A novel class of Plasmodial ClpP protease inhibitors as potential antimalarial agents

Article abstract of DOI:10.1016/j.bmc.2017.08.049

The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria parasite, represents a potential drug target. In the present study, we utilized in silico structure-based screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC?? 9.0 ± 0.2 μM), a 9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P. falciparum cultures with compound 33 caused morphological and developmental abnormalities in the parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of apicoplast.

Full text of DOI:10.1016/j.bmc.2017.08.049

Accepted Manuscript
A novel class of Plasmodial ClpP protease inhibitors as potential antimalarial
agents
Sourabh Mundra, Vandana Thakur, Angelica M. Bello, Sumit Rathore, Mohd
Asad, Lianhu Wei, Jane Yang, Sai Kumar Chakka, Radhakrishnan Mahesh,
Pawan Malhotra, Asif Mohmmed, Lakshmi P. Kotra
PII:
S0968-0896(17)31340-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.049
BMC 13953
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 June 2017
17 August 2017
24 August 2017
Please cite this article as: Mundra, S., Thakur, V., Bello, A.M., Rathore, S., Asad, M., Wei, L., Yang, J., Kumar
Chakka, S., Mahesh, R., Malhotra, P., Mohmmed, A., Kotra, L.P., A novel class of Plasmodial ClpP protease
inhibitors as potential antimalarial agents, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.08.049
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Manuscript#: BMC-2017-981 revision
A novel class of Plasmodial ClpP protease inhibitors as potential antimalarial agents
Sourabh Mundra,1,3† Vandana Thakur,2† Angelica M. Bello,1,5 Sumit Rathore,4 Mohd Asad,2 Lianhu
Wei,1 Jane Yang,5 Sai Kumar Chakka,1 Radhakrishnan Mahesh,3 Pawan Malhotra,2 Asif Mohmmed,2*
and Lakshmi P. Kotra1,5*
1Center for Molecular Design and Preformulations, and Toronto General Research Institute, University
Health Network, Toronto, Ontario, Canada, M5G 1L7,
2International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi,
India,
3Department of Pharmacy, Birla Institute of Technology & Science, Pilani, Rajasthan, India, 333031.
4Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110 029, India.
5Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto,
Toronto, Ontario, Canada, M5S 3M2,
RECEIVED DATE (to be automatically inserted)
*Corresponding Authors: AM: ICGEB, Aruna Asif Ali Marg, New Delhi- 100 067, India, Email:
amohd@icgeb.res.in; LPK: #5-356, TMDT/MaRS Center, 101 College Street, Toronto, Ontario,
Canada M5G 1L7. Tel. (416) 581-7601, E-mail: lkotra@uhnres.utoronto.ca.
†These authors contributed equally to this work.
1

Abstract.
The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria
parasite, represents a potential drug target. In the present study, we utilized in silico structure-based
screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds
inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-
activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-
oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with
antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC₅₀ 9.0 ± 0.2 μM), a
9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P.
falciparum cultures with compound 33 caused morphological and developmental abnormalities in the
parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of
apicoplast.
Key words: Malaria, ClpP protease, in silico docking, apicoplast, antimalarial agents.
2

Introduction
Malaria is a parasitic infection in humans caused primarily by Plasmodium falciparum and/or
P. vivax, with a minor fraction (<10%) of the clinical cases caused by other plasmodia species.
Malaria continues to burden the developing world and remains a disease of concern in the developed
world despite intense efforts over the past decade to eliminate this disease. The World Health
Organization (WHO) estimates that as many as 214 million cases of malaria were reported in 2015
resulting in 438,000 deaths worldwide.1 Parasite resistance has rendered previously effective
antimalarial agents ineffective in most parts of the world. Currently, artemisinin-based combination
therapies (ACTs) are the standard of care for malaria. In 2009, it was confirmed that P. falciparum had
acquired resistance to artemisinins too; therefore urgent efforts are needed to combat these drug-
resistant parasites.2,3 A recent report that surveyed a defined region in the endemic areas of India
estimated that adult and child mortalities due to malaria are vastly underreported, and that the
estimates of the WHO are at least 10 times less than may occur in the field at the time of the survey,
suggesting a massive gap between the WHO estimates and the reality in endemic areas.4 These
combined factors underscore the need for new measures in the prevention and treatment of malaria.
Variants of known drug classes are no longer acceptable because there is a higher chance for
the development of cross-resistance to these drugs. The campaign for malaria treatments stresses the
need for new and different classes of antimalarial drugs that will be effective in treating current drug
resistance parasites,5 and in accordance with this, the efforts in the past year have shifted towards
developing novel classes of antimalarial drugs. Another attractive approach would be to develop drugs
directed against novel targets that have not yet been fully exploited for therapeutic use.
ATPase dependent protease machineries such as eukaryotic 26S proteasome and the
prokaryotic caseinolytic proteases (ClpP) systems are large protein degradation complexes that play an
essential role in cell cycle regulation.6,7 The P. falciparum genome also harbors cyanobacterial ClpP
3

protease, PfClpP, and its putative ATPase partner. The ATPases assemble into large multi-subunit
complexes with the ClpP proteases and act as chaperons to unfold the substrate proteins which
subsequently get degraded by the protease component. The PfClpP is localized in the apicoplast as
determined by the GFP targeting approach, immunoelectron microscopy and by immunofluorescence
assays.8 The apicoplast is a reduced cyanobacterial plastid in the parasite; the metabolic pathways in
the mitochondrion and the apicoplast, two parasite organelles of prokaryotic origin, may represent
suitable drug targets in the parasite. Apicoplast plays an important role in biosynthesis of haeme,
isopentenyl diphosphate and fatty-acids,9 thus the apicoplast is considered to be important for parasite
survival. Indeed, antibacterial agents such as ciprofloxacin, rifampicin and thiostrepton that target
DNA replication, transcription and translation of the apicoplast respectively, have been also shown to
kill the parasite.10,11,12,13
P. falciparum contains two ClpP proteases, a PfClpP (gene ID: PF3D7_0307400), and one
PfClpR (gene ID PF3D7_1436800); the parasite also contains four Clp ATPases (PfClpB1, PfClpB2,
PfClpC, and PfClpM), all these proteins are expressed in blood stages of the parasite.8,14 The ClpP and
ClpR proteases are shown to be localized in parasite apicoplast.8,14 Earlier, we also screened cell-
permeable -lactones that bind at the active site of prokaryotic ClpP, and identified compounds that
inhibit the development of apicoplast leading to parasiticidal activities.8 The crystal structure of
PfClpP shows the presence of the conserved active site triad of serine proteases (PDB: 2F6I),15 and it
forms compacted tetra-decamer; such oligomerization was also observed in the case of ClpP proteases
from Streptococcus pneumoniae and Mycobacterium tuberculosis.14 In the present study, we
conducted in silico screening of chemical compound libraries using the active site of PfClpP to
identify a small set of hit compounds; novel series of compounds were designed and synthesized
based upon the structures of the hit compounds. In vitro screening of selected compounds using
enzyme activity assays and parasite growth inhibition assays identified potential lead compound
4

targeting PfClpP with antimalarial activities.
Results.
The structures of ClpP proteases from different organisms have shown two distinct
conformational states. An extended state three-dimensional structure is gleaned from the ClpP
protease structures of E. coli, Homo sapiens, and Helicobacter pylori. In the extended state, the
catalytic triad is generally organized and pre-positioned for a catalytic reaction. The second state, a
compact state is observed in the three-dimensional structures of ClpP protease from S. pneumonia, P.
falciparum and M. tuberculosis, which is an inactive state. Three-dimensional structure of PfClpP
protease in its extended state is not available. The extended state of E. coli crystal structure (RCSB
code: 2FZS)16 was used as a template to model the P. falciparum ClpP protease and was used for in
silico screening. In silico screening was focused on the S1 and S1’pockets in the active site of ClpP
protease (Figure 1), and it was
carried out using commercially
K205
E202
available compounds library
S2
containing
molecule structures; this study
ultimately led to the
450,000
small
S236
S1’
S264
S3
identification of 13 hits (1-13)
meeting all the screening criteria
including their potential for
S1
H289
D338
further medicinal chemistry (Table 1).
These 13 hit compounds,
Figure 1. Illustration of the features defined in the active site
of Pf ClpP protease. Protein sequence is shown as a cartoon
model, and the amino acid residues are shown in capped-stick
representation (C: white, O: red, N: blue). Active site features
are depicted over the active site of the enzyme using the
following color-coding: hydrophobic feature: yellow sphere;
1-13 were evaluated for their
anionic center: red sphere; cationic center: blue sphere;
5
magenta sphere: hydrogen bond acceptor corresponding to
S264; green ring: hydrogen bond donor. S1’, S1, S2, S3
indicate the protein binding site positions of Pf ClpP.

potential to inhibit PfClpP protease followed by their antiparasitic activity. Five compounds 1, 2, 4, 6,
and 11 exhibited appreciable inhibition of PfClpP protease activity with inhibition constants (IC50)
lower than 100 µM. These five compounds were then evaluated for their antiparasitic activities against
P. falciparum. Compounds 1, 6 and 11 inhibited parasite growth with inhibition constants (IC50) in the
range of 25-75 µM. Upon comparison of the structures of these three active compounds, and the ease
of synthetic feasibility to functionalize the core structural elements, the common pyrimidine core of
compounds 6 and 11 was further pursued (Figure 2).
B
A
S
ubstit
utions
on 2-
thio
moiety of the
Figure 2. Design of analogs based on compounds 6 (panel A) and 11 (panel B).
pyrimidin-2-
thione, including p-methoxyphenyl, p-diethylaminophenyl, morpholinyl and 4-benzonitrile moieties,
were synthesized in order to investigate structure-activity relationship (Scheme 1). Synthesis of
compounds 22-26 was initiated starting from 6-methyl-2-thio-2-pyrimidinone (14) (Scheme 1A).
Alkylation of compound 14 using an appropriate halide in the presence of potassium carbonate in
DMF yielded the thioethers 15A-15E. Compounds 15A-15E were subjected to chlorination using
phosphorous oxychloride to obtain the corresponding chlorothioethers 16A-16E, which were used as
intermediates en route to the target compounds 22-26 (Scheme 1B). Thus phenyl methylacetate (17)
6

was treated with hydrazine hydrate to obtain the hydrazine 18, which was further treated with
chloroacetyl chloride to obtain compound 19 in 66% yield. Compound 19 was then treated with
potassium thioacetate, followed by saponification to yield compound 21 in approximately 70% yield
for each transformation. Compound 21 was then coupled to compounds 16A-16E by the substitution
of the chloro moiety to yield compounds 22-26, respectively.
7

Scheme 1. Synthesis of compounds 22-26. Reagents and conditions: (i) A-E halides, K2CO3, DMF,
16 h, rt; (ii) POCl3, 70 °C, 1 h; (iii) NH2NH2·H2O, MeOH, 16 h, rt; (iv) chloroacetyl chloride,
DIPEA, CH2Cl2, -10 °C to rt, 30 min; (v) AcS-K+, EtOH, rt; (vi) K2CO3, MeOH, rt, 1 h; (vii)
compounds 16A-16E, Et3N, AcCN.
8

Compounds 30-38, designer compounds based on the core structure derived from compound
11 (Figure 2B) were synthesized using the chemical synthesis illustrated in Scheme 2. Benzyl thiourea
(27) and diethyl (ethoxymethylene)malonate were cyclized to yield 2-thio-3-benzyl uracil derivative
28 in 55% yield. Compound 28 was transformed into the phenylpropyl amine derivative 29;
compound 29 was alkylated using appropriate alkyl halides in the presence of potassium carbonate to
yield compounds 30-38 in 30-50% yield. Additional derivatives bearing a morpholine moiety such as
40-42 and 44 were synthesized from 2-mercapto-5-carboxyethyl uracil (39). Alkylation of the thio
moiety with acetohydrazide or bromoethylmorpholine gave compounds 40 and 41, respectively
(Scheme 3). Alkylation of N3 position on 41 with bromoacetophenone yielded compound 42.
Hydrolysis of ethyl ester 42 led to the cyclized bicyclic thiazine derivative 43, and did not provide the
intended product of monocyclic 5-carboxy-2,3-substituted pyrimidine derivative. Thus, compound 41
was then derivatized at C5 position introducing phenylpropylamine moiety to yield compound 44 in
60% yield. It was also evident that introduction of substitutions onto 2-thiouracil at 2, 3 and 5
positions is more efficient via thiourea-malonate condensation pathway (Scheme 2), and introduction
of acetophenyl moiety makes the compounds susceptible to cyclization (such as 42 to 43).
9

Scheme 2. Synthesis of compounds 30-38. Reagents and conditions: (i) Diethyl
(ethoxymethylene)malonate, K2CO3, DMF, 120 ºC, 1 h; (ii) 3-Phenyl-1-propylamine, trimethyl
aluminum, 16 h, 0 °C to rt; (iii) A-I halides, K2CO3, DMF, 16 h, rt.
Scheme 3. Synthesis of compounds 40-44. Reagents and conditions: (i) 2-Chloro-N'-(2-
phenylacetyl)acetohydrazide or 4-(2-bromoethyl)morpholine, K2CO3, acetone, 16 h, rt; (ii) 2-
Chloroacetophenone, K2CO3, acetone, 16 h, rt; (iii), LiOH in THF/MeOH (3:1) 16 h, rt; (iv) 3-
Phenylpropylamine, trimethyl aluminium, 16 h, 0 °C- rt.
10

Identification of specific PfClpP inhibitors with parasiticidal efficacies. All synthesized compounds
were evaluated for their inhibitory activities against recombinant PfClpP protease. Compounds that
exhibited PfClpP enzyme inhibition (IC50) at less than 100 µM concentration were then evaluated for
their antiparasitic activities. Compounds 1, 2, 4, 6 and 11, which were the hits from the in silico
screening showed promising enzyme inhibition with inhibition constants (IC50) in the range of 22-92
M and the corresponding apparent Ki in the range of 9-37 M. These compounds were subsequently
evaluated for their antiparasitic activities; compounds 1, 6 and 11 showed moderate inhibition with
IC50 ranging from 25-75 M. These three compounds were also tested for cytotoxicity in HeLa cell
line, and all three compounds showed IC50 > 5 mM.
Compounds 22-26, which are based on the core structure 6 inhibited the PfClpP protease
activity with moderate potency with IC50 in the range of 28-35 M. Compounds 22-26 inhibited P.
falciparum parasite cultures with IC50s in the range of 11.8-17.3 M. Compounds 30-32, 40, 42 and
44 showed moderate to good inhibition of PfClpP protease activity, with IC50s in the range of 10-72
M. Compound 30 is the most active compound in this group of inhibitors, with an IC50 of 24±8 M,
and the apparent Ki = 10±3 M (Table 1). Compounds 41 and 43 did not exhibit appreciable inhibition
of PfClpP protease, upto 100 µM concentration. Compound 33 exhibited the most potent inhibitory
activity of all tested compounds against PfClpP protease, with an IC50 of 10.5 ± 0.2 M (apparent Ki =
2.3 ±0.2 M). Compounds 34-38 exhibited either weak or no inhibition of PfClpP protease activity;
specifically compounds 34, 36 and 38 inhibited the protease activity at concentrations (IC50) in the
range of 45-58 M. Compounds 35 and 37 did not inhibit the protease activity upto 100 M
concentration.
Table 1. ClpP Enzyme inhibition and parasite growth assay.
11

P.
PfClpP Protease
Inhibition
falciparum
inhibition
IC50 ± SE
(μM)
Compound
Structure
IC50 ± SE
Ki (μM)
(μM)
30.0 ± 4.0 12.1 ± 0.2
27.0 ± 2.0
1
92.0 ±
105.0
38.0 ±
43.0
NA
NA
2
3
>100.0
NA
88.0 ± 31.0 36.0 ±13.0
NA
4
>100.0
NA
NA
5
6
26.0 ± 6.0 11.0 ± 2.0
75.0 ± 9.0
>100.0
NA
NA
7
12

>100.0
NA
NA
8
>100.0
>100.0
NA
NA
NA
NA
9
10
23.0 ± 10.0
9.0 ± 4.0
25.0 ± 4.0
11
>100.0
>100.0
NA
NA
NA
NA
12
13
34.7 ± 0.5 14.2 ± 0.2 11.80 ± 0.03
22
23
24
25
28.3 ± 0.4
11.6 ±0.2
12.14±0.03
17.3 ± 0.1
14.4 ± 0.1
28.8 ± 0.4 11.8 ± 0.2
30.3± 0.1
12.3 ± 0.1
13

37.4± 0.2
15.3 ±0.1
12.4 ±0.1
20.0 ± 2.0
26
30
24.0 ± 8.0 10.0 ± 3.0
45.0 ± 13.0 18.0 ± 5.0
2.4 ± 1.5
31
29.5 ±
72.6 ± 44.4
NA
32
33
18.0
10.5± 0.2
2.3 ± 0.2
9.0 ± 0.2
58.3 ± 0.1 23.8 ± 0.1
34
35
15.9±0.1
9.2 ± 0.1
>100.0
NA
53.0 ± 0.1 21.6 ± 0.1
9.2 ±0.1
13.7 ± 0.1
11.7 ± 0.1
82.5 ± 0.5
36
37
38
40
>100.0
NA
46.0 ± 0.7 18.8 ± 0.3
42.0 ± 8.0 17.0 ± 3.0
14

>100.0
NA
NA
41
42
40.0 ± 12.0 16.0 ± 5.0
19.0 ± 2.0
>100.0
NA
11.0 ± 2.0
22.0 ± 0.5
43
44
44.0 ± 7.0 18.0 ± 3.0
Compounds 30-33, 40, 42 and 44, which are the active compounds against the protease
activity, were then evaluated for their inhibition of P. falciparum growth in the culture. Compound 30
inhibited P. falciparum growth at a concentration (IC50) of 20.0±2.0 M, as expected based on its
activity against ClpP protease. Compound 31 inhibited P. falciparum cultures at a concentration (IC50)
of 2.4 ± 1.5 M, about 20-fold higher potency than the IC50 for the inhibition of PfClpP protease.
Compound 32 did not inhibit P. falciparum growth even at 100 M concentration. Compound 33
inhibited P. falciparum at a concentration of 9.0±0.2 M, and compounds 34-38 similarly inhibited P.
falciparum growth with IC50s in the range of 9.2-15.9 M, somewhat higher potency than that for the
inhibition of the target enzyme. Compound 40 inhibited the parasite growth with moderate potency of
IC50 at 82.5±0.5 M. Compounds 42 and 44 inhibited the parasite growth at 19.0±1.5 M and
21.9±0.5 M, respectively, which are about 2-fold higher potency than their corresponding in vitro
PfClpP protease activity inhibition (Table 1).
Compound 33 was further studied for cytotoxicity using HeLa cell line. This compound
exhibited an IC50 >5 mM in this cell line indicating high selectivity for its parasiticidal effects.
Effect of ClpP inhibitors on plasmodial parasite development. We investigated the effect of one of
15

the most active compounds in the series, compound 33, on the morphology and development of P.
falciparum during the asexual stages (Figure 3). The ring stage parasites were treated with the protease
inhibitor 33 or no treatment. The parasites developed from ring stage to trophozoite stage and
subsequently developed into the schizont stage in the first 48 h, when there was no inhibitor present in
the growth culture; the merozoites also invaded fresh erythrocytes and completed the second cycle
during 48-96 h of incubation. The compound 33-treated parasites were developmentally arrested at the
transition from early to late stage schizont. The abnormal parasites in cultures were not able to develop
into mature schizonts, and were seen as abnormal parasites even at 60 hpi (Figure 3). These
phenotypes in second cell-cycle after treatment with compound 33 are similar to those seen in
parasites treated with other inhibitors known to be targeting apicoplast targets.17,18
52 hpi
68hpi
76hpi
92hpi
110hpi
Tr
ea
te
d
Co
ntr
ol
Figure 3. Effect of compound 33 on development and morphology of P. falciparum parasites. Growth
of trophozoite stage parasite and development of schizonts was inhibited in 33 treated parasite
cultures.
Effect of PfClpP inhibitors on apicoplast development. Since ClpP is an apicoplast protease, the
effect of inhibitor 33 was assessed on growth and development of the apicoplast, we utilized the
transgenic parasite line expressing nuclear-encoded GFP fusion protein, which is targeted to the
apicoplast. The growth and development of the apicoplast during the first cell cycle in the parasites
treated with 33 at 10 μM concentration were indistinguishable from that of untreated parasite; the
16

apicoplast showed elongated and branched pattern in the early schizont-stage parasites and divided
normally (Figure 4). However, during the second cell cycle the growth and development of apicoplast
was severely affected by the treatment
Compound 33
with compound 33. In the trophozoite
stages of the second cell cycle, the
apicoplast was seen as spherical
structure in the treated cultures, as in
case of the control set; however, the
apicoplast was not able to grow and
divide during the schizogony in the
Compound 33
treated cultures. In the early schizont-
stage (72 h after the drug treatment) and
the late schizont-stage parasites (90–96
h after drug treatment), the apicoplast
appeared as a round stump-like structure
without any branching and segregation.
Figure 4. Effect of compound 33 on apicoplast
However, the nuclear divisions in these
parasites proceeded normally and at 96
h the multinucleate parasites were
observed with a single apicoplast
development in P. falciparum. Fluorescence microscopic
images of D10-ACP-GFP parasite showing development
of apicoplast at 36hpi (A) and 72hpi (B) in parasite
cultures treated with 33 at IC50 concentration. The
apicoplast development in treated parasite was severely
inhibited in treated cultures.
(Figure 4). Overall, the compound 33, a specific inhibitor of PfClpP, showed targeted effect on
apicoplast development and segregation during the second cell-cycle after treatment.
Discussion.
The apicoplast is a specialized organelle in the malaria parasite with prokaryotic origin. Several
metabolic pathways in this organelle are unique to the parasite as compared to the host and thus these
17

pathways are good targets to design new antimalarials. The P. falciparum ClpP serine protease
(PfClpP), homologue of a cyanobacterial ClpP, is one such potential drug target. The ClpP is localized
in the apicoplast and plays an essential role in the apicoplast growth and development.8 Parasite
proteases have been considered as potential drug targets for malaria as they play crucial roles in
different metabolic pathways and can be inhibited by specific inhibitors.19,20 The crystal structure of
PfClpP (PDB ID: 2F6I) shows the presence of the conserved active site triad of serine proteases; this
protease forms a compact, inactive tetradecamer as in the case of ClpP proteases from Streptococcus
pneumoniae and Mycobacterium tuberculosis.14 Three dimensional structure of “extended” PfClpP
protease in its active state is not resolved. Therefore, we used the extended state of E. coli crystal
structure (PBD code: 2FZS) as a template to model the extended state of P. falciparum ClpP. This
energy minimized 3D extended state PfClpP model was used for in silico high throughput screening of
large compound libraries to identify potential inhibitors. Initial in silico screening combined with
activity assay based screening identified five compounds, 1, 2, 4, 6 and 11, showed inhibitory activity
on PfClpP enzyme and also showed promising effect on parasite growth with moderate potency (IC50 in
the range of 5 M) (Table 1). Other compounds did not show appreciable activities against either ClpP
protease or the parasite growth.
Based on the structures of compound 6 and 11, two series of substituted pyrimidine derivatives
were synthesized (Figure 2). Initially, we examined the role of C2 substitution on the pyrimidine
moiety using the first set of compounds 22-26. The substitutions included morpholine moiety and
substituted phenyl moieties. None of these compounds exhibited superior inhibition of ClpP protease
when compared to compound 6, but were almost equipotent. Based on this small set of compounds, it
was concluded that p-substituted benzyl moieties at C2 position via a thioether linker (compounds 22,
23 and 26) did not contribute towards any changes in the activities, as was the case for the morpholine
substitution (compounds 24 and 25).
18

The second set of compounds, 30-38 and 40-44 were evaluated using the primary and
secondary assays, i.e. ClpP protease assay and the P. falciparum culture assay, respectively. Inhibition
of ClpP protease activities and the corresponding antiparasitic activities of compounds 30-33 indicated
that the morpholinoethylthio substitution at C2 position rendered good activity to compound 33, and
the aromatic substitutions at C2 on compounds 30-32 only offered moderate to poor potency.
Compound 34, with a C2-ethylacetyl substitution, and compounds 35-37 and 40, with C2-aromatic
substitutions were only weak or inactive compounds against ClpP protease. Compounds 42 and 44,
both carrying a morpholino moiety at C2 but no bulky substitution either at N3 or C5 (42 vs 44),
exhibited only moderate inhibition of ClpP protease activities (IC50 40±12 and 44±7 µM, respectively),
and moderate antiplasmodial activities. Compounds 38, 41 and 43 did not show any inhibition of ClpP
protease, at upto 100 µM concentration, despite the presence of morpholino moiety on the C2 side
chain. Close inspection of the structures of compounds 33, 38 and other morpholine carrying sidechains
at C2 indicate that the presence of a tertiary amide involving morpholine compromises the potency
against the inhibition of ClpP protease. The length of the side chain at C2 is optimal at 3 atoms (i.e.
thioethyl) in the most active compounds. C4-Methyl substitution as in compounds 22-26 did not
provide any advantage over 4-ketone (compounds 30-38 and 40-44) on the pyrimidine moiety; in
addition benzyl substitution at N3 provided important hydrophobic character with improved potency,
somewhat supporting the core structure based on compound 11 (Figure 2B).
Upon comparing the C6 substitutions on compounds 22-26, and C5 substitutions on compounds
30-38 and 40-44, a general trend towards hydrophobic groups substituted at C5 position may enhance
activity. Although this needs to be investigated further, C6 substitution alone did not help improve the
biological activities, but two substitutions at C5 (ethoxycarbonyl and phenylpropylaminocarbonyl
moieties) helped maintain the antiparasitic activities and is not a prohibitive substitution. Above
observations are summarized in Figure 5, illustrating the importance of each substitution on the
19

pyrimidine moiety (Figure 5).
Figure 5. General structure-activity observations based on the pyrimidine core structure, and the
derivatives of 6 and 11.
Among the active compounds, most effective compound 33 was selected to study detailed effect
on parasite growth and development (Figures 3 and 4). A number of apicoplast targeting drugs are
shown to severely affect ability of the apicoplast to grow and segregate in the subsequent cycle leading
to death of the parasite.21,22,23,24,25 Since ClpP is an apicoplast protein, its inhibitors are expected to
specifically effect the development of apicoplast in the subsequent cycle of the parasites. Our data
show that inhibition of the PfClpP by selected inhibitor 33, inhibited the growth and segregation of
apicoplast in the subsequent cell cycle after treatment, which hampered the development of schizonts in
that cycle, ultimately causing parasite death (Figure 3).
Currently, the most effective compounds utilized in antimalarial therapy programs belong to
single class of compounds, the artemisinins; however, resistance is already emerging against
artemisinins. To avoid generation of drug-resistance and for effective malaria cure, combinations
therapies are developed; however, most of these combination therapies utilize artemisinin along with
other anti-malarial drugs for which resistance in parasite is already reported. Therefore, there is a need
to develop new drugs, which can be utilized in combination therapy with artemisinins. Understanding
20

the mechanisms by which a new drug kills the parasite may help to assess its potential in combination
therapies. It has been suggested that slow acting, apicoplast targeting drugs can be effectively used as
combination therapy with fast acting artemisinin based drugs; further, these drugs in combination with
artemisinin may even slow the spread of artemisinin resistance.26,27 Overall our studies using in silico
screening and structure activity relationship analysis led to identification of novel pyrimidine class of
compounds targeting PfClpP protease. We reveal that these molecules specifically target the
apicoplast in the P. falciparum parasite and inhibit parasite growth, these compounds can be
developed further to design new antimalarials which can be used in combination therapy strategy.
Experimental Section.
Molecular modeling and docking procedures: Three-dimensional structure of the tetra-decamer form
of P. falciparum ClpP protease was generated based on the X-ray crystal structure of E. coli ClpP
protease. Then the structure was subjected to energy minimization using Amber version 11.0 suite
molecular simulation package.28 An all-atom force field and the default atomic charges for the
residues were used as implemented in Amber software package (ff03 force field). The molecule was
immersed in a rectangular TIP3P water box that is at least 9 Å from the surface of the protein before
performing energy minimization. During energy minimization, non-bonded interactions were
computed for up to a distance of 12.0 Å. The molecular systems were first energy-minimized using
steepest decent method for 50 cycles and then using conjugate gradient method up to 20,000 cycles.
Then the energy minimized 3D extended PfClpP model was used for in silico high throughput
screening.
The in silico screening focused on the active site of PfClpP protease; S1 and S1’pockets in the
active site of ClpP protease are hydrophobic, and are surrounded by I237, M265, V268, P291, L316, and
L320. S3 pocket is also hydrophobic, which is ensconced by the side chains of L241, I237, L312, and L261
(spheres in yellow, Figure 1). Thus, three hydrophobic centers were defined representing S1, S3, and
21

S1 pockets’ surface properties. A hydrogen bond donor feature is defined on the side chain of S236
(green ring in Figure 1), and a hydrogen bond acceptor feature was defined using the side chain of S264
(sphere in magenta, Figure 1), which is the catalytic residue for proteolysis. An anionic center
representing the side chain of K205, a cationic center representing the side chain of E202 were defined
as the active site features for the in silico screening. A total of seven active site features were defined
and were used as a template for UNITY database search. The Unity3D flexible search was performed
using the following rules for the filtering of the small molecules: (1) match the hydrophobic features
S1 and S3, and the hydrogen bond acceptor feature; (2) match at least one of the remaining four
features. Commercial chemical libraries from Asinex, Otava, and Pharmeks containing a total of over
450,000 compounds, customized in-house, were used for this screening and total 1,768 hits from the
UNITY search were obtained. There are 251 hits out of Asinex Platinum library, 257 hits out of
Asinex Gold library, 58 hits out of Asinex Elite library, 575 hits from Pharmeks library, 278 hits from
Otava in-house library, and 349 hits from Otava backorder library. These were then docked to PfClpP
protease active site using GOLD program from CCDC (http://www.ccdc.cam.ac.uk). In the docking
procedure, all the active site residues were kept rigid. Gold scoring function was used to choose the
top ranking hits and all ligands were used 40 GA runs. The top 225 hits (GOLD fitness scores cut-off
is 73) was chosen for the next round of manual selection, which were narrowed down to 13
compounds based on their chemical structures, and potential to synthesize analogs for optimization.
Chemistry. All solvents and chemical reagents were obtained from commercial sources. All anhydrous
reactions were performed under a nitrogen atmosphere; anhydrous solvents were prepared following
standard procedures. Reaction progress was monitored by TLC plates (Silica gel-60 F254).
Chromatographic purifications were performed using silica gel (60 Å, 70-230 mesh). The purchased
library compounds were characterized by 1H NMR, UV and mass spectral analyses. Purity data for the
synthesized compounds were obtained using two independent methods using a Waters® HPLC system
22

equipped with a photodiode array detector employing reverse phase chromatography (XBridge
analytical C18 column, 4.6x150 mm2, 5 m) (determined to be >95%, supplemental information).
Mass spectra for all compounds were recorded using Waters® MS3100 mass spectrometer in the ESI
+ve mode. NMR spectra for all compounds were recorded on a Bruker NMR spectrometer (400 MHz
1
for H and 101 MHz for 13C nuclei). Chemical shifts are reported in δ ppm using tetramethylsilane
(TMS) as a reference.
N-(5-Ethyl-1,3,4-thiadiazol-2-yl)-2-((5-(3-hydroxynaphthalen-2-yl)-4-(2-methoxyethyl)-4H-1,2,4-
1
triazol-3-yl)thio)acetamide (1). H NMR (CDCl3+CD3OD)  1.40 (t, J = 7.6Hz, 3H), 3.05 (q, J =
7.6Hz, 2H), 3.25 (s, 3H), 3.59 (t, J = 5.4Hz, 1H), 4.21 (s, 2H), 4.30 (t, J = 5.4Hz, 1H), 7.34 (s, 1H),
7.36 (dt, J = 8, 0.8Hz, 1H), 7.49 (dt, J = 8, 0.8Hz, 1H), 7.71 (d, J = 8.4Hz, 1H), 7.79 (d, J = 8Hz, 1H),
8.10 (s, 1H). Mass (ESI, +ve mode) calculated for [C21H23N6O3S2]+ = 471.13; found 471.16.
1
2,2',2''-((1,3,5-Triazin-2,4,6-triyl)tris(sulfanediyl))-tris-(N-(furan-2-ylmethyl)acetamide) (2). H
NMR (CDCl3+CD3OD)  3.98 (s, 6H), 4.42-4.40 (m, 6H), 6.22 (d, J = 3.2Hz), 6.30 (dd, J = 3.2,
0.8Hz, 3H), 7.33 (d, J = 0.8Hz, 3H), 7.98 (t, 1H). Mass (ESI, +ve) calculated for [C24H25N6O6S3]+ =
589.09; found 589.07.
1
Dibenzyl ((benzyloxy)carbonyl)glycylglutamate (3). H NMR (CDCl3)  2.10-1.90 (m, 1H), 2.20-
2.29 (m, 1H), 2.33-2.48 (m, 2H), 3.80-3.92 (m, 2H), 4.67 (q, J = 5.2Hz, 1H), 5.08 (d, J = 2.4, 2H),
5.15 (s, 2H), 5.12 (s, 2H), 5.30 (bs, 1H), 6.71 (d, J = 7.6H, 1H), 7.29-7.40 (m, 15H). Mass (ESI, +ve
mode) calculated for [C29H31N2O7]+ = 519.2131; found 519.2327.
N,N'-(5-(Thiazol-2-ylcarbamoyl)-1,3-phenylene)bis(2-phenoxyacetamide)
(4).
1H
NMR
(CDCl3+CD3OD)  4.69 (s, 4H), 6.96-7.05 (m, 6H), 7.08 (d, J = 3.6Hz, 1H), 7.32 (t, J = 7.2 Hz, 4H),
7.49 (d, J = 3.6Hz, 1H), 8.03-8.07 (m, 2H), 8.32-8.35 (m, 1H). Mass (ESI, +ve mode) calculated for
[C26H23N4O5S]+ = 503.14; found 503.10.
(E)-2-(2-((5-(((4-Bromophenyl)sulfonamido)methyl)furan-2-yl)methylene)hydrazinyl)-N-(4-
23

1
methoxybenzyl)-2-oxoacetamide (5). H NMR (DMSO-d6+CD3OD)  3.58 (s, 2H), 3.63 (s, 3H),
3.84 (s, 2H), 6.78 (d, J = 8.8Hz, 1H), 7.27-7.39 (m, 5H), 7.52 (d, J = 8.8Hz, 2H), 7.61 (s, 1H), 8.05 (d,
J = 7.6Hz, 2H). Mass (ESI, +ve mode) calculated for [C22H22BrN4O6S]+ = 549.0443 and 551.0423;
found 549.06 and 551.17.
2-((6-Methyl-2-((2-oxo-2-phenylethyl)thio)pyrimidin-4-yl)thio)-N'-(2-
phenylacetyl)acetohydrazide (6). 1H NMR (CDCl3)  2.27 (s, 3H), 3.58 (s, 2H), 3.71 (s, 2H), 3.85 (s,
2H), 6.78 (s, 1H), 7.25-7.36 (m, 5H), 7.50 (t, J = 7.2Hz, 2H), 7.61 (t, J = 7.2 Hz, 1H), 8.04 (d, J = 7.2
Hz, 2H). Mass (ESI, +ve mode) calculated for [C23H23N4O3S2]+ = 467.12; found 467.13.
(E)-3-(3-(4-Butoxy-2-methylphenyl)-1-phenyl-1H-pyrazol-4-yl)-2-cyano-N-(furan-2-ylmethyl)
1
acrylamide (7). H NMR (CDCl3)  1.00 (t, J=7.2Hz, 3H), 1.57-1.48 (m, 1H), 1.83-1.76 (m, 1H),
2.27 (s, 3H), 4.01 (t, J=6.8Hz, 2H), 4.55 (d, J=5.6 Hz, 2H), 6.28 (d, J=3.2Hz, 1H), 6.33 (dd, J=2.8,
1.6Hz, 1H), 6.47 (t, J=5.6Hz, 1H), 6.81 (dd, J=8.4, 2.4 Hz, 1H), 6.86 (d, J=2Hz, 1H), 7.18 (d,
J=8,4Hz, 1H), 7.38 (s, 1H), 7.39 (t, J=7.6Hz, 1H), 7.506 (t, J=7.6 Hz, 2H), 7.81 (d, J=7.6 Hz, 2H),
8.04 (s, 1H), 9.01 (s, 1H). Mass (ESI, +ve mode) calculated for [C29H29N4O3]+ = 481.22; found
481.21.
(Z)-5-((3-(3-Fluoro-4-propoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-phenethyl-2-
thioxothiazolidin-4-one (8). 1H NMR (CDCl3)  1.09 (t, J = 7.2Hz), 1.86-1.95 (m, 2H), 2.99-3.04 (m,
2H), 4.08 (t, J = 6.4Hz, 2H), 4.31-4.36 (m, 2H), 7.25-7.34 (m, 5H), 7.40 (t, J = 7.6Hz, 1H), 7.45 (dd, J
= 11.6, 2Hz, 1H), 7.53 (t, J = 3.6Hz,2H), 7.72 (s, 1H), 7.79 (d, J = 3.6Hz, 2H), 8.16 (s, 1H). Mass
(ESI, +ve mode) calculated for [C30H27FN3O2S2]+ = 544.15; found 544.13.
5-Chloro-N-(3-((4-chlorophenyl)amino)-3-oxopropyl)-2-((4-ethylbenzyl)thio)pyrimidine-4-
carboxamide (9). 1H NMR (CD3OD)  1.21 (t, J = 7.6Hz, 3H), 2.59 (q, J = 7.6Hz, 2H), 2.70 (t, 6Hz,
2H), 3.36 (s, 2H), 3.74 (t, J = 6Hz, 2H), 7.07 (d, J = 8.4Hz, 2H), 7.26 (d, J = 8.4Hz, 2H), 7.36 (d, J =
8.4Hz, 2H), 7.41 (d, J = 8.4Hz, 2H), 7.48 (s, 1H), 8.64 (s, 1H). Mass (ESI, +ve mode) calculated for
24

[C23H23Cl2N4O2S]+ = 489.09; found 489.08.
2-((6-Isopropyl-4-oxo-3-phenethyl-3,5,6,8-tetrahydro-4H-pyrano[4',3':4,5]thieno[2,3-
1
d]pyrimidin-2-yl)thio)-N-(3-methoxyphenyl)acetamide (10). H NMR (CDCl3)  1.04 J = 6.8Hz,
3H), 1.07 (d, J = 6.8Hz, 3H), 1.85-1.95 (m, 1H), 2.80-2.90 (m, 1H), 3.05 (t, J = 8Hz, 2H), 3.18 (dt, J =
14.8, 2.8Hz, 1H), 3.39-3.44 (m, 1H), 3.79 (s, 3H), 3.96 (s, 2H), 4.28 (dt, J = 9.2, 2.8Hz, 2H), 4.81 (dt,
J = 15, 2.4Hz, 1H), 4.91 (d, J = 15Hz, 1H), 6.65 (dd, J = 8, 1.6Hz, 1H), 6.94 (dd, J = 8, 1.2Hz, 1H),
7.19 (t, J = 8Hz, 1H), 7.21-7.32 (m, 6H), 9.20 (s, 1H). Mass (ESI, +ve mode) calculated for
[C29H32N3O4S2]+ = 550.18; found 550.15.
2-((6-Isopropyl-4-oxo-3-phenethyl-3,5,6,8-tetrahydro-4H-pyrano[4',3':4,5]thieno[2,3-
1
d]pyrimidin-2-yl)thio)-N-(4-phenylbutan-2-yl)acetamide (11). H NMR (CDCl3)  1.03 (d, J =
6.8Hz, 3H), 1.06 (d, J = 6.8Hz, 3H), 1.15 (d, J = 6.4Hz, 3H), 1.65-1.78 (m, 2H), 1.85-1.95 (m, 1H),
2.55 (t, J = 8.4Hz, 2H), 2.78-2.87 (m, 1H), 3.04 (t, J = 7.6Hz, 2H), 3.16 (d, J = 17.6Hz, 1H), 3.35-3.42
(m, 1H), 3.75-3.87 (m, 2H), 3.98-4.05 (m, 1H), 4.22-4.35 (m, 2H), 4.75 (d, J = 14.4Hz, 1H), 4.84 (d, J
= 14.4Hz, 1H), 6.63 (bt, J = 7.6Hz, 1H), 7.07-7.10 (m, 2H), 7.15 (t, J = 8Hz, 1H), 7.23 (t, J = 7.2Hz,
2H), 7.30-7.36 (m, 5H). Mass (ESI, +ve mode) calculated for [C32H38N3O3S2]+ = 576.24; found
576.26.
N-Benzyl-2-((6-isopropyl-4-oxo-3-phenethyl-3,5,6,8-tetrahydro-4H-pyrano[4',3':4,5]thieno[2,3-
d]pyrimidin-2-yl)thio)acetamide (12) . 1H NMR (CDCl3)  1.03 (d, J = 6.8Hz, 3H), 1.07 (d, J =
6.8Hz, 3H), 2.84-2.94 (m, 1H), 2.77-2.88 (m, 1H), 3.03 (t, J = 8Hz, 2H), 3.16 (dt, J = 16.4, 2.4Hz,
1H), 3.36-3.42 (m, 1H), 3.91 (s, 2H), 4.27 (dt, J = 8, 4.8Hz, 2H), 4.44 (d, J = 5.6Hz, 2H), 4.77 (dt, J =
14.8, 2.4Hz, 1H), 4.86 (d, J = 14.8Hz, 2H), 6.93 (bt, 1H), 7.19-7.24 (m, 2H), 7.24 (d, J = 5.2Hz, 4H),
7.29 (d, J = 4.4Hz, 4H). Mass (ESI, +ve mode) calculated for [C29H32N3O3S2]+ = 534.19; found
534.21.
1
S-Benzyl-N-(2-((2-oxo-4-propyl-2H-chromen-7-yl)oxy)acetyl)cysteine (13). H NMR (CDCl3) 
25

1.05 (t, J = 7.6Hz), 1.66-1.77 (m, 2H), 2.70 (t, J = 7.6Hz, 2H), 2.93-3.03 (m, 2H), 4.61 (s, 2H), 3.73
(s, 2H), 4.86 (q, J = 7.6Hz, 1H), 6.17 (s, 1H), 6.90 (d, J = 2.4Hz, 1H), 6.93 (dd, J = 8, 1.6Hz, 1H),
7.19-7.33 (m, 6H), 7.56 (d, J = 9.2Hz, 1H). Mass (ESI, +ve mode) calculated for [C24H26NO6S]+ =
456.15; found 456.12.
2-(2-(4-Methoxyphenyl)-2-oxoethylthio)-6-methylpyrimidin-4(3H)-one (15A). A mixture of
compound 14 (300 mg, 2,1 mmol), 2-bromo-1-(4-methoxyphenyl)ethanone (483 mg, 2.1 mmol) and
potassium carbonate (291 mg, 2.1 mmol) was stirred in anhydrous N,N-dimethylformamide (3 mL) at
room temperature for 12 h. Completion of the reaction was monitored by TLC. The reaction content
was poured on cold water (5 mL) and extracted with ethyl acetate (3 x 10 mL). The organic layers
were combined together, washed with brine (1 x 10 mL), dried over anhydrous sodium sulfate and the
solvent was evaporated to dryness. The crude mixture was purified by column chromatography
1
(hexanes:ethyl acetate gradient) to obtain pure compound 15A as a pale yellow (306 mg, 50%). H
NMR (DMSO-d6)  12.659 (s, 1H), 8.0235 (d, J = 9.2 Hz, 2H), 7.0745 (d, J = 9.2 Hz, 2H), 5.931 (s,
1H), 4.699 (s, 2H), 3.855 (s, 3H), 1.981 (s br., 3H).
2-(2-(4-(Diethylamino)phenyl)-2-oxoethylthio)-6-methylpyrimidin-4(3H)-one (15B). Compound
15B was synthesized using the method described for compound 15A starting from compound 14 (200
mg, 1.4 mmol) and 2-bromo-1-(4-(diethylamino)phenyl)ethanone (380 mg, 1.4 mmol). The product
1
was obtained as an orange solid (256 mg, 55%). H NMR (DMSO-d6) δ 7.843 (d, J = 8.8 Hz, 2H),
6.714 (d, J = 9.2 Hz, 2H), 5.918 (s, 1H), 4.638 (s, 2H), 3.433 (q, J = 7.2 Hz, 4H), 2.046 (s br., 3H),
1.122 (t, J = 7.0 Hz, 6H).
6-Methyl-2-(2-morpholino-2-oxoethylthio)pyrimidin-4(3H)-one (15C). Compound 15C was
synthesized using the method described for compound 15A starting from compound 14 (300 mg, 2.1
mmol) and 2-bromo-1-morpholinoethanone (345 mg, 2.1 mmol) heating at 70 °C for 5 h. The product
1
was obtained as a yellow solid (307 mg, 54%). H NMR (DMSO-d6) δ 5.987 (s, 1H), 4.153 (s, 2H),
26

3.64-3.60 (m, 2H), 3.58-3.54 (m, 4H), 3.48-3.43 (m, 2H), 2.152 (s, 3H).
6-Methyl-2-(2-morpholinoethylthio)pyrimidin-4(3H)-one (15D). Compound 15D was synthesized
using the method described for compound 15A starting from compound 14 (250 mg, 1.7 mmol) and 4-
(2-bromoethyl)morpholine (327 mg, 1.7 mmol) heating at 70 °C for 5.5 h. The product was obtained
as a pale yellow solid (256 mg, 57%). 1H NMR (DMSO-d6) δ 5.943 (s, 1H), 3.571 (t, J = 4.6 Hz, 4H),
3.250 (t, J = 7.0 Hz, 4H), 2.46-2.40 (m, 4H), 2.145 (s, 3H).
4-(2-(4-Methyl-6-oxo-1,6-dihydropyrimidin-2-ylthio)acetyl)benzonitrile (15E). Compound 15E
was synthesized using the method described for compound 15A starting from compound 14 (300 mg,
2.1 mmol) and 4-(2-bromoacetyl)benzonitrile (472 mg, 2.1 mmol). The product was obtained as a pale
1
yellow solid (325 mg, 54%). H NMR (DMSO-d6) δ 7.844 (d, J = 8.4 Hz, 2H), 7.614 (d, J = 8.4 Hz,
2H), 5.917 (s, 1H), 4.721 (s, 2H), 1.897 (s br., 3H).
2-(4-Chloro-6-methylpyrimidin-2-ylthio)-1-(4-methoxyphenyl)ethanone (16A). POCl3 (1.0 mL,
1.645g, 10.7mmol) was added to compound 15A (100 mg, 0.38 mmol) and the mixture was heated at
70°C with stirring. After 15 min, the reaction was quenched with sodium bicarbonate saturated
solution (10 mL) and extracted with ethyl acetate (10 mL). The organic phase was washed with water
(3 x 10 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude
mixture which was purified by column chromatography (chloroform/methanol gradient) to yield
1
compound 16A as an yellow solid (85 mg, 80%). H NMR (DMSO-d6) δ 8.054 (d, J = 9.2 Hz, 2H),
6.973 (d, J = 8.8 Hz, 2H), 6.871 (s, 1H), 4.604 (s, 2H), 3.893 (s, 3H), 2.391 (s, 3H).
2-(4-Chloro-6-methylpyrimidin-2-ylthio)-1-(4-(diethylamino)phenyl)ethanone (16B). Compound
16B was synthesized using the method described for compound 16A heating at 70°C for 20 min
1
obtained as a solid (90 mg, 85%). H NMR (DMSO-d6) δ 7.954 (d, J = 8.8 Hz, 2H), 6.843 (s, 1H),
6.644 (d, J = 9.2 Hz, 2H), 4.590 (s, 2H), 3.438 (q, J = 7.0 Hz, 4H), 2.388 (s, 3H), 1.216 (t, J = 7.0 Hz,
6H).
27

2-(4-Chloro-6-methylpyrimidin-2-ylthio)-1-morpholinoethanone (16C). Compound 16C was
synthesized using the method described for compound 16A heating at 70 °C for 10 min obtained as a
1
solid (83 mg, 78%). H NMR (DMSO-d6) δ 6.889 (s, 1H), 4.064 (s, 2H), 3.758-3.646 (m, 8H), 2.441
(s, 3H).
4-(2-(4-Chloro-6-methylpyrimidin-2-ylthio)ethyl)morpholine (16D). Compound 16D was
synthesized using the method described for compound 16A heating at 75°C for 20 min obtained as a
1
white solid (75 mg, 70%). H NMR (CDCl3) δ 6.862 (s, 1H), 3.76-3.70 (m, 4H), 3.32-3.26 (m,
2H),2.74-2.68 (m, 2H), 2.59-2.53 (m, 4H), 2.433 (s, 3H).
4-(2-(4-Chloro-6-methylpyrimidin-2-ylthio)acetyl)benzonitrile (16E). Compound 16E was
synthesized using the method described for compound 16A and the product was obtained as a pale
1
yellow solid (74 mg, 75%). H NMR (DMSO) δ 8.156 (d, J = 8.8 Hz, 2H), 7.813 (d, J = 8.8 Hz, 2H),
6.874 (s, 1H), 4.549 (s, 2H), 2.351 (s, 3H).
2-Phenylacetohydrazide (18). To a solution of methyl 2-phenylacetate 17 (500 mg, 3.3 mmol) in
anhydrous methanol (25 mL), hydrazine hydrate (533 mg, 16.6 mmol) was added in portions over ten
minutes period under nitrogen atmosphere at room temperature, and the reaction mixture was stirred
for further 16 h. After completion of the reaction (monitored by TLC), the solvent was evaporated
under reduced pressure. The crude residue was treated with diethyl ether (10 mL) to afford compound
1
18 as white precipitate that was collected by filtration and dried under vacuum (400 mg, 80 %). H
NMR (CDCl3) δ 3.57 (s, 2H), 3.84 (s, 2H), 6.66 (bs, 1H), 7.25 – 7.38 (m, 5H).
2-Chloro-N'-(2-phenylacetyl)acetohydrazide (19). 2-Chloroacetyl chloride (0.23 mL, 2.9 mmol)
was added to a solution of compound 18 (400 mg, 2.6 mmol) and N,N-di-isopropyl ethylamine (0.46
mL, 2.6 mmol) in anhydrous dichloromethane (20 mL) at –10 °C slowly under inert atmosphere. The
reaction mixture was stirred at 0 °C and after 30 min, it was quenched with saturated sodium
bicarbonate solution (10 mL), the organic phase was collected and washed with water (3 x 10 mL) and
28

dried over anhydrous sodium sulfate. After evaporation of the solvent, the crude mixture was purified
by column chromatography (chloroform/methanol gradient) to yield compound 19 as a white solid
(400 mg, 66%). 1H NMR (DMSO-d6) δ 3.48 (s, 2H), 4.12 (s, 2H), 7.212 – 7.26 (m, 1H), 7.28-7.33 (m,
4H), 10.28 (bs, 1H), 10.30 (bs, 1H).
S-2-(2-(2-Phenylacetyl)hydrazinyl)ethyl ethanethioate (20). To a solution of compound 19 (400
mg, 1.76 mmol) in ethanol (12 mL), potassium thioacetate (705 mg, 6.17 mmol) was added and the
mixture was stirred at room temperature for 16 h. After completion of the reaction (TLC monitored),
the mixture was diluted with ethyl acetate and THF, washed with water and brine, and dried over
anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the crude was
purified by column chromatography (chloroform methanol gradient) to yield compound 20 as a brown
1
solid (320 mg, 68%). H NMR (DMSO-d6) δ 2.36 (s, 3H), 3.45 (s, 2H), 3.65 (s, 2H), 7.21 - 7.25 (m,
1H), 7.27-7.31 (m, 4H), 10.15 (bs, 1H), 10.18 (bs, 1H).
2-Mercapto-N'-(2-phenylacetyl)acetohydrazide (21). To a solution of compound 20 (200 mg, 0.75
mmol) in methanol (5 mL), K2CO3 (207 mg, 1.5 mmol) was added and the mixture was stirred at
room temperature for 1 h. The reaction mixture was diluted with ethyl acetate (10 mL) and THF (10
mL), washed with water and brine, and dried over anhydrous sodium sulphate. The solvent was
evaporated and the crude mixture was purified by column chromatography (chloroform - methanol
1
gradient) to obtain compound 21 as a white solid (120 mg, 71.4 %). H NMR (DMSO-d6) δ 2.79 (t, J
= 8 Hz, 1H), 3.15 (d, J = 8 Hz, 2H), 3.47 (bs, 2H), 7.20-7.26 (m, 1H), 7.28-7.36 (m, 4H), 10.06 (d, J =
2 Hz, 1H), 10.20 (d, J = 2 Hz,, 1H).
2-(2-(2-(4-Methoxyphenyl)-2-oxoethylthio)-6-methylpyrimidin-4-ylthio)-N'-(2-
phenylacetyl)acetohydrazide (22). To a solution of compound 21 (68 mg, 0.22 mmol) and 16A (50
mg, 0.22 mmol) in anhyd acetonitrile (10 mL), triethylamine (0.034 mL, 0.24 mmol) was added and
the reaction mixture was stirred at 75 °C for 6 h. Reaction mixture was quenched with cold water (5
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