gem-Dithioacetylated indole derivatives as novel antileishmanial agents

Article abstract of DOI:10.1016/j.bmcl.2017.09.018

In this communication we report a serendipitously discovered hybrid molecule 1, combining fragment of 3 (an in vivo active antileishmanial molecule) with H₂S donor moiety (known for bimodal behavior of cytoprotection and apoptosis), as antileis

Full text of DOI:10.1016/j.bmcl.2017.09.018

Accepted Manuscript
gem-Dithioacetylated Indole Derivatives as Novel Antileishmanial Agents.
Sharad Porwal, Suman Gupta, Prem M.S. Chauhan
PII:
S0960-894X(17)30903-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.018
BMCL 25282
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
10 May 2016
5 September 2017
7 September 2017
Please cite this article as: Porwal, S., Gupta, S., Chauhan, P.M.S., gem-Dithioacetylated Indole Derivatives as Novel
Antileishmanial Agents., Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.09.018
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gem-Dithioacetylated Indole Derivatives as Novel Antileishmanial Agents.
Sharad Porwal a, 1, Suman Gupta b, Prem M. S. Chauhan a, *
a Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute,
Lucknow 226031, U.P., India
b Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226031,
U.P., India
Corresponding author: Tel.: +91 522 2771940, +91 522 2771942; fax: +91 522
2771941.
E-mail addresses: prem_chauhan_2000@yahoo.com, premsc58@hotmail.com
(P.M.S. Chauhan).
Abstract:
In this communication we report a serendipitously discovered hybrid molecule 1,
combining fragment of 3 (an in vivo active antileishmanial molecule) with H2S donor
moiety (known for bimodal behavior of cytoprotection and apoptosis), as antileishmanial
agent. Compound 1 suppresses 99.82% parasitemia of L. donovani infected macrophages
at 12.5 µg/ml without even deforming them (CC50 > 100 µg/ml). This compound appears
cytotoxic for intracellular amastigotes while cytoprotective to host macrophages. The
concept can be utilized to develop high therapeutic index NCE (New Chemical Entities)
for other macrophage mediated diseases like tuberculosis and cancer.
Key words: Hybrid, H2S donor, antileishmanial, cyctoprotection.
1
Current Address: Department of Chemistry, Lovely Professional University, Phagwara 144411, Punjab,
India
1

Leishmaniasis is a vector born disease caused by protozoa of genus Leishmania
which is transmitted by female phlebotomine sand fly to human host. Depending on
causative protozoan species it is manifested in four clinical forms: visceral leishmaniasis,
cutaneous leishmaniasis, mucocutaneous leishmaniasis and post kala azar dermal
leishmaniasis (PKDL). Among these four clinical forms of leishmaniasis, visceral
leishmaniasis (VL, also known as kala azar) is fatal if left untreated. There are 0.2 to 0.3
million cases of VL appeared every year worldwide and an estimated 20000 to 40000
people die annually due to VL.1
Treatment of VL relied on set of four drugs; sodium stibogluconate (antimonial
therapy), liposomal amphotericin B (polyene antibiotic), paromomycin (aminoglycoside
antibiotic) and miltefosin (phospholipid).2 All the four drugs have well documented
toxicity and suffer from problem of resistance. The antimonial therapy induces cardiac
arrhythmia and pancreatitis especially in malnourished and immunocompromised
patients.3 Liposomal amphotericin B, though a highly effective option for treatment of
VL, however this drug formulation is very expensive and suffered from life threatening
+
side effects such as hypokalemia (low K level in the blood), nephrotoxicity and first dose
anaphylaxis.3 The third option miltefosin is contraindicated in pregnant woman due to
teratogenicity and cause mild to severe gastrointestinal side effects.4 Similarly,
paromomycin is suffered from high tone ototoxicity and increased hepatic transaminases.5
In spite of toxicity of prevalent treatments, the wide spread treatment failure are observed
with antimonials 3 and the other three drugs are also prone to development of resistance.6
Climate change, mass movement of population and the emergence of PKDL have further
increased the risk of spread of leishmaniasis.1 The development of new safe and efficacious
drugs are thus required for VL therapy.
H2S is recently recognized as endogenous gasotransmitter like NO and CO. It has
multiple roles in normal physiology and pathogenesis of diseases. At low concentration, H2S
activates many intracellular signaling pathway promoting vasodilation and cyctoprotection,
while at high concentration it becomes detrimental to viability of cell through various
mechanisms (mitochondrial inhibition, intracellular acidification etc.).7 To exploit the role of
H2S in cellular biology, slow H2S donors have emerged as important chemotype to treat
various pathological conditions.16 The slow H2S donors; diallyl trisulfide, GYY4137 and
S-propargyl-cystein were found active in animal models of cancer (Fig. 1).7 Furthermore,
conjugation of H2S donating moiety with known drug (naproxen) was found to reduce the
toxicity of parent drug.8
Previously, our group has developed novel antileishmanial agents combining natural
product scaffold with drug fragments or other privileged structures.9 In one of our report we
combined pentamidine fragment with 2-thio analogue of aplysinopsin to create novel
antileishmanial chemotype 3.9a Herein, we report that the fragment of above chemotype 3
when conjugated to H2S donor moiety, can provide an antileishmanial agent with better
activity profile (Fig. 1).
Compound 1 was serendipitously formed during one pot muticomponent synthesis of
3.10 After discovering 1 as potential antileishmanial agent, we tried to find out the mechanism
of unusual reactivity of 1-alkylated indole-3-carbaldehyde to increase the yield of product. It
was found that the role of glycine in multicomponent synthesis of gem-dithioacetylated
indole derivative was just to create acidic condition during reaction, instead of generating
acetylthiocyanate (CH3CO-SCN) species (through intermediate 1-acetylthiohydantoin) as we
previously proposed.10 Since tetrabutylammonium tribromide (TBATB) was used as acid
2

catalyst in gem-diacetyl protection of aldehyde and ketone,11 we also used the same catalyst
for synthesis of gem-dithiolacetylated indole derivatives. Use of TBATB was found slightly
more effective in comparison to glycine in above KSCN and acetic anhydride mediated gem-
dithiolacylation of indole-3-carbaldehyde (Scheme 1).
S
H3CO
S
O
NH2
S
S
H3CO
P S H2N
N
O
S
O
Diallyl trisulfide
ABT-346 (naproxen derivative)
O
GYY4137
CN
O
O
O
N
S
O
S
O
S
OH
NH2
NH
HN
S-proparyl-cystein
S
Cell permeable H2S donor
Antileishmanial agent with in vivo activity
2
3
CN
O
S
O
N
O
S
Hybrid molecule combining fragment of 3 with H2S donor
1
Fig 1. Structures of various H2S donors and basis of structure of hybride molecule 1.
This protocol is specific only to N-alkylated indole carbaldehyde since N-alkylation
increases electron density at 2-position of indole.10 It was further confirmed when we
observed no reaction with 3-formylazaindole and poor yield with 5-bromo substituted
1-alkylindole under this multicomponent protocol (Supplementary data). It indicates that
TBATB catalyzed KSCN based gem-dithioacetylation is very sensitive to electron density at
2-position of indole.
O
O
S
S
H
KSCN, TBATB
Ac2O, 70 oC
O
N
N
5 O
5 O
1
CN
CN
Scheme 1: Synthesis of gem-dithioacetylated indole using TBATB instead glycine.
To determine role of various fragments of 1 in its antileishmanial activity we
synthesized compounds 4, 5 and 6. Syntheses of all these compounds are shown in
Scheme 2.
3

O
H
O
(A)
a
b
HO
CN
O
Br
O
CN
n
Br
Br
n
N
n
S
S
9, n=5
c
CN
O
N
O
n
1, n=5
4, n=6
5, n=4
CN
(B)
N
N
d
HN
N
Br
Br
N
N
10
(C)
O
O
H
H
d
Br
Br
N
H
N
Br
11
O
S
O
S
H
c
O
e
N
N
N
N
N
N
6
Scheme 2: Reagents and conditions: (a) Acetone, K2CO3, dibromoalkane (n = 4, 5, 6; 5
equiv.), p-cyanophenol (1.0 equiv.); (b) 3-formylindole, NaOH (aq), TBAB, toluene; (c)
KSCN (2.0 equiv.), Ac2O, TBATB, 70 oC; (d) 1,5-dibromopentane (5.0 equiv.), N-
phenylpiperazine/3-formylindole (1.0 equiv., added dropwise), acetone, K2CO3; (e)
DIPEA, AcCN, N-phenylpiperazine.
In synthesis of N-phenylpiperazine analogue 6 we have to change the reaction
sequence (through intermediate 11) since alkylation of N-phenylpiperazine with
dibromopentane provided exclusively the bis-substituted product 10 (Scheme 2B, 2C).
4

Methyl and butyl substituted analogues (7 and 8 respectively) were synthesized following
reaction sequence shown in Scheme 2A.
Initially compound 7, 8 and 1 were screened for antileishmanial activity against
promastigote and intracellular amastigote stages of parasite. Firefly luciferase transfected
L. donovani promastigotes were used for screening against promastigotes. For screening
against intracellular amastigote stage of parasite, mouse macrophages were infected with
firefly luciferase expressing promastigotes, which transformed to amastigotes in
macrophages. The inhibition was determined by comparison of luciferase activity of the
drug treated parasites with that of the untreated control (Supplementary data).
Table 1: Antileishmanial activity of gem-dithioacetylated indole derivatives
O
S
O
S
H
O
N
N
R
R
9
1-8
% Inhibition
intracellular
amastigote
stage a
% Inhibition
promastigote
stage a, b
Cytotoxcity
CC50
Comp.
R
(µg/ml)
1
4
5
6
7
8
9
p-cyanophenoxypentyl
p-cyanophenoxyhexyl
p-cyanophenoxybutyl
96
91.56
90.87
60.10
31
99.82
40.16
97.79
86.77
NDd
ND
NTc
NT
NT
N-phenylpiperazinylpentyl
methyl
13.83
ND
butyl
74
ND
p-cyanophenoxypentyl
NIe
NI
ND
a inhibition measured at 12.5 µg/ml. b promastigote strain: MHOM/IN/Dd8, c NT: not toxic i.e. CC50 > 100 µg/ml. d ND: not
determined, e NI: no inhibition.
As shown in Table 1 methyl and butyl substituted compounds 7 and 8 have shown
only moderate activity against promastigote stage of parasite. The compound 1 with p-
cyanophenoxypentyl chain was found active against both stages of parasites. Compound
1 suppressed 99.82% parasitemia of L. donovani infected macrophages at 12.5 µg/ml
concentration, while similar compound 3 with same p-cyanophenoxypentyl indole
fragment (Fig. 1) had suppressed only 62.0% parasitaemia.9a Surprisingly, there was no
visible deformation of host macrophages treated with compound 1 (CC50 > 100 µg/ml).
Encouraged by good activity profile of 1, additional compounds 4, 5 (variation of linker)
and 6 (variation of p-cyanophenoxy group) were synthesized (Scheme 2) to understand
SAR of this novel gem-dithioacetylated indole scaffold.
5

The SAR study (Table 1) shows that removal of gem-dithioacetyl group from 1
(the compound 9) resulted in almost no activity. It highlights the importance of H2S
donor functionality in compound 1 for suppression of parasitemia. Also, the moderate
activities of 7 and 8 show the importance of p-cyanophenoxy moiety for anti parasitic
activity. The importance of linker chain joining the above two fragments was shown by 4
and 5. Changing the linker from pentyl chain to butyl (compound 5) produce slight drop
in bioactivity in comparison to 1, while making this chain longer (compound 4) resulted
in substantial reduction in bioactivity. The pentyl chain is most suitable linker for
bioactivity.
We also replaced cyanophenoxy moiety of 1 with N-phenylpiperazine, an
important fragment of many bioactive compounds. The combination of N-
phenylpiperazine with gem-dithioacetylated indole (compound 6) though retained
antiparasitic activity, but it resulted in toxic molecule. Similar replacement of p-
cyanophenoxy moiety in 3 by N-phenylpiperazine (which is more polar than p-
cyanophenol) had resulted in an inactive molecule towards intracellular amastigotes. 9a In
our previous manuscript we reported the involvement of lipophilicity in enhancement of
antiparasitic activity of compound 3, since N-phenylpiperazine is a polar fragment so it
abolishes bioactivity of 3.9a However for compound 1, it looks its lipophilicity is
sufficient that replacement of p-cyanophenoxy fragment by more polar N-
phenylpiperazine is easily tolerated.
Following mechanisms may explain the efficient killing of intracellular
amastigotes within macrophages. As par the nature of H2S donors, which are cytotoxic at
high concentration and cytoprotective at low concentration7, compound 1 show almost
complete inhibition of parasite at 12.5 µg/ml while no inhibition at 6.5µg/ml.12 This H2S
generation may occur either inside or outside of amastigote in a macrophage. The
generation of H2S inside amastigote would be possible by selective accumulation of 1
inside intracellular amastigotes, where it may be triggered to release H2S most probably
by thiols of trypanothione,13 which is a key molecule involved in parasite defense against
oxidative stress.14 At high concentration of H2S inside amastigotes the parasite survival
could be hampered probably due to inhibition of mitochondrion bioenergetics,
acidification of intracellular environment, activation of caspase 9 and apoptosis.15
However, small amount of H2S that may have been generated in host macrophages could
have provided the cyctoprotective effect for host macrophages by enhancing cellular
bioenergetics and reducing reactive oxidative species ROS.16 This bimodal
pharmacological effect of H2S, 7 may be the probable cause of selective suppression of
parasitemia without deforming the host macrophages.
However, if 1 does not accumulate inside amastigote and H2S is generated outside
amastigote, then it may trigger generation NO in macrophages17 to kill intracellular
amastigotes. In addition to that, the pharmacological actions of some slow H2S donors7
extend beyond the biological effect of H2S as these were found to act on other targets
such as HDAC. It is likely that 1 might also act as inhibitor of HDAC. A detailed study is
needed to determine whether 1 donates H2S inside or outside intracellular amastigote
(and at what rate) or it acts via inhibition of other targets that would be reported in our
future communication.
In conclusion we have developed a hybrid molecule conjugating H2S donor
moiety with fragment of in vivo active antileishmanial compound. This hybrid shows
6

bimodal pharmacological character of H2S, killing intracellular amastigotes while
protecting host macrophages. The concept can be utilized in developing therapeutics for
other macrophage associated diseases like TB and cancers by designing the H2S donor
conjugated hybrid molecules that selectively accumulate in mycobacterium or cancer
cells while protecting or stimulating host macrophages.18 Efforts are underway to
incorporate other hydrogen sulfide donor moieties 19 with above bioactive fragment to
obtain antileismanial candidate with good in vivo antileishmanial profile.
Acknowledgements
We are thankful to Sophisticated Analytical Instruments Facility, CDRI for providing
analytical data and CSIR, New Delhi, for providing financial assistance to S.P.
CDRI Communication No: 9557
Supplementary data
Synthesis procedures, analytical data of synthesized compounds and screening protocols
can be found in the online version.
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7

Graphical Abstract
Cytoprotective & apoptotic
H2S Donor
CN
S
O
N
S
O
O
Fragment of in vivo active
antileishmanial 3
99.82% suppression of parasitamia at 12.5 µg/ml.
No deformation of host macrophases (CC50 > 100 µg/ml).