosphine) palladium (0) (5.1 lmg,
0.004426 mmol) was taken in a mixed solvent (2.0 ml, 1:1 dioxane/triethyl amine) and
stirred under reflux for 36 hours. Upon checking for completion, the solvent was
removed, and to the residue added 4-5ml of water. The reaction mixture was then
extracted using ethyl acetate, separated, dried and evaporated. The residue was then
purified using the HPLC system. However, once purified, the compound could not be
isolated from the solvent as the molecule is not stable once removed from it. The
confirmation of product formation was from the LC/MS system with a single peak with
m/z 565 (M+H)+.

Claims
(1) A compound of Formula I:
wherein:
5 R1 is absent or is CM alkyl;
R2 is a halogen isotope; and,
X is -O- or -NH-.
(2) The compound as defined in Claim 1 wherein R1 is methyl.
(3) The compound as defined in either Claim 1 or Claim 2 wherein X is -0-.
10 (4) The compound as defined in any one of Claims 1-3 wherein R2 is a radioactive
halogen.
(5) The compound as defined in Claim 4 wherein said radioactive halogen is a gammaemitting
radioactive halogen selected from I, I and Br.
(6) The compound as defined in Claim 5 wherein said gamma-emitting radioactive
15 halogen is 123I.
(7) The compound as defined in Claim 4 wherein said radioactive halogen is a positronemitting
radioactive halogen selected from 17F, 18F, 75Br, 76Br and 124I.
(8) The compound as defined in Claim 7 wherein said positron-emitting radioactive
halogen is selected from 18F and 124I.
20 (9) The compound as defined in any one of Claims 1-3 wherein R2 is a non-radioactive
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halogen selected from 1271,79Br, 81Br, 19F.
(10) The compound as defined in Claim 9 wherein said non-radioactive halogen is
selected from 127I and 19F.
(11) The compound as defined in any one of Claims 1-10 which is of Formula la:
wherein
R11 is as defined for R1 in either Claim 1 or Claim 2;
R12 is as defined for R2 any one of Claims 1 or 4-10; and,
X1 is as defined for X group in either Claim 1 or Claim 3.
(12) A precursor compound for the preparation of compound of Formula I as defined in
Claim 4, which is a compound of Formula II:
wherein:
R21 is as defined for R1 in either Claim 1 or Claim 2;
R22 is a non-radioactive iodine or bromine, an organometallic derivative such as a
trialkylstannane or a trialkylsilane, an organoboron compound such as a boronate ester
or an organotrifluoroborate, or is selected from amino, hydroxy, nitro, bromo, iodo, tri-
Ci-3-alkylammonium, quaternary ammonium, diazonium, iodonium, tosylate, mesylate
and triflate; and,
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X2 is as defined for X in either Claim 1 or Claim 3.
(13) The precursor compound as defined in Claim 12 which is of Formula Ha:
wherein:
R31 is as defined for R1 in either Claim 1 or Claim 2;
R32 is as defined for R22 in Claim 12;
X3 is as defined for X in either Claim 1 or Claim 3.
(14) A method for the preparation of the compound as defined in Claim 4 wherein said
method comprises reaction of the precursor compound as defined in either Claim 12 or
10 Claim 13 with a suitable source of said radioactive halogen.
(15) The method as defined in Claim 14 wherein R22 of said precursor compound is an
organometalhc derivative such as a trialkylstannane or a trialkylsilane, and wherein said
suitable source of said radioactive halogen comprises I-radioiodide ( I").
(16) The method as defined in Claim 14 wherein R22 of said precursor compound is a
15 leaving group selected from hydroxyl, nitro, bromo, iodo, tri-Ci-3-alkylammonium,
quaternary ammonium, diazonium, iodonium, tosylate, mesylate and triflate, and wherein
said suitable source of said radioactive halogen is 18F-fluoride (18F~).
(17) A kit for carrying out the method as defined in any one of Claims 14-16 wherein said
kit comprises a vial containing said precursor compound.
20 (18) A cassette for the automated performance of the method as defined in Claim 16,
wherein said cassette comprises:
(i) a vessel containing said precursor compound; and
(ii) means for eluting the vessel with 18F-fluoride (18F~).
(19) The cassette as defined in Claim 18 which further comprises:
(iii)an ion-exchange cartridge for removal of excess 18F-fluoride (18F~).
(20) A pharmaceutical composition comprising the compound as defined in any one of
5 Claims 1-11 together with a biocompatible carrier in a form suitable for mammalian
administration.
(21) An in vivo imaging method comprising:
(a) administration of the compound as defined in Claim 4;
(b) allowing said compound to bind to the cell wall of any mycobacteria present in said
10 subject;
(c) detecting by an in vivo imaging procedure signals emitted by said radioactive
halogen;
(d) generating an image representative of the location and/or amount of said signals;
and,
15 (e) determining the distribution of mycobacteria in said subject wherein said distribution
is directly correlated with said signals.
(22) The in vivo imaging method as defined in Claim 21 wherein said administration step
is carried out by intravenous injection.
(23) The in vivo imaging method as defined in either Claim 21 or Claim 22 wherein said
20 mycobacterium is Mycobacterium tuberculosis.
(24) The in vivo imaging method as defined in Claim 23 which is carried out repeatedly
during the course of a treatment regimen for said subject, said regimen comprising
administration of a drug to combat tuberculosis caused by Mycobacterium tuberculosis.
(25) A method for diagnosis of a mycobacterial infection in a subject wherein said
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method comprises the in vivo imaging method as defined in any one of Claims 21-23,
together with a further step (vi) of attributing the distribution of mycobacterium to a
mycobacterial infection.
(26) The method of diagnosis as defined in Claim 25 wherein said mycobacterial infection
5 is tuberculosis caused by Mycobacterium tuberculosis.
(27) A method for the treatment of a mycobacterial infection wherein said method
comprises administration of the compound as defined in Claim 9.
(28) The compound as defined in any one of Claims 1-11 for use in a medical method.
(29) The compound as defined in Claim 28 wherein said medical method is as defined in
10 any one of Claims 21-27.