A new Heck reaction modification using ketone Mannich bases as enone precursors: Parallel synthesis of anti-leishmanial chalcones

Article abstract of DOI:10.1016/j.bmcl.2008.01.112

A new Heck-type reaction for the synthesis of chalcones has been established using Mannich bases as enone precursors. The novel reaction proceeds rapidly in air atmosphere under ligandless conditions and can be adapted for library synthesis in a parallel reactor station. Screening of the synthesized chalcones revealed N-{4-[(1E)-3-oxo-3-(3-pyridinyl)-1-propenyl]phenyl}benzamide (3f) to be a potent anti-leishmanial agent.

Full text of DOI:10.1016/j.bmcl.2008.01.112

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1985–1989
A new Heck reaction modification using ketone Mannich bases
as enone precursors: Parallel synthesis of anti-leishmanial chalcones
Christina Reichwald,a Orly Shimony,b Nina Sacerdoti-Sierra,b
Charles L. Jaffeb and Conrad Kunicka,*
aTechnische Universita¨t Braunschweig, Institut fu¨r Pharmazeutische Chemie, Beethovenstraße 55, 38106 Braunschweig, Germany
bDepartment of Parasitology, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel
Received 14 January 2008; revised 28 January 2008; accepted 29 January 2008
Available online 2 February 2008
Abstract—A new Heck-type reaction for the synthesis of chalcones has been established using Mannich bases as enone precursors.
The novel reaction proceeds rapidly in air atmosphere under ligandless conditions and can be adapted for library synthesis in a par-
allel reactor station. Screening of the synthesized chalcones revealed N-{4-[(1E)-3-oxo-3-(3-pyridinyl)-1-propenyl]phenyl}benzamide
(3f) to be a potent anti-leishmanial agent.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Kala azar or visceral leishmaniasis (VL) is caused by
infection with Leishmania donovani parasites that are
transmitted by the bite of sandflies. Typical symptoms
of this disease include hypergammaglobulinemia, ane-
mia, weight loss, hepatosplenomegaly, fever, and immu-
nosuppression. Without early diagnosis and proper
treatment VL is fatal. Due to the epidemiological situa-
tion and therapeutic options available to populations in
endemic countries affected by this disease, the WHO has
designated leishmaniasis a ‘neglected and emerging dis-
ease’. All existing medical treatments against leishmani-
asis have major drawbacks (resistance against antimony
drugs, side effects of pentamidine and amphotericin B,
reproductive toxicity of miltefosine) and there is an ur-
gent need for additional effective medicines.1,2 Chal-
cones (=1,3-diarylpropenones) of natural or synthetic
origin have repeatedly been mentioned as anti-leishman-
ial agents.3–6 It has been shown that licochalcone A, a
chalcone from the roots of Glycyrrhizae inflata,7 alters
the ultrastructure of mitochondria in Leishmania para-
sites8 and inhibits the leishmanial fumarate reductase.9
Oxygenated chalcones exhibited in vivo anti-leishmanial
activity in a hamster model.10 Detailed structure–activ-
ity relationships (SAR) of anti-leishmanial chalcones
have been published by several groups.11–14 Chalcones
have also been found to show anti-plasmodial activity
in vitro.15 The majority of the chalcones used in these
studies were either extracted from natural sources or
prepared by a classical aldol condensation reaction
employing an aromatic aldehyde and an appropriate
aromatic ketone. For a systematic exploitation of the
published SAR and the generation of chalcone libraries
additional synthetic procedures are desirable. These
methods should be broadly applicable, start with inex-
pensive educts and allow the rapid generation of cong-
eners in a parallel fashion. For example, a variety of
substituted haloarenes are commercially available that
could serve as educts for carbon–carbon-cross coupling
reactions.
For a synthesis strategy involving haloarenes, the palla-
dium catalyzed Heck reaction16–18 using aryl vinyl ke-
tones as reactants for the construction of the enone
substructure is an obvious option. However, a survey
of the literature revealed that examples for such reac-
tions are extremely rare,19–21 presumably because of
the low stability of aryl vinyl ketones under the condi-
tions typically used for Heck reactions. We therefore
considered the use of suitable stable precursors liberat-
ing the aryl vinyl ketones in the course of the reaction.
Although ketone Mannich bases have been applied as
enone substitutes for the reaction with various nucleo-
philes,22 they have to our best knowledge not been em-
ployed in transition-metal catalyzed carbon–carbon
coupling reactions. Since it has been reported that the
Keywords: Leishmania; Chalcones; Heck reaction.
*
Corresponding author. Tel.: +49 531 391 2751; fax: +49 531 391
2799; e-mail: c.kunick@tu-braunschweig.de
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.112

1986
C. Reichwald et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1985–1989
Table 1. Modification of catalyst in synthesis of chalcone 3a according
to Scheme 1
corresponding Z-diastereomers. Because of similar
chromatographic behaviour of main and side products,
the purification by column chromatography was diffi-
cult. Therefore, crystallization from ethanol was applied
as standard work-up procedure to yield the desired
(E)-chalcones. Since the crystallization caused some loss
of material, the yields indicated in Table 2 still bear some
optimization potential.23
Catalyst
Yielda (%)
2.5 mol% Pd(OAc)2
5 mol% Pd(OAc)2
28
44 (72)
37
43
5 mol% Pd(OAc)2/5 mol % triphenylphosphine
10 mol% Pd(OAc)2
5 mol% [Ph3P]2PdCl2
21
13
5 mol% Pd[Ph3P]4
5 mol% PdCl2dppf2 · CH2Cl2
37
The reaction was also applicable for the reaction of aro-
matic ketone Mannich bases with bromoarenes, albeit
the reactions furnished lower yields (data not shown).
Chloroarenes showed reactivity only when activated by
an additional strongly electron-withdrawing substituent,
for example, a nitro group (data not shown). This grad-
uated reactivity of haloarenes allowed the synthesis of
the chalcone 3c, because the chloro substituent of the
corresponding educt ketone Mannich base remained
unaffected during the reaction.
a Yield after work-up and crystallization from ethanol. Yield in
brackets determined by HPLC (area% method).
toxicity of anti-leishmanial chalcones against the human
host cells is decreased by a large substituent in position
4,14 we chose for synthetic studies the preparation of
4-benzoylaminochalcone 3a from N,N-dimethyl-3-oxo-
3-phenylpropan-1-aminium chloride 1a and N-(4-iodo-
phenyl)benzamide 2a as a model reaction (Scheme 1).
The reactions were performed in 20 mL standard vials
of a parallel synthesis reactor to establish a method use-
ful for a rapid generation of structure analogues. Initial
attempts were carried out in DMF under air atmosphere
in the presence of triethylamine as base. Of the four
palladium catalysts employed, 5 mol% palladium(II)ace-
tate gave the best result, presumably because of its air
stability. The addition of triphenylphosphine as ligand
or the modification of catalyst loading (2.5 or
10 mol%) failed to increase the yield of isolated product.
The small exemplary chalcone library generated by the
novel parallel synthesis approach was screened for
anti-leishmanial activity. Depending on the stage of
the life cycle, Leishmania exist in two forms: extracellu-
lar promastigotes residing in the insect vectors and
intracellular amastigotes that multiply in human host
macrophages. The anti-leishmanial activity of the
chalcones reported here was screened using a fluorescent
viability microplate assay with L. donovani axenic
amastigotes.29 In an initial screening, the inhibition of
the amastigote growth by a 15 lM chalcone concentra-
tion was assessed (refer to Table 2). For chalcones show-
ing more than 90% inhibition the GI50 (concentration
for 50% growth inhibition) was also determined. In
addition, the general cytotoxicity of the active chalcones
was determined using an alamarBlue viability assay on
the human macrophage cell line THP-1 (Table 3).30
The described method was then implemented for the syn-
thesis of a small exemplary chalcone library starting from
ketone Mannich bases 1with aryl iodides 2 (Scheme 2/
Tables 2 and 3). In all cases, the reaction proceeded
rapidly and was completed within less than 30 min.
Although HPLC analyses of the raw reaction mixtures
before work-up indicated product yields between 63%
and 72%, the isolated yields of the pure products appear
as moderate. The HPLC traces showed the presence of
side products in low concentrations, among them the
The initial results revealed that four compounds (3a, 3d,
3e, 3f) with the p-benzoylamino-substituent at the
1-aryl-ring and the p-acetyl derivative 3i exhibited strong
H
N
Cl
CH3
N+
H
N
i
+
H
O
CH3
O
O
I
O
3a
1a
2a
Scheme 1. Modification of reaction conditions for synthesis of chalcone 3a. Reagents and conditions: Palladium catalyst, DMF, triethylamine,
140 ꢁC, parallel reactor station, 30 min. For catalysts and yields refer to Table 1.
Cl
CH3
N+
Aryl2
Aryl1
i
Aryl1
H
I
Aryl2
+
CH3
O
O
1
2
3
Scheme 2. General procedure for palladium-catalyzed synthesis of chalcones 3 from Mannich bases 1 and iodoarenes 2. For residues Aryl1 and Aryl2
refer to Table 2. Reagents and conditions: 5 mol% Pd(OAc)2, DMF, triethylamine, 140 ꢁC, parallel reactor station, 30 min.

C. Reichwald et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1985–1989
1987
Table 2. Synthesized chalcones 3a–ka
Chalcone
Structure
Yieldb
Inhibition of axenic L. donovani amastigotes
at 15 lMc (% s.e.)
H
N
3a
44 (72)
91.6 1.2
O
O
H
MeO
N
3b
3c
29 (73)
2.1 5.1
O
O
H
Cl
N
37 (66)
59.8 2.6
O
O
H
N
3d
3e
65
95.3 0.73
O
S
O
H
N
40 (72)
92.4 0.89
O
O
O
H
N
3f
43 (67)
98.7 0.34
54.7 6.7
51.0 2.0
N
O
O
H
N
CH3
MeO
MeO
3g
41
O
O
O
3hd
50
O
CH3
3i
3j
31
24
93.6 0.71
O
O
N
O
63.7 1.9
(continued on next page)

1988
C. Reichwald et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1985–1989
Table 2 (continued)
Chalcone
Structure
Yieldb
Inhibition of axenic L. donovani amastigotes
at 15 lMc (% s.e.)
MeO
N
O
3k
45
42.0 4.8
O
DMSOe
—
—
0.0 2.0
Amphotericin Bf
99.0 0.33
a Compounds 3a–b,24 3d–e,25 3g,26 3h,12,15 and 3i27 have been described before. For melting points of 3c, 3f, 3j, 3k refer to note.28
b Yields after work-up and a single crystallization from ethanol. Values in brackets represent yields determined by HPLC from raw reaction mixtures
(area% method).
c Mean of two experiments (triplicates standard error).
d Compound 3h has been reported to show an IC50 > 200 lM in an assay measuring the viability of L. donovani amastigotes using a tetrazolium dye-
based reagents.12
e 1% DMSO in medium was used as negative control.
f Amphotericin B used as positive control.
References and notes
Table 3. Biological activities of selected chalcones
Chalcone
GI50 on axenic
L. donovani amastigotesa
% Killing of THP-1
macrophages at 1 lMb
1. Mishra, J.; Saxena, A.; Singh, S. Curr. Med. Chem. 2007,
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2. Pechan, P.; Jaffe, C. L. Dosis 2006, 22, 158.
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(lM s.e.)
3a
3d
3e
3f
3i
7.6 0.30
5.5 0.30
6.0 0.0
37
6
ˆ
4. Lunardi, F.; Guzela, M.; Rodrigues, A.; Correa, R.; Eger-
Mangrich, I.; Steindel, M.; Grisard, E.; Assreuy, J.;
Calixto, J.; Santos, A. Antimicrob. Agents Chemother.
2003, 47, 1449.
0
2.5 0.44
5.6 0.25
0
45.7 6.3
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6. Torres-Santos, E.; Moreira, D.; Kaplan, M.; Meirelles,
M.; Rossi-Bergmann, B. Antimicrob. Agents Chemother.
1999, 43, 1234.
a Mean of duplicate experiments (3f: triplicate) standard error.
b Determined as single experiments (3i: triplicate standard error).
7. Nielsen, S. F.; Chen, M.; Theander, T. G.; Kharazmi, A.;
Christensen, S. B. Bioorg. Med. Chem. Lett. 1995, 5, 449.
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Kharazmi, A. Antimicrob. Agents Chemother. 2001, 45,
2023.
10. Zhai, L.; Chen, M.; Blom, J.; Theander, T.; Christensen,
S.; Kharazmi, A. J. Antimicrob. Chemother. 1999, 43, 793.
11. Boeck, P.; Bandeira Falcao, C.; Leal, P.; Yunes, R.; Filho,
V.; Torres-Santos, E.; Rossi-Bergmann, B. Bioorg. Med.
Chem. 2006, 14, 1538.
12. Gutteridge, C.; Vo, J.; Tillett, C.; Vigilante, J.; Dettmer, J.;
Patterson, S.; Werbovetz, K.; Capers, J.; Nichols, D.;
Bhattacharjee, A.; Gerena, L. Med. Chem. 2007, 3, 115.
13. Liu, M.; Wilairat, P.; Croft, S.; Tan, A. L.-C.; Go, M.-L.
Bioorg. Med. Chem. 2003, 11, 2729.
anti-leishmanial activity at 15 lM. The following dose–
response studies distinguished the pyridinyl derivative
3f,31 which showed a GI50 of 2.5 lM and proved to be
at least twice as potent as the other chalcones tested.
While 3f showed no toxicity at 1 lM in initial studies
on the human macrophage cell line THP-1, the chal-
cones 3a and 3i caused a definite cell killing. In conclu-
sion, we have established a novel method for the rapid
preparation of chalcones from Mannich Bases and halo-
arenes. The procedure is suitable for chalcone library
generation in a parallel reactor station device. Among
the chalcones prepared by the new method, 3f proved
to be a compound with interesting anti-leishmanial
properties. This structure will be the basic scaffold for
a focused chalcone library we intend to prepare in the
search of novel effective antiparasitic agents.
14. Nielsen, S.; Christensen, S.; Cruciani, G.; Kharazmi, A.;
Liljefors, T. J. Med. Chem. 1998, 41, 4819.
15. Gutteridge, C. E.; Nichols, D. A.; Curtis, S. M.; Thota, D.
S.; Vo, J. V.; Gerena, L.; Montip, G.; Ashe, C. O.; Diaz,
D. S.; Ditusa, C.; Smith, K. S.; Bhattacharjee, A. K.
Bioorg. Med. Chem. Lett. 2006, 16, 5682.
Acknowledgment
16. Alonso, F.; Beletskaya, I.; Yus, M. Tetrahedron 2005, 61,
11771.
17. Heck, R. F.; Nolley, J. P. J. Org. Chem. 1972, 37, 2320.
18. Knowles, J.; Whiting, A. Org. Biomol. Chem. 2007, 5, 31.
Funding by a grant of the European Commission (Con-
tract No. LSHB-CT-2004-503467; to C.R., C.L.J., and
C.K.) is gratefully acknowledged.

C. Reichwald et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1985–1989
1989
19. Barabanov, I. I.; Fedenok, L.; Polyakov, N. E.; Shvartz-
berg, M. S. Russ. Chem. Bull., Int. Ed. 2001, 9, 1663.
20. Bianco, A.; Cavarischia, C.; Farina, A.; Guiso, M.;
Marra, C. Tetrahedron Lett. 2003, 44, 9107.
21. Bianco, A.; Cavarischia, C.; Guiso, M. Eur. J. Org. Chem.
2004, 2894.
22. Tramontini, M.; Angiolini, L. Tetrahedron 1990, 46, 1791.
23. Synthesis of chalcones by Heck-type reaction, general
procedure: 1 mmol iodoarene, 1.1 mmol ketone Mannich
base hydrochloride, 11.25 mg (0.05 mmol) palla-
dium(II)acetate and 2 mL triethylamine are suspended in
5 mL DMF and stirred (20 mL vials, carousel 12 place
reactor station, Radley Discovery Technologies, UK) for
30 min at 140 ꢁC vessel reactor block temperature. After
filtration, silica gel (1.5 g) is added to the filtrate. After
evaporation of the solvent the remaining silica gel/reaction
product mixture is added onto a silica gel pad in a glass frit
and then eluted with ethylacetate (200 mL). After evapo-
ration of the solvent the remaining solid is purified by
crystallization from ethanol.
24. Pfeiffer, P. Liebigs Ann. Chem. 1925, 441, 228.
25. Rtishchev, N. I.; Nosova, G. I.; Solovskaya, N. A.;
Luk’ysahina, V. A.; Galaktionova, E. F.; Kudryavtsev, V.
V. Russ. J. Gen. Chem. 2001, 71, 1272.
26. Edwards, M.; Stemerick, D.; Sunkara, P. J. Med. Chem.
1990, 33, 1948.
27. Meier, H.; Aust, H.; Ickenroth, D.; Kolshorn, H. J. Prakt.
Chem. (Weinheim, Germany) 1999, 341, 529.
28. Melting points of new compounds: 3c, 198 ꢁC; 3f, 192 ꢁC;
3j, 111 ꢁC; 3k, 134 ꢁC.
in complete amastigote medium, containing 1% DMSO,
and were aliquoted in triplicate (125 ll/well) into 96-well
flat-bottom plates. Amastigotes (5.0 · 105 cells/ml; 125 ll/
well) were added to each well and incubated for 24 h at
37 ꢁC in a 5% CO2 incubator. The alamarBlue viability
indicator was added (25 ll/well) and the plates incubated
for an additional 24 h at which time the fluorescence
(kex = 544 nm; kem = 590 nm) was measured in a micro-
plate reader. Complete medium both with and without
DMSO was used as negative controls (0% inhibition of
amastigote growth). Amphotericin B was included as a
positive control on each plate and gave >90% inhibition of
parasite growth at 1 lM. Standardization and optimiza-
tion of the assay will be described elsewhere (Shimony and
Jaffe, in preparation).
30. For the toxicity assay, compounds (2 lM) were diluted in
the complete medium containing 1% DMSO and ali-
quoted in triplicate (125 ll/well) into 96-well plates. THP-
1 macrophages in complete RPMI-1640 were added
(8 · 105 cells/ml, 125 ll/well) and incubated for 48 h
(37 ꢁC, 5% CO2). AlamarBlue (25 ll) was added, the
plates incubated for an additional 3 h and the fluorescence
read. Medium both with and without DMSO was used as
negative controls (0% inhibition).
1
31. Analytical data of compound 3f: H NMR 7.54–7.58 (m,
2H, ArH), 7.60–7.64 (m, 2H, ArH), 7.79 (d, 1H, J = 15.7,
(C@O)CH@C), 7.97–7.99 (m, 2H, ArH), 7.92 (d, 5H, ArH
and (C@O)C@CH), 8.47 (dt, 1H, J = 7.9/2.2/1.8, ArH), 8.84
(dd, 1H, J = 4.8/1.7 Hz, ArH), 9.34 (dd, 1H, J = 2.2/0.7 Hz,
ArH), 10.49 (s, 1H, NH); 13C NMR 120.1 (2·), 120.2, 123.9,
127.7 (2·), 128.4 (2·), 129.9 (2·), 131.8, 135.8, 144.5, 149.6,
153.2 (tert. arom. C), 129.6, 133.0, 134.8, 141.7, 165.8, 188.2
(quat. arom. C); Anal. (C21H16N2O2) calcd. C 76.81, H 4.91,
N 8.53; found C 76.77, H 4.99, N 8.45.
29. Screening was carried out using an assay similar to one
reported for leishmanial promastigotes (Mikus, D.; Ste-
verding, D. Parasitol. Internat. 2000, 48, 265). Compounds
to be assayed were diluted to twice the final concentration