Anilinopyrimidines as novel antituberculosis agents

Article abstract of DOI:10.1016/S0960-894X(03)00241-5

A selection of novel anilinopyrimidine analogues have been found to have micromolar activity against Mycobacterium tuberculosis. This could potentially generate new lead compounds in the fight against multi-drug resistant tuberculosis.

Full text of DOI:10.1016/S0960-894X(03)00241-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1755–1757
Anilinopyrimidines as Novel Antituberculosis Agents
Jody Morgan,a Rachada Haritakulb and Paul A. Kellera,*
aDepartment of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia
bNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency
(NSTDA), 113 Phaholyothin Rd., Klong 1, Klong Luang, Pathumthani 12120, Thailand
Received 20 February 2003;revised 25 February 2003;accepted 26 February 2003
Abstract—A selection of novel anilinopyrimidine analogues have been found to have micromolar activity against Mycobacterium
tuberculosis. This could potentially generate new lead compounds in the fight against multi-drug resistant tuberculosis.
# 2003 Elsevier Science Ltd. All rights reserved.
Tuberculosis (TB) is the leading infectious cause of
death today1 and is rapidly becoming a global epi-
demic.2 It is estimated that worldwide 100 million peo-
ple are infected annually.3 Approximately ten million
develop the disease, with five million of these progres-
sing to the infectious stage and ultimately three million
dying.4 The increase in TB is a result of two major fac-
tors;the susceptibility of people infected with the
acquired immune deficiency syndrome (AIDS), which
augments the risk of developing TB 100-fold,5 and the
increase in resistant strains of the disease6 with some
shown to be resistant to as many as nine drugs.5
Mycobacterium tuberculosis. The details of our pre-
liminary results are outlined in Table 1.
The short synthetic pathway involved can lead to the
generation of a large range of these anilinopyrimidine
analogues quickly and easily so they may be tested
against TB in order to determine an accurate structure–
activity relationship for these compounds.
To the best of our knowledge, there has been no pre-
vious reports of anilinopyrimidines as antituberculosis
agents. However, there are numerous examples of
nitrogen containing heterocycles being used to treat TB,
with example structures shown in Table 2. These com-
pounds provide structural precedence that our anilino-
pyrimidine analogues may lead to the generation of
novel anti-TB therapeutics.
Although one possible long term solution to the prob-
lem is a better vaccine, in the short term, the major
reliance will be on chemotherapy4 requiring the devel-
opment of novel, effective, non-toxic antituberculosis
agents.1,4,7
The mechanism of action of both isoniazid and pyr-
azinamide is known to be inhibition of cell-wall synth-
esis. Isoniazid acts as an inhibitor of InhA, a NADH-
specific enoylase-reductase, involved in the synthesis of
Our work focused upon the synthesis and biological
testing of some novel anilinopyrimidine compounds.
These compounds were generated via two simple sub-
stitution reactions of substituted amines onto a chloro-
pyrimidine derivative.8 An example reaction is outlined
in Scheme 1.
Seven of these synthesized derivatives were tested
against malaria and tuberculosis. None of these showed
any activity against Plasmodium falciparum, however,
six of the analogues showed micromolar activity against
*Corresponding author. Tel.: +61-2-4221-4692;fax: +61-2-4221-
4287;e-mail: paul_keller@uow.edu.au
Scheme 1. Pathway for the synthesis of anilinopyrimidine analogues.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00241-5

1756
J. Morgan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1755–1757
Table 1. The structure and anti-TB activity (MIC) against Myco-
bacterium tuberculosis for a range of anilinopyrimidine compounds
Table 2. Nitrogen containing heterocycles as known anti-TB agents
Drug
Structure
Remarks
Ref
Compds
Structure
MIC mg/mL
Active against
MDR-TB,
7
Clofazimine
1
50
MIC 2.0 mg/mL
3
Inactive
Most widely used
agent, however it is
hepato-toxic.
6,9
Isoniazid
9
Pyrazinamide
MIC 20 mg/mL
2
4
5
25
to inhibit fatty acid synthase type I, which provides the
precursors for mycolic acid biosynthesis.12 At this stage,
it is too premature to predict the mode of action of our
anilinopyrimidine compounds.
50
12.5
Biological Testing
The antimycobacterial activity was assessed against M.
tuberculosis H37Ra using the Microplate Alamar Blue
Assay (MABA). Standard drugs, isoniazid (MIC of
0.040–0.090 mg/mL) and kanamycin sulfate (MIC of
2.0–5.0 mg/mL) were used as reference compounds for
the antimycobacterial assay. The cytotoxicity assay
against the BC (human breast cancer cells), KB (oral
human epidermal carcinoma) and NCl-187 (human
small cell lung cancer) was performed employing the
colorimetric method,13 the reference substance was
ellipticine with an IC50 of 0.1–0.4 mg/mL. The anti-
malarial activity was evaluated against the parasite
P. falciparum (K1, multidrug-resistant strain), which
was cultured continuously according to the method of
Trager and Jensen.14 Quantitative assessment of anti-
malarial activity in vitro was determined by means of
the microculture radioisotope technique based upon
the method described by Desjardins et al.15 The
inhibitory concentration (IC50) represents the concen-
tration that cause 50% reduction in parasite growth as
indicated by the in vitro uptake of [3H]-hypoxanthine
by P. falciparum. An IC50 value of 1 ng/mL was
observed for the standard compound, artemisinin, in
the same test system.
6
12.5
7
3.12
fatty acids in mycobacteria.10 Initially this compound is
converted into its active form via KatG catalase-perox-
idase enzyme.11 The active component then inhibits
mycolic acid biosynthesis via the InhA enzyme. Mycolic
acids have been shown to be a key component in the
mycobacterial cell wall.12 Pyrazinamide has been shown
Conclusions
We have synthesised a novel series of anilinopyrimidine
compounds, via a short and easy three step procedure.
Six of these analogues have been found to have reason-

J. Morgan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1755–1757
1757
able activity against tuberculosis. These compounds
while not active enough to be considered as therapeutics
are definitely lead compounds in the search for novel
agents to combat resistance.
mmol, 42%) as a pale yellow solid, mp. 116–117 ꢀC. MS (CI);
m/z 222 (32%) [M+H 37Cl]+;220 (100) [M+H 35Cl]+;
1
HRMS for C11H11N335Cl, calc 220.0642, found 220.0635; H
0
NMR d 7.64, bs, 1H, NH;7.32, m, 5H, ArH2 ,30,40,50,60;6.43,
s, 1H, ArH5;2.32, s, 3H, CH
;
13C NMR d 168.7, ArC2;
3
163.0, ArC10;160.1, ArC6;137.1, ArC4;129.6, ArC2
0,60;
0
0
125.9, ArC5;123.5, ArC3 ,50;100.7, ArC4 ;23.8, CH 3. To a
solution of 4-anilino-2-chloro-6-methylpyrimidine (1) (0.12 g,
0.53 mmol) in dry THF (5 mL) was added diethylamine (0.6
mL, 0.54 mmol). The reaction mixture was flushed with nitro-
gen, and stirred in a sealed tube at 160 ꢀC for 24 h. The reac-
tion mixture was concentrated in vacuo. The crude residue was
subjected to gravity silica column chromatography, and elu-
tion with 10% ethyl acetate:hexanes gave 4-anilino-2-diethyl-
amino-6-methylpyrimidine (2) (0.10 g, 0.39 mmol, 79%) as
beige crystals, mp. 78–80 ꢀC. MS (CI); m/z 257 (100%)
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C 15H21N4, calc 257.1766, found
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1H, NH;5.82, s, 1H, ArH5;3.61, q, J=7.2 Hz, 4H, CH2CH3;
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1
0
166.6, ArC4;161.9, ArC1 ;161.2, ArC2;139.7, ArC6;129.0,
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CH2CH3;24.4, Ar–CH ;13.3, CH 2CH3.
3
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mixture was heated at reflux for 40 min. The cooled reaction
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