Cell-based optimization of novel benzamides as potential antimalarial leads

Article abstract of DOI:10.1016/j.bmcl.2009.10.050

Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid

Full text of DOI:10.1016/j.bmcl.2009.10.050

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6970–6974
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cell-based optimization of novel benzamides as potential antimalarial leads
Tao Wu a, Advait Nagle a, Tomoyo Sakata a, Kerstin Henson a, Rachel Borboa a, Zhong Chen a, Kelli Kuhen a,
David Plouffe a, Elizabeth Winzeler a, Francisco Adrian a, Tove Tuntland a, Jonathan Chang a,
Susan Simerson a, Steven Howard a, Jared Ek a, John Isbell a, Xianming Deng b, Nathanael S. Gray b,
David C. Tully a, Arnab K. Chatterjee a,
*
a Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA
b Harvard Medical School, 250 Longwood Ave., Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay
we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to
a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial
activity. The evolution of this compound series leading to optimized candidates with good cellular
potency against multiple strains as well as decent in vivo profile is described in this Letter.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 28 August 2009
Revised 9 October 2009
Accepted 12 October 2009
Available online 29 October 2009
Keywords:
Malaria
Kinase
Cellular assay
Cyclic amines
Pharmacokinetics
Resistant strains
Malaria is an infectious disease that profoundly affects many
developing countries. With hundreds of million cases and one mil-
lion deaths each year, malaria poses a tremendous health and eco-
nomic burden to the affected regions.1 There is still no effective
antimalarial vaccine available and we still heavily depend on low
molecular weight entities to treat the affected population. Quinine,
chloroquine, mefloquine and artemisinin derivatives have play an
important role in the treatment of malaria. However, widespread
drug resistance has made many of these compounds less effective.
Artemisinin is the only anti-malarial for which there are yet no
reported cases of clinical resistance. However, parasite tolerance
to artemisinin has been observed recently2 and it seems likely that
resistance will emerge soon. Therefore, it is important to discover
new chemotherapies that are effective against the multi-drug
resistant parasite strains.3 In this Letter, we discuss an effort4 to
find and optimize novel antimalarial entities using a cell-based
screening strategy.
to a cell-based of Plasmodium falciparum proliferation assay5,6 We
envisioned that the hits arising from this screen can be rapidly
optimized by leveraging our past experiences with these com-
pounds series in alternative target-classes/indications. Further-
more, we thought that it would be prudent to remove the human
kinase activity early on during the compound optimization phase
to negate the possibility of toxicity arising from host-related off-
target activities (Scheme 1).
Our starting point is compound 1, which was originally de-
signed as a pan-kinase Bcr-Abl inhibitor7,8 Compound 1 shows a
moderate EC50 of 200 nM against the chloroquine sensitive 3D7
P. falciparum parasite strain. By switching the solubility enhancing
group, compound 2 exhibits a ꢀ3-fold improvement in potency.
When the 2-methyl group in the left phenyl ring of 2 is replaced
with a 3-methoxy group, compound 3 is obtained which is equipo-
tent on malaria parasite. In addition, 3 no longer has any human
kinase activities of 2, as measured in a Ba/F3 transformed cell-line
RTK panel9 Since preserving the pharmocophore necessary for
inhibiting human kinases is not necessary, we speculated that
the benzamide portion of the molecule might be responsible for
the antimalarial activity of 3 and decided initiate a broad SAR
investigation.
Currently there is a need for novel chemical scaffolds with dif-
ferent mechanisms of action, since most of the current approved
antimalarial drugs belong to the aminoquinoline family. In order
to find new chemical scaffolds, we initiated a compound screen
using our in-house kinase inhibitor collection and subjected them
Schemes 2–4 describe the synthetic strategies used to study the
three key portions of compound 3. Scheme 2 outlines the synthesis
of the amide connectivity of 3. The synthesis starts from 3-fluoro-
* Corresponding author. Tel.: +1 8583324751; fax: +1 8588121648.
E-mail address: akc@gnf.org (A.K. Chatterjee).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.050

T. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6970–6974
6971
Scheme 1. Proprietary kinase scaffolds offered hits: piperidine benzamides.
Scheme 4. General synthesis scheme of piperidyl benzamides amine SAR. Reagents
and conditions: (a) (i) SOCl2, CHCl3, reflux; (ii) aniline, pyridine, 72–79%; (b)
Pd2(dba)3, BINAP, t-BuOK, toluene, 100 °C, 45–84%.
Scheme 2. General synthesis scheme of piperidyl benzamides amides. Reagents
and conditions: (a) K2CO3, DMSO, 80 °C; (b) 50% H2SO4, reflux, 78%, 2 steps; (c)
RNH2, HATU, DIEA, DMF, 23 °C, 60–70%.
amide bond formation is executed using various amines and HATU
as the activating agent. We were satisfied with the straightforward
synthesis for these compounds given the need for low cost of goods
is one of the essential criteria for the antimalarial target product
profile (TPP).12
5-(trifluoromethyl)benzonitrile. A SNAr reaction followed with a
H2SO4 mediated hydrolysis provides the acid in good yield. The
Scheme 3 outlines the synthesis for the reverse amides, sulfon-
amides and ureas. Curtius rearrangement of the corresponding
benzoic acids illustrated in Scheme 2 serves as the key step in
the aniline synthetic route.
Scheme 4 outlines the synthesis for determining the SAR on the
3-position on CF3 bearing phenyl ring. We started with commer-
cially available 3-bromo-5-(trifluoromethyl)benzoic acid and car-
ried out a palladium catalyzed amination reaction on a variety of
substrates to afford the final compounds in moderate to good
yields.
Table 1 outline our SAR determination on the amide portion of
compound 3. While linear alkyl (compound 4) and pyrimidines
(compound 5) are incompatible, a wide variety substituted phenyl
groups are well tolerated. The substitution pattern favors meta-
and para- mono-substitution, with 3,4-disubstitution (compounds
11) and 3,5-disubstitution (compound 13) modestly enhancing po-
tency as well.
Scheme 3. General synthesis scheme of piperidyl benzamides reverse amides,
sulfonamides and ureas. Reagents and conditions: (a) (i) DPPA, Et3N, t-BuOH, reflux;
(ii) TFA, DCM, 23 °C, 33%; (b) R0CO2H, HATU, DIEA, DMF, 23 °C; or R0SO2Cl, DIEA,
CH2Cl2; or R0NCO, toluene, 80 °C, 40–80%.

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T. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6970–6974
Table 1
Table 2
Piperidyl benzamides SAR: aniline modifications
Piperidyl benzamides SAR: linker modifications
P. falciparum 3D7 strain EC50, l
Ma
Compds
R
P. falciparum 3D7 strain EC50, l
Ma
Compds
R
4
6.48
14
0.201
5
6
2.16
1.72
15
0.402
7
0.114
16
17
18
8.25
4.68
0.452
8
9
0.134
0.146
0.174
10
19
20
21
0.328
1.98
11
12
0.054
0.098
0.638
13
0.048
a
Values are means of two experiments. Each assay plate has mefloquine, sulfa-
22
23
0.734
0.423
doxine and artimesinin as internal standards. The EC50 values for standard com-
pounds match the literature values.
Table 2 summarizes how varying the amide linkage affects the
potency using compound 8 and 10 as our template since they were
potent starting points in our early SAR. The sulfonamide (com-
pound 16) was not tolerated nor was removal of the carbonyl
and replacement with a methylene unit (compound 20). A free
amide NH was essential for activity as the N-methyl compound
loses potency by 20-fold (compound 17). Ureas do not seem to of-
fer any advantage over amides, moreover both amide orientations
(compound 10 vs 14) are tolerated and offer advantage in terms of
physiochemical properties (e.g., improved solubility). Therefore we
decided to explore additional structure–activity relationships with
the amide in place.
We next turned our attention to the center benzamide ring to
explore the possibility of incorporating heterocycles in that ring.
As depicted in Table 3, attempts to make substituent changes
(compound 24 and 25) or incorporate heteroatoms (compound
26, 27, 28 and 29) failed to enhance the potency of the compounds
demonstrating tight SAR in that region of the pharmocophore.
a
Values are means of two experiments. Each assay plate has mefloquine, sulfa-
doxine and artimesinin as internal standards. The EC50 values for standard com-
pounds match the literature values.
One important aspect of antimalarial drug discovery is to
identify agents that are active against the drug-resistant parasites.
Table 4 outlines our results on chloroquine sensitive 3D7 strain
and multidrug resistant W2 strain which is resistant to chloro-
quine, quinine, pyrimethamine, cycloguanil, and sulfadoxine.
While most of these resistance phenotypes are due to point muta-
tions in the drug target, the W2 strain also contains an amplifica-
tion of the P. falciparum multidrug resistance transporter.10
Interestingly, while compound 8 show a ꢀ6-fold shift in potency
between 3D7 and W2, replacement of the bicyclic amine part pyr-
rolidinyl piperidine to bipiperidine (compound 30, 31, and 32) led
to much improved potency against W2 strain. Although 34 was the
most potent compound, it was deprioritized since the introduction

T. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6970–6974
6973
Table 3
Table 4
Piperidyl benzamides SAR: center ring modifications
Piperidyl benzamides SAR: bicyclic amine modifications
CF3
H
N
R1
R2
O
Compds
X
P. falciparum 3D7 strain EC50, l
Ma
Compds R1
R2
P. falciparum 3D7 P. falciparum W2
strain EC50,
l
Ma
strain EC50
, l
Ma
24
0.766
8
Cl
Cl
0.134
0.888
25
26
5.88
30
0.082
0.149
0.140
0.296
0.299
0.323
0.674
31
32
CF3
27
28
29
1.67
2.98
2.08
Cl
33
CF3
1.49
1.41
a
Values are means of two experiments. Each assay plate has mefloquine, sulfa-
doxine and artimesinin as internal standards. The EC50 values for standard com-
pounds match the literature values.
34
35
CF3
CF3
0.058
0.175
0.211
0.635
of a stereocenter in the molecule increases the complexity of the
synthesis and cost of goods.
Compound 32 was profiled in an extended panel of 15 drug
resistant strains (Table 5) and against a 6-cell line toxicity panel.
We were delighted to find that all the potencies are within 3-fold
of each other. The observed cytotoxicity TC50s (293T, Ba/F3, CHO,
a
Values are means of two experiments. Each assay plate has mefloquine, sulfa-
doxine and artimesinin as internal standards. The EC50 values for standard com-
pounds match the literature values.
Table 5
Potencies of compound 32 against 15 P. falciparum strains
HEp2, HeLa, Huh7) were greater than 8
good selectivity index (SI >20).
lM which translates to a
Some of the more potent compounds against both 3D7 and W2
strains were selected to assess their in vivo pharmacokinetic pro-
files in mice. Mice were dosed a single dose of 20 mg/kg orally
and their exposure levels were monitored over a period of 5 h
and results are summarized in Table 6.11
Most compound demonstrated good oral exposure, with com-
pound 32 exhibited the highest AUC(0–5h) as well as a high Cmax
(ꢀ13-fold above the 3D7 potency). We also performed preliminary
solubility and metabolic stability measurements on compound 32.
Compound 32 exhibits moderate solubility (HT-thermodynamic
solubility at pH6.8 0.026 mg/mL free base) and modest metabolic
stability (extraction ratio in mouse microsomes = 0.383 0.04
P. falciparum strain
EC50, lM
3BAG
7G8
C188
D10
D6
Dd2
Camp R
FCB
FCR3
HB3
K1
NF54
3D7
0.268
0.309
0.083
0.094
0.081
0.141
0.138
0.306
0.218
0.146
0.150
0.104
0.140
0.120
0.323
and CLint (lL/min/mg) = 21.399) that are consistent with the
in vivo pharmacokinetic results.
In conclusion, we have successfully transformed a pan human
kinase inhibitor scaffold (compound 2) into a selective anti-malar-
ial series. SAR studies revealed that the amide linkage is preferred
for the antimalarial activity. Switching the bicyclic amine moiety
from a 6,5 system to a substituted 6,6 system significantly im-
proves the potency shift across the drug-resistant strains. The se-
lected lead compound 32 exhibited good potency across different
strains, favorable physiological properties and good in vivo phar-
TM91C235
W2
a
Values are means of two experiments. Each assay plate has mefloquine, sulfa-
doxine and artimesinin as internal standards. The EC50 values for standard com-
pounds match the literature values.

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T. Wu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6970–6974
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AUC(0–5 h)
(h nM)
Cmax
(nM)
Tmax
(h)
AUC(0–5h)/dose
[(min ug/mL)/(mg/kg)]
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8
13
14
30
32
35
3916
4806
4766
4365
7932
1672
1355
1643
1170
1368
2279
672
1.00
5.00
3.00
1.00
5.00
0.50
5.31
7.01
6.94
6.10
11.42
2.33
Dose (mg/kg): 20; strain: mice balb/c.
Formulation: 2.5 mg/mL in: PEG300 /D5 W, 3:1, solution.
Salt form: free base.
macokinetic profile. The further studies on this scaffold are war-
ranted and would be reported in due course.
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Acknowledgments
This work was supported by a joint grant to Genomics Institute
of the Novartis Research Foundation, Biomedical Primate Research
Center, Swiss Tropical Institute and Novartis Institute for the Trop-
ical Diseases from the Wellcome Trust and Medicines for Malaria
Venture.
10. Kidgell, C.; Winzeler, E. A. Chromosome Res. 2005, 13, 225.
11. Snapshot PK is developed and used by GNF pharmacology group as a rapid
pharmacokinetic screening approach. The pharmacokinetic parameters of the
compounds are studied in a single mode of administration (generally PO arm)
over a period of 5 h. The study is carried out on two animals and the samples
are pooled together at each time point. This high throughput approach offers
exposure levels with reasonable confidence without committing to extensive
resources. For details, see: Liu, B.; Chang, J.; Gordon, W. P.; Isbell, J.; Zhou, Yi.;
Tuntland, T. Drug Discovery Today 2008, 13, 360–367.
References and notes
12. Medicines forMalariaVenture‘SelectionandReview:MMVCompoundProgression
Criteria’, June 17, 2009, http://www.mmv.org/article.php3?id_article=503.
1. WHO ‘World Malaria Report 2008’, June 5, 2009, http://apps.who.int/malaria/
wmr2008/malaria2008.pdf.