Synthesis and evaluation of substituted 4-(N-benzylamino)cinnamate esters as potential anti-cancer agents and HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.05.023

Encouraging selectivity and low micromolar activity against HeLa cervical carcinoma (IC₅₀? 3.0 μM) and the aggressive MDA-MB-231 triple negative breast carcinoma (IC₅₀? 9.6 μM) cell lines has been exhibited by a number of readily acc

Full text of DOI:10.1016/j.bmcl.2016.05.023

Accepted Manuscript
Synthesis and Evaluation of Substituted 4-(N-Benzylamino)cinnamate Esters as
Potential Anti-Cancer Agents and HIV-1 Integrase Inhibitors
Faridoon, Adrienne L. Edkins, Michelle Isaacs, Dumisani Mnkandhla, Heinrich
C. Hoppe, Perry T. Kaye
PII:
S0960-894X(16)30507-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.023
BMCL 23882
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 March 2016
6 May 2016
7 May 2016
Please cite this article as: Faridoon, Edkins, A.L., Isaacs, M., Mnkandhla, D., Hoppe, H.C., Kaye, P.T., Synthesis
and Evaluation of Substituted 4-(N-Benzylamino)cinnamate Esters as Potential Anti-Cancer Agents and HIV-1
Integrase Inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2
016.05.023
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Evaluation of Substituted 4-(N-Benzylamino)cinnamate
Esters as Potential Anti-Cancer Agents and HIV-1 Integrase Inhibitors
a
Faridoon, Adrienne L. Edkins,
b,c,d
d
Michelle Isaacs, Dumisani Mnkandhla,
d
b,d a,d
Heinrich C. Hoppe, and Perry T. Kaye *
a
b
Department of Chemistry, Department of Biochemistry and Microbiology, Biomedical Biotechnology
c
d
Research Unit (BioBRU), and Centre for Chemico- and Biomedicinal Research, Rhodes University,
Grahamstown, 6140, South Africa.
Abstract. Encouraging selectivity and low micromolar activity against HeLa cervical
carcinoma (IC50 ≥ 3.0 µM) and the aggressive MDA-MB-231 triple negative breast carcinoma
(
IC50 ≥ 9.6 µM) cell lines has been exhibited by a number of readily accessible 4-(N-
benzylamino)cinnamate esters. The potential of the ligands as HIV-1 integrase inhibitors has
also been examined.
Keywords: 4-(N-benzylamino)cinnamate esters, anti-cancer agents, HIV-1 integrase inhibitors
_
*
__________
Corresponding author:
Phone: + 27 46 6037030; Fax: +27 46 6225109; E-mail: P.Kaye@ru.ac.z
1

Biologically active cinnamic acid derivatives are widely distributed in nature, and cinnamic
1
acid itself plays important roles in phenylpropanoid and shikimate biosynthetic pathways.
Baccharin 1, a cinnamic acid derivative isolated from Brazilian green propolis, has been
shown to selectively inhibit Human Type-5 17β-Hydroxysteroid Dehydrogenase (AKR1C3),
2
a proposed target for breast and prostate cancer therapy, at nanomolar levels. O-Prenylated
3
cinnamate esters (e.g., compound 2) have shown modest activity against K562 cancer cells,
while cinnamyl hydroxyamides and 2-aminoanilides have been reported to exhibit potent
4
apoptotic and cytodifferentiation activity. The potential of cinnamic acid derivatives has
been recognised not only as therapeutics for the treatment of tuberculosis, malaria,
5
,6
7
cardiovascular diseases and cancer, but also as HIV-1 integrase (IN) inhibitors. In our
research on novel HIV-1 enzyme inhibitors, we have reported the preparation and bioassay of
cinnamate ester−AZT conjugates 3 as potential, dual-action HIV-1 integrase/reverse
8
transcriptase (IN/RT) inhibitors. We now report the HIV-1 IN inhibition- and anti-cancer
,9
activities of a series of novel 4-(N-benzylamino)cinnamate esters.
CH3
O
O
O
1
R
H3C
OH
OCH3
OMe
NH
OH
O
O
O
R2 Bn
O
O
O
N
O
N
N
NH
N
CH3
1
2
3
H
O
The 4-(N-benzylamino)cinnamate esters 7a-i were accessed from 4-nitrocinnamic acid 4 via a
1
0
11
four-step pathway, involving the methyl ester 5 and its 4-amino analogue 6 (Scheme 1).
Esterification of 4-nitrocinnamic acid 4 afforded the methyl ester 5 in 84% yield and
subsequent reduction of the nitro group using stannous chloride dihydrate gave methyl 4-
aminocinnamate 6 in 77% yield. Condensation of the amino derivative 6 with a series of
benzaldehyde and salicylaldehyde derivatives gave the corresponding imines, which were not
isolated but reduced in situ using sodium cyanoborohydride to afford, following column
chromatography, the targeted 4-(N-benzylamino)cinnamate esters 7a-i in yields ranging from
6
0% to 84%. The 4-(N-benzylamino)cinnamate esters 7a-i are all new compounds and were
fully characterised using NMR and HRMS techniques. Assignment of an (E)-configuration to
2

the cinnamate double bond is supported by the large coupling constant (J = ca. 15.8 Hz)
between the vinylic protons which typically resonate at ca. 6.2 and 7.5 ppm.
O
O
1
R
2
R
3
R
4
R
OH
H2SO4/MeOH
Reflux
OCH3
7
7
7c
7d
a
b
OH
OH
H
OH
OH
OH
OH
OH
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
Cl
Br
NO
H
Cl
Br
O2N
O N
2
OH
OH
H
H
H
OMe
Cl
Br
4
SnCl2.2H2O
5
HCl, EtOH
Reflux, 2h
7e
O
i. ArCHO,
AcOH, MeOH
Reflux, 2h
O
7f
2
OCH3
7g
7h
7i
OCH3
R1 HN
ii. NaCNBH3
room temp
over night
R2
R3
H2N
6
7
R4
Scheme 1
The 4-(N-benzylamino)cinnamate esters 7a-i were each tested against both the HeLa cervical
carcinoma and MDA-MB-231 triple negative breast carcinoma cell lines, using paclitaxel as a
positive control. The results are summarised as IC50 values in Table 1, and it is apparent that
all of the ligands exhibit a measure of activity against the HeLa cells. Five of them (7a, 7d,
7
e, 7f and 7g), however, have IC50 values well below 20 µM. Each of these five compounds
1
contain an ortho-hydroxy group (R = OH); three of them contain an electron-withdrawing
4
4
4
2
substituent (7d: R = Cl; 7e: R = Br; 7f: R = NO ), while the most active ligand 7g (IC50 =
2
.04 µM) contains a meta-methoxy substituent (R = OMe). The meta-hydroxy analogue 7b
3
2
R = OH), however, exhibits a much higher IC50 value (68.2 µM).
(
Perhaps, even more significant is the observed activity of most of the ligands against the
aggressive MDA-MB-231 triple negative breast carcinoma cells. All three of the compounds
1
7f, 7h and 7i) which exhibit IC50 values below 20 µM contain an ortho-hydroxy group (R =
(
4
OH) and an electron-withdrawing substituent on the aromatic carbon C-5 (7f: R = NO
2
; 7h:
4 4
R = Cl; 7i: R = Br); the slightly more active ligands 7h (IC50 = 9.61 µM) and 7i (IC50 = 11.5
3
µM), however, contain an additional, matching para-halogen substituent (7h: R , R = Cl; 7i:
4
3
R , R = Br). Of particular interest are the ligands which exhibit:
4
i)
promising activity against both cell lines, viz., 7a, 7f, 7h and 7i; and
a measure of selectivity against the HeLa cells relative to the MDA-MB-231 cells,
viz., 7d, 7e and 7g.
ii)
3

The structural features which typically characterise the most active ligands thus include the
1
presence of an ortho-hydroxy group (R = OH) and appropriately located, electronegative
2
4
2
aromatic substituents (R = Br, Cl and/or R = Br, Cl, NO ).
Table 1.
Anti-cancer activity (IC50 at µM) of compounds 7a-i against HeLa cervical
carcinoma and MDA-MB-231 triple negative breast carcinoma cell lines.
HeLa cervical
carcinoma cell activity
MDA-MB-231 triple negative
breast carcinoma cell activity
2
2
Compound
IC50 (µM)
R
IC50 (µM)
R
7
7
7
7
7
7
7
7
a
b
c
d
e
f
7.168
68.19
78.12
8.558
7.821
15.97
3.042
25.88
35.4
0.9302
0.9677
0.9248
0.7284
0.8617
0.8976
0.86
31.69
32.6
0.8918
0.8839
0.9754
0.7402
0.9095
0.9104
0.9665
0.9713
0.8533
36.63
86.41
109.4
17.4
g
h
73.68
9.611
11.5
0.917
0.09815
7
i
a
Paclitaxel
19.87 nM
0.9409
3.388 nM
0.9141
a
Positive control.
The 4-(N-benzylamino)cinnamate esters 7a-i, all of which showed no significant toxicity
against HEK293 human embryonic kidney cells (Table 2), were also examined for their
capacity to inhibit the HIV-1 integrase (IN) enzyme. Disappointingly, only one of the ligands
(
7c) showed statistically significant activity against HIV-1 IN ( an average of 43.2% residual
enzyme activity at a concentration 20 µM), relative to the established inhibitor, raltegravir,
1
2
which was used as a positive control (0.67% residual activity).
Access to a series of nine, novel 4-(N-benzylamino)cinnamate esters has been achieved in
good yield in four steps. A number of the compounds show encouraging activity, at low
micromolar concentrations, against HeLa cervical carcinoma and the aggressive MDA-MB-
2
31 triple negative breast carcinoma cell lines – in some cases with interesting selectivity
against the HeLa cells relative to the MDA-MB-231 cells. The compounds, generally, exhibit
minimal HEK293 toxicity at 100 µM, but only one shows > 50% inhibition of HIV-1 integrase
at a concentration of 20 µM.
4

Table 2.
HIV-1 IN enzyme activity at inhibitor concentrations of 20 µM and HEK 293
cell viability data (at 100 µM) for compounds 7a-i .
a
HIV-1 IN activity at 20 µM
HEK293 viability at 100 µM
Compound
Negative control
% Activity
SD
% Cell viability
SD
b
100
0.67
105.9
73.6
43.2
78.6
73.8
87.3
90
0.07
0.01
0.06
0.19
0.09
0.08
0.16
0.001
0.02
0.08
0.08
c
Positive control
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
94.4
82.35
113.6
0.17
1.32
0.44
0.04
0.94
3.14
0.9
92.36
98.11
108.01
114.12
106.67
114.33
g
h
i
102.5
96.7
11.69
10.09
a
b
c
Mean of duplicate results. No inhibitor present. Raltegravir for HIV-1 IN; emetine for HEK 293
cells.
ACKNOWLEDGEMENTS
The authors thank Rhodes University for a Postdoctoral Fellowship (Faridoon) and the South
African Medical Research Council (MRC) and Rhodes University for generous financial
support. This research project was funded by the South African Medical Research Council
(
MRC) with funds from National Treasury under its Economic Competitiveness and Support
Package.
5

REFERENCES and NOTES
1
2
. Sharma, P., J. Chem. Pharm. Res. 2011, 3(2):403.
. Endo,S.; Matsunaga, T.; Kanamori, A.; Yoko Otsuji, Y.; Nagai, H.; Sundaram, K.; El-
Kabbani, O.; Toyooka, N.; Ohta, S.; Hara, A. J. Nat. Prod. 2012, 75, 716.
3
2
4
. Koolaji, N.; Abu-Mellal, A.; Tran, V.H.; Duke, R.K.; Duke, C.C. Eur. J. Med. Chem.,
013, 63, 415.
. Valente, S.; Tardugno, M.; Conte, M.; Cirilli, R.; Perrone, A.; Ragno, R.; Simeoni, S.;
Tramontano, A.; Massa, S.; Nebbioso, A.; Miceli, M.; Franci, G.; Brosch, G.; Altucci, L.;
Mai, A. Chem. Med. Chem. 2011, 6, 698.
5
. De, P.; Bedos-Belval, F.; Vanucci-Bacque, C.; Baltas, M. Curr. Org. Chem. 2012, 16, 747.
. De, P.; Baltas, M.; Bedos-Belval, F. Curr. Med. Chem. 2011, 18, 1672.
. Desideri, N.; Sestili, I.; Stein, M.L.; Tramontano, E.; Corrias, S.; La Colla, P. Antivir.
Chem. Chemother. 1998, 9, 497.
. Olomola, T. O.; Klein, R.; Kaye, P. T. Tetrahedron 2014, 70, 9449.
. Manyeruke, M.H.; Olomola, T.O.; Majumder, S.; Abrahams, S.; Isaacs, M.; Mautsa, N.;
6
7
8
9
Mosebi, S.; Mnkandhla, D.; Hewer, R.; Hoppe, H.C.; Klein, R.; Kaye, P.T. Bioorg. Med.
Chem. 2015, 23, 7521.
1
0. Pinto, O.; Sardinha, J.; Vaz, P. D.; Piedade, F.; Calhorda, M. J.; Abramovitch, R.;
Nazareth, N.; Pinto, M.; Nascimento, M. S. J.; Rauter, A. P. J. Med. Chem. 2011, 54, 3175.
1. Suárez-Meneses, J.V.; Bonilla-Reyes, E.; Blé-González, E.A.; Ortega-Alfaro, M.C.;
Toscano, R.A.; Cordero-Vargas, A.; López-Cortés, J.G. Tetrahedron 2014, 70, 1422.
2. Grobler, J.A; Stillmock, K.; Hu, B.; Witmer, M.; Felock, P.; Espeseth, A.S.; Wolfe, A.;
1
1
Egbertson, M.; Bourgeois, M.; Melamed, J.; Wai, J.S.; Young, S.; Vacca, J.; Hazuda, D.J.
Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (10), 6661.
1
3. Experimental (synthetic and bioassay) details and NMR spectroscopic data are provided in
Supplementary Data files I and II, respectively.
6

7