Synthesis and evaluation of 2-pyridyl pyrimidines with in vitro antiplasmodial and antileishmanial activity

Article abstract of DOI:10.1016/j.bmcl.2008.11.098

A series of 2-pyridyl pyrimidines, reported inhibitors of Plasmodium falciparum methionine aminopeptidase 1b were synthesized and evaluated for their antiplasmodial activities. An analysis of physicochemical properties demonstrated a link between lipophil

Full text of DOI:10.1016/j.bmcl.2008.11.098

Bioorganic & Medicinal Chemistry Letters 19 (2009) 401–405
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 2-pyridyl pyrimidines with in vitro
antiplasmodial and antileishmanial activity
b
c
c
Chitalu C. Musonda a,b, Gavin A. Whitlock b, , Michael J. Witty , Reto Brun , Marcel Kaiser
*
a University of Cape Town, Department of Chemistry, P/B Rondebosch 7700, Cape Town, South Africa
b Department of Chemistry, Pfizer Global R&D, Ramsgate Road, Sandwich, Kent CT13 9NJ, United Kingdom
c Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-pyridyl pyrimidines, reported inhibitors of Plasmodium falciparum methionine aminopepti-
dase 1b were synthesized and evaluated for their antiplasmodial activities. An analysis of physicochem-
ical properties demonstrated a link between lipophilicity and antiparasitic activity. Cross screening of the
library against cultured Leishmania donovani parasites revealed this class of compounds as potent inhib-
itors of parasite development in vitro.
Received 22 September 2008
Revised 18 November 2008
Accepted 18 November 2008
Available online 3 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Plasmodia
Malaria
Leishmania
P. falciparum
L. donovani
Hit-to-lead
Annual global clinical cases of malaria infection exceed 300
million, resulting in over 1 million deaths.1 Among the four species
of the parasite that cause malaria in man, Plasmodium falciparum is
the most virulent and is responsible for 90% of reported malaria
cases. Many first line defenses against malaria, such as chloroquine
(CQ) have been rendered ineffective due to development of drug
resistance in the parasite. Although the discovery of new treat-
ments such as artemisinin has been a significant achievement in
the fight against malaria,2 there is still an urgent need for the
discovery, development and delivery of new chemotherapies to
combat the disease.
Other tropical diseases also cause significant morbidity and
mortality in the developing world. For example, leishmaniasis
causes debilitating effects, severely attacking visceral, cutaneous
and mucocutaneous organs.3 Caused by more than 20 different
species of Leishmania, leishmaniasis has a prevalence of an esti-
mated 12 million cases annually, the majority of which are
reported in the tropics and subtropics.4 The disease is further com-
pounded by low efficacies of current therapies as well as resistance
and toxicity. As with malaria, new chemotherapeutic agents are
urgently needed for the treatment of leishmaniasis.
(PfMetAP) 1b and arrest parasite development in vitro.5 For exam-
ple, XC11 (Fig. 1) was found to be a potent inhibitor of PfMetAP 1b
enzyme activity and XC11 also inhibited P. falciparum proliferation
of both the chloroquine-sensitive 3D7 and chloroquine-resistant
Dd2 strains. XC11 was also shown to possess in vivo activity in
two mouse models of malaria, and was shown to have a synergistic
effect when dosed in vivo with CQ.
As part of a collaboration between Pfizer and WHO-TDR to dis-
cover new hits and leads to treat neglected tropical diseases,6 we
sought to expand the SAR governing the activity of the 2-pyridyl
pyrimidine scaffold reported by Chen et al.5 In addition to explor-
ing antiplasmodial SAR, we also determined the activity for this
series against other parasites where MetAP enzymes have been
shown to be important in parasite survival. For example it has
Cl
H
N
P. falciparum MetAP1b IC50 0.11μM
N
N
N
P. falciparum Dd2 IC50 3.1μM
Active in in vivo plasmodia models
Synergisitc in vivo activity with CQ
It has recently been disclosed that 2-pyridyl pyrimidines act as
selective inhibitors of P. falciparum methionine aminopeptidase
XC11
* Corresponding author. Tel.: +44 1304 649174; fax: +44 1304 651987.
E-mail address: gavin.whitlock@pfizer.com (G.A. Whitlock).
Figure 1. Structure and biological activity of XC11.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.098

402
C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 401–405
R1
recently been demonstrated that MetAP2 inhibitors such as fum-
R1
agillin and TNP-470 arrest parasite growth in Leishmania donovani
parasites.7 Herein, we present the results of a synthesis and screen-
ing programme of 2-pyridyl pyrimidines against malaria and leish-
mania parasites.
The medicinal chemistry design strategy focused on under-
standing which parts of the XC11 structure were important for
antiparasitic activity (the 2-pyridine had previously been shown
to be crucial for Pf MetAP 1b and antiparasitic activity).5 A series
of compounds was then designed to test where polarity could be
tolerated, the effect of conformational constraint and to inform
on the relationship between activity and lipophilicity (as estimated
by calculated LogP values).
R
Cl
NR2R3
R
N
N
N
(i)
N
N
N
30-80%
1 R = Me R1 = Cl
2 R = Me R1 = H
3 R = CF3 R1 = H
4 - 33
Scheme 1. Reagent and condition: (i) R2R3NH, Et3N, EtOH, reflux.
Table 1
Chemical structures and in vitro P. falciparum K1, L. donovani activity and rat myoblast L6 cytotoxicity for compounds 4–33
R1
NR2R3
R
N
N
N
Compound
R
R1
NR2R3
cLogP
P. falciparum IC50
(l
M)
L. donovani IC50
(l
M)
Cytotoxicity IC50 (lM)
Chloroquine
Artemisinin
Miltefosine
4
5
6
—
—
—
Me
Me
Me
—
—
—
Cl
Cl
Cl
—
—
—
NH2
NMe2
NEt2
0.25
0.005
nda
20.8
15.1
9.0
nda
nda
0.7
3.3
1.1
70.1
>170
nda
85.3
115
1.8
2.7
3.8
1.22
148
7
8
Me
Me
Cl
Cl
2.9
4.1
8.1
3.2
0.53
0.47
80
N
70.1
HN
9
Me
Cl
4.8
9.8
0.5
25.5
HN
HN
10
11
Me
Me
Cl
Cl
5.1
4.8
1.9
3.5
0.48
0.32
73.5
62.5
N
12
13
14
15
16
Me
Me
Me
Me
Me
Cl
Cl
Cl
Cl
Cl
5.3
5.8l
2.5
2.5
3.1
2.4
2.3
0.31
0.26
10.5
10.7
7.4
42.4
49.5
125
HN
HN
15.3
10.1
8.2
O
HN
HN
SO2Me
SO2Me
93.6
103.5
HN
HN
Cl
17
Me
Cl
5.5
6.3
1.2
91.6

C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 401–405
403
Table 1 (continued)
Compound
R
R1
Cl
NR2R3
cLogP
P. falciparum IC50
(l
M)
L. donovani IC50
(l
M)
Cytotoxicity IC50
32.6
(lM)
Cl
18
Me
5.5
2.5
0.44
HN
HN
Cl
19
20
Me
Me
Cl
Cl
5.5
4.7
2.0
2.5
0.45
0.43
40.4
8.7
OMe
HN
N
21
22
Me
Me
Cl
Cl
3.3
4.7
9.5
3.7
14.1
0.42
110
HN
HN
33.2
23
Me
Cl
4.8
8.9
1.1
183
N
N
24
25
Me
Me
Cl
Cl
4.4
4.4
4.7
4.1
0.39
0.70
133
76.5
N
N
26
27
28
Me
Me
Me
Cl
Cl
Cl
4.8
4.9
3.7
4.3
3.1
0.63
0.38
92.3
88
N
N
O
N S
O
11.6
54.4
210
O
S
29
Me
Cl
3.3
10.4
11.4
72.8
HN
O
H
N
CF3
30
31
Me
Me
Cl
Cl
4.7
1.9
2.74
7.5
0.28
156
5.2
HN
N
N
OH
N
270
32
Me
CF3
H
H
4.0
4.5
3.7
8.2
3.0
2.0
18.3
74.2
HN
HN
33
a
nd, not determined.
The target compounds were synthesized according to the gen-
eral route in Scheme 1. The commercially available chloro-pyrim-
idines 1–3 were aminated with the desired amines in EtOH in
presence of Et3N to afford compounds 4–33 (Table 1).8
The antiplasmodial activity of compounds 4–33 was deter-
mined in a CQ-resistant K1 P. falciparum strain using chloroquine
as standard (Table 1).9 Simple N-alkylated analogues 4–7 had weak
to moderate P. falciparum activity. Extension of the side chain and
incorporation of an aryl group (8–11) gave a trend towards increas-
ing activity, although with a rise in lipophilicity also. In our hands,
XC11 (9) had similar activity in a CQ-resistant P. falciparum strain
when compared to data reported in the literature.5 Saturation of
the aryl ring in 12–13 was well tolerated. However, incorporation
of a more polar aliphatic heterocycle (14) reduced activity signifi-
cantly. Polar substitution on the aryl ring (15–16) also reduced
activity, whereas chloro and methoxy analogues 17–20 were more
active. Swapping the aryl for a 4-pyridyl ring (21) led to a drop in
P. falciparum potency. Conformational constraints on the side chain
were then investigated. Analogues 22–27 all had similar activity,
despite locking the aryl ring in a number of different orientations.

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C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 401–405
More polar linker systems were then tested (28–31), and all
showed reduced P. falciparum activity. Having spanned many
changes on the 4-N side chain, the SAR of other substituents was
briefly explored. Deletion of the 5-Cl (32), and swapping R to a
trifluoromethyl group (33) retained activity when compared to
XC11 (9).
Overall the in vitro plasmodia screening results demonstrated a
very flat SAR, with all analogues spanning only one order of
magnitude in activity. Additionally all the activity appeared to be
driven by increases in lipophilicity (Fig. 2), and none of the
analogues containing polar groups possessed good in vitro activity.
This was a cause for concern, due to the potential for more lipo-
philic compounds to have higher attrition risks associated to poly-
pharmacology, pharmacokinetics and toxicology.10 The calculated
LogP/potency relationship for P. falciparum K1 indicated that a very
high cLogP (>9) would be required to achieve CQ levels of in vitro
P. falciparum K1 activity in this series.
The antileishmanial activity of compounds 4–33 was then deter-
mined in L. donovani using miltefosine as standard (Table 1).9
Activity was still linked to lipophilicity (Fig. 3), however activity
spanned three orders of magnitude and a significant number of
highly active compounds were identified. In particular, analogues
7–13, 18–20, 22, 24–27 and 30 were as active or more active than
the standard agent miltefosine, and most had excellent selectivity
over cytotoxic effects in rat myoblast L6 cells.9
Of particular interest were analogues 4–7, which did not pos-
sess a large 4-N substituent (highlighted in red in Fig. 3). This result
indicated that a large lipophilic group in this position was not
required for good L. donovani activity. Analogues 5 and 7 had the
best combination of low cLogP and L. donovani activity, and could
form the basis for a hit-to-lead program to identify additional com-
pounds with increased L. donovani potency.
The relationship between antiparasitic activity and cytotoxicity
was then explored, to determine if such a relationship existed
(Figs. 4 and 5). There was a general trend towards the most potent
antiparasitic compounds also exhibiting more cell-based cytotox-
icity. However, within a given potency range (for example L. dono-
vani IC50 < 0.5 lM) the cytotoxicity value varied >10-fold,
indicating that antiparasitic activity was not intimately linked to
cell-based cytotoxicity.
In conclusion, we have extended the SAR of the 2-pyridine
pyrimidines with regard to their antiplasmodial activity, giving
an insight into the structural features that drive potency. To this
end, we have shown that lipophilicity is correlated with antiplas-
modial activity.
We have further demonstrated the efficacy of the 2-pyridyl
pyrimidines against L. donovani parasite development in vitro.
Figure 4. Plot showing relationship between cytotoxicity and P. falciparum activity.
Coloured by cLogP (red = polar to blue = lipophilic).
Figure 2. Plot showing relationship between cLogP and P. falciparum activity.
Figure 5. Plot showing relationship between cytotoxicity and L. donovani activity.
Coloured by cLogP (red = polar to blue = lipophilic).
Figure 3. Plot showing relationship between cLogP and L. donovani activity.

C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 19 (2009) 401–405
405
Compound 5: 1H NMR (400 MHz, CDCl3) d ppm 2.68 (s, 3H), 3.27 (s, 6H), 7.28–
7.32 (m, 1H), 7.72–7.77 (m, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.75 (d, J = 3.9 Hz, 1H).
Compound 7: 1H NMR (400 MHz, CDCl3) d ppm 1.92–2.11 (m, 4H), 2.64 (s, 3H),
3.57–3.66 (m, 4H), 7.28–7.34 (m, 1H), 7.76–7.81 (m, 1H), 8.41 (d, J = 7.8 Hz,
1H), 8.77 (d, J = 4.7 Hz, 1H).
The low molecular weight, ease of synthesis and low cLogP of com-
pounds 5 and 7 lend themselves to being interesting starting
points for a hit-to-lead generation project against L. donovani.
Compound 10: 1H NMR (400 MHz, CDCl3) d ppm 2.03–2.10 (m, 2H), 2.62 (s,
3H), 2.78 (t, J = 7.4 Hz, 2H), 3.67–3.71 (m, 2H), 5.44 (br s, 1H), 7.20–7.24 (m,
3H), 7.29–7.37 (m, 3H), 7.76–7.82 (m, 1H), 8.33 (d, J = 8.2 Hz, 1H), 8.81 (d,
J = 4.7 Hz, 1H).
Acknowledgements
We thank the WHO-TDR Special Programme for Research and
Training in Tropical Diseases for a postdoctoral fellowship (C.M.)
and for support for the biological screening of the compounds
(R.B.).
Compound 21: 1H NMR (400 MHz, CD3OD)
d ppm 2.52 (s, 3H), 3.05 (t,
J = 7.0 Hz, 2H), 3.94 (t, J = 7.0 Hz, 2H), 7.35 (d, J = 6.2 Hz, 2H), 7.48–7.52 (m, 1H),
7.93–7.98 (m, 1H), 8.35 (d, J = 7.9 Hz, 1H) 8.37 (d, J = 6.2 Hz, 2H) 8.68 (d,
J = 3.9 Hz, 1H).
Compound 25: 1H NMR (400 MHz, CDCl3) d ppm 1.90–2.02 (m, 2H), 2.03–2.10
(m, 1H), 2.37–2.46 (m, 1H), 2.59 (s, 3H), 4.01–4.06 (m, 1H), 4.29–4.35 (m, 1H),
5.61 (t, J = 6.6 Hz, 1H), 7.16–7.19 (m, 1H), 7.24–7.30 (m, 5H), 7.62–7.67 (m, 1H),
7.87 (d, J = 7.8 Hz, 1H), 8.72 (d, J = 3.9 Hz, 1H).
References and notes
Compound 26: 1H NMR (400 MHz, CDCl3) d ppm 1.76–1.86 (m, 2H), 1.92–2.01
(m, 2H), 2.62–2.69 (m, 1H), 2.68 (s, 3H), 3.34 (dd, J = 13.3, 3.5 Hz, 1H), 3.50 (d,
J = 5.1 Hz, 1H), 3.81–3.87 (m, 1H), 4.01–4.08 (m, 1H), 7.23 (m, 1H), 7.28–7.32
(m, 4H), 7.35–7.38 (m, 1H), 7.81 (m, 1H), 8.41 (d, J = 7.8 Hz, 1H) 8.83 (d,
J = 3.9 Hz, 1H).
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