Antiplasmodial activity of piperazine sulfonamides

Article abstract of DOI:10.1016/j.bmcl.2009.10.130

A high-throughput screening program identified two piperazine sulfonamides with activity against Plasmodium falciparum. Both screening positives had three structural features with potential liabilities: furanyl, thiourea and nitrophenyl groups. The furan could be replaced with no loss of activity, replacement of the nitrophenyl led to some loss of activity, and any attempt to replace the thiourea led to a significant decrease in activity, which implicates this reactive functional group's role in the antiplasmodial activity of this compound class.

Full text of DOI:10.1016/j.bmcl.2009.10.130

Bioorganic & Medicinal Chemistry Letters 20 (2010) 218–221
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiplasmodial activity of piperazine sulfonamides
Derek C. Martyn a, Joseph F. Cortese a, Erin Tyndall a, Justin Dick a, Ralph Mazitschek a,b
,
Benito Munoz a, Jon Clardy a,c,
*
a Broad Institute Infectious Diseases Initiative, 7 Cambridge Center, Cambridge, MA 02142, USA
b Center for Systems Biology, Massachusetts General Hospital, MA 02142, USA
c Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 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:
A high-throughput screening program identified two piperazine sulfonamides with activity against Plas-
modium falciparum. Both screening positives had three structural features with potential liabilities: fura-
nyl, thiourea and nitrophenyl groups. The furan could be replaced with no loss of activity, replacement of
the nitrophenyl led to some loss of activity, and any attempt to replace the thiourea led to a significant
decrease in activity, which implicates this reactive functional group’s role in the antiplasmodial activity
of this compound class.
Received 31 August 2009
Revised 26 October 2009
Accepted 29 October 2009
Available online 31 October 2009
Keywords:
Malaria
Ó 2009 Elsevier Ltd. All rights reserved.
Antiplasmodial
Thiourea
Malaria is a major parasitic disease in many areas of Africa, Asia
and South America. Plasmodium falciparum causes the majority of
cases, and its resistance to many contemporary antimalarials
throughout Africa and parts of Asia has compounded the problem
of disease control.1 To address the discovery of novel chemothera-
peutic strategies, the Broad Institute’s Infectious Diseases
Initiative, the Genzyme Corporation and the Medicines for Malaria
Venture have formed the Malaria Drug Discovery Initiative to cre-
ate a collaborative pipeline for candidate therapeutics. Our primary
measured by an in vitro DAPI staining-based method for determin-
ing P. falciparum cell viability after exposure to the compound for
72 h.2 Compounds were tested against the laboratory strain 3D7
and the multidrug resistant southeast Asian isolate Dd2,6 using
chloroquine as a control to validate the assay results. Replacement
of the furanyl with tetrahydrofuranyl or methoxymethyl groups
resulted in some decrease in activity, whereas replacement with
a phenyl group (2e) resulted in a compound equipotent to screen-
ing positive 1a. An analog of 2e with an additional methylene at R1
(2g) displayed a two-fold decrease in activity against Dd2. Com-
pounds 2a and 2h revealed that bulky groups at R1 and R2 are re-
quired for activity, as methyl groups at either position eliminated
activity.
Replacement of the nitrophenyl group was investigated with
compounds 3a–i (Table 2). The toxicological properties of nitroa-
renes have been studied intensively.7 While several marketed
drugs possess nitroarenes, many suffer from a range of side ef-
fects.8 Compounds 3a–i displayed decreased antiplasmodial activ-
ity compared to 1a. Only two compounds displayed
submicromolar activity (3c and 3g), and both of these compounds
means of identifying small molecule candidates utilizes
a
high-throughput screen to uncover scaffolds amenable to the
development of novel antimalarials.2 Two closely related screening
positives are displayed below (1a–b, Fig. 1). While these piperazine
sulfonamide-based compounds displayed excellent antiplasmodial
activity, the furanyl, thiourea and nitrophenyl functionalities have
noted metabolic liabilities, and we undertook a stepwise strategy
to determine whether potency was maintained when they were re-
placed with more benign functional groups.
We initially addressed replacement of the furanyl group using
readily available compounds3 (2a–h, Table 1). The furanyl group
can be metabolically transformed to
or
a,b-unsaturated dicarbonyls
c
-ketocarboxylic acids via a furan-2,3-epoxide intermediate that
is capable of reacting with a range of biomolecules.4 The carcinoge-
nicity of aflatoxin B1, which results from a furan-derived epoxide
reacting with N7 of guanine,5 provides one well-known example.
The antiplasmodial activity of the commercial compounds was
* Corresponding author. Tel.: +1 617 432 2845; fax: +1 617 738 3702.
E-mail address: jon_clardy@hms.harvard.edu (J. Clardy).
Figure 1. Structure and antiplasmodial activity of screening positives 1a–b.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.130

D. C. Martyn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 218–221
219
had R1 at the 4-position (cf. 1a–b). However, the only other analog
Table 3
with R1 at C4 (3i) was >10-fold less active than 1a, demonstrating
that not all functionalities at this position are well tolerated. The
bicyclic compound (3a) had the most significant decrease in activ-
ity compared to 1a, suggesting that bulkier groups are not toler-
ated at this position.
Antiplasmodial activity of 4a–b
Compd
R1
Pf Dd2 EC50 (lM)
We then focused on the replacement of the thiourea. This func-
tionality has a range of known toxicological effects, such as thyroid
depression, pulmonary edema, and liver necrosis.9 Teramoto et al.
demonstrated that a range of structurally diverse thioureas were
teratogenic in rats, whereas the corresponding ureas were not.10
The adverse effects of thioureas are attributed to the formation
of S-oxide intermediates catalyzed primarily by CYP450 enzymes
and FAD-containing monooxygenases.11 The resulting S-oxide can
be hydrolyzed to the corresponding urea, or react with a range of
biomolecules to induce toxicity.12 Two compounds that lacked a
thiourea but retained the distal phenyl and 4-nitrophenyl groups
of 2e were tested against the Dd2 strain (4a–b, Table 3). Both com-
pounds were significantly less active than 2e, which revealed that
heteroatom-containing groups needed to be investigated as a
replacement for the thiourea.
4a
4b
Ph
>10
6.05
PhCH@CH
ties.13 To this end, N-Boc piperazine (5) was reacted with 4-nitro-
phenylsulfonyl chloride to afford the desired sulfonamide in 92%
yield, which was deprotected and isolated as the free amine 6 in
99% yield (Scheme 1). Reaction with diphenyl cyanocarbonimidate
gave intermediate 7, which was converted by reaction with benzyl-
amine to afford 8, the cyanoguanidine analog of 2e. Purification by
mass-directed reverse phase HPLC14 gave this material in 12% yield
over the final two steps. Testing of 8 in the P. falciparum live-dead
assay revealed that it had no activity against both the 3D7 and Dd2
strains (EC50s >20 lM), revealing that a cyanoguanidine was not a
The synthesis of analogs of 2e was undertaken to determine if a
suitable replacement for the thiourea could be found. Our initial ef-
fort focused on replacement of the thiourea with the more benign
cyanoguanidine moiety, an approach that has been used previously
to give compounds with more favorable pharmacokinetic proper-
suitable replacement for the thiourea.
We next investigated whether the thiourea could be replaced by
a urea. The synthesis of two ureas with the furanyl and aryl func-
tionalities possessed by 1a–b is displayed in Scheme 2. Piperazine
5 was reacted with furfuryl isocyanate to afford urea 9,15 which
was then deprotected and reacted separately with two sulfonyl
chlorides to give 10a–b. While these compounds displayed greater
antiplasmodial activity than cyanoguanidine 8, they were signifi-
cantly less potent than screening positives 1a–b (see Table 4).
Table 1
Antiplasmodial activity of 2a–h
Compd
R1
R2
Pf EC50
3D7
(
l
M)
Dd2
2a
2b
2c
2d
2e
2f
Furan-2-yl
4-ClPh
Tetrahydrofuran-2-yl
MeOCH2
Me
4-ClPh
4-ClPh
4-NO2Ph
4-NO2Ph
4-NO2Ph
4-NO2Ph
2-CF3Ph
>10
>10
NDa
0.400
0.860
0.354
0.103
0.337
0.211
>10
0.667
0.310
0.056
0.313
ND
Ph
Tetrahydrofuran-2-yl
Bn
Me
2g
2h
>10
a
ND = not determined.
Scheme 1. Reagents and conditions: (i) 4-Nitrophenylsulfonyl chloride, C5H5N, rt,
2 h (92%); (ii) HCl/dioxane, 0 °C?rt, 4 h (99%); (iii) diphenyl cyanocarbonimidate,
C5H5N, reflux, 18 h. (iv) BnNH2, C5H5N, reflux, 18 h (12% over two steps).
Table 2
Antiplasmodial activity of 3a–i
Compd
R1
Pf EC50 (lM)
3D7
Dd2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3,4-(OCH2CH2O)
4-Br-2,6-Cl
4-F
2,3,4-F
3-F
H
4-Br-2-Cl
2-F
4-OEt
6.49
2.67
0.447
1.52
1.47
1.86
0.830
2.41
1.23
>10
4.41
0.492
1.61
1.97
1.92
0.873
3.13
1.96
Scheme 2. Reagents and conditions: (i) Furfuryl isocyanate, hexanes, 60 °C, 2 h; (ii)
HCl/dioxane, 0 °C?rt, 4 h; (iii) C5H5N, rt, 2 h and either 4-nitrophenylsulfonyl
chloride (10a, 31% over three steps) or 4-chlorophenylsulfonyl chloride (10b, 20%
over three steps).

220
D. C. Martyn et al. / Bioorg. Med. Chem. Lett. 20 (2010) 218–221
With the cyanoguanidine and urea functionalities proven to be
unsuitable replacements for the thiourea, we decided to synthesize
a selection of compounds with a range of moieties in place of the
thiourea to determine whether they would have activity approach-
ing that of 1a. Amine 6 was subjected to a range of conditions to
achieve this goal (Scheme 3). First, 6 was reacted with benzylcya-
namide in (CF3)2CHOH16 to afford guanidine 11 in 18% yield. Amine
6 was also reacted with methyl isothiocyanate,17 then benzyl bro-
mide to give 12 in 8% yield over two steps after purification by re-
verse phase HPLC. Reaction of 6 with hydrocinnamoyl chloride
afforded tertiary amide 13 in 75% yield, which was subsequently
reacted with Lawesson’s reagent18 to give thioamide 14 in 74%
yield. Finally, 6 was irradiated under microwave conditions with
two chloropyrimidines in separate reactions to afford 15a–b in
92% and 88% yield respectively.
Two additional compounds were prepared to afford analogs
with a different amide/thioamide backbone to the tertiary amide/
thioamide 13 and 14 (Scheme 4). To this end, carboxylic acid 16
was coupled to benzylamine, and subsequent deprotection gave
amide 17 as the free amine in 66% yield over two steps. Exposure
to 4-nitrophenylsulfonyl chloride afforded the secondary amide
18 in 65% yield, which was converted to thioamide 19 in 67% yield
with Lawesson’s reagent.
Scheme 4. Reagents and conditions: (i) BnNH2, PyBOP, DIEA, CH2Cl2, rt, 18 h; (ii)
HCl/dioxane, 0 °C?rt, 4 h (66% over 2 steps); (iii) 4-nitrophenylsulfonyl chloride,
C5H5N, rt, 2 h (65%); (iv) Lawesson’s reagent, THF, reflux, 2 h (67%).
Table 5
Antiplasmodial activity of 11–14, 15a–b, 18–19
None of the compounds tested (11–14, 15a–b and 18–19,
Table 5) displayed activity near that of thioureas 1a or 2e. Replace-
ment of the sulfur with NH, as in guanidine 8, completely abro-
gated activity. It is interesting to note that two of the four
Compd
Pf EC50 (lM)
3D7
>25
11.4
13.5
1.25
11.1
3.97
Dd2
>25
13.0
13.6
1.69
10.0
5.81
11
12
13
14
15a
15b
18
Table 4
Antiplasmodial activity of 10a–b
5.95
6.06
3.74
6.19
19
Compd
R1
Pf EC50 (lM)
3D7
Dd2
10a
10b
NO2
Cl
6.41
2.55
4.48
2.30
compounds with EC50s < 10 lM (thioamides 14 and 19) possessed
a sulfur atom at the same position as 1a–b and 2e.
In summary, a high-throughput screening program to identify
compounds amenable to the development of novel antimalarials
discovered piperazine sulfonamides 1a–b. These compounds dis-
played potent activity against P. falciparum in a live-dead assay,
however they possessed furanyl, thiourea and nitrophenyl groups,
all of which have metabolic liabilities. The furan could be replaced
with no loss of activity, and the nitrophenyl could be replaced with
some loss of activity. A number of analogs were synthesized that
replaced the thiourea with a range of functional groups or hetero-
cycles, but in all cases there was a significant decrease in activity,
highlighting the thiourea’s importance for antiplasmodial activity.
Acknowledgments
The authors received funding from the Medicines for Malaria
Venture and Genzyme Corporation. Genzyme Corporation funded
these studies on a not-for-profit basis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.130.
References and notes
Scheme 3. Reagents and conditions: (i) BnNHCN, (CF3)2CHOH, 110 °C, 16 h (18%);
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N-phenylpyrimidin-4-amine (15a, 92%) or 2-chloro-6-methyl-N-benzylpyrimidin-
4-amine (15b, 88%).

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