Discovery of triazine mimetics as potent antileishmanial agents

Article abstract of DOI:10.1021/ml400317e

The World Health Organization has classified the leishmaniasis as a major tropical disease. The discovery of new compounds for leishmaniasis is therefore a pressing concern for the anti-infective research program. We have synthesized 19 compounds of triazine dimers as novel antileishmanial agents. Most of the synthesized derivatives exhibited better activity against intracellular amastigotes (IC₅₀ ranging from 0.77 to 10.32 μM) than the control, pentamidine (IC₅₀ = 13.68 μM), and are not toxic to Vero cells. Compounds 14 and 15 showed significant in vivo inhibition of 74.41% and 62.64%, respectively, in L. donovani/hamster model. Moreover, expansion of Th1-type and suppression of Th2-type immune responses proved that compound 14 stimulates mouse macrophages to prevent the progression of leishmania parasite. The molecular docking studies involving PTR1 protein PDB further validated the concepts involved in the design of these compounds. Among the investigated analogues, compound 14 has emerged as the potential one to enlarge the scope of the study.

Full text of DOI:10.1021/ml400317e

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Letter
Discovery of Triazines Mimetics as potent antileishmanial agents
Kuldeep Chauhan, Moni Sharma, Rahul Shivahare, Utsab Debnath,
Suman Gupta, Yenamandra S. Prabhakar, and Prem M.S. Chauhan
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 01 Oct 2013
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Discovery of Triazines Mimetics as potent antileishmanial agents
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Kuldeep Chauhan,† Moni Sharma,† Rahul Shivahare,‡ Utsab Debnath†, Suman Gupta, ‡
Yenamandra S. Prabhakar† and Prem M. S. Chauhan†*
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†Medicinal and Process Chemistry Division and ‡Division of Parasitology, CSIRꢀ Central Drug Research Institute,
Lucknowꢀ226031, U.P., India
Abstract: The World Health Organization has classified the leishmaniasis as a major tropical
disease. The discovery of new compounds for leishmaniasis is therefore a pressing concern for
the antiꢀinfective research program. We have synthesized 19 compounds of triazine dimers as
novel antileishmanial agents. Most of the synthesized derivatives exhibited better activity against
intracellular amastigotes (IC50 ranging from 0.77 to 10.32 ꢁM) than the control, pentamidine
(IC50 = 13.68 ꢁM) and not toxic to vero cells. Compounds 14 and 15 showed significant in vivo
inhibition of 74.41% and 62.64% respectively in L. donovani/hamster model. Moreover,
expansion of Th1ꢀtype and suppression of Th2ꢀtype immune responses proved that compound 14
stimulate mouse macrophages to prevent the progression of leishmania parasite. The molecular
docking studies involving PTR1 protein PDB further validated the concepts involved the design
of these compounds. Among the investigated analogues, compound 14 has emerged as potential
one to enlarge the scope of the study.
Keywords: L. donovani, Triazine, Pentamidine, In vitro/in vivo, Pteridine reductase 1.
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Leishmaniasis is a group of tropical disease resulting from infection of macrophage by obligate
intracellular parasites of genus Leishmania, and it is a major health problem worldwide.1 It may
manifest in three clinical forms, cutaneous, mucoꢀcutaneous and visceral leishmaniasis (VL).
Among all, VL is the most severe form of the disease.2 Out of the annual 2 million cases reported
every year, 500,000 are due to VL thus causing a significant health problem.3 Leishmania
infection is classically associated with a depression of T helper type 1 (Th1) cells and
preferential expansion of T helper type 2 (Th2) cells. Th1 type immune responses play an
important role in mediating protection against Leishmania,4 including roles of interferon (IFN)ꢀγ,
interleukin (IL)ꢀ12, tumor necrosis factor (TNF)ꢀα and nitric oxide (NO), whereas inhibitory
effects have been reported for Th2 cytokines, ILꢀ10 and transforming growth factor (TGF)ꢀβ.5, 6
Control of Leishmania infection depends on ILꢀ12ꢀdriven expansion of Th1 cells, macrophage
activation through production of IFNꢀγ and the subsequent generation of nitric oxide (NO).7 The
serious problems associated with the treatment of leishmania infections, such as expensive and
toxic drugs, emerging resistance and the limited funding, have recently led many research groups
to design and synthesize safer, inexpensive and more efficient novel antileishmanial compounds.
In this regard, pentamidine8 has been used to treat early stage Trypanosoma brucei
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gambiense related Human African Trypanosomiasis,9, 10 and antimony resistant VL which was
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caused by Leishmania donovani.11,
Due to fewer side effects and low propensity to
development of resistance, pentamidine may be a viable starting point for the development of
novel antileishmanial agents.13ꢀ15 On the other side, triazine scaffold provides basis for the design
of biologically relevant molecules with extensive application as therapeutics. Previously, it was
found that triazine based scaffolds showed various biological activity such as antimalarial,
antileishmanial and antimicrobial activity.16 We have recently gained interest in the synthesis of
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pentamidine, pyrimidine, triazine and quinazolinone derivatives that showed a marked
antileishmanial activity.17ꢀ19
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Dihydrofolate reductase (DHFR) is a well established drug target for a wide range of
diseases.20 This pathway has been effectively used against cancer, bacterial infections and
malaria, but with limited success against Leishmaniasis.21 Interestingly, Leishmania is known to
proficiently overcome DHFR inhibition by over expressing pteridine reductase 1 (PTR1, EC
1.5.1.33). Pteridine reductase (PTR) is associated with the pterin and folate metabolism and
essential for the growth of Leishmania. Methotrexate and triaminoquinazolines are some known
potential inhibitors of this protozoan enzyme.22 Hence, the PTR1 pathway may offer unique
opportunity to develop new chemotherapeutics which could be effective against Leishmania.23, 24
As part of our ongoing antiꢀinfective research program,25, 26 considering the structural similarity
between the key structural units of PTR1 inhibitors (pteridines and quinazolines) and
benzimidamide moiety of pentamidine (a well known antileishmanial agent), we have
synthesized the triazine dimers and evaluated them for their antileishmanial activity. In view of
aforesaid, we have also carried out docking studies of these compounds to investigate their
plausible mode of binding and affinity to the PTR1.
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We have used cyanuric chloride, an inexpensive commercially available reagent as the
starting material for the synthesis of compounds (5ꢀ23). Herein, we report synthetic procedure
for triazine compounds (5ꢀ23) which is depicted in Scheme 1. Triꢀsubstituted triazines
derivatives were synthesized; starting from the treatment of aniline 1 with cyanuric chloride at
0°C to give monoꢀsubstituted triazine derivatives 2 in excellent yield (>95%). In next step,
compound 2 reacted with aminoalkanol at room temperature to give compound 3 and 4 in good
yield. Further, the compounds 3 and 4 with 2 were reacted in the presence of sodium hydride in
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dry THF to afford the heterodimeric intermediates 5 and 6 in moderate yield (~50%). Thus,
isolated compound 5 and 6 were treated with different amines at 60°C to give the final products
in high yield (80ꢀ95%).
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Scheme1. Synthesis of triazine mimetics (5 23):
Cl
N
Cl
NH2
N
N
N
N
a
b
( )
N
NH nOH
3
4
N
H
N
2
Cl
H
n = 5
n = 4
1
c
Cl
N
Cl
N
R2
R1
N
N
N
N
N
d
N
N
N
N
( )n
( )
NH nO
N
NH
O
N
H
N
N
N
H
H
H
n = 5
n = 4
5
6
7, 8 n = 5
9-23 n = 4
HN
HN
(13) R1 = R2 =
(14) R1 = R2 =
(15) R1 = R2 =
(16) R1 = R2 =
(19) R1 = R2 =
(20) R1 = R2 =
N
N
(7) R1 = R2 =
N
O
(8) R1 = R2 =
(9) R1 = R2 =
(10) R1 = R2 =
(11) R1 = R2 =
HN
N
HN
HN
N
O
(21) R1 = R2 =
N
HN
N
O
HN
(22) R1 =
(23) R1 =
R2 =
HN
HN
HN
(17) R1 = R2 =
(18) R1 = R2 =
NH
HN
N
O
HN
R2 =
(
12) R = R =
1
2
N
N
Reagents and conditions: (a) Cyanuric chloride, 0°C; (b) aminoalkanol, K2CO3, THF, rt; (c) Monoꢀ
substituted triazine; (2), NaH, THF, rt; (d) RNH2, THF, 60°C.
In this endeavor to identify the activity of triazines, we have synthesized a series of 19
compounds and screened them against L. donovani. The results are summarized in Table 1.
Table 1 In vitro antileishmanial activity and cytotoxicity of triazine mimetics (5ꢀ23)
Entry
Antiamastigote activity
Cytotoxicity
Selectivity Indexc
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IC50a (ꢁM)
CC50b (ꢁM)
17.10 ± 2.61
7.74 ± 1.2
NDd
(SI)
1.90
8.98 ± 0.73
10.32 ± 1.87
>40
5
0.75
NAe
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>40
ND
NA
8
3.15 ± 0.80
2.91 ± 0.32
1.70 ± 0.54
6.46 ± 1.20
3.21 ± 0.67
1.99 ± 0.31
0.77 ± 0.15
3.51 ± 0.81
>40
275.63 ± 11.78
115.27 ± 5.98
13.80 ± 2.14
>400
87.50
39.61
8.11
9
10
11
>61.92
>124.61
108.58
257.48
>113.96
NA
12
>400
13
216.08 ± 5.89
198.26 ± 9.98
>400
14
15
16
ND
17
1.33 ± 0.51
0.86 ± 0.06
8.98 ± 0.98
7.52 ± 1.19
2.65 ± 0.54
8.26 ± 0.62
46.70 ± 2.75
13.68 ± 1.57
8.10 ± 0.70
20.06 ± 2.52
16.23 ± 2.27
17.10 ± 1.10
>400
15.08
18.87
1.90
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19
20
>53.19
>150.94
2.20
21
22
>400
18.23 ± 4.37
>400
23
SSGf
8.56
85.86 ± 5.27
53.12 ± 4.75
6.27
Pentamidine
Miltefosine
6.56
IC50 and CC50 values are the average of two independent assays expressed as average ± standard
deviation; The Selectivity Index (SI) is defined as the ratio of bCC50 on vero cells to aIC50 on L. donovani
intracellular amastigotes; dND: not determined. eNA: not available; fSSG: Sodium stibogluconate.
Initially, we have synthesized the triazine derivatives (5, 7 and 8) with 5ꢀaminopentanꢀ1ꢀ
ol linker, considering the importance of standard drug pentamidine but unfortunately, these
compounds were found to be inactive against Leishmania donovani. Further to improve the
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activity of these compounds, we have synthesized the triazine analogues with shorter chain
length. Interestingly, all the synthesized derivatives showed significant to moderate
antiamastigote activity with IC50 ranging from 0.77 to 10.32 ꢂM. Most of the synthesized
compounds having 4ꢀaminobutanꢀ1ꢀol linker responded better than the control drugs in the test
system and displayed significant activity. First line drug, sodium stibogluconate (SSG) displayed
a poor in vitro activity against Leishmania amastigotes (IC50 = 46.70 ꢁM) although it is not toxic
to vero cells (CC50 > 400 ꢁM). Other standard drugs, pentamidine and miltefosine displayed
moderate in vitro antiamastigote activity (IC50 = 13.6 and 8.10 ꢁM respectively) and low
cytotoxicity towards vero cells (CC50 values of 85.86 and 53.12 ꢁM). Our synthesized
intermediate 6 which have chloro substituent at R1 and R2 exhibited moderate activity (IC50 =
10.32 ꢁM) and cytotoxicity towards vero cells (CC50 = 7.74 ꢁM). In compound 6, when we
replaced the chloro with morpholine (9) (IC50 = 3.15 ꢁM, CC50 = 275.6 ꢁM), amino ethyl
morpholine (10) (IC50 = 2.91ꢁM, CC50 = 115.27 ꢁM) and amino propyl morpholine (11) (IC50 =
1.70 ꢁM, CC50 = 13.80 ꢁM), the resulting compounds showed successive increase in the
antileishmanial activity with concomitant reduction in cytotoxicity. For the structure activity
relationship (SAR), we have introduced the aliphatic amines at R1 and R2 such as nꢀpentyl amine
containing analogues (12) which displayed good antiamastigote activity (IC50 = 6.46 ꢁM) and
better selectivity (> 61.92). However, decreasing the chain length by one carbon (R1 = R2 = nꢀ
butyl), enhanced the activity of compound 13 (IC50 = 3.21 ꢁM), and interestingly, further
decrease in chain length by one carbon (R1 = R2 = nꢀpropyl, 14) demonstrated further
enhancement in antiamastigote activity (IC50 = 1.99 ꢁM) and selectivity (108.58). Surprisingly,
tertꢀbutyl substituted derivative 15 exhibited better activity (IC50 = 0.77 ꢁM) and selectivity
(257.48) than isoꢀpropyl substituted derivative 16 (IC50 = 3.51 ꢁM; SI > 113.96) and found best
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among all the synthesized analogues. Notably, all the nꢀamino alkyl substituted derivatives were
found to be nonꢀtoxic to vero cells (CC50 ranging from 198.26 ꢀ >400 ꢁM). Unexpectedly, in the
case of nꢀcyclopropyl substituted compound 17, no significant activity was obtained.
Compounds 18 and 19, in which chlorine atom were replaced by Nꢀmethylpiperazine and Nꢀ
ethylpiperazine showed excellent activity with IC50 = 1.33 ꢁM and 0.86 ꢁM and CC50 = 20.06
ꢁM and 16.23 ꢁM, respectively. Additionally, tetrahydroquinoline containing analogue 20
exhibited moderate antiamastigote activity (IC50 = 8.98 ꢁM) and poor selectivity (> 1.90),
whereas the tetrahydroisoquinoline containing analogue 21 displayed comparable antiamastigote
activity (IC50 = 7.52 ꢁM) and better selectivity (SI > 53.19) than 20 representing that the position
of nitrogen atom in the tetrahydro ring is crucial for selectivity. Above observations tempted us
to check the effect of nꢀcyclopropyl on SAR. For this purpose, we have synthesized compounds
22 and 23 which bear different alkyl amine at R1 and R2. Interestingly, compound 22 (R1 = nꢀ
propyl, R2 = nꢀcyclopropyl) was found to be effective (IC50 = 2.65 ꢁM) with good selectivity (SI
= >150.94) and compound 23 (R1 = nꢀcyclopropyl, R2 = nꢀpropyl) showed significant but lesser
potency (IC50 = 8.26 ꢁM) and poor selectivity (2.20) than compound 22.
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On the basis of excellent in vitro potency and selectivity index, we have selected
compounds 14 and 15 as potential candidates for further evaluation; accordingly, in vivo tests
were performed in the L. donovani/golden hamster model. The aqueous suspensions of test
compounds were administered for five consecutive days at 50 mg/kg/day by intraperitoneal (IP)
route. The postꢀtreatment (p.t.) splenic biopsies were done on day 7 of the last dose
administration and amastigote counts were assessed by Giemsa staining. Compound 14 showed
good percentage inhibition (74.41 ± 10.26 %) in parasite multiplication and compound 15 has
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shown moderate in vivo efficacy (62.64 ± 10.74 %). Furthermore, we have not observed any
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abnormal behavior of animals at the time of dosing (Figure 1).
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Figure 1: In vivo antileishmanial efficacy in L. donovani / golden hamster model. Each bar represents
pooled data (mean ± SD value) of two experiments. Five animals were used in each experiment.
Pentamidine (PM), SSG (Sodium stibogluconate) and Miltefosine (MF) are used as reference drugs. SD,
Standard deviation; i.p., intraperitoneal; p.o., per oral.
Potent in vitro and in vivo efficacy of compound 14 prompted us to explore its
immunostimulatory properties whether it is able to reverse immunosuppression in Leishmania
infected mouse macrophages (Jꢀ774A.1). Our investigations clearly indicate that compound 14
treated cells induce the Th1 type immune responses by remarkable production of ILꢀ12, TNFꢀα
and nitric oxide (NO) and effective suppression of Th2 type cytokines, ILꢀ10 and TGFꢀβ (Figure
2). We have observed significant (p<0.05) increase in the production of ILꢀ12 (3 fold more than
infected control), TNFꢀα (2 fold more than infected control) and NO (p<0.01, 2 fold higher than
untreated infected macrophages) at 24 hr post treatment (p.t.). In contrast, among the Th2 type
cytokines, there was considerable (p<0.05) suppression of ILꢀ10 was observed at 24 hr p.t. The
level of TGFꢀβ was also downꢀregulated in compound 14 treated cells (1.8 fold less production
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than infected cells). Our study also revealed that after 24 hr p.t., all the cytokines and nitric oxide
started attaining its basal level at 48 hr p.t. (Figure 2)
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Figure 2: Compound 14 mediated upꢀregulation of Th1 cytokines (ILꢀ12 and TNFꢀα), downꢀregulation of
Th2 cytokines (ILꢀ10 and TGFꢀβ) and generation of nitric oxide in infected mouse macrophages (Jꢀ
774A.1) cell line. Each bar represents pooled data of two individual experiments (mean ± SD value).
Miltefosine (MF) is used as reference drug. The asterisks indicate statistically significant increase and
decrease (*, p<0.05; **, p<0.01; and ***) in cytokine production and NO generation compared between
compound 14 treated versus untreated infected macrophages. ns; nonꢀsignificant.
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Enzyme assay along with physical studies is the best way to determine the mechanism of
action of new compounds. However, high structural similarity between PTR1inhibitors and
present compounds has been considered for carrying out the physical investigation through
hypothetical docking studies.27 The docking experiments were performed in Autodock 4.0.28
Prior to the docking, the molecule/ligand and protein structure were prepared by geometry
optimization using a combination of the standard Tripos molecular mechanics force field of the
SYBYL molecular modeling package29 with Powell energy minimization algorithm, Gasteigerꢀ
Huckel charges, and 0.001 kcal/(mol.Å) energy gradient convergence criterion. The Lamarckian
genetic algorithm was used for docking the molecules. In docking experiments pentamidine and
the most potent molecule 14 occupied a location in the PTR1 binding pocket (PDB id: 1E7W)
which is comparable to its ligand, methotrexate. Here, pentamidine (IC50 = 13.68 ꢂM) and
compound 14 (IC50 = 1.99 ꢂM) showed ꢀ6.95 and ꢀ8.54 kcal/mol as binding energies,
respectively. They shared Phe113, Tyr114 and Tyr191 as common binding residues. Figure 3
gives a 2Dꢀview of the binding pocket interactions. The docking interactions indicated that
compound 14 showed Hꢀbonding and piꢀstacking with Tyr191 (Figure 4b). In case of
pentamidine, its interaction with Tyr191 is limited to piꢀstacking alone (Figure 4a). Along with
compound 14, we also examined the docked poses of a less active compound (compound 17).
Interestingly, in docking experiments the less active compound did not mimic the active
analogue (14) as well as reference compound pentamidine (Figure 4d). Moreover, compound 17
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is protruding from the PTR1binding domain (Figure 4c). These studies inferred that compound
14 is more potent compare to others due to its favorable hydrophobic and Hꢀbond interactions
with the binding domain of the PTR1.
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Pentamidine
14
17
Figure 3: 2Dꢀview of pentamidine, compound 14 and 17 with their common binding residues and
possible hydrophobic interactions
a
b
c
d
Figure 4: (a, b and c) specify the docked conformations of pentamidine, compound 14 and 17. Figure (d)
indicate the superimposition of pentamidine, most active (14) and less active (17) compounds which is
used to differentiate the binding mode of most active and less active compound compare with reference
(pentamidine).
In conclusion, Most of the synthesized compounds exhibited better potency against the
intracellular amastigotes of L. donovani than the standard drug pentamidine and were not found
to be cytotoxic. Compound 14 showed very consistent and promising leishmanicidal activity
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against intracellular amastigotes in vitro. This compound also displayed in vivo potential in the L.
donovani/golden hamster model, as the hamster model of VL closely mimic the active human
VL condition. Furthermore, compound 14 stimulate cellꢀmediated immune responses to prevent
progression of leishmania parasite. Molecular docking analysis further validated the concepts
involved the exploring these compounds. Interestingly antileishmanial compounds also show
activity against other protozoan parasites. It makes compound 14 more interesting for further
investigation. These results give scope to further exploration of triazine mimetics as new
antileishmanial leads.
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ASSOCIATED CONTENT
Supporting Information: Final compounds characterization data, material and methods of
docking study associated with this article. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION:
*Corresponding author. Tel.: 91 522 2612411; fax: +91 522 2623405,
Eꢀmail addresses: prem_chauhan_2000@yahoo.com; premsc58@hotmail.com
ACKNOWLEDGEMENT: We are grateful to Council of Scientific and Industrial Research
(K.C) and Indian Council of Medical Research (M.S) for the financial support in the form of
fellowship and also thankful to S.A.I.F. Division of CDRI, Lucknow, for providing the
spectroscopic data.
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Discovery of Triazines Mimetics as potent antileishmanial agents
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Kuldeep Chauhan,† Moni Sharma,† Rahul Shivahare,‡ Utsab Debnath†, Suman Gupta, ‡
Yenamandra S. Prabhakar† and Prem M. S. Chauhan†*
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