Antileishmanial lead structures from nature: Analysis of structure-activity relationships of a compound library derived from caffeic acid bornyl ester

Article abstract of DOI:10.3390/molecules19021394

Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC₅₀ = 48.8 μM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization.

Full text of DOI:10.3390/molecules19021394

Molecules 2014, 19, 1394-1410; doi:10.3390/molecules19021394
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antileishmanial Lead Structures from Nature: Analysis of
Structure-Activity Relationships of a Compound Library
Derived from Caffeic Acid Bornyl Ester
Jan Glaser 1, Martina Schultheis 2, Sudipta Hazra 3, Banasri Hazra 3, Heidrun Moll 2,
Uta Schurigt 2,* and Ulrike Holzgrabe 1,*
1
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, Würzburg 97074,
Germany; E-Mail: jan.glaser@uni-wuerzburg.de
2
Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str. 2,
Würzburg 97080, Germany; E-Mails: martina.schultheis@uni-wuerzburg.de (M.S.);
heidrun.moll@uni-wuerzburg.de (H.M.)
3
Department of Pharmaceutical Technology, Jadavpur University, Kolkata-700032, India;
E-Mails: sudipta_manu@rediffmail.com (S.H.); banasrihazra@yahoo.co.in (B.H.)
* Authors to whom correspondence should be addressed;
E-Mails: uta.schurigt@uni-wuerzburg.de (U.S.); u.holzgrabe@pharmazie.uni-wuerzburg.de (U.H.);
Tel.: +49-931-318-4671 (U.S.); +49-931-318-5461 (U.H.).
Received: 23 December 2013; in revised form: 13 January 2014 / Accepted: 13 January 2014 /
Published: 27 January 2014
Abstract: Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii
(V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester
(1) as the active component against Leishmania major (L. major) promastigotes
(IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of
compounds based on 1 was synthesized and tested in vitro against L. major and
L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a
murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some
compounds showed antileishmanial activity in the concentration range of pentamidine and
miltefosine which are the standard drugs in use. In the L. major amastigote assay
compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against
BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic
hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM.
The Michael system seems not to be essential for antileishmanial activity. Based on the
results compound 27 can be regarded as new lead structure for further structure optimization.

Molecules 2014, 19
Keywords: Valeriana wallichii; leishmaniasis; caffeic acid bornyl ester; structure-activity
1395
relationship
1. Introduction
Leishmaniasis is one of the most dreadful infectious diseases worldwide. It is endemic in 98 countries
and almost 350 million people on five continents are at risk of infection [1,2]. The disease has an
especially high impact on the population of the disadvantaged regions of South America, Africa and
Asia (especially India), all of them suffering from poverty and low health care standards.
Leishmaniasis is caused by protozoan parasites, which are transmitted by the bite of the sand fly. It
comprises a variety of diseases. Cutaneous leishmaniasis mostly caused by L. major produces skin
sores, ulcers and eventually heals by leaving defacing scars. The most severe form is the visceral
leishmaniasis also known as kala-azar or black fever. Here the parasites invade internal organs,
especially the liver and the spleen. This is fatal if left untreated. The species known to cause visceral
leishmaniasis is L. donovani which is prevalent in India and East Africa. 70% of the global burden of
kala-azar is concentrated in India [1,3]. The estimated incidence of visceral leishmaniasis in India is
146,700 to 282,800 cases per year [1].
The infection can be treated but the drugs in use like miltefosine or amphotericin B show severe
side effects. Additionally these drugs are expensive and require adequate medicinal care which is not
readily available in the most affected regions of India or Africa. Therefore, the need for new
antileishmanial drugs is evident. As reported previously the chloroform extract of V. wallichii roots
showed antileishmanial activity [4]. In this paper we describe the isolation and structure elucidation of
one active ingredient, which was assigned to be caffeic acid bornyl ester (1). In order to analyse
structure-activity relationship for activity optimization a library of derivatives has been synthesized.
The respective cytotoxicity against diverse species of Leishmania promastigotes, amastigotes, and
macrophages was determined.
2. Results and Discussion
2.1. Isolation
The crude chloroform extract obtained from the pulverized rhizomes of V. wallichii was subjected
to bioactivity-guided fractionation. The extract was roughly fractionated into twelve fractions by
means of semi-preparative HPLC using reverse phase material and a gradient of H2O/MeOH as the
eluent. The most active fraction was repeatedly partitioned by normal phase silica gel column
chromatography with CHCl3/MeOH (4.8:0.2 v/v) as mobile phase. From a relatively polar sub-fraction
with an IC50 of 11.8 µg/mL against L. major promastigotes a pure compound could be isolated.
1H- NMR data showed two doublets for vinyl protons at 6.3 and 7.6 ppm, respectively, with the typical
coupling constants of 15.9 Hz indicating a double bond with trans configuration. Signals for three
aromatic protons with a characteristic coupling pattern suggested meta and para substitution in relation
to the side chain. Characteristic for the borneol moiety were the three -CH3 singlets at 0.90, 0.95 and

Molecules 2014, 19
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0.99 ppm respectively. ESIMS data confirmed the assumed ester structure with two hydroxyl groups as
substituents on the aromatic ring by giving a mass of m/z 315 [M−H]−. The obtained spectroscopic data
are in accordance to literature data [5–7] for caffeic acid bornyl ester. Similar bornyl esters have been
found in Conocephalum conicum (liverwort) [8,9] and some Verbesina species [7,10,11].
2.2. Chemistry
Since the caffeoyl-skeleton has been described to be a valuable pharmacophore for antiviral [5,12],
antibacterial [13] and antiprotozoal activity [14] we tried to enhance the antileishmanial activity and to
decrease the cytotoxicity by the synthesis of a library of derivatives of 1 with systematic structure
variations with regard to the terpenoid part on the one hand and the caffeic acid part on the other hand.
To investigate structure-activity relationship borneol was replaced by moieties varying in size and
bulkiness, and substituents with different properties such as hydroxyl, methoxy and nitro groups as
well as halogen atoms were attached to the aromatic ring in varying positions. The general synthesis of
phenolic compound 1 and its derivatives 2–13 via Knoevenagel-Doebner condensation is outlined
in Scheme 1.
Scheme 1. Preparation of compounds 1–13 (substituents are listed in Table 1).
Activation of the corresponding alcohol with Meldrum’s acid without isolation of the activated
product and subsequent aldol condensation using the substituted aldehyde lead to the cinnamic acid
derivatives 1–13 in acceptable yields [5]. Compounds 14–26 were accessible by facile esterification
using the Steglich concept composed of N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethyl-
aminopyridine (DMAP) in CHCl3 or THF (Scheme 2).
Scheme 2. Preparation of compounds 14–26 (substituents are listed in Table 2).
The α,β-unsaturated carbonyl moiety of the caffeic acid is a Michael acceptor which is highly
reactive to nucleophilic attacks, e.g., by amino or thiol groups. To investigate whether the double bond
is essential for antileishmanial activity compound 27 has been synthesized via a different synthesis
route as described before [15]. By esterification of 3-phenyl propanoic acid with borneol using tosyl
chloride, Et3N and DMAP as a catalyst (Scheme 3) product 27 was obtained.

Molecules 2014, 19
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Scheme 3. Preparation of compound 27.
It might be possible that the catechol moiety of compound 1 contributes to cytotoxicity or
antileishmanial activity. Hence both phenolic hydroxyl groups were acetylated by adding acetic acid
anhydride to caffeic acid bornyl ester 1 (isolated from the plant) in pyridine at 0 °C and stirring for 24 h
at room temperature (Scheme 4) to give compound 28.
Scheme 4. Preparation of compound 28.
2.3. Structure-Activity Relationship Investigation
Antileishmanial activities of the synthesized compounds were evaluated in L. major promastigotes as
described before [16] and in L. donovani promastigotes according to Hazra et al. [17]. Cytotoxicity was
determined on a J774.1 murine cell line [16]. The antiprotozoal activities are presented in Tables 1–3 as
inhibitory concentrations (IC50). The most active compound is the cinnamic acid bornyl ester 15
having an IC50 value of 39.6 µM against L. major and 15.6 µM against L. donovani promastigotes,
which is in the concentration range of pentamidine and miltefosine; both are drugs currently in use.
2.3.1. Influence of the Bornyl Moiety
The bornyl moiety is of particular importance of preserving antileishmanial activity. The most
active compounds 1, 7, 15 and 27 are all esters of borneol. There is no significant change in activity
using isoborneol (1) instead of borneol (7). Substituting borneol with a less sterically demanding
alcohol e.g., cyclohexanol (23) or eugenol (24) gives IC50 values higher than 100 µM (L. major).
Using thymol (10, 11, 19), menthol (12, 13, 20) or other bulky substituents like naphthol (22) or
adamantol (21) preserves the activity. A similar pattern is found with L. donovani promastigotes.
Again the bornyl esters show the best IC50 values, and the activity is at a similar level using thymol,
menthol or naphthol. In contrast to L. major some of the sterically less demanding compounds exhibit
activity against L. donovani promastigotes (e.g., 23.4 μM (23), 41.9 μM (26)). This might be due to
biological differences between the two species. However borneol seems to be the most advantageous
substituent fulfilling the requirement of bulkiness in this position.

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2.3.2. The Catechol Moiety
The caffeic acid derivative (1) isolated from V. wallichii having two hydroxyl groups in position 3
and 4 of the aromatic ring shows an IC50 value of 48.8 μM against L. major promastigotes and
relatively high cytotoxicity (8.3 μM) against a J774.1 cell line. The toxicity is an attribute of all meta
and para dihydroxylated cinnamic acid derivatives (7, 10, 12) and has been reported in the literature
for similar compounds [14,18,19]. The catechol moiety is prone to oxidation resulting in an o-quinone
which can easily react with amino groups of proteins [20]. Hence, omitting the hydroxyl groups lead to
15 with antileishmanial activity in the same concentration range as 1 but with a fivefold lower cytotoxicity.
Table 1. Antileishmanial activity and cytotoxicity (compounds 1–13).
IC50 (μM) *
L. donovani
promastigotes promastigotes
Compd.
R1
R2
R3
L. major
J774.1
1
2
3
4
5
-OH
-OH
48.8
64.4
27.3
41.3
8.3
-OH
-H
-OCH3
-Cl
48.7
49.5
54.6
>100
>100
71.2
>100
>100
>100
>100
-H
-Br
>100
>100
>100
-H
-N(CH3)2
6
7
8
-H
-OH
-OH
-OH
45.8
60.6
34.8
74.5
8.8
-OCH3
44.3
9
-H
-NO2
-OH
>100
57.6
>100
42.1
>100
9.5
10
-OH
11
-OH
-OCH3
59.8
79.6
45.6
-OH
-OH
-OH
59.5
54.2
79.4
1.95
44.6
12
13
-OCH3
>100
* Positive control: pentamidine 82 μM (L. major), 38.6 μM (J774.1); miltefosine: 36.2 μM (L. major),
56.5 μM (J774.1); amphotericin B 0.4 μM (L. donovani).

Molecules 2014, 19
Table 2. Antileishmanial activity and cytotoxicity (compounds 14–26 and 28).
1399
IC50 (µM) *
L. donovani
promastigotes promastigotes
Compd.
R1
R2
R3
R4
R5
L. major
J774.1
14
15
16
17
18
-H
-H
-H
-H
-H
-H
-H
-Cl
-H
-H
-H
-H
-Cl
-H
-H
-Cl
-H
65.6
39.6
60.9
80.5
>100
15.6
58.9
45.2
42.6
60.6
-OCH3
-Cl
>100
>100
-H
64.3
51.2
46.0
19
-H
-H
-H
-H
-H
-H
-H
-H
55.0
53.1
>100
79.7
44.8
44.8
20
21
-H
-H
-H
-H
-H
-H
-H
-H
60.0
67.0
>100
28.7
46.7
44.5
22
23
-H
-H
-H
-H
>100
23.4
>100
24
25
-H
-H
-H
-H
-H
-H
-H
-H
>100
>100
>100
80.3
>100
32.2
-H
-H
-H
-H
-H
-H
26
28
>100
30.7
41.9
-
>100
2.1
-OAc
-OAc
* Positive control: pentamidine 82 μM (L. major), 38.6 μM (J774.1); miltefosine: 36.2 μM (L. major),
56.5 μM (J774.1); amphotericin B 0.4 μM (L. donovani).

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The same effect is observed by replacement of the hydroxyl group in 4-position with a methoxy
group (compounds 2, 8, 11, 13). However, the cytotoxicity is not correlating with antileishmanial
activity, the latter stays in the same concentration range for compounds whether with or without
hydroxyl groups. Acetylation to “mask” the catechol structure (compound 28) slightly increased
antileishmanial activity but cytotoxicity persisted in the single-digit micromolar range.
2.3.3. Substitution on the Aromatic Ring
In order to analyse the influence of the substituents of the aromatic ring on antileishmanial activity
and cytotoxicity, bornyl esters are compared in the following. Compound 15 with an unsubstituted
benzene ring is the most promising compound with an antileishmanial activity in a low micromolar
concentration and low cytotoxicity. The comparison of all derivatives with substituents in para
position reveals compounds 3 (-Cl) and 16 (-OMe) to have minor antileishmanial activity against
L. major (71.2 μM and 60.9 μM). Larger substituents are not tolerated in this location
(compounds 4–6, 9) whereas chlorination in position 2 and 3 (compound 14), and 2 and 4 (compound 17),
respectively, is acceptable. None of these compounds exhibited any activity against L. donovani
promastigotes. Thus, a non-substituted aromatic ring is advantageous for a good antiprotozoal activity
against both species.
2.3.4. Michael System
All compounds are characterized by the highly reactive enone Michael system, which is prone to
unspecific covalent reactions with proteins of both parasite and host. In order to check whether the
Michael moiety is necessary for antileishmanial activity, compound 27 characterized by a simple
carbonyl group was synthesized. This compound is as active as the corresponding compound 15 and
did not show cytotoxicity (Table 3). This is in contrast to the results observed for caffeic acid alkyl
ester derivatives which were found to be inactive without the double bond [14].
Table 3. Antileishmanial activity of compounds 15 and 27.
L. major
promastigotes
IC50 (µM)
Cytotoxicity
J774.1
Selectivity index
SI a
Compound
IC50 (µM)
39.6
45.2
1.1
50.2
>100
>2.0
a SI = IC50 for J774.1/IC50 for L. major.
2.4. Amastigote Results
The amastigote is the intracellular pathogenic form of the parasite in the vertebrate host and
therefore the important target. Compounds 1, 2, 7, 10, 15, 19, 20 showing high activity against

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L. major promastigotes were selected for screening against L. major amastigotes and cytotoxicity
against BMDM. The amastigote assay was conducted as reported by Bringmann et al. [16] and the
results are presented in Table 4. The cytotoxicity against BMDM mirrors those against the
macrophages J774.1. With regard to antileishmanial activity cinnamic acid menthyl (20) and thymyl
ester (19) show good activities combined with low cytotoxicity. Again the cinnamic acid bornyl ester
(15) is the most active compound. In general, the activity lies in the range of miltefosine with an IC50
value of 33.0 μM. Based on these results, the compounds should be tested for in vivo activity against
Leishmania infections.
Table 4. Antileishmanial activity and cytotoxicity of selected compounds.
L.major
amastigotes
IC50 (µM)
47.6
Cytotoxicity
BMDM
IC50 (µM)
5.3
Selectivity index
SI a
Compound
1
0.1
0.2
7
47.0
10.9
15
10.9
54.3
5.0
2
39.2
49.0
1.2
19
20
49.1
>148
>3.0
>6.4
0.2
19.5
>126
10
54.1
9.8
Miltefosine
33.0
65.5
2.0
a SI = IC50 for BMDM/IC50 for L. major.
3. Experimental
3.1. General Procedures
Starting materials and reagents were purchased from Sigma-Aldrich (Taufkirchen, Germany) and
VWR (Darmstadt, Germany). Solvents were of synthetic or analytical grade. Melting points were
determined on a Stuart melting point apparatus SMP10 (Bibby Scientific, Stone, United Kingdom) and
are uncorrected. Optical rotations were measured on a CHIRALYSER 6.3 (IBZ Messtechnik, Springe,
Germany). IR spectra were acquired on a JASCO (Gross-Umstadt, Germany) FT/IR-6100 Fourier
1
Transformation Infrared Spectrometer equipped with an ATR unit. H (400.132 MHz) and 13C
(100.613 MHz) NMR spectra were recorded on a Bruker Avance 400 Ultra Shield™ (Bruker Biospin,
Ettlingen, Germany) spectrometer. The signals of the deuterated solvents were used as internal standards
(CDCl3: 1H 7.26 ppm, 13C 77.0 ppm; MeOD: 1H 4.84 ppm, 13C 49.05 ppm). LC/MS was conducted on an
Agilent 1100 analytical HPLC with DAD detection and an Agilent LC/MSD Trap (Agilent Technologies,
Böblingen, Germany). ESIMS data was conducted in positive and negative mode. For LC/MS, following
conditions were used: Nucleodur Sphinx RP-18 (Macherey-Nagel, Düren, Germany, 150 mm × 4.6 mm,
5 µm, A) CH3CN + 0.1% FA, B) H2O + 0.1% formic acid (FA), gradient: 10% B (0–2 min), 30% B
(2–3 min), 100% B (3–25 min), 80% B (25–33 min), 40% B (33–37 min), 10% B (38–40 min),
0.8 mL/min, MS-detection: ESI, nebulizer pressure: 50 psi, drying gas: 10 L/min, drying gas
temperature: 350 °C, capillary voltage: 3500 V. An Agilent 1100 preparative HPLC with fraction
collector and multiple wavelength detector (MWD) was used for fractionation of the extract.

Molecules 2014, 19
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3.2. Plant Material and Preparation of Extract
The plant material was obtained as described previously [4]. The rhizomes were pulverized and a
portion of 10 g was refluxed for 2 h with 100 mL chloroform. Evaporation of the solvent yielded a
black syrup with a characteristic smell.
3.3. Bioactivity-Guided Fractionation and Isolation of 1
The crude chloroform extract (10.9 g) was subjected to a bioactivity-guided fractionation (Figure 1).
The extract was slurried in MeOH and the insoluble parts were removed by filtration over
LiChroprep© RP-18 material (Yield: 9.5 g).
Figure 1. Overview of bioactivity-guided fractionation.
The extract was subjected to semi-preparative HPLC (250 mm × 10 mm, 5 µm, Macherey-Nagel
Nucleosil 100-5; (A) H2O, (B) MeOH, gradient: 70% B (0 min), 75% B (7 min), 100% B (25 min),
70% B (30 min); flow rate: 3.3 mL/min) and partitioned into 12 fractions. Fraction 4–8 exhibited
significant antileishmanial activity against L. major promastigotes. Fraction 5 (559 mg) was further
fractionated by column chromatography using silica gel and MeOH/CHCl3 (4.8:0.2 v/v) as mobile
phase. Fraction 5β3 (258 mg) was partitioned a second time by column chromatography using silica gel
and MeOH/CHCl3 (4.8:0.2 v/v) to yield three fractions (5β3a, 5β3b, 5β3c) from which the last fraction
afforded 135 mg of caffeic acid (−)-bornyl ester (IC50 = 48.8 µM). The structure was identified by
means of NMR and LC/MS data.
Caffeic Acid (−)-bornyl Ester (1). Brownish solid (MeOH); m.p. 150 °C (lit.[6] 148–151 °C); [α]426
1
−5.1° (c 0.1, MeOH); IR: 3444, 3165, 1661, 1604, 1273, 1186 cm−1; H-NMR (MeOD) δ (ppm)
J (Hz): 0.90 (s; 3H); 0.94 (s; 3H); 0.98 (s; 3H); 1.06 (dd; 3.7; 13.7; 1H); 1.26–1.35 (m;2H); 1.70 (t;
4.5; 1H); 1.79–1.88 (m; 1H); 2.07–2.13 (m; 1H); 2.38–2.46 (m; 1H); 5.00 (ddd; 2.2; 3.4; 9.9; 1H); 6.30

Molecules 2014, 19
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(d; 15.9; 1H); 6.80 (d; 8.2; 1H); 7.00 (dd; 2.1; 8.2; 1H); 7.05 (d; 2.1; 1H); 7.54 (d; 15.9; 1H); 13C-NMR
(MeOD) δ (ppm) 13.9; 19.2; 20.1; 28.2; 29.0; 37.9; 46.4; 48.9; 49.5; 81.2; 115.1; 115.5; 116.5; 122.9;
127.8; 146.7; 146.8; 149.6; 169.7; ESIMS: m/z 315 [M−H]−, 339 [M+Na]+.
3.4. Synthesis
3.4.1. General Procedure for the Synthesis of Compounds 1–13
Synthesis was conducted according to the method of Xia et al. [5]. The alcohol (2.94 mmol) and
Meldrum’s acid (2.94 mmol) were dissolved in toluene (20 mL) and refluxed for 4 h. To the resulting
activated alcohol the corresponding aldehyde (2.94 mmol), pyridine (2.5 mL) and piperidine (250 µL)
were added. This mixture was stirred at room temperature for 24–48 h. The solvent was evaporated,
the residue dissolved in diethyl ether (20 mL) and washed three times with an aqueous saturated
NaHCO3 solution (10 mL), 20% HCl (10 mL), and H2O (10 mL), respectively. After drying over
MgSO4 the solvent was evaporated and the residue was chromatographed using silica gel (petroleum
ether/ethyl acetate 60:40) to yield the desired product (overall yields of the two steps are reported).
Synthesized Caffeic Acid (−)-bornyl Ester (1). Yield 19%; off-white solid; m.p. 150 °C; [α]426 −4.5° (c
0.1, MeOH); IR and NMR data in accordance with above and literature [6].
Isoferulic Acid (−)-bornyl Ester (2). Yield 9%; colorless syrup; IR: 3384, 2952, 2877, 1698, 1631,
1
1263, 1172, 1155 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz): 0.88 (s; 3H); 0.90 (s; 3H); 0.95 (s; 3H);
1.04–1.08 (m; 1H); 1.25–1.32 (m; 2H); 1.71 (t 4.5; 1H); 1.75–1.85 (m; 1H); 2.02–2.09 (m; 1H);
2.38–2.46 (m; 1H); 3.94 (s; 3H); 5.02 (ddd; 2.1; 3.4; 9.9; 1H); 6.30 (d; 16.0; 1H); 6.92 (d; 8.2; 1H); 7.05 (d;
13
1.9; 1H); 7.09 (dd; 8.2; 1.9; 1H); 7.60 (d; 16.0; 1H); C-NMR (CDCl3) δ (ppm): 13.6; 18.9; 19.7; 27.3;
28.1; 36.9; 45.0; 47.9; 48.9; 56.0; 79.8; 109.3; 114.7; 116.2; 123.0; 127.1; 144.3; 146.7; 147.8; 167.6.
4-Chlorocinnamic Acid (−)-bornyl Ester (3). Yield 14%; slightly yellow solid; m.p. 80–83 °C; IR:
2952, 2876, 1704, 1636, 1490, 1308, 1184, 820 cm−1; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.88 (s; 3H);
0.90 (s; 3H); 0.94 (s; 3H); 1.03–1.08 (m; 1H); 1.25–1.32 (m; 2H); 1.71 (t; 4.6; 1H); 1.74–1.84 (m; 1H);
2.01–2.06 (m; 1H); 2,38–2.46 (m; 1H); 5.02 (ddd; 2.2; 3.4; 10.0; H); 6.44 (d; 16.0; 1H); 7.36 (d; 8.5;
13
2H); 7.47 (d; 8.5; 2H); 7.61 (d; 16.0; 1H); C-NMR (CDCl3) δ (ppm): 13.5; 18.9; 19.7; 27.2; 28.1;
36.9; 45.0; 47.9; 49.0; 80.2; 119.4; 129.1 (2C); 129.2 (2C); 133.1; 136.0; 142.7; 167.1.
4-Bromocinnamic Acid (−)-bornyl Ester (4). Yield 9%; yellow solid; m.p. 71–74 °C; IR: 2953, 2875,
1705, 1635, 1203, 1158, 816 cm−1; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.88 (s;3H); 0.90 (s;3H); 0.94 (s;
3H); 1.03–1.08 (m; 1H); 1.25–1.32 (m;2H); 1.71 (t; 4.6; 1H); 1.74–1.84 (m;1H); 2.01–2.06 (m;1H);
2.38–2.46 (m; 1H); 5.03 (ddd; 2.2; 3.4; 9.9; 1H); 6.44 (d; 16.0; 1H); 7.36 (d; 8.5; 2H); 7.47 (d; 8.5;
13
2H); 7.61 (d; 16.0; 1H); C-NMR (CDCl3) δ (ppm): 13.5; 18.9; 19.7; 27.2; 28.1; 36.9; 45.0; 47.9;
48.9; 80.2; 119.4; 124.4; 129.4 (2C); 132.1 (2C); 133.4; 142.7; 167.1.
4-N-Dimethylaminocinnamic Acid (−)-bornyl Ester (5). Yield 25%; yellowish solid; m.p. 114–116 °C;
1
IR: 2950, 2877, 1696, 1603, 1524, 1151, 810 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz): 0.88 (s; 3H);
0.90 (s; 3H); 0.94 (s; 3H); 1.04–1.08 (m; 1H); 1.25–1.37 (m; 2H); 1.70 (t; 4.5; 1H); 1.74–1.84 (m; 1H);

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2.03–2.10 (m; 1H); 2.37–2.45 (m; 1H); 3.02 (s; 6H); 5.01 (ddd; 2.0; 3.3; 9.9; 1H); 6.25 (d; 15.9; 1H);
6.67 (d; 8.8; 2H); 7.43 (d; 8.8; 2H); 7.61 (d; 15.9; 1H); 13C-NMR (CDCl3) δ (ppm): 13.6; 18.9; 19.8; 27.3;
28.1; 36.9; 40.1 (2C); 45.0; 47.8; 48.9; 79.4; 111.8 (2C); 113.2; 123.0; 129.7 (2C); 144.7; 151.7; 168.2.
4-O-Benzoylcinnamic Acid (−)-bornyl Ester (6). Yield 13%; lightly yellow solid; m.p. 93–95 °C; IR:
2951, 2878, 1695, 1627, 1600, 1510, 1255, 1171, 998 cm−1; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.88 (s;
3H); 0.90 (s; 3H); 0.95 (s; 3H); 1.04–1.08 (m; 1H); 1.25–1.37 (m; 2H); 1.70 (t; 4.5; 1H); 1.73–1.84 (m;
1H); 2.02–2.08 (m; 1H); 2.37–2.45 (m; 1H); 5.01 (ddd; 2.0; 3.4; 9.9; 1H); 5,10 (s; 2H); 6.34 (d; 16.0;
1H); 7.00 (d; 8.7; 2H); 7.34–7.44 (m; 5H); 7.49 (d; 8.7; 2H); 7.60 (d; 16.0; 1H); 13C-NMR (CDCl3) δ
(ppm): 13.5; 18.9; 19.7; 27.2; 28.1; 36.9; 45.0; 47.8; 48.9; 70.1; 79.8; 115.2 (2C); 116.5; 127.4 (2C);
127.5; 128.0; 128.7 (2C); 129.7 (2C); 136.5; 143.8; 160.4; 167.6.
Caffeic Acid Isobornyl Ester (7). Yield 12%; brownish solid; m.p. 160–161 °C; IR: 3444, 3168, 2956,
1666, 1604, 1439, 1277, 1182 cm−1; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.86 (s; 3H); 0.89 (s;3H); 1.05
(s; 3H); 1.09–1.25 (m; 2H); 1.55–1.62 (m; 1H); 1.68–1.88 (m; 4H); 4.80 (dd; 4.6; 6.9; 1H); 6.23 (d;
13
15.9; 1H); 6.87 (d; 8.2; 1H); 6.99 (dd; 1.7; 8.2; 1H); 7.12 (d; 1.7; 1H); 7.53 (d; 15.9; 1H); C-NMR
(CDCl3) δ (ppm) 11.2; 19.7; 19.8; 26.7; 33.4; 38.5; 44.8; 46.7; 48.6; 81.2; 114.1; 115.2; 115.7; 122.1;
127.1; 143.6; 144.5; 146.3; 167.4.
Isoferulic Acid Isobornyl Ester (8). Yield 11%; colourless syrup; IR: 3387, 2952, 2876, 1696, 1631,
1591, 1512, 1263, 1154 cm−1; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.87 (s;3H); 0.90 (s;3H); 1.07 (s;3H);
1.09–1.26 (m;2H); 1.54–1.61 (m;1H); 1.68–1.90 (m;4H); 3.94 (s;3H); 4.80 (dd;4.3;7.4;1H); 6.25
(d;15.9;1H); 6.91 (d;8.2;1H); 7.02 (d;1.9;1H); 7.07 (dd;1.9;8.2;1H); 7.55 (d;15.9;1H); 13C-NMR
(CDCl3) δ (ppm) 11.5; 20.0; 20.1; 27.0; 33.8; 38.9; 45.1; 47.0; 48.9; 56.0; 81.0; 109.3; 114.7; 116.3;
123.0; 127.1; 144.3; 146.8; 147.8; 167.0.
4-Nitrocinnamic Acid Isobornyl Ester (9). Yield 16%; yellowish solid; m.p. 121–123 °C; IR: 2950,
1
2876, 1700, 1516, 1340, 1301, 1163, 842 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz): 0.87 (s; 3H); 0.90 (s;
3H); 1.06 (s; 3H); 1.07–1.25 (m; 2H); 1.54–1.61 (m; 1H); 1.70–1.92 (m; 4H); 4.83 (dd; 4.1; 7.5; 1H); 6.53
13
(d; 16.0; 1H); 7.65 (d; 16.0; 1H); 7.67 (d; 8,7; 2H); 8.24 (d; 8.7; 2H); C-NMR (CDCl3) δ (ppm) 11.5;
20.0; 20.1; 27.0; 33.8; 38.8; 45.1; 47.0; 49.0; 81.8; 123.2; 124.1 (2C); 128.6 (2C); 140.7; 141.3; 165.6.
Caffeic Acid Thymyl Ester (10). Yield 14%; brownish solid; m.p. 115–117 °C; IR: 3339, 2960, 1726,
1
1695, 1616, 1514, 1235, 1136, 1114 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz): 1.20 (s; 3H); 1.21 (s;
3H); 2.32 (s; 3H); 3.03 (sept; 6.9; 1H); 5.84 (br s; -OH); 5.89 (br s; -OH); 6.48 (d; 15.9; 1H); 6.87 (d;
8.2; 1H); 6.87 (d; 0.9; 1H); 7.04 (dd; 0.9; 7.9; 1H); 7.07 (dd; 2.0; 8.2; 1H); 7.22 (d; 7.9; 1H); 7.77 (d;
15.9; 1H); 13C-NMR (CDCl3) δ (ppm) 20.5; 22.7 (2C); 26.9; 114.2; 114.4; 115.2; 122.4; 122.5; 126.2;
126.9; 127.0; 136.3; 136.9; 143.5; 146.3; 146.4; 147.6; 166.2.
1
Isoferulic Acid Thymyl Ester (11). Yield 15%; colourless syrup; IR according to ref. [21]; H-NMR
(CDCl3) δ (ppm) J (Hz): 1.21 (s; 3H); 1.23 (s; 3H); 2.32 (s; 3H); 2.34 (s; 3H); 3.05 (sept; 6.9; 1H);
3.96 (s; 3H); 6.52 (d; 15.9; 1H); 6.89 (d; 0.9; 1H); 6.96 (d; 8.2; 1H); 7.05 (dd; 0.9; 7.9; 1H); 7.11 (d;
13
1.9; 1H); 7.12 (dd; 1.9; 8.2; 1H); 7.23 (d; 7.9; 1H); 7.81 (d; 15.9; 1H); C-NMR (CDCl3) δ (ppm)

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21.2; 23.4 (2C); 27.5; 56.3; 109.8; 114.9; 115.1; 123.2; 123.7; 126.7; 127.1; 127.4; 136.8; 137.5;
146.8; 147.1; 148.3; 148.6; 166.3.
Caffeic Acid Menthyl Ester (12). Yield 25%; colourless syrup; IR: 3384, 2953, 2868, 1694, 1632,
1
1591, 1511, 1264, 1170 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz): 0.78 (d; 6.9; 3H); 0.90 (d; 2.7; 3H);
0.90 (d; 2.7; 3H); 1.00–1.15 (m; 2H); 1.42–1.55 (m; 2H); 1.69–1.72 (m; 2H); 1.87–1.95 (m; 1H);
2.03–2.06 (m; 1H); 4.8 (dt; 4.4; 10.9; 1H); 6.25 (d; 15.9; 1H); 6.87 (d; 8.2; 1H); 6.99 (dd; 1.9; 8.2;
13
1H); 7.11 (d; 1.9; 1H); 7.57 (d; 15.9; 1H); C-NMR (CDCl3) δ (ppm) 16.8; 21.1; 22.4; 24.0; 26.8;
31.8; 34.7; 41.4; 47.6; 75.1; 114.8; 115.8; 116.2; 122.7; 127.8; 144.4; 145.4; 147.0; 168.2.
Isoferulic Acid Menthyl Ester (13). Yield 24%; colourless syrup; IR: 3398, 2953, 2926, 2868, 1695,
1513, 1263, 1158 cm−1; 1H-NMR (CDCl3) δ (ppm) J (Hz) 0.79 (d; 6.9; 3H); 0.91 (d; 2.9; 3H); 0.92 (d;
2.6; 3H); 1.00–1.16 (m; 2H); 1.40–1.55 (m; 2H); 1.67–1.73 (m; 2H); 1.90–1.99 (m; 1H); 2.03–2.09
(m; 1H); 3.93 (s; 3H); 4.82 (dt; 4.4; 10.9; 1H); 6.28 (d; 15.8; 1H); 6.91 (d; 8.2; 1H); 7.04 (d; 1.8; 1H);
13
7.07 (dd; 1.8; 8.2; 1H); 7.60 (d; 15.8; 1H); C-NMR (CDCl3) δ (ppm) 16.4; 20.8; 22.0; 23.6; 26.3;
31.4; 34.3; 41.1; 47.3; 55.9; 74.1; 109.2; 114.7; 116.1; 123.0; 127.1; 144.4; 146.7; 147.8; 166.8.
3.4.2. General Procedure for the Synthesis of Compounds 14–27
Cinnamic acid (500 mg, 3.37 mmol) and the respective alcohol (3.37 mmol) were dissolved in THF or
CHCl3 (20 mL). After addition of DCC and DMAP the resulting mixture was stirred for 16 h at room
temperature. After evaporation and column chromatography on silica gel (CHCl3/MeOH 4.8:0.2) the
desired product was obtained.
2,3-Dichlorocinnamic acid (−)-bornyl ester (14). Yield 18%; colourless crystals; m.p. 86–87 °C; IR:
1
2930, 2875, 2118, 1711, 1635, 1315, 1178 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz) 0.89 (s; 3H); 0.90
(s; 3H); 0.95 (s; 3H); 1.05–1.09 (m; 1H); 1.25–1.38 (m; 2H); 1.72 (t; 4.5; 1H); 1.75–1.83 (m; 1H);
2.00–2.06 (m; 1H); 2.39–2.47 (m; 1H); 5.03 (ddd; 2.2; 3.4; 9.9; 1H); 6.44 (d; 16.0; 1H); 7.22 (t; 7.9;
13
1H); 7.48 (dd; 1.5; 7.9; 1H); 7.54 (dd; 1.5; 7.9; 1H); 8.09 (d; 16.0; 1H); C-NMR (CDCl3) δ (ppm)
13.2; 18.6; 19.4; 26.9; 27.7; 36.5; 44.6; 47.6; 48.6; 80.1; 122.3; 125.5; 127.0; 131.1; 132.6; 133.7;
134.9; 139.7; 166.2.
Cinnamic Acid (−)-Bornyl Ester (15). Yield 7%; colourless syrup; spectral data are in accordance with
data reported in ref. [22,23].
4-Methoxycinnamic Acid (−)-Bornyl Ester (16). Yield 25%; crystalline solid; m.p. 87–90 °C; IR: 2952,
1
2929, 2117, 1701, 1627, 1602, 1513, 1152 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz) 0.87 (s; 3H); 0.88
(s; 3H); 0.94 (s; 3H); 1.03–1.08 (m; 1H); 1.24–1.39 (m; 2H); 1.70 (t; 4.5; 1H); 1.73–1.80 (m; 1H);
2.02–2.08 (m; 1H); 2.37–2.45 (m; 1H); 3.84 (s; 3H); 5.02 (ddd; 2.2; 3.4; 9.9; 1H); 6.34 (d; 15.9; 1H);
13
6.90 (d; 8.7; 2H); 7.49 (d; 8.7; 2H); 7.63 (d; 15.9; 1H); C-NMR (CDCl3) δ (ppm) 13.2; 18.6; 19.4;
26.9; 27.7; 36.6; 44.7; 47.5; 48.6; 55.0; 79.4; 114.0 (2C); 116.0; 127.0; 129.3 (2C); 143.5; 160.9; 167.3.
2,4-Dichlorocinnamic Acid (−)-bornyl Ester (17). Yield 85%; colourless crystalline solid; m.p. 90–92 °C;
IR: 2930, 2882, 2118, 1712, 1638, 1469, 1312, 1177 cm−1; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.90 (s; 3H);

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0.92 (s; 3H); 0.97 (s; 3H); 1.07–1.11 (m; 1H); 1.27–1.40 (m; 2H); 1.74 (t; 4.5; 1H); 1.76–1.85 (m; 1H);
2.02–2.09 (m; 1H); 2.41–2.49 (m; 1H); 5.03 (ddd; 2.2; 3.4; 9.9; 1H); 6.44 (d; 16.0; 1H); 7.22 (t; 7.9; 1H);
7.48 (dd; 1.5; 7.9; 1H); 7.54 (dd; 1.5; 7.9; 1H); 8.09 (d; 16.0; 1H); 13C-NMR (CDCl3) δ (ppm) 13.5; 18.9;
19.7; 27.2; 28.0; 36.8; 44.9; 47.9; 49.0; 80.4; 121.8; 127.5; 128.3; 130.0; 131.4; 135.5; 136.2; 138.8; 166.5.
Cinnamic Acid Isobornyl Ester (18). Yield 14%; colourless syrup; IR: 2952, 2877, 1708, 1637, 1309,
1
1160 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz) 0.88 (s; 3H); 0.90 (s; 3H); 1.07 (s; 3H); 1.09–1.24 (m;
2H); 1.55–1.62 (m; 1H); 1.68–1.91 (m; 4H); 4.81 (dd; 4.3; 7.3; 1H); 6.41 (d; 16.0; 1H); 7.36–7.40 (m;
13
3H); 7.00–7.54 (m; 2H); 7.63 (d; 16.0; 1H); C-NMR (CDCl3) δ (ppm) 11.5; 20.0; 20.1; 27.0; 33.8;
38.9; 45.1; 47.0; 48.9; 81.1; 118.9; 128.0 (2C); 128.8 (2C); 130.1; 134.5; 144.2; 167.0.
Cinnamic Acid Thymyl Ester (19). Yield 24%; colourless solid; m.p. 64 °C; spectral data are in
accordance with data reported in ref. [24].
Cinnamic Acid Menthyl Ester (20). Yield 25%; colourless crystals; m.p. 51–53 °C; spectral data are in
accordance with data reported in ref. [25–27].
Cinnamic Acid Adamantyl Ester (21). Yield 10%; colourless powder; m.p. 66 °C [lit. [28]: 63–65 °C];
1
IR: 2896, 2866, 2848, 1703, 1687, 1641, 1170 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz) 1.70 (m; 6H);
13
2.20 (s; 9H); 6.36 (d; 15.9; 1H); 7.36–7.38 (m; 3H); 7.49 (m; 2H); 7.57 (d; 15.9; 1H); C-NMR
(CDCl3) δ (ppm) 30.9 (3C); 36.2 (3C); 41.4 (3C); 80.6; 102.4; 128.0; 128.8; 129.9; 134.7; 143.4; 166.0.
Cinnamic Acid Naphthyl Ester (22). Yield 15%; colourless crystalline solid; m.p. 109–110 °C [lit. [24]
106–107 °C]; spectral data are in accordance with data reported in ref. [24].
Cinnamic Acid Cyclohexyl Ester (23). Yield 33%; colourless liquid; spectral data are in accordance
with data reported in ref. [29–31].
Cinnamic Acid Bisabolyl Ester (24). Yield 11%; yellowish syrup; IR: 2962, 2924, 1703, 1636,
1
1160 cm−1; H-NMR (CDCl3) δ (ppm) J (Hz) 1.28–1.33 (m; 1H); 1.41 (s; 3H); 1.53 (s; 3H); 1.58 (s;
3H); 1.59 (s; 3H); 1.74–2.00 (m; 9H); 5.04 (br m; 1H); 5.31 (br m; 1H); 6.32 (d; 16.0; 1H); 7.29–7.32
13
(m; 3H); 7.43–7.46 (m; 2H); 7.52 (d; 16.0; 1H); C-NMR (CDCl3) δ (ppm) 17.6; 20.6; 22.1; 23.3;
23.7; 25.7; 26.4; 30.9; 35.7; 40.5; 87.3; 120.1; 120.3; 124.1; 128.0 (2C); 128.8 (2C); 130.0; 131.6;
134.1; 134.7; 143.5; 166.1.
Cinnamic Acid Eugenyl Ester (25). Yield 50%; colourless plates; all data are in accordance with data
reported in ref. [21,32].
Cinnamic Acid Geranyl Ester (26). Yield 28%; colourless liquid; spectral data are in accordance with
data reported in ref. [33].
3.4.3. Synthesis of Phenyl Propanoic Acid (−)-Bornyl Ester (27)
Propanoic acid (700 mg, 4.66 mmol) was dissolved in NEt3 (3.2 mL, 5 eq.) and cooled in an ice
bath. 4-Dimethylaminopyridine (114 mg, 0.2 eq.) in CH3CN (2 mL) and tosyl chloride (1.07 g, 1.2 eq.)

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in CH3CN (3 mL) were added and the solution stirred for 30 min until a red colour developed. Then
(−)-borneol (719 mg, 4.66 mmol) dissolved in CH3CN (2 mL) was added to the solution. The mixture
was allowed to warm up to room temperature and stirred for two hours. After evaporation of the
solvent the reaction mixture was suspended in water (10 mL) and extracted three times with diethyl
ether (10 mL). The organic phase was washed three times each with water, brine and Na2SO4. After
drying the organic phase over MgSO4 the product was purified twice by column chromatography on
silica gel using ethyl acetate/petroleum ether (60:40) as eluent to yield 278 mg of 27. Yield 21%;
colourless liquid; IR: 2952, 2877, 1730 cm−1; 1H-NMR (MeOD) δ (ppm) J (Hz) 0.78 (s; 3H); 0.89 (s;
3H); 0.91 (s; 3H); 0.84–0.88 (m; 2H); 1.14–1.20 (m; 1H); 1.26–1.34 (m; 1H); 1.63 (t; 4.5; 1H);
1.72–1.80 (m; 1H); 1.87–1.94 (m; 1H); 2.26–2.34 (m; 1H); 2.67 (t; 7.4; 2H); 2.95 (t; 7.4; 2H); 4.84 (m;
13
1H); 7.11–7.29 (m; 5H); C-NMR (MeOD) δ (ppm) 13.8; 19.2; 20.1; 28.1; 28.9; 32.2; 37.0; 37.7;
46.3; 48.8; 49,7; 81.4; 127.3; 129.4 (2C); 129.5 (2C); 141.8; 175.0.
3.4.4. Synthesis of 3-(3,4-Bis(acetyloxy))phenyl Propenoic Acid (−)-Bornyl Ester (28)
Compound 1 (20 mg) was suspended in pyridine (2 mL) at 0 °C. One equivalent of acetic acid
anhydride was added and the solution stirred for 2 h at room temperature. After evaporation the residue
was subjected to column chromatography on silica gel using CHCl3/MeOH (4.8:0.2 v/v) as eluent to
1
yield the desired product. Yield 51%; orange syrup; IR: 2953, 2877, 1771, 1707 cm−1; H-NMR
(CDCl3) δ (ppm) J (Hz) 0.87 (s; 3H); 0.90 (s; 3H); 0.94 (s; 3H); 1.03–1.07 (m; 1H); 1.25–1.35 (m;
2H); 1.71 (t; 4.5; 1H); 1.75–1.81 (m; 1H); 1.99–2.05 (m; 1H); 2.30 (s; 3H); 2.31 (s; 3H); 2.37–2.47 (m;
1H); 5.01 (ddd; 2.1; 3.4; 9.9; 1H); 6,41 (d; 16.0; 1H); 7.22 (d; 8.4; 1H); 7.38 (d; 2.0; 1H); 7,42 (dd; 2.0;
8.4; 1H); 7.60 (d; 16.0; 1H); 13C-NMR (CDCl3) δ (ppm) 13.5; 18.9; 19.7; 20.6; 20.7; 27.2; 28.0; 36.8; 45.0;
47.9; 49.0; 80.2; 120.0; 122.7; 123.9; 126.3; 133.5; 142.2; 142.4; 143.4; 166.9; 168.0; 168.1.
3.5. Biological Assays
Materials and methods for the biological assays have been described before by the authors.
AlamarBlue assays for investigation of antileishmanial activities against L. major promastigotes,
amastigotes and J774.1 and BMDM cytotoxicity were conducted as previously reported [16]. The
protocol for the L. donovani quantitative colorimetric assay is outlined by Hazra et al. [17]. The IC50
values are presented as mean values of two independent experiments against the parasite and macrophages.
4. Conclusions
In summary, caffeic acid bornyl ester (1) was isolated as the antileishmanial component of the
chloroform extract of V. wallichii rhizome. Structure-activity relationships of a compound library of
27 derivatives were analysed and revealed the size of the non-cinnamyol part of the molecule to have a
significant influence on antileishmanial activity. Hydroxyl groups in the 3 and 4 positions of the
aromatic ring increase cytotoxicity and the Michael system in the side chain is not essential for
antiprotozoal activity against L. major promastigotes (Table 3). The cinnamic acid bornyl ester 15
showed the best activity with regards to L. major and L. donovani promastigotes with acceptable

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cytotoxicity. Compound 27 with no Michael acceptor moiety was almost as active as compound 15. Since
27 is less toxic it presents an attractive new lead structure derived from nature for further optimization.
Additionally esters of caffeic and ferulic acid under certain conditions may face the problem of
limited bioavailability because of the possible formation of a polar phenolate ion which prevents the
compound from penetrating the cell membrane. By omitting the hydroxyl groups and thereby
increasing the lipophilicity this problem should be avoided.
Since bornyl hydroxycinnamic esters have been shown to inhibit the trypanosomal rhodesain [10] it
is tempting to speculate whether they are able to inhibit the corresponding leishmanial proteases.
Preliminary experiments using L. major promastigote full-lysate in a cysteine-cathepsin fluorescence
activity assay [16,34] showed protease inhibitory activity of compound 1 and 15 and, thus, point to
this target.
Acknowledgments
Thanks are due to the Deutsche Forschungsgemeinschaft (SFB 630) for financial support. Sudipta
Hazra was awarded a fellowship from ICMR, New Delhi (3/1/3/WL/JRF-2008/MPD). This publication
was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the
funding programme Open Access Publishing.
Author Contributions
The listed authors contributed to this work as described in the following. Jan Glaser fractionated the
plant extract and isolated and identified the antileishmanial compound. He also carried out the
synthesis and prepared the manuscript. As corresponding author for infection biology Uta Schurigt
together with Martina Schultheis conducted assays against L. major amastigotes and promastigotes and
helped interpreting the results. Sudipta Hazra and Banasri Hazra were in charge of the L. donovani
promastigote assays. As corresponding author for medicinal chemistry Ulrike Holzgrabe proposed the
subject and monitored the progress of the ongoing research. Banasri Hazra, Heidrun Moll and Ulrike
Holzgrabe contributed with valuable discussions and scientific input. All authors helped preparing the
manuscript and approved the final version.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–28 are available from the authors.
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