Discovery of novel 1H-imidazol-2-yl-pyrimidine-4,6-diamines as potential antimalarials

Article abstract of DOI:10.1016/j.bmcl.2010.05.095

A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2010.05.095

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4027–4031
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 1H-imidazol-2-yl-pyrimidine-4,6-diamines as potential
antimalarials
Xianming Deng a,b, Advait Nagle c, Tao Wu c, Tomoyo Sakata c, Kerstin Henson c, Zhong Chen c, Kelli Kuhen c,
David Plouffe c, Elizabeth Winzeler c, Francisco Adrian c, Tove Tuntland c, Jonathan Chang c, Susan Simerson c,
Steven Howard c, Jared Ek c, John Isbell c, David C. Tully c, Arnab K. Chatterjee c, Nathanael S. Gray a,b,
*
a Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave., SGM 628, Boston, MA 02115, USA
b Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Ave., SGM 628, Boston, MA 02115, USA
c Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity
against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria.
A systematic SAR study resulted in the identification of compound 40 which exhibits good potency
against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 23 April 2010
Revised 21 May 2010
Accepted 24 May 2010
Available online 2 June 2010
Keywords:
1H-Imidazol-2-yl-pyrimidine-4,6-diamines
Antimalarial
Malaria infection infects nearly 500 million people causing
nearly 1 million deaths per year, with highest mortality among
pregnant women and young children, despite the recent introduc-
tion of artemisinin-based combination therapies (ACTs) as first line
therapy.1,2 Reports of increased drug tolerance to ACTs along the
Thai–Cambodian border3 as well as widespread resistance to quin-
oline based therapies mandates the development of novel thera-
pies against Plasmodium falciparum (Pf) parasitic infections.4 Here
we report our discovery of novel 1H-imidazol-2-yl-pyrimidine-
4,6-diamines with potent activity against the both wild-type and
drug resistant parasite strains of Pf.
In our efforts to identify new molecular scaffolds structurally
unrelated to known antimalarials that could either target parasite
or host processes, we performed a cell-based proliferation assay
against the erythrocyte stage of Pf.5–7 We screened a collection of
kinase inhibitor scaffolds, primarily developed against human ki-
nases with the idea that host cell kinases or nucleotide requiring
parasite enzymes could serve as potential targets. Here we report
on our medicinal chemistry efforts starting from screening ‘hit’ 1
(Fig. 1) which possessed a moderate EC50 of 436 nM against the
chloroquine sensitive 3D7 parasite strain.
lian kinases suggesting that it is not a promiscuous kinase
inhibitor.
In order to systematically investigate the structure–activity
relationships with respect to proliferation of Pf in erythrocytes,
we developed a variety of synthetic routes to this structure
(Schemes 1–4). The first route starts with the reaction of variously
substituted anilines with cyanamide under acidic conditions to af-
ford the corresponding substituted phenyl guanidine nitrates.8
Cyclization was accomplished by reaction with 2-chloroacetalde-
hyde to afford the substituted phenyl-1H-imidazol-2-amines as
key intermediates. Final products were obtained by sequential acid
or base-catalyzed displacement of dichloro-pyrimidines or di-
chloro-quinazoline with the imidazol-2-amine and a second amine
nucleophile of choice.
In order to diversify the phenyl appended to the imidazoyl
N1-position, palladium catalyzed amination were performed on
CF3
N
H
N
Compound 1 appeared to be an attractive lead because it is
structurally unrelated to known antimalarials and did not exhibit
significant activity when screened against a panel of 40 mamma-
N
N
N
HN
OCF3
1
3D7 EC50 = 436 nM
* Corresponding author. Tel.: +1 617 582 8590.
E-mail address: nathanael_gray@dfci.harvard.edu (N.S. Gray).
Figure 1. Imidazolyl pyrimidine screening hit compound 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.095

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X. Deng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4027–4031
R1
R1
Table 1
R1
R1
SAR of the central pyrimidine core
a
c
b
Compd Structure
P. falciparum 3D7
strain EC50 (lM)
N
a
H
N
N
HN
NH2
N
N
CF3
NH2
NH
R2
Ar2
=
N
N
H2N HNO3
Ar1
=
Cl
N
R1
OCF3
N
d
N
N
H
N
2
3
0.432
0.268
N
N
X
HN
Ar
N
N
NH
Ar
R2
1
2
N
OCF3
N
(X = NH, O)
HN
Ar
NH
1
2
Ar
HN Ar
Scheme 1. General synthesis scheme of phenyl-1H-imidazol-2-yl-pyrimidine-
diamines. Reagents and conditions: (a) NH2CN (2.4 equiv, 50% aq), concd HNO3
(1.1 equiv), EtOH, 100 °C, 78–86%; (b) ClCH2CHO (2.0 equiv, 50% aq), Na2CO3 (satd
aq), EtOH, 80 °C, 60–85%; (c) substituted di-chloro pyrimidines or di-chloroquinaz-
oline, DIEA, dioxane, 100 °C, 60–80%; (d) 4-(trifluoromethoxy)benzenamine, TFA, 2-
PrOH, 80 °C, 78–85%; or 4-(trifluoromethoxy)phenol, NaH, dioxane, rt to 80 °C, 75%.
1
N
4
5
0.564
1.330
HN
2
N
Ar
N
N
HN
Ar
N
NH
Ar
N
N
N
N
N
N
1
2
a
N
H
NH
N
N
H
NH
Me
N
N
N
6
7
0.114
4.680
F3C
R
F3C
Br
HN
Ar
NH
Ar
OCF3
1
2
OCF3
N
N
Scheme 2. General synthesis of amine-substituted phenyl-1H-imidazol-2-yl-
pyrimidine-4,6-diamines. Reagents and conditions: (a) secondary amine, Pd(OAc)2
(10%), BINAP (15%), t-BuONa (3.0 equiv), toluene, 100 °C, 50–75%; or primary amine,
Pd2(dba)3 (10%), BINAP (15%), t-BuONa (3.0 equiv), toluene,100 °C, 50–70%.
HN
Ar
NH
2
1
Me Ar
N
N
Me
8
9
3.830
4.240
N
Ar
NH
Ar
arylbromide containing substrates (Scheme 2) or amide bond for-
mation using arylcarboxylate containing substrates (Scheme 3).9
Introduction of functionality at C2 of the central pyrimidine
core was accomplished by SNAr substitution of a C2-methylsulfo-
nyl group with various amines or alkyl alcohols (Scheme 4).10
The compound potencies were determined in a Pf infected hu-
man red blood cell assay using Sybr green staining as the readout
for parasite proliferation.5 Our initial SAR investigation was fo-
1
2
N
N
HN
Ar
O
Ar
1
2
a
Values are means of two experiments. As internal standards, each assay plate
contains mefloquine, sulfadoxine and artemisinin which possess EC50 values of 20,
30 and 10 nM, respectively.
R1
R1
R1
N
N
N
N
N
N
N
N
N
a
b
N
H
NH
N
H
NH
N
H
NH
N
N
N
R2
EtO2C
R1 = H, Me
HO2C
OCF3
OCF3
OCF3
O
Scheme 3. General synthesis of amide-substituted phenyl-1H-imidazol-2-yl-pyrimidine-4,6-diamines. Reagents and conditions: (a) LiOH, MeOH/THF/H2O, rt, 85–95%;
(b) primary or secondary amines, HATU, DIEA, DMSO, rt, 75–88%.
SMe
N
R
SO2Me
N
N
N
N
N
N
N
N
a
b
N
H
NH
N
H
NH
N
N
N
H
NH
N
F3C
F3C
F3C
OCF3
OCF3
OCF3
Scheme 4. General synthesis of 2-substituted-N4-(4-(trifluoromethoxy)phenyl)-N6-(1-(3-(trifluoromethyl)phenyl)-1H-imidazol-2-yl)pyrimidine-4,6-diamines. Reagents and
conditions: (a) oxone (4.0 equiv), CH2Cl2/MeOH/H2O, rt, 85%; (b) amine, DIEA, dioxane, 80–100 °C, 60–80%; or ROH, NaH, 80–100 °C, 60–70%.

X. Deng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4027–4031
4029
cused on the central pyrimidine core as these modifications were
anticipated to dramatically affect the conformational preferences
of the inhibitor (Table 1). Replacing the 4,6-pyrimidine of 1 with
the two 2,4-pyrimidine regioisomers and 2,4-quinazoline resulted
in approximately equipotent compounds 2, 3 and 4. The corre-
sponding triazine analog 5 exhibited ꢀthree-fold decrease in po-
tency. Introduction of a methyl group to the pyrimidine C2 (6)
resulted in an approximate four-fold improvement in potency rel-
ative to 1 while a methyl group at the pyrimidine C5 (7) resulted in
a compound ten-fold less active compared to 1. Both NH groups of
1 appear to be essential as methylation of the imidazolyl NH (8) or
replacement of the 4-trifluoromethoxy aniline NH with an oxygen
(9) resulted in a ten-fold loss of activity.
2-(piperazin-1-yl)ethanol amide with a free OH resulted in a de-
crease in potency (compounds 20, 21 and 25). Interestingly, we
again observed that a C2-methyl substituent on the pyrimidine
core could improve the potency (19 vs 23, 20 vs 25).
Based upon the favorable effects of a C2-pyrimidine methyl
substitutent, we decided to further explore this position through
the synthesis of a focused library (Table 4). Various functionalities
were installed, ranging from electron withdrawing groups (30, 33)
to electron donating groups (6, 32 and 34) and alkyl substituent (6)
to aryl substituents (31 and 36). Most of them were well tolerated
with the exception of the hydroxyl substituent (37). Amine substit-
uents increased potency dramatically (38–42), with optimal
activity being obtained for the simple NH2 (38) and piperazine
(40) substituted compounds which possessed EC50 values of
approximately 30 nM.
One liability of compound 1 was its very poor aqueous solubil-
ity (water solubility of 34 lg/mL at pH 6.8) which we sought to ad-
dress through the introduction of tertiary amines to the imidazoyl
N1 phenyl substituent (Table 2). Introduction of primary, mono-
cyclic and bi-cyclic amine substituents resulted in compounds
(11, 14, 16, 17 and 18) that possessed EC50 values below 100 nM
which represented an improvement relative to unsubstituted com-
pound 1 and bromo-substituted compound 10.
Since the meta-amine substituent appeared to be favorable and
the trifluoromethyl group was not essential (data not shown), we
next explored the possibility of incorporating amines linked via
one, two or three-carbon spacer to a benzamide amide (Table 3).
The most potent compound 22 substituted with a (1-ethylpyrroli-
din-2-yl)methan-amide possessed an EC50 of 60 nM. Other
favorable substituents included ethyl (23) and propyl (27) mor-
pholinyl containing amides. Histamine amide with a free NH and
Table 3
SAR of amide-substituted phenyl-1H-imidazol-2-yl-pyrimidine-4,6-diamines
1
R
N
N
N
N
H
NH
N
2
R
OCF
O
3
Compd
R1
H
R2
P. falciparum 3D7
a
strain EC50
(lM)
O
19
N
0.249
N
H
Table 2
SAR of amine-substituted phenyl-1H-imidazol-2-yl-pyrimidine-4,6-diamines
NH
N
N
20
21
H
1.777
0.768
N
N
N
H
N
H
NH
N
N
Me
OH
N
F C
3
R
H
N
OCF
N
3
22
Me
0.060
a
Compd
R
P. falciparum 3D7 strain EC50 (lM)
O
Br
N
10
0.223
23
24
25
Me
Me
Me
N
N
0.069
0.168
0.822
N
H
N
O
11
12
0.065
0.532
N
N
H
OH
13
0.175
NH
N
N
N
N
N
N
N
H
14
15
16
0.088
0.090
0.092
N
O
N
N
N
26
Me
0.046
N
N
H
O
N
H
N
27
28
29
Me
Me
Me
0.067
0.224
0.239
O
N
N
N
17
18
N
N
0.087
0.091
N
H
N
H
N
H
N
N
H
a
a
Values are means of two experiments. As internal standards, each assay plate
Values are means of two experiments. As internal standards, each assay plate
contains mefloquine, sulfadoxine and artemisinin which possess EC50 values of
20 nM, 30 nM and 10 nM, respectively.
contains mefloquine, sulfadoxine and artemisinin which possess EC50 values of
20 nM, 30 nM and 10 nM, respectively.

4030
X. Deng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4027–4031
Table 4
Table 5
SAR of C2-substituent of pyrimidine core
Potencies of compound 40 against 15 P. falciparum strains
R
N
N
N
N
N
N
NH
N
H
N
N
N
N
H
NH
N
F C
3
OCF
3
a
F C
Compd
R
P. falciparum 3D7 strain EC50 (lM)
3
OCF
3
6
0.114
0.353
Me
CF
a
P. falciparum strain
EC50
0.06
(lM)
30
3
3BAG
Camp R
C188
D10
0.050
0.14
31
0.074
0.015
0.05
0.071
0.119
0.05
0.139
0.095
0.046
0.088
0.033
0.058
0.12
D6
Dd2
3D7
FCB
FCR3
7G8
HB3
32
33
34
0.107
0.291
0.190
SMe
SO Me
2
OMe
35
36
0.060
0.116
K1
O
NF54
TM91C235
W2
O
a
Values are means of two experiments. As internal standards, each assay plate
contains mefloquine, sulfadoxine and artemisinin which possess EC50 values of 20,
30 and 10 nM, respectively.
37
38
5.440
0.049
OH
NH
2
Me
N
H
39
40
0.054
0.034
Table 6
Metabolic stability and solubility of selected compounds
N
N
Compd Metabolic
stability
Metabolic
stability
ER_Rata
Metabolic
stability
Thermodynamic
solubility at pH 6.8b
(mg/mL)
N
41
42
0.113
0.053
ER_Mousea
ER_Humana
O
15
22
23
26
40
<0.17
<0.17
<0.17
<0.17
<0.17
NDc
0.316
NDc
<0.42
<0.31
<0.28
0.11
O
0.275
0.243
0.367
0.264
0.089
0.090
0.080
0.040
N
N
H
a
Values are means of two experiments. As internal standards, each assay plate
a
b
c
Values of extraction ratio in mouse, rat or human microsomes.
Values of thermodynamic solubility at pH 6.8.
Not done (assay not performed).
contains mefloquine, sulfadoxine and artemisinin which possess EC50 values of
20 nM, 30 nM and 10 nM, respectively.
We next evaluated the potency of our new lead compound 40
against a panel of 15 parasite strains (Table 5). The EC50 values of
compound 40 were all lower than 140 nM, which suggest that this
compound may provide a good lead candidate to overcome this set
of drug resistant strains. To evaluate a therapeutic index relative to
toxicity activity against mammalian cells, compound 40 was tested
for antiproliferative activity against a six cell line panel including
293T, Ba/F3, CHO, HEp2, HeLa, Huh7. The EC50 values were all
microsome of less than 0.17, 0.264, and 0.28, respectively. An oral
and intravenous pharmacokinetic study of compound 40 in mice
demonstrated good oral exposure with AUCinf of 14,201 h nM, Cmax
of 946 nM with a half-life of 12 h. The compound was found to
have oral bioavailability of 30%.
In summary, we have identified a new imidazolyl pyrimidine
scaffold with potent cellular antimalarial activity. SAR studies
demonstrated that amine substituent or amide substituent on
imidazolyl N1-phenyl ring were favorable. A 4,6-diamine pyrimi-
dine with a free NH appears to be essential for achieving submi-
cromolar potency. A C2-substituent on central pyrimidine core is
preferred. The optimized compound 40 displays broad-spectrum
activity relative to a panel of 15 parasite strains and displays favor-
able physiological and pharmacokinetic properties. Our results
demonstrate the utility of cell-based screening to identify and opti-
mize new molecular scaffolds possessing antimalarial activity. Fur-
ther work will be required to establish the molecular target(s) of
this compound class and to further investigate their potential as
antimalarial drugs.
greater than 2 lM. As lead compound 1 was originally derived
from a kinase inhibitor library, we also tested compound 40
against a panel of 40 cellular tyrosine kinase assays11 and a panel
of 150 biochemical kinase assays at a concentration of 10 l
M.12
This profiling did not reveal significant inhibition of any mamma-
lian kinase.
Some of the more potent compounds were selected to assess
their metabolic stabilities and aqueous solubilities (Table 6). Most
compounds demonstrated good metabolic stability and exhibited
high solubility. Among them, compound 40 exhibited the best met-
abolic stability with extraction ratio in mouse, rat and human

X. Deng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4027–4031
4031
J.; Yan, S. F.; Trager, R.; Caldwell, J. S.; Schultz, P. G.; Zhou, Y.; Winzeler, E. A.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 9059.
Acknowledgments
6. Kato, N.; Sakata, T.; Breton, G.; Le Roch, K. G.; Nagle, A.; Andersen, C.; Bursulaya,
B.; Henson, K.; Johnson, J.; Kumar, K. A.; Marr, F.; Mason, D.; McNamara, C.;
Plouffe, D.; Ramachandran, V.; Spooner, M.; Tuntland, T.; Zhou, Y.; Peters, E. C.;
Chatterjee, A.; Schultz, P. G.; Ward, G. E.; Gray, N.; Harper, J.; Winzeler, E. A. Nat.
Chem. Biol. 2008, 4, 347.
7. Wu, T.; Nagle, A.; Sakata, T.; Henson, K.; Borboa, R.; Chen, Z.; Kuhen, K.; Plouffe,
D.; Winzeler, E.; Adrian, F.; Tuntland, T.; Chang, J.; Simerson, S.; Howard, S.; Ek,
J.; Isbell, J.; Deng, X.; Gray, N. S.; Tully, D. C.; Chatterjee, A. K. Bioorg. Med. Chem.
Lett. 2009, 19, 6970.
8. Ren, P.; Wang, X.; Zhang, G.; Ding, Q.; You, S.; Zhang, Q.; Chopiuk, G.; Albaugh,
P. A.; Sim, T.; Gray, N. S. WO 2005123719; Chem. Abstr. 2005, 144, 88305.
9. Cho, G. Y.; Okamura, H.; Bolm, C. J. Org. Chem. 2005, 70, 2346.
10. Gray, N. S.; Zhang, J.; Okram, B.; Deng, X.; Chang, J.; Wojciechowski, A. WO
2009073153; Chem. Abstr. 2009, 151, 49314.
This work was supported by a joint grant to the Genomics Insti-
tute of the Novartis Research Foundation, the Biomedical Primate
Research Center, the Swiss Tropical Institute and the Novartis Insti-
tute for the Tropical Diseases from Wellcome Trust and Medicines
for Malaria Venture.
References and notes
1. World Health Organization 2009 Malaria Report. http://www.who.int/malaria/
world_malaria_report_2009/en/index.html.
2. White, N. J. J. Clin. Invest. 2004, 113, 1084.
3. Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K. M.;
Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.; Silamut, K.; Imwong, M.;
Chotivanich, K.; Lim, P.; Herdman, T.; An, S. S.; Yeung, S.; Singhasivanon, P.; Day,
N. P. J.; Lindegardh, N.; Socheat, D.; White, N. J. N. Engl. J. Med. 2009, 361, 455.
4. Gelb, M. Curr. Opin. Chem. Biol. 2007, 11, 440.
11. Melnick, J. S.; Janes, J.; Kim, S.; Chang, J. Y.; Sipes, D. G.; Gunderson, D.; Jarnes,
L.; Matzen, J. T.; Garcia, M. E.; Hood, T. L.; Beigi, R.; Xia, G.; Harig, R. A.;
Asatryan, H.; Yan, S. F.; Zhou, Y.; Gu, X.-J.; Saadat, A.; Zhou, V.; King, F. J.; Shaw,
C. M.; Su, A. I.; Downs, R.; Gray, N. S.; Schultz, P. G.; Warmuth, M.; Caldwell, J. S.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3153.
5. Plouffe, D.; Brinker, A.; McNamara, C.; Henson, K.; Kato, N.; Kuhen, K.; Nagle, A.;
Adrian, F.; Matzen, J. T.; Anderson, P.; Nam, T.; Gray, N. S.; Chatterjee, A.; Janes,
12. (a) Jia, Y. Expert Opin. Drug Discovery 2008, 3, 1461–1474; (b) http://
www.invitrogen.com/kinaseprofiling.