Synthesis and Antileishmanial activity of Piperoyl-Amino Acid Conjugates

Article abstract of DOI:10.1016/j.ejmech.2010.04.033

Based on reported antileishmanial activity of naturally occurring alkaloid piperine and amino acid esters,their conjugates were synthesized by the hydrolysis of piperine to piperic acid followed by reaction with amino acid methyl esters. These conjugates were further converted to compounds with free carboxyl group and those with reduced double bonds. The synthesized compounds were evaluated for activity against promastigote and amastigote forms of L. donovani in vitro. All the compounds showed better activity than either piperine or the amino acid methyl esters. Piperoyl-valine methyl ester was the most active compound showing an IC₅₀ of 0.075 mM against the amastigotes. Two active compounds were evaluated for in vivo activity in golden hamster model of leishmaniasis.

Full text of DOI:10.1016/j.ejmech.2010.04.033

European Journal of Medicinal Chemistry 45 (2010) 3439e3445
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and Antileishmanial activity of Piperoyl-Amino Acid Conjugates
,
a
a
b
c
Inder Pal Singh a , Shreyans Kumar Jain , Amandeep Kaur , Sukhvinder Singh , Rajender Kumar ,
*
Prabha Garg c, Shyam Sundar Sharma d, Sunil Kumar Arora b
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER)1, Sector 67, S.A.S. Nagar, Punjab 160062, India
b Molecular Immunology Laboratory, Department of Immunopathology, Postgraduate Institute of Medical Education and Research (PGIMER), Sector 12, Chandigarh 160012, India
c Computer Centre, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160062, India
d Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on reported antileishmanial activity of naturally occurring alkaloid piperine and amino acid esters,
their conjugates were synthesized by the hydrolysis of piperine to piperic acid followed by reaction with
amino acid methyl esters. These conjugates were further converted to compounds with free carboxyl
group and those with reduced double bonds. The synthesized compounds were evaluated for activity
against promastigote and amastigote forms of L. donovani in vitro. All the compounds showed better
activity than either piperine or the amino acid methyl esters. Piperoyl-valine methyl ester was the most
active compound showing an IC50 of 0.075 mM against the amastigotes. Two active compounds were
evaluated for in vivo activity in golden hamster model of leishmaniasis.
Received 16 February 2010
Received in revised form
23 April 2010
Accepted 26 April 2010
Available online 23 May 2010
Keywords:
Leishmaniasis
Amastigote
Amino acid
Piperine
Ó 2010 Elsevier Masson SAS. All rights reserved.
Promastigote
1. Introduction
Piperine 1, the most common and abundant alkaloid of the
genus Piper, has shown inhibitory activity against promastigote
Leishmaniasis is a neglected protozoal disease and is a major
cause of concern in developing countries. The disease is caused by
different species of protozoa Leishmania and transmitted by bite of
Phlebotomine sand flies. It is mainly manifested in three forms -
visceral leishmaniasis (VL), also known as kala-azar caused by L.
donovani; cutaneous leishmaniasis (CL) caused by L. major, L.
donovani, L. tropica and L. aethiopica; muco-cutaneous leishmani-
asis (MCL) caused by L. braziliensis. VL is the most severe form of
leishmaniasis and is usually fatal in the absence of treatment. Most
of the first line drugs available for the treatment of leishmaniasis
such as sodium stibogluconate, miltefosine, pentamidine etc cause
serious side effects and toxicity [1e3]. Moreover, resistance
developing to first line agents is now increasing even up to an
extent of >60% of the patients in some endemic areas [1]. Thus,
there is an urgent need for safe, inexpensive and orally available
drugs. Various classes of natural products such as alkaloids, flavo-
noids, lignans, terpenes etc have been shown to possess anti-
leishmanial activity [4].
form of L. donovani in vitro [5]. Reports have suggested that this
activity is enhanced if piperine is delivered in oil-in-water emulsion
form or as mannose-coated liposomes [6,7]. More recently,
piperine and its derivatives have been evaluated for inhibitory
effects against epimastigote and amastigote forms of the protozoan
parasite Trypanosoma cruzi [8,9].
Amino acid esters and amides have been found to possess
inhibitory activity against intracellular as well as isolated amasti-
gotes of L. amazonensis and L. mexicana. Amastigotes of leishmania
protozoa replicate within the macrophage phagolysosome. Amino
acid esters have been found to accumulate in these phag-
olysosomes. Once inside, the acidic pH of these organelles causes
accumulation of charged amino acids, which leads to osmotic
disruption of these phagolysosomes resulting in killing of the
infected cells. Inside the amastigotes, amino acid esters are
hydrolyzed by parasitic enzymes resulting in free amino acids
accumulation which kills the parasite [10]. However there are no
reports of antileishmanial activity of amino acid esters against L.
donovani.
In our continuous search for new antileishmanial compounds,
we aimed to make use of abundantly available natural product
piperine for generation of new antileishmanial compounds. We
report in this paper, synthesis and evaluation of antileishmanial
* Corresponding author. NIPER Communication Number 470. Tel.: þ91 172
2214682/7X2144; fax: þ91 172 2214692.
E-mail addresses: ipsingh@niper.ac.in, ipsingh67@yahoo.com (I.P. Singh).
1
NIPER Communication Number 470.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.033

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I.P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3439e3445
activity of i) piperoyl-amino acid methyl ester conjugates 3-7 ii)
piperoyl-amino acid conjugates 8-12 and iii) tetrahydropiperoyl-
amino acid methyl ester conjugates 13-17. Lee et al reported central
nervous system depressant action of conjugates of piperic acid with
alanine and phenyl alanine together with some non-natural amino
acids [11].
the conversion of ester group into free carboxyl group in
compounds 8-12.
The third series comprised of saturated derivatives of
compounds 3-7 i.e. tetrahydropiperoyl-amino acid methyl ester
conjugates 13-17. Two alternate strategies were tried for prepara-
tion of these compounds (i) hydrogenation of compounds 3-7 of
series 1, (ii) hydrogenation of piperic acid to tetrahydropiperic acid
followed by reaction with amino acid methyl esters. Compounds
13-15 and 17 were synthesized by catalytic hydrogenation of
compounds 3-5 and 7. Efforts to prepare 16 by catalytic hydroge-
nation of 6 were not successful; this could be ascribed to catalytic
poisoning by sulfur. Therefore, for synthesizing 16, tetrahy-
dropiperic acid (2a) was prepared by catalytic hydrogenation of
piperic acid (2), it was then coupled to methionine methyl ester by
the same method as used for preparation of compounds of series 1
[9]. As a result of reduction of conjugated double bonds, proton
signals in the olefinic region of compounds 3-7 disappeared while
additional signals in the aliphatic region for eight protons were
observed confirming hydrogenation of the double bond.
2. Results and discussion
2.1. Chemistry
Reported activities of piperine and amino acid esters suggested
that their conjugates may show enhanced antileishmanial activity.
The amide bond of piperine can be hydrolyzed to obtain piperic
acid (2) and the resulting carboxylic acid group can be used as a site
for linking it covalently with amino acid esters via amide bond
formation.
Piperine (1) was isolated from chloroform extract of Piper nig-
rum by column chromatography using n-hexane-ethyl acetate
gradient elution (0-30%). Piperic acid (2) was prepared by alkaline
hydrolysis of piperine by reported procedures [12]. An alternative
method for preparation of piperic acid involved direct saponifica-
tion of the chloroform extract of P. nigrum. The piperic acid liber-
ated was washed with water and recrystallized from methanol. The
yield of piperic acid obtained by two methods was similar, no
chromatographic purification was required in the alternative
method.
2.2. Antileishmanial activity
Parasites of the Leishmania genus are known to exist as flagel-
lated extracellular promastigotes and intracellular amastigotes. All
the synthesized compounds were evaluated for antileishmanial
activity against both these forms in vitro. The results are summa-
rized in Table 1.
Piperoyl-amino acid conjugates with carboxyl protected amino
acids (amino acid methyl esters) were prepared via piperic acid
mesylate obtained by reaction of piperic acid with methanesulfonyl
chloride (CH3SO2Cl) in dry CH2Cl2 at 0 ꢀC. This mesylate was reac-
ted with amino acid methyl esters to yield the desired piperoyl-
amino acid methyl ester conjugates 3-7 in yields ranging from 40-
75% [12] (scheme1).
Compounds 8-12 were prepared by deprotection of carboxyl
group of compounds 3-7 by microwave assisted solid phase
reaction with Al2O3 (40% KF) in 70-80% yields [13,14]. Disappear-
ance of methoxy signal in 1H NMR of compounds 3-7 confirmed
All the compounds showed higher activity against the amasti-
gote form compared to promastigote form (Fig. 1); IC50 values were
approximately three times lower against amastigotes. Piperine 1
showed an IC50 value of 0.752 and 2.558 mM against promastigotes
and amastigotes, respectively. Piperoyl-amino acid ester conjugates
(3-7) showed substantial increase in activity as compared to either
piperine or amino acid esters. All the compounds were found to be
active in IC50 range of 0.075-0.310 mM against amastigotes while
IC50 was considerably higher i.e. 0.473-1.312 mM against promas-
tigotes. This indicates that amastigotes are more sensitive to these
compounds than promastigotes. Compounds 3-7 were found to be
O
O
O
COOMe
R
5
3
2'
1'
O
O
O
O
O
O
1
N
a
OH
b, c
N
H
2
4
6'
1
2
3-7
O
O
COOMe
R
O
COOH
R
O
O
O
O
d
e
N
N
H
H
3, 8, 13 R = CH2C6H5
4, 9, 14 R = CH2C6H4p-OH
5, 10, 15 R = CH(CH3)2
6, 11, 16 R = CH2CH2SCH3
7, 12, 17 R = CH2(3-indolyl)
3-7
8-12
COOMe
R
O
COOMe
R
O
O
O
O
N
H
N
H
3, 4, 5, 7
13, 14, 15, 17
O
O
COOMe
R
O
O
O
e
b, c
N
H
OH
2
O
2a
16
Scheme 1. Reagents and conditions: (a) 20% KOH, CH3OH, 80 ꢀC, 3 days, 88% (b) CH3SO2Cl, Et3N, DCM, 0 ꢀC, 30 min, 85% (c) Amino acid methyl ester, Et3N, DCM, RT, 40-75% (d) KF-
Al2O3 (40%), Microwave, 1000 W, 3-4 min, 70-80% (e) 5% Pd/C, H2 (40 Psi), MeOH, 30 min, 75-80%

I.P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3439e3445
3441
Table 1
Reduction of double bonds
decreases activity against amastigotes,
increases against promastigotes
Antileishmanial activity and cytotoxicity of piperoyl-amino acid conjugates.
Compound
IC50 (mM)
Cytotoxicity (%)
Hydrolysis of ester functionality
decreases activity against both
promastigotes & amastigotes
O
COOCH3
Amastigotes
Promastigotes
At IC50
At 2 ꢁ IC50
O
O
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0.752
0.395
0.197
0.210
0.075
0.168
0.119
0.256
0.249
0.310
0.237
0.236
0.212
0.202
0.240
0.221
0.176
1.537
2.558
1.756
0.834
0.791
0.875
0.823
0.855
0.927
0.761
1.030
1.312
1.025
0.669
0.536
0.722
0.607
0.473
2.161
7
4.5
4
10
8
7
N
H
R
3.5
3
4
3
1.2
4
6
Piperine subunit
Amino acid subunit
3.8
7.5
5.2
8.5
6
Fig. 2. Piperoyl-amino acid methyl ester conjugates
antileishmanial activity of the conjugates against both the forms of
parasite (Fig. 2).
5.5
1.8
3.6
5.5
5
9
7.7
10
7.2
7.6
7.7
5
Cytotoxicity of all the synthesized compounds was determined
against PBMC (peripheral blood mononuclear cells) at two
concentrations (IC50 and twice that of IC50). None of the compounds
showed more than 10% killing at a concentration twice that of IC50
(Table 1).
Compounds 3 and 5, which showed higher activity against the
amastigote form, were evaluated in vivo and the results are shown
in Table 2. A dose of 250 mg/kg was chosen based on pilot studies.
Since the oral absorption of these compounds is not known,
therefore these were administered intraperitoneally (i.p.).
Compound 3 showed significant reduction in spleen parasitic
burden (35%) and spleen weight (30%) in in vivo assay. Though 5
showed reduction in parasitic burden (24%) but reduction in spleen
weight was not observed. Standard antileishmanial, miltefosine
showed significant reduction in spleen parasitic burden (87%) and
spleen weight (43%) in in vivo study.
4.5
3
17
2.5
5.7
5
8.2
L-Phenylalanine
methyl ester (18)
L-Tyrosine methyl
ester (19)
L-Valine methyl
ester (20)
L-Methionine methyl
ester (21)
L-Tryptophan methyl
ester (22)
1.396
1.760
1.898
1.421
0.018
2.091
2.348
1.931
1.366
0.033
0.3
4
5
6
1.5
1.8
29
7.3
5.3
35
Miltefosine
more active against amastigotes while compounds 13-17 were
more active against promastigotes. Maximum effectiveness against
amastigotes was found with compound 5, conjugate of piperic acid
and valine methyl ester, which showed an IC50 of 0.075 mM against
amastigotes. Other than valine, conjugates of piperic acid with
methionine methyl ester (6) and tryptophan methyl ester (7) also
showed promising activity against amastigotes with IC50 values of
0.168 and 0.119 mM, respectively. Modification by deprotection of
carboxyl group of amino acid functionality in compounds 8-12
resulted in decrease in antileishmanial activity against amastigotes
(IC50 range 0.236-0.310 mM). Though much more potent than
piperine and amino acid methyl esters, compounds of series 2 (8-
12) were less active than compounds of series 1. Compounds of
series 3 with reduced double bonds were more active than their
unsaturated analogues 3-7 against promastigotes, tetrahy-
dropiperoyl tryptophan methyl ester 17 being most active with an
IC50 value of 0.473 mM. These observations indicate that reduction
of conjugated double bonds in piperine subunit decreases the
antileishmanial activity of the conjugates against amastigotes
where as activity against promastigotes is enhanced. Hydrolysis of
ester functionality in amino acid subunit decreases the
Table 2
Effect of compounds on spleen weight and parasitic burden in hamster model of
visceral leishmaniasis.
Groups
Spleen weight in mg (% Spleen parasitic burden ꢁ 109
reduction)a
(% reduction)a
Vehicle
503 ꢂ 34
0.54 ꢂ 0.04
3 (250 mg/kg, i.p.)
5 (250 mg/kg, i.p.)
Miltefosine
353 ꢂ 42b (30%)
516 ꢂ 20
0.35 ꢂ 0.06b (35%)
0.41 ꢂ 0.02 (24%)
0.07 ꢂ 0.00b (87%)
287 ꢂ 23b (43%)
(12.5 mg/kg, p.o.)
a
Results are expressed as mean ꢂ SEM (n ¼ 5)
p<0.05 vs vehicle treated group
b
2.3. Molecular docking against putative targets
Compound 5 which showed good antileishmanial activity was
docked against L. donovani targets to generate the appropriate
binding orientation and conformation to the active site of these
targets. The docking study of compound 5 was performed against
the target adenine phosphoribosyltransferase (APRT), PDB ID 1QB8
[15] using AutoDock 4.2 [16] program. The enzyme 1QB8 has role in
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Amastigotes Promastigotes
Fig. 1. IC50 values of piperoyl-amino acid conjugates and standard

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I.P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3439e3445
3. Conclusions
In the present study, several piperoyl-amino acid ester conju-
gates were synthesized and evaluated for antileishmanial activity in
vitro and in vivo. Piperoyl-valine methyl ester showed an IC50 of
0.075 mM against the amastigotes and was further evaluated in
vivo in the golden hamsters. Molecular docking studies indicated
that 5 binds to the active site of L. donovani protein 1QB8.
4. Experimental
4.1. Chemicals and instruments
All solvents used were of analytical grade (CDH Laboratory
Reagents, India). TLC plates of silica gel GF254 (Merck, Germany)
were used for analysis. Silica gel 60-120 mesh (CDH Laboratory
Reagents, India) was used for column chromatography. Melting
points were recorded on capillary melting point apparatus and are
uncorrected. 1H/13C NMR spectra are recorded on 300/75 MHz
Bruker FT-NMR (Avance DPX300) spectrometer using tetrame-
thylsilane as internal standard and the chemical shifts are reported
Fig. 3. Docked pose of Compound 5 into active site of 1QB8 protein. Green color sticks
represent co-crystallized AMP structure and white color sticks represent AutoDock 4.2
conformation. Magenta color area represents Mg2þ ion (generated by MOLCAD
program)
in
d units. Mass spectra were recorded on auto sampler/direct
salvage pathway by converting adenine into adenosine-5-mono-
phosphate (AMP). APRT is not essential for purine salvage in L.
donovani promastigote form (found in sandfly vector), but may play
an essential role in the infective amastigote form of the L. donovani
that lives in the host macrophage cell. So, for this parasite APRT is
a target for antileishmanial chemotherapy [17,18]. The compound 5
was built using SYBYL7.1 molecular modeling package installed on
a Silicon Graphics Fuel Work station running IRIX 6.5. Tripos force
field, Gasteiger Huckel, partial atomic charges [19] and Powell’s
conjugate gradient method were used for minimization of all
molecules with 0.05 kcal/mol energy gradient convergence crite-
rion [20]. The magnesium (Mg2þ) is required for activity of this
enzyme. The compound 5 was showing H-bonding interaction to
Ala150, Thr151, Gly152, Thr154 and Ala155 residues. It was found
that Mg2þ ions coordinated with electron-rich carbonyl oxygen and
-NH group (distance from Mg2þ 2.04 Ǻ and 3.09 Ǻ, respectively) of
compound 5. These interactions have good agreement to experi-
mental data [15]. The Compound 5 docked well in terms of location
(near to AMP and Mg2þ) and H-bond interactions (shown in Figs. 3
and 4) that implies these interactions are important for binding of
this compound to the active site of L. donovani protein 1QB8.
injection LCMS (APCI/ESI).
4.2. Chemistry
4.2.1. Procedure for preparation of piperic acid (2) from piperine (1)
Piperine 1 (11.8 g) was isolated from 20 g chloroform extract of
P. nigrum. Piperine 1 (11.4 g, 0.04 mol) was refluxed with meth-
anolic KOH (20%, 500 mL) for 3 days. After completion of hydrolysis,
methanol was distilled under reduced pressure. The resulting
reaction mixture was suspended in hot water and acidified with
HCl to pH<1. Yellow precipitate obtained was collected by filtra-
tion, washed with cold water and recrystallized from methanol to
yield crystals of piperic acid 2 (7.9 g, yield 88%); Rf 0.30 (CH2Cl2:
MeOH ¼ 9.5:0.5); m.p. 212-215 ꢀC (Lit. 215-216 ꢀC). The yield of
piperic acid from crude extract was 39.5%. This procedure was then
scaled up to 50 g of piperine to obtain 36 g of piperic acid which
was used for further reactions.
4.2.2. Alternative method for preparation of piperic acid (2) from
chloroform extract of P. nigrum
Chloroform extract of P. nigrum (20 g) was dissolved in chloro-
form (150 mL), washed with cold aqueous NaOH (2 N,150 mL) three
times. Remaining organic layer was evaporated to dryness. Dried
extract was refluxed with methanolic KOH (40%, 200 mL) for 2 days
and the reaction mixture was washed with ether and acidified with
conc. HCl to pH<1. Precipitates of piperic acid obtained were filtered
and washed with water. Piperic acid was recrystallized from meth-
anol (8 g, yield 40%) and confirmed by co-TLC with standard.
4.2.3. General procedure for preparation of amides (3-7) from
piperic acid
Piperic acid 2 (218 mg, 1 mmol) was stirred in dry CH2Cl2
(10 mL) for 10 minutes at -15 ꢀC. Triethylamine (0.26 mL, 2 mmol)
was added to the reaction mixture followed by methanesulfonyl
chloride (0.15 mL, 1.5 mmol) and reaction mixture was stirred for
1 h. Solution of amino acid methyl ester (1 mmol) in CH2Cl2 was
then slowly added to the reaction mixture. It was then allowed to
stir for 30 min at 0 ꢀC followed by stirring at room temperature for
4-5 h. Reaction was monitored by TLC using mobile phase - CHCl3:
MeOH (9.5:0.5). Reaction mixture was extracted with CH2Cl2 and
was washed with NaHCO3 and 2 N HCl. CH2Cl2 layer was dried over
anhydrous sodium sulfate. The crude product was purified by silica
gel column chromatography using CHCl3:MeOH with increasing
Fig. 4. Docking interactions of Compound 5 into active site of 1QB8 protein. White
color-sticks represent AutoDock 4.2 conformation and purple color sticks represents
co-crystallized AMP structure. The dotted lines show H-bonding with active site
residues (Green color). Magenta, Red and Cyan colors represent Mg2þ, Citrate and
water molecule respectively (generated by Sybyl7.1 program).

I.P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3439e3445
3443
gradient (0-0.5%). All the conjugates were characterized by 1H, 13
NMR, IR and MS data.
C
solid support KF/Al2O3 with substrate. The round bottom flask was
then fitted with septum, placed in microwave oven and irradiated
at 100% power for 3-4 min. After cooling, 5 mL water was added to
the reaction mixture, stirred for 5 min and filtered. The filtrate was
neutralized by adding aqueous HCl and the precipitate thus
obtained was filtered and dried to obtain compounds 8-12. All the
compounds were characterized by 1H, 13C NMR, IR and MS data.
4.2.3.1. Piperoyl-L-phenylalanine methyl ester (3). Off-white solid;
yield 70%; m.p. 117-118 ꢀC; IR (KBr) ymax 3314, 1749, 1646, 1608,
1531, 1504, 1247, 1218, 997, 928 cmꢃ1 1H NMR (CDCl3, 300 MHz)
;
d
7.40 (dd, 1H, J ¼ 10.5 Hz, 14.8 Hz, eCH]CHeCO), 7.31-6.69 (9H,
olefinic and Ar-H), 6.65 (dd, 1H, J ¼ 10.5 Hz, 15.4 Hz, ]CHeCH]
CHeCO), 5.91 (s, 2H, OCH2O), 5.89 (d, 1H, J ¼ 14.8 Hz, ]CHeCO),
5.03-4.97 (m, 1H, NHeCHeCO), 3.73 (s, 3H, eCOOCH3), 3.20 (d, 2H,
4.2.4.1. Piperoyl-L-phenylalanine (8). Yellow solid; yield 78%; m.p.
100-101 ꢀC; IR (KBr) ymax 3466,1717,1264 cmꢃ1; 1H NMR (DMSO-d6,
J ¼ 5.5 Hz, CH2eAr); 13C NMR (CDCl3, 75 MHz)
d
172.6, 166, 148.7
300 MHz)
and Ar-H), 5.31 (d,1H, J ¼ 14.9 Hz, olefinic), 3.8-3.6 (m,1H, CH), 2.31-
2.05 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
173.5,165.8,148.2,
d
7.62 (d,1H, J ¼ 7.8 Hz), 6.43-5.98 (m,12H, OCH2O, olefinic
(2C), 142.4, 140, 136.4, 131.3, 129.8 (2C), 129.1, 127.6 (2C), 125, 123.2,
122.8, 109, 106.3, 101.8, 53.7, 52.8, 38.4; CIMS: m/z 379 [Mþ1]þ
d
148.1, 140.5, 138.7,137.9, 131,129.3 (2C),128.5 (2C),126.8, 125.3,124,
4.2.3.2. Piperoyl-L-tyrosine methyl ester (4). Light yellow solid; yield
50%; m.p. 188-189 ꢀC; IR (KBr) ymax 3518, 1724, 1608, 1506,
123, 108.8, 105.9, 101.5, 54, 37.1; CIMS: m/z 366.1 [Mþ1]þ
1244 cmꢃ1
;
1H NMR (DMSO-d6, 300 MHz)
d
7.36 (dd, 1H, J ¼ 15.2
4.2.4.2. Piperoyl-L-tyrosine (9). Brown solid; yield 77%; m.p. 127-
Hz, 10.6 Hz, eCH]CHeCO), 7.14-6.77 (m, 8H, olefinic and Ar-H),
6.70 (dd, 1H, J ¼ 10.6 Hz, 15.2 Hz, ]CHeCH]CHeCO), 6.10 (d, 1H,
J ¼ 15.2 Hz, ]CHeCO), 5.98 (s, 2H, OCH2O), 5.03-4.99 (m, 1H,
NHeCHeCO), 3.74 (s, 3H, COOCH3), 3.19 (d, 2H, J ¼ 5.0 Hz, CH2eAr);
128 ꢀC; IR (KBr) ymax 3100, 1595, 1253, 1193, 1148, 1036 cmꢃ1 1H
;
NMR(DMSO-d6, 300 MHz)
olefinic and Ar-H), 6.04 (s, 2H, OCH2O), 4.47-4.40 (m, 1H, NHCHCO),
3.01-2.74 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz)
173.6, 165.5,
d 9.24 (brs), 8.38 (1H, NH), 7.16-6.12 (11H,
d
13C NMR (DMSO-d6, 75 MHz)
d
173.1, 166.2, 156.7, 148.7 (2C), 141.1,
156.2, 148.2, 148.1, 140.2, 138.5, 131.1, 130.3 (2C), 128, 125.5, 124.3,
139.3, 131.6, 130 (2C), 128.1, 125.9, 124.3, 123.6, 115.9 (2C), 109.3,
123, 115.3 (2C), 108.7, 106, 101.6, 54.3, 36.4; CIMS: m/z 382.1 [Mþ1]þ
106.5, 102.1, 54.9, 52.6, 36.8; CIMS: m/z 418 [MþNaþ1]þ
4.2.4.3. Piperoyl-L-valine (10). Light yellow solid; yield 81%; m.p.
4.2.3.3. Piperoyl-L-valine methyl ester (5). Light green solid; yield
75%; m.p. 124-125 ꢀC; IR (KBr) ymax 3293, 1741, 1647, 1606, 1253,
151-152 ꢀC; IR (KBr) ymax 3200, 1705, 1608, 1255 cmꢃ1 1H NMR
;
(CDCl3, 300 MHz)
d
7.36 (dd, 1H, J ¼ 10.9 Hz, 14.8 Hz, eCH]CHCO),
1037 cmꢃ1; 1H NMR (CDCl3, 300 MHz)
d
7.36 (dd,1H, J ¼ 10.4 Hz,14.4
6.94-6.51 (5H, olefinic and Ar-H), 6.02 (d, 1H, J ¼ 14.9 Hz, ]CHCO),
5.95 (s, 2H, OCH2O), 4.70-4.66 (m, 1H, NHCHCO), 2.31-2.24 (m, 1H),
Hz, eCH]CHeCO), 7.08-6.63 (5H, olefinic and Ar-H), 6.01 (d, 1H,
J ¼ 14.7 Hz, ]CHeCO), 5.99 (s, 2H, OCH2O), 4.71-4.67 (m, 1H,
NHeCHeCO), 3.75 (s, 3H, COOCH3), 2.23-2.15 (m, IH, CH(CH3)2), 0.96
(d, 3H, J ¼ 8.1 Hz, CH3), 0.92 (d, 3H, J ¼ 7.3 Hz, CH3); 13C NMR (CDCl3,
1.01-0.96 (m, 6H); 13C NMR (CDCl3, 75 MHz)
d 173.5, 165.8, 148.2,
148.1, 140.2, 138.5, 131.1, 125.5, 124.4, 123, 108.7, 106, 101.6, 52.6,
30.2, 19.5, 18.4; CIMS: m/z 318.1 [Mþ1]þ
75 MHz) d 173.2, 166.4, 148.7 (2C), 142.4, 139.9, 131.3, 125, 123.2, 123,
109,106.3,101.8, 57.6, 52.7, 32.1,19.4,18.4;CIMS:m/z354 [MþNaþ1]þ
4.2.4.4. Piperoyl-L-methionine (11). White solid; yield 75%; m.p.
169-170 ꢀC; IR (KBr) ymax 3300, 3273, 1707, 1612, 1512, 1255,
4.2.3.4. Piperoyl-L-methionine methyl ester (6). Off-white solid;
yield 68%; m.p. 121-122 ꢀC; IR (KBr) ymax 3286, 1720, 1610, 1252,
1039 cmꢃ1
;
1H NMR (DMSO-d6, 300 MHz)
d
8.44 (d, 1H, J ¼ 7.6 Hz,
NH), 7.19 (dd, 1H, J ¼ 9.4 Hz, 14.8 Hz, eCH]CHCO), 7.03-6.85 (5H,
olefinic and Ar-H), 6.20 (d, 1H, J ¼ 14.8 Hz, ]CHCO), 6.05 (s, 2H,
OCH2O), 4.44-4.41 (m, 1H, NHCHCO), 2.53 (m, 2H, CH2), 2.05-1.93
1149, 1097, 1038 cmꢃ1 1H NMR (CDCl3, 300 MHz)
; d 7.37 (dd, 1H,
J ¼ 10.5 Hz, 14.8 Hz, eCH]CHeCO), 6.97 (s, 1H, AreH), 6.89 (d, 1H,
J ¼ 7 Hz, AreH), 6.97-6.63 (m, 2H, olefinic and Ar-H), 6.36 (d, 1H,
J ¼ 7.7 Hz, AreH), 6.00 (d, 1H, J ¼ 14.8 Hz, ]CHeCO), 5.92 (s, 2H,
OCH2O), 4.87-4.80 (m,1H, NHeCHeCO), 3.77 (s, 3H, COOCH3), 2.60-
2.47 (m, 2H, eCH2eCH2Se), 2.23-2.20 (m, 2H, eCH2Se), 2.10 (s, 3H,
(5H, SCH2, SCH3); 13C NMR (DMSO-d6, 75 MHz)
d 173.8, 165.8, 148.2,
148.1, 140.4, 138.6, 131.1, 125.4, 124.1, 123, 108.8, 106, 101.6, 51.4, 31,
30, 14.8; CIMS: m/z 349.9 [Mþ1]þ
eSCH3); 13C NMR (CDCl3, 75 MHz)
d
172.6, 166.1, 148.7 (2C), 142.5,
4.2.4.5. Piperoyl-L-tryptophan (12). Brown solid; yield 76%; m.p.
148-149 ꢀC; IR (KBr) ymax 3402, 1738, 1648, 1488, 1441, 1245,
140.1, 131.2, 125, 123.2, 122.7, 109, 106.3, 101.8, 53.1, 52.1, 32.4, 30.5,
16; CIMS: m/z 386.1 [MþNaþ1]þ
1037 cmꢃ1 1H NMR (CD3OD, 300 MHz)
; d 7.57-6.72 (11H, olefinic
and Ar-H), 6.07 (d, 1H, J ¼ 15.1 Hz, ]CHCO), 5.90 (s, 2H, OCH2O),
4.2.3.5. Piperoyl-L-tryptophan methyl ester (7). Off-white solid;
yield 61%; m.p. 129-130 ꢀC; IR (KBr) ymax 2919, 2853, 1739, 1613,
4.96-4.85 (m, 1H, NHCHCO), 3.34 (d, 2H, J ¼ 2.3 Hz, eCH2 e3-
Indolyl); 13C NMR (CD3OD, 75 MHz)
d 173.1,166.6,147.5,140.6,138.3,
1281, 1062 cmꢃ1; 1H NMR (CDCl3, 300 MHz)
d
8.09 (NH), 7.55-6.59
135.8, 130, 126.7, 123.6, 122.8, 122.6, 122.2, 121.6, 121.4, 120.2, 117.6,
(11H, olefinic and Ar-H), 6.04 (d, 1H, J ¼ 14.9 Hz, ]CHCO), 5.97 (s,
2H, OCH2O), 5.10-5.04 (m, 1H, NHeCHeCO), 3.70 (s, 3H, COOCH3),
3.38 (d, 2H, J ¼ 4.9 Hz, eCH2 e3-Indolyl); 13C NMR (CDCl3, 75 MHz)
117.1, 110.1, 108.8, 107.1, 100.5, 52.7, 26.4; CIMS: m/z 405.1 [Mþ1]þ
4.2.5. Synthesis of compounds (13-17)
d
173.3, 166.3, 148.8, 148.7, 142.3, 139.9, 136.7, 131.6, 128.2, 125,
Amides (3-5 and 7) were dissolved in methanol and Pd/C (5%)
was added to it. The reaction mixture was subjected to catalytic
hydrogenation at 40 psi for 30 min. The reaction mixture was
filtered, the filtrate was collected and dried to yield compounds 13-
15 and 17. For synthesis of compound 16, tetrahydropiperic acid 2a
was obtained by catalytic hydrogenation of piperic acid. Piperic acid
2 (4.36 g, 20 mmol) was treated with 5% Pd/C in methanol and
subjected to catalytic hydrogenation at 40 psi for 30 min. The
solution was filtered and the filtrate was concentrated to obtain
tetrahydropiperic acid 2a in 85% yield, it was coupled with
methionine methyl ester using the same method as used for the
preparation of compounds 3-7.
123.4, 123.2, 123, 122.7, 120.2, 119.2, 111.8, 110.5, 109, 106.3, 101.8,
53.7, 52.9, 28.3; CIMS: m/z 419 [Mþ1]þ
4.2.4. General procedure for deprotection of carboxyl moiety:
Synthesis of compounds (8-12)
Neutral alumina (15 g) was mixed with KF (10 g) in 200 mL of
water for the preparation of KF/Al2O3. Water was then removed at
50-60 ꢀC in rotary evaporator, this reagent was further dried in
vacuum oven for 12 h. Compounds 3-7 (1 mmol) were added to KF/
Al2O3 (1 g, 40% KF) and this dry mixture was stirred at room
temperature in a round bottom flask to ensure uniform mixing of

3444
I.P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3439e3445
4.2.5.1. Tetrahydropiperoyl-L-phenylalanine methyl ester (13).
Yellow oil; yield 80%; IR (KBr) ymax 3407, 1728, 1603, 1255,
and the resultant formazan formed in live promastigotes was dis-
solved in DMSO (100 L) and absorbance was read at OD492 nm. The
mean percentage of post treatment viable cells was calculated
relative to control and results were expressed as concentration
inhibiting the parasitic growth.
m
1054 cmꢃ1
;
1H NMR (CDCl3, 300 MHz)
d
7.28-6.58 (8H, Ar-H), 5.90
(s, 2H, OCH2O), 4.92-3.86 (m, 1H, NHeCHeCO), 3.72 (s, 3H,
COOCH3), 3.18-3.04 (m, 2H, CH2ePh), 2.54-2.49 (m, 2H, eCH2Ar),
2.20-2.15 (m, 2H, eCH2CO), 1.59-1.58 (m, 4H, eCH2eCH2e); 13C
NMR (CDCl3, 75 MHz)
d
172.9, 172.7, 147.5 (2C), 136.5, 136.3, 129.7
4.3.2. In vitro amastigote assay
(2C),129.1 (2C),127.6,121.6,109.3,108.6, 101.2, 53.4, 52.8, 38.4, 36.8,
L. donovani amastigotes isolated from hamster spleen or axenic
culture of amastigotes (transformed from promastigotes in vitro)
was acclimatized to gradual change in temperature (from 26 ꢀC to
37 ꢀC) and pH (from 7.2 to 5.5) [22]. L. donovani amastigotes (5ꢁ105
cells/mL) from logarithmic phase culture were grown in 96 well
plate for 48 h at 37 ꢀC before the treatment with drugs. Drug
dilutions were prepared in DMSO and appropriate concentration of
35.8, 31.6, 25.5; CIMS: m/z 384.1 [Mþ1]þ
4.2.5.2. Tetrahydropiperoyl-L-tyrosine methyl ester (14). Colorless
oil; yield 75%; IR (KBr) ymax 3481, 3284, 1729, 1646, 1506,
1290 cmꢃ1; 1H NMR (CDCl3, 300 MHz)
d 6.93-6.57 (7H, Ar-H), 5.90
(s, 2H, OCH2O), 4.89-4.82 (m, 1H, NH-CH-CO), 3.72 (s, 3H, COOCH3),
3.10-2.93 (m, 2H, CH2Ph), 2.57-2.47 (m, 2H, CH2eAr), 2.38-2.30 (m,
2H, CH2CO), 1.65-1.55 (m, 4H, eCH2eCH2e); 13C NMR (CDCl3,
each drug was used in triplicate (20-100 mg/mL). Modified MTT
assay as described above was used for evaluation of drug activity
against L. donovani amastigotes. The mean percentage of post
treatment viable amastigotes was calculated relative to control and
results were expressed as concentration inhibiting the parasitic
growth.
75 MHz)
d 178.9, 173, 156.1, 148, 146.1, 136.5, 136.7, 130.7, 128.4,
127.4, 121.6, 116.1, 109.3, 108.6, 101.2, 53.7, 52.9, 35.7, 32, 31.5, 31.3,
25.5; CIMS: m/z 400 [Mþ1]þ
4.2.5.3. Tetrahydropiperoyl-L-valine methyl ester (15). Yellow oil;
yield 80%; IR (KBr) ymax 3457, 1741, 1653, 1506, 1253, 1154 cmꢃ1; 1H
4.3.3. In vitro cytotoxicity assay
NMR (CDCl3, 300 MHz)
d
6.72-6.59 (3H, Ar-H), 5.90 (s, 2H, OCH2O),
In vitro cytotoxicity was determined against PBMC (peripheral
blood mononuclear cells) separated from heparinized blood of
a normal healthy individual by Ficoll-Hypaque (Sigma, USA)
density gradient centrifugation. Modified MTT ([3-(4, 5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay [21] was
used for in vitro cytotoxicity. Briefly, the assay was performed in 96-
well tissue culture plates. Cells were seeded to the wells of 96-well
plate (1ꢁ105 cells/well) and were exposed to drug compounds (IC50
concentration and twice the concentration of IC50 were used) in
triplicate. After treatment with drugs, plate was kept at 37 ꢀC for
4.59-4.55 (m, 1H, NHeCHeCO), 3.73 (s, 3H, COOCH3), 2.56-2.52 (m,
2H, eCH2Ar), 2.27-2.08 (m, 2H, eCH2CO), 2.19-2.08 (m, 1H, eCH
(CH3)2), 1.67-1.60 (m, 4H, eCH2eCH2e), 0.93-0.88 (m, 6H, 2CH3);
13C NMR (CDCl3, 75 MHz)
d 173.2 (2C), 148, 146.1, 136.5, 121.6, 109.3,
108.6, 101.2, 57.3, 52.6, 37, 35.9, 31.7 (2C), 25.6, 19.4, 18.3; CIMS: m/z
336.1 [Mþ1]þ
4.2.5.4. Tetrahydropiperoyl-L-methionine methyl ester (16). Brown
oil; yield 67%; IR (KBr) ymax 3304, 1737, 1644, 1532, 1444, 1250,
1038 cmꢃ1; 1H NMR (CDCl3, 300 MHz)
d
6.72-6.59 (m, 3H, Ar-H), 5.90
48 h. MTT to a final concentration of 400 mg/mL was added and
(s, 2H, OCH2O), 4.75-4.68 (m,1H, NHeCHeCO), 3.75 (s, 3H, COOCH3),
2.57-2.47 (m, 2H, CH2Ar), 2.26-2.00 (m, 9H, eCH2eCH2eSeCH3,
eCH2CO), 1.67-1.60 (m, 4H, eCH2eCH2); 13C NMR (CDCl3, 75 MHz)
incubated for 4 h at 37 ꢀC. These cells were centrifuged at 2000 rpm
for 5 min, supernatant was removed and resultant formazan
formed in live PBMC was dissolved in DMSO (100 mL) and absor-
d
173 (2C), 148 (2C), 136.5, 121.6, 109.3, 108.6, 101.2, 53, 52, 36.9, 35.8,
bance was read at OD492 nm. The mean percentage of post treat-
ment viable cells was calculated relative to control (unexposed to
drugs).
32.3, 31.7, 30.5, 25.5, 16; CIMS: m/z 368 [Mþ1]þ
4.2.5.5. Tetrahydropiperoyl-L-tryptophan methyl ester (17). Brown
oil; yield 89%; IR (KBr) ymax 3402, 3306, 1738, 1657, 1652, 1501, 1245,
4.3.4. In vivo testing
1038 cmꢃ1
;
1H NMR (CDCl3, 300 MHz)
d
8.51 (NH), 7.51-6.04 (8H,
Male Golden Syrian Hamsters, weighing 50-60 g were obtained
from Central Animal Facility, National Institute of Pharmaceutical
Education and Research (NIPER). Animals were housed in a room at
a temperature of 24 ꢂ 2 ꢀC and 12 h dark and light cycle. Standard
diet and water were allowed ad libitum. All the procedures per-
formed in the study were approved by Institutional Animal Ethics
Committee (IAEC), NIPER.
Ar-H), 5.87 (s, 2H, OCH2O), 4.96-4.90 (m, 1H, NHeCHeCO), 3.66 (s,
3H, COOCH3), 3.35-3.24 (m, 2H, eCH2e3-Indolyl), 2.47-2.43 (m, 2H,
CH2Ar), 2.13-2.09 (m, 2H, eCH2CO),1.56-1.49 (m, 4H, eCH2eCH2e);
13C NMR (CDCl3, 75 MHz)
d 173.2, 173.1, 148, 146, 136.7, 136.6, 128.2,
123.3, 122.7, 121.6, 120.1, 119, 111.9, 110.4, 109.4, 108.6, 101.2, 53.4,
52.9, 36.8, 35.8, 31.6, 28.1, 25.4; CIMS: m/z 423 [Mþ1]þ
Visceral leishmaniasis (VL) was induced by intracardiac injec-
tion of L. donovani amastigotes (1ꢁ107) in golden hamsters. After 20
days of infection, hamsters were administered compounds by
intraperitoneal route at 250 mg/kg for a period of ten days.
Hamsters were sacrificed next day after the last dose for spleen
weight and spleen parasitic burden. Spleen parasitic burden was
assessed from geimsa stained impression smears [23]. Compounds
were dissolved in 50% DMSO and vehicle treated control group
received DMSO. Miltefosine was used as standard antileishmanial
drug.
4.3. Antileishmanial activity
4.3.1. In vitro promastigote assay
Antileishmanial activity of the compounds was tested in vitro
using modified MTT ([3-(4, 5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide]) assay [21] against a culture of L. donovani
promastigotes grown in phenol red free RPMI-1640 (Sigma, USA),
supplemented with 10% FCS (Sigma, USA) at 26 ꢀC. L. donovani
(5ꢁ105 cells/mL) promastigotes from logarithmic phase culture
were grown in 96 well plate for 48 h before treatment with drugs.
Drug dilutions were prepared in DMSO and concentration (100-
4.3.5. Statistical analysis
500
m
g/mL) of each drug was used in triplicate. The standard mil-
Data on spleen weight and parasitic burden are expressed as
mean ꢂ SEM. Data was analyzed using one way analysis of variance
followed by Dunnett’s test. Differences were considered to be
significant if p<0.05. Statistical analysis was performed using Sigma
Stat 2 statistical software.
tefosine was used at reported IC50 value. After treatment with drugs
plate was kept at 26 ꢀC for 48 h. MTT to a final concentration of
400
were centrifuged at 2000 rpm for 5 min, supernatant was removed
m
g/mL was added and incubated for 4 h at 37 ꢀC. These cells

I.P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 3439e3445
3445
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Authors acknowledge financial support from International
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agreement no. F/3971-1. dt 2005/12/19). Authors acknowledge Mr
Jung Bahadur for assistance with animal experiments and Mr
Rakesh Kumar for isolation of piperine.
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