Design and synthesis of guanylthiourea derivatives as potential inhibitors of Plasmodium falciparum dihydrofolate reductase enzyme

Article abstract of DOI:10.1016/j.bmcl.2013.12.009

A new class of compounds based on S-benzylated guanylthiourea has been designed as potential PfDHFR inhibitors using computer aided methods (molecular electrostatic potential, molecular docking). Several compounds in this class have been synthesized start

Full text of DOI:10.1016/j.bmcl.2013.12.009

Bioorganic & Medicinal Chemistry Letters 24 (2014) 613–617
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of guanylthiourea derivatives as potential
inhibitors of Plasmodium falciparum dihydrofolate reductase enzyme
Legesse Adane a, Shweta Bhagat a, Minhajul Arfeen a, Sonam Bhatia a, Rachada Sirawaraporn b,
Worachart Sirawaraporn b, Asit K. Chakraborti a, Prasad V. Bharatam a,
⇑
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Mohali 160062, Punjab, India
b Department of Biochemistry, Mahidol University, Bangkok 10400, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of compounds based on S-benzylated guanylthiourea has been designed as potential PfDHFR
inhibitors using computer aided methods (molecular electrostatic potential, molecular docking). Several
compounds in this class have been synthesized starting from guanylthiourea and alkyl bromides. In vitro
Received 5 September 2013
Revised 22 November 2013
Accepted 2 December 2013
Available online 8 December 2013
studies showed that two compounds from this class are active with the IC50 value of 100 lM and 400 nM.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Antimalaria
Guanylthiourea
PfDHFR
MESP
Molecular docking
Plasmodium falciparum dihydrofolate reductase (PfDHFR) is one
of the validated targets to develop potential therapeutic agents for
the treatment of malaria. Inhibition of PfDHFR by typical antifo-
lates such as trimethoprim, cycloguanil and pyrimethamine pre-
vents biosynthesis of thymidine, and consequently interrupts
DNA biosynthesis.1 However, point mutations at amino acid resi-
dues such as Ala16, Ile51, Cys59, Ser108 and Ile164 in the active
site of wild-type PfDHFR enzyme has resulted in widespread resis-
tance of the parasite to these drugs.2 Thus, discovery of new poten-
tial PfDHFR inhibitors to overcome drug-resistant parasites, is an
urgent need.
unfavorable steric clashes with Asn108 in the active site of the mu-
tant PfDHFR enzyme.3,4
Dasgupta et al.5 reported the X-ray crystallographic structures
of the wild-type (PDB code: 3DGA) and quadruple mutant (PDB
code: 3DG8) PfDHFRs with biguanide based bound ligands
(RJF01302 and RJF670). Summerfield et al.6 suggested, based on
the results of crystal structure analysis of Escherichia coli DHFR en-
zyme complexed with amidinoisothiouronium salts (PDB code:
2ANO, 2ANQ) that guanylthiourea (GTU) derivatives can mimic
The known antifolate based PfDHFR inhibitors have 2,4-diami-
nopyrimidine or 1,3,5-triazine moiety that interact with amino
acids in the active site via hydrogen bond and hydrophobic inter-
actions. Molecular modeling studies have shown that potential
PfDHFR enzyme inhibitors must fulfill at least three criteria re-
quired for chemical and geometrical complementarity of ligands
with the active site of the enzyme.3 These are (i) H-bond donor
head group that can form H-bond interaction with Asp54, Ile14
and Leu164, (ii) hydrophobic aromatic tail which occupies the
hydrophobic pocket of the active site (Phe58, Met55, Phe116,
Pro113, Ile112 and Ser111) to enhance inhibitory activity,
and (iii) linker unit between the H-bond donor head groups and
hydrophobic aromatic tail to provide flexibility, in order to avoid
Figure 1. The MESP surfaces of (a) protonated pyrimethamine core and (b)
protonated GTU derivative. Red color refers negative potential whereas blue color
refers positive potential. All the structures were optimized using B3LYP method and
the MESP analysis was generated using SPARTAN software.10
⇑
Corresponding author.
E-mail address: pvbharatam@niper.ac.in (P.V. Bharatam).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.009

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L. Adane et al. / Bioorg. Med. Chem. Lett. 24 (2014) 613–617
Table 1
The synthesized S-alkylated GTU derivatives (Fig. 3, structure III)
Compounds
WR99210
1
R
Gscore
Yield (%)
—
1J3 K
1J3I
3DG8
3DGA
—
À8.708
À8.707
À6.665
À7.837
À9.075
À8.851
À8.498
À7.327
12.0
2
3
À8.832
À8.754
À8.167
À8.430
À7.514
À8.099
À6.489
À7.876
12.0
90.0
4
5
À8.620
À8.347
À8.766
À8.255
À8.028
À7.102
À7.031
À6.087
11.0
15.0
6
À8.192
À8.041
À7.745
À7.731
54.0
7
8
À8.160
À8.016
À7.728
À7.843
À7.288
À6.673
À6.319
À6.444
50.0
11.5
9
À7.964
À8.271
À6.841
À5.743
57.0
10
11
À7.878
À7.790
À7.673
À7.615
À7.281
À6.750
À7.026
À6.678
65.8
90.0
12
13
14
15
À7.694
À7.651
À7.620
À7.577
À7.932
À7.757
À8.081
À7.467
À6.911
À7.186
À7.540
À6.318
À6.236
À6.975
À7.057
À6.162
40.0
86.0
42.0
72.1
16
À7.526
À7.686
À7.074
À6.344
35.0
17
18
À7.076
À7.058
À6.894
À7.528
À6.414
À6.428
À6.306
À6.949
51.0
48.0

L. Adane et al. / Bioorg. Med. Chem. Lett. 24 (2014) 613–617
615
Table 1 (continued)
Compounds
R
Gscore
Yield (%)
1J3 K
1J3I
3DG8
3DGA
19
À7.033
À7.883
À6.733
À6.108
90.0
42.0
20
À6.977
À6.797
À6.583
À6.512
* Position of R group.
the binding interactions of 2,4-diaminopyrimidine moieties of
known DHFR inhibitors such as methotrexate.
(DHFR) with cofactor NDP610 was retained; all water molecules
and the rest of the chains were removed. A radius of 15 Å was se-
lected for active site cavity during receptor grid generation. The
reproducibility of the docking calculation was evaluated by dock-
ing the bound ligands into the prepared active sites. Table 1 shows
Gscore of important compounds (which show reasonable docking
scores) on both protein crystal structures of quadruple mutant
type (1J3K and 3DG8) and wild type (1J3I and 3DGA) PfDHFR en-
zyme. Glide scores of GTU derivatives showing most stable GTU
conformation were taken into consideration.
The results of molecular docking analysis indicated that, similar
to pyrimethamine (3QG2),13 WR99210 (1J3K and 1J3I)14 and
biguanide derivatives (3DG8 and 3DGA),5 GTU moiety was found
to form the expected hydrogen bond interactions with Asp54,
Leu164, Ile14 and hydrophobic interactions with Phe58 and
Phe116 in the active site of PfDHFR enzymes. Flexibility was main-
tained by introducing linker unit with 1, 2 or 3 carbon atoms be-
tween the hydrophobic aromatic tail and GTU moiety in order to
prevent potential steric clashes with the amino acid residue
Asn108 in the active site of the mutant PfDHFR enzyme. The de-
signed compounds were cross docked on biguanide based crystal
structures (PDB code: 3DG8 and 3DGA). The Glide scores obtained
were comparable to that of the co-crystallized ligands RJF670 and
RJF01302. In Table 1 (arranged according the descending docking
scores in 1J3K), compound 1 showed highest Gscore which may be
attributed to additional hydrophobic interactions with Phe116.
Bi-substitued guanylthiourea structure (Table 1, compd 3) showed
In order to investigate the similarities of GTU moiety with the
2,4-diaminopyrimidine moiety of the known antifolate drugs such
as pyrimethamine (Pyr), molecular electrostatic potential (MESP)
analysis was carried. Rastelli et al. reported that the antifolate
drugs get protonated at physiological pH.3c Our results indicated
that S-alkylated GTU derivatives are basic in nature and get pro-
tonated.7,8 Also, conformational analysis indicated that the most
preferred conformer of the GTU prefers to exist in a conformation
comparable to that of pyrimethamine.7 Therefore, the MESP analy-
sis was carried out on the protonated, most stable conformer of
GTU and protonated pyrimidine moiety (representative unit of
pyrimethamine) to compare the electrostatic potentials (Fig. 1).
The most significant feature of these two MESP surfaces is defined
by the –(H2N)2-C-N-C-N(H2)– unit of these molecules. The MESP
analysis of two protonated species showed that blue color (hydro-
gen bond donating property) extends over the nitrogen and carbon
atom (1st and 7th) in Pyr (Fig. 1a). The similar nature of surface is
observed over the nitrogen atoms (5th and 6th) in case of proton-
ated GTU (Fig. 1b). The negative surface (red color, hydrogen bond
acceptor) is observed over N(3) in both the cases, while there is
partial blue color over N(7) in case of GTU. The alternate electron
deficient - electron rich - electron deficient potential surface of
–(H2N)2-C-N-C-N(H2)– region in these two species is required for
molecular recognition interaction with the target macromolecule
(PfDHFR). The above analysis showed that under protonated condi-
tion the surface of Pyr and GTU are similar in nature. These results
are in accordance with our research work on biguanides and other
related molecules.3,9
Gscore comparable to that of WR99210 (Fig. 2a and c). Compound 3
also showed additional interactions with Ser111, Lys49 and Trp48
(Fig. 2a). These additional interactions are possible only because of
the six rotatable bonds between the two guanylthiourea moieties,
facilitating stronger interactions. Compound 11 (Table 1) showed
hydrogen bonding interaction with Asp54 and hydrophobic inter-
action with Phe58 (Fig. 2b). The bulky iodine group at the meta-
position of the benzyl ring occupies the hydrophobic pocket of
the enzyme.
Twenty S-alkylated derivatives (Table 1) were taken up for syn-
thesis as per the Schemes 1 and 2. The compounds were synthe-
sized in pure and good to excellent yields. The observation from
this experiment also confirmed that the mechanism of S-alkylation
of guanylthiourea which was proposed to be based on molecular
modeling study is acceptable (Fig. 3).20
Synthesized compounds were tested for their inhibitory activity
using wild-type and quadruple mutant PfDHFR enzymes expressed
in E. coli. The expression constructs2c harboring the synthetic
PfDHFR genes (wild type and quadruple type) were used as a
source of recombinant PfDHFR for inhibition testing of the com-
pounds. Briefly the plasmids were transformed into E. coli strain
BL21 (DE3)pLysS[F-, ompT, hsdSB,(rB- ,mB-), dcm, gal, k9(DE3),
pLysSCmR]. The transformed bacteria were then plated on LB agar
Sirichaiwat et al.11 reported the design and synthesis of tri-
methoprim derivatives. Derivatives with benzyloxy substituents
at the 3 and 4 positions of the benzyl ring of the trimethoprim
showed better hydrophobic interactions with amino acids Phe58,
Phe116 and Pro113. They showed that antimalarial activity in both
the wild-type and mutant varieties of PfDHFR enzymes is improved
in case of benzyloxy derivatives as compared to other derivatives
with no aromatic substituents. It was suggested that such substit-
uents would increase the binding affinity via hydrophobic interac-
tions with the amino acid residues near the opening of the active
site of the enzyme. Based on the above results obtained and sug-
gestions made by Sirichaiwat et al. and Summerfield et al. several
GTU derivatives (mono- and bi-subtituted) were designed and
their binding potentials were examined using molecular docking
methodology.
The Glide docking program was used to study the binding poses
of the compounds.12 The docking calculations were carried out
using X-ray crystal structures of wild type (PDB code: 1J3I and
3DGA) and quadruple mutant type (PDB code: 1J3K and 3DG8)
PfDHFR enzymes. The crystal structures are in the dimeric form
of the DHFR–TS complex and they are co-crystals with cofactors
and ligands. During the protein preparation step, only chain A
plate and then supplemented with 100
lg/ml ampicillin and incu-
bated overnight at 37 °C. When E. coli colonies appear, an isolated

616
L. Adane et al. / Bioorg. Med. Chem. Lett. 24 (2014) 613–617
Scheme 1. Reagents and conditions: (i) GTU, EtOH, NaOH, 80 °C, reflux (Table 1, 15
and 20);15 (ii) GTU, conc. HBr, 100 °C, reflux (Table 1, 11 and 19);16 (iii) GTU, CH3CN,
MW, 100 °C, 15 min (Table 1, 1, 2, 5–10, 14, 16).17,18
Scheme 2. Reagents and conditions: (i) anhyd K2CO3, CH3CN, 80 °C, reflux;19 (ii)
GTU, EtOH, NaOH, 80 °C, reflux (Table 1 (13, 17, n = 2; 12, 18, n = 1)).15
E. coli was then used to inoculate 500-ml culture. Unless
otherwise indicated, the culture was grown at 37 °C until A600
reached ꢀ0.5-0.6, at which time IPTG was added to a final concen-
tration of 1 mM. The culture was then allowed to grow with shak-
ing at 37 °C for additional 10-12 h. The E. coli cells expressing
recombinant PfDHFR were harvested by centrifugation at 3,000xg
for 15 min at 4 °C. The resulting cell pellet obtained was resus-
pended in 20 ml ice-cold buffer (20 mM potassium phosphate buf-
fer, pH 7.0; 1 mM EDTA; 1 mM DTT; 20% glycerol) and the cells
were passed through French pressure cell (American Instrument
Co., Inc., Silver Spring, MD, USA) at 15000 psi twice to lyse the cells.
Clear extract was separated from the particulate debris by centrifu-
gation at 12000 rpm for 30 min at 4 °C.
The protein concentration of the crude extract was deter-
mined21 and the DHFR activity was determined spectrophotomet-
rically by measuring the decrease in absorbance at 340 nm upon
NADPH utilization. The assay reaction (1 ml) is composed of
1ÂDHFR buffer, 100
lM H2 folate, 100 lM NADPH, 1 mg/ml BSA,
and the reaction was initiated by addition of ꢀ0.01 units of en-
zyme. One unit of enzyme activity is defined as amount of DHFR
that produces 1
lmol of product/min at 25 °C. To determine the
inhibition of DHFR activity, stock solution of compounds to be
tested were first dissolved with dimethyl sulfoxide (DMSO). Two-
fold serial dilutions of the compounds were prepared, and 10
ll
of each of the diluted compounds was added in the assay reaction
to test for the enzyme inhibition. The IC50 values were then calcu-
lated as the concentrations of the compounds which inhibit 50% of
the DHFR activity.
The preliminary enzyme assay tests using crude enzyme indi-
cated that two compounds (Table 1, 3 and 11) could inhibit the
Figure 2. Molecular docking in the active site of quadruple mutant PfDHFR enzyme
(a) Guanylthiourea derivatives 3 (Table 1) showing H-bond interaction with Trp48,
Lys49, Asp54 and Ser111; (b) compound 11 (Table 1) showing H-bond interaction
with Asp54, Phe58 and (c) WR99210 showing H-bond interaction with Asp54,
Leu164, Ile14 and Cys15.
PfDHFR enzyme at
400 nm respectively.
lM range with IC50 values ꢀ100
lM and
In conclusion, the in vitro activity showed that the compound
which is meta-substituted with a bulky atom is active. Bi-substi-
tuted guanylthiourea compound showed good activity. This may
colony was picked and inoculated in 5 ml LB medium supple-
mented with 100 lg/ml ampicillin. Fresh overnight culture of

L. Adane et al. / Bioorg. Med. Chem. Lett. 24 (2014) 613–617
617
Figure 3. Proposed reaction mechanism for S-alkylation of GTU.7,20
4. (a) Maitarad, P.; Kamchonwongpaisan, S.; Vanichtanankul, J.; Vilaivan, T.;
Yuthavong, Y.; Hannongbua, S. J. Comput. Aided Mol. Des. 2009, 23, 241; (b)
Adane, L.; Patel, D. S.; Bharatam, P. V. Chem. Biol. Drug Des. 2010, 75, 115.
5. Dasgupta, T.; Chitnumsub, P.; Kamchonwongpaisan, S.; Maneeruttanarungroj,
C.; Nichols, S. E.; Lyons, T. M.; Tirado-Rives, J.; Jorgensen, W. L.; Yuthavong, Y.;
Anderson, K. S. ACS Chem. Biol. 2009, 4, 29.
6. Summerfield, R. L.; Daigle, D. M.; Mayer, S.; Mallik, D.; Hughes, D. W.; Jackson,
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be due to increase in hydrogen bond interactions in the active site
of PfDHFR enzyme. This additional feature of bi-substituted guan-
ylthiourea derivatives can be further studied to improve the activ-
ity of this class of compounds. More work is being carried out in
relation to mono-substitued and bi-substituted guanylthiourea
derivatives.
7. Mehdi, A.; Adane, L.; Patel, D. S.; Bharatam, P. V. J. Comput. Chem. 2010, 31,
1259.
Acknowledgments
8. (a) Adane, L.; Bharatam, P. V. Curr. Med. Chem. 2008, 15, 1552; (b) Adane, L.
Computer-Aided Design and Synthesis of Potential Antimalarial Agents:
Targeting PfDHFR Enzymes, Ph.D. thesis, Feb 2010, NIPER, Mohali, Punjab,
India.
9. (a) Bharatam, P. V.; Patel, D. S.; Iqbal, P. J. Med. Chem. 2005, 48, 7615; (b) Patel,
D. S.; Bharatam, P. V. J. Phys. Chem. A 2011, 115, 7645; (c) Patel, D. S.; Bharatam,
P. V. Chem. Commun. 2009, 1064; (d) Patel, D. S.; Bharatam, P. V. J. Org. Chem.
2011, 76, 2558; (e) Bhatia, S.; Bagul, C.; Kasetti, Y.; Patel, D. S.; Bharatam, P. V. J.
Phys. Chem. A 2012, 116, 9071; (f) Bhatia, S.; Malkhede, Y. J.; Bharatam, P. V. J.
Comput. Chem. 2013, 34, 1577.
The authors thank Department of Science and Technology, New
Delhi for financial support. L. Adane also thanks Ministry of Educa-
tion of the government of Federal Democratic Republic Ethiopia for
financial support to study in India.
Supplementary data
10. Hehre, W. J. A guide to molecular mechanics and quantum chemical calculations;
Wavefunction: Irvine, 2001.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.009.
11. Sirichaiwat, C.; Intaraudom, C.; Kamchonwongpaisan, S.; Vanichtanankul, J.;
Thebtaranonth, Y.; Yuthavong, Y. J. Med. Chem. 2004, 47, 345.
12. (a) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D.
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