Synthesis and biological evaluation of bis-N2,N2′-(4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccinodihydrazides

Article abstract of DOI:10.1016/j.bmcl.2019.126911

A series of N²,N²′-bis[4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccino-hydrazides, containing 4-hydroxycoumarin, hydrazine and tartaric acid moieties, have been prepared and examined for possible biological activity. Several of these compounds exhibit promising HIV-1 integrase inhibition (IC₅₀ = 3.5 μM), and anti-T. brucei (32% viability) and anti-mycobacterial (Visual MIC90 = 15.63 μM) activity.

Full text of DOI:10.1016/j.bmcl.2019.126911

Journal Pre-proofs
Synthesis and Biological Evaluation of Bis-N 2,N 2’ -(4-hydroxycoumarin-3-
yl)ethylidene]-2,3-dihydroxysuccinodihydrazides
Meloddy H. Manyeruke, Thendamudzimu Tshiwawa, Heinrich C.Hoppe,
Michelle Isaacs, Ronnett Seldon, Digby F. Warner, Rui W.M. Krause, Perry T.
Kaye
PII:
S0960-894X(19)30889-3
DOI:
https://doi.org/10.1016/j.bmcl.2019.126911
BMCL 126911
Reference:
Bioorganic & Medicinal Chemistry Letters
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24 October 2019
13 December 2019
14 December 2019
Please cite this article as: Manyeruke, M.H., Tshiwawa, T., C.Hoppe, H., Isaacs, M., Seldon, R., Warner, D.F.,
2
2’
Krause, R.W.M., Kaye, P.T., Synthesis and Biological Evaluation of Bis-N ,N -(4-hydroxycoumarin-3-
yl)ethylidene]-2,3-dihydroxysuccinodihydrazides, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://
doi.org/10.1016/j.bmcl.2019.126911
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Synthesis and Biological Evaluation of
Bis-N2,N2′-(4-hydroxycoumarin-3-yl)ethylidene]-2,3-
dihydroxysuccinodihydrazides
Meloddy H. Manyerukea, Thendamudzimu Tshiwawaa, Heinrich C.Hoppeb,c, Michelle
Isaacsc, Ronnett Seldond, Digby F. Warner,e Rui W.M. Krausea,c*and Perry T. Kayea,c*
aDepartment of Chemistry, b Department of Biochemistry and Microbiology and
cCentre for Chemico- and Biomedicinal Research, Rhodes University, Grahamstown,
6140, South Africa.
eDrug Discovery and Development Centre (H3-D), Department of Chemistry, University of Cape
Town, Rondebosch 7701, South Africa
fMolecular Mycobacteriology Research Unit, Department of Pathology and Institute of Infectious
Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
Abstract. A series of N2,N2′-bis[4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccino-
hydrazides,containing 4-hydroxycoumarin, hydrazine and tartaric acid moieties, have been
prepared and examined for possible biological activity. Several of these compounds exhibit
promising HIV-1 integrase inhibition (IC50=3.5M), and anti-T.brucei (32% viability) and
anti-mycobacterial (Visual MIC90 = 15.63 M)
Keywords. 4-Hydroxycoumarins, succinodihydrazides, synthesis, HIV-1 integrase, anti-
trypanosomal, anti-mycobacterial.
8a: HIV-1 IN
IC50=3.5 M
.
_________
Corresponding authors: Prof Perry Kaye. E-mail: P.Kaye@ru.ac.za and
Prof Rui Krause. E-mail: R.Krause@ru.ac.za
1

Numerous coumarin derivatives have been found to be present as secondary metabolites in
bacteria, fungi and plants, thus prompting research into their isolation and the study of their
biological activities.1 Such derivatives exhibit wide varieties of medicinal properties, including
anti-parasitic, anti-HIV, anti-bacterial, as well as serine protease, cholinesterase and
lipoxygenase monoamine oxidase inhibitory activities.2-4 The antibiotics, novobiocin (isolated
from Streptomyces niveus and Streptomycesspheroides) and chartreusin (isolated from
Streptomyces chartreusis) are examples of coumarin derivatives isolated from bacteria,5 while
aflatoxins are highly toxic, coumarin-containing, fungal metabolites found in Aspergillus
species.6
Various biologically active coumarin derivatives have also been synthesised,7 including:
coumarin-2-carboxamides,8 which exhibit ant-cancer activity; the 4-hydroxycoumarin
derivative 1, which exhibits HIV-1 protease (PR) inhibition activity;9 and coumarin-containing
hydrazine derivatives, such as compound 2, which exhibit minimum inhibitory concentrations
(MIC) of 15-17 μM against M.tuberculosis.10 In their highly cited paper, Zhao et al.,11 discuss
coumarin-based HIV Integrase (IN) inhibitors. More recently, 4-hydroxycoumarin dimers12
and coumarin-3-carbohydrazide derivatives13 have been reported to exhibit HIV-1 IN inhibitory
activities with IC50 values in the low micromolar and nanomolar ranges, respectively. In earlier
studies, we have explored the synthesis and biological activity of various coumarin
derivatives,14-17 including furocoumarins as potential HIV-1 (IN) inhibitors14 and coumarin-
AZT conjugates 3 as potential dual-action HIV-1 PR and reverse transcriptase (RT)
inhibitors.17 We now report the synthesis and bioactivity of a series of 4-hydroxy-coumarin
derivatives generated by the conjunction of 4-hydroxycoumarin, hydrazine and tartaric acid
moieties.
H
O
O
N
O
H
N
O
HO
N
N
HO
N
O
S
O
O
2
N
O
O
H
N
R1
OH
O
N
N
N
O
O
1
3
R2
HO
O
Figure 1. Structures of synthetic, biologically active coumarin derivatives.
2

The wide spectrum of applications of coumarin derivatives has led to considerable interest in
their synthesis and numerous methods have been developed. These include the classic Perkin,
Knoevenagel and Pechmann syntheses as well applications of Morita-Baylis-Hillman (MBH)
methodology developed by our group.18 However, our attempts to use MBH methodology to
access the 4-hydroxycoumarins derivatives required in this study proved unsatisfactory and,
consequently, the 4-hydroxycoumarins 5a-g (Scheme 1) were prepared using the method
reported by Zhao et al.19 but with minor modifications, viz., i) the use of dimethyl carbonate,
in some cases, instead of diethyl carbonate; and ii) omitting recrystallisation from ethanol when
NMR analysis indicated formation of the products in satisfactory purity. The 2-
hydroxyacetophenones 4a-g were reacted with either diethyl or dimethyl carbonate in the
presence of sodium hydride to afford the corresponding 4-hydroxycoumarins 5a-g in yields of
60-87%.
R2 OH
R2
O
R2 OH
racemic-tartaric acid
NaH,
(EtO)2CO,
R1
R1
R1
O
POCl3
AcOH
NH2NH2,
HCl or AcOH
O
5a-g
O
OH
Toluene
R1
O
O
4a-g
6a-g
OH
O
H
R2
NH2
N
H2N
N
H
AcOH,
EtOH
a
b
c
d
e
f
H
F
H
H
H
H
H
H
O
O
OH
7
Cl
Br
Me
O
N
R2 OH
OH O
H
N
MeO
R1
g
R1
5,6-Benzo
N
N
H
OH R2
8a-g
O
OH
O
O
Scheme 1. Synthesis of bis-(4-hydroxycoumarinyl)succinohydrazides.
Acetylation of the 4-hydroxycoumarins 5a-g was effected following the method described by
Sukdolak et al.20 It was observed, however, that the yields improved with longer reaction times
(at least 1 hour rather than the reported 30 minutes); moreover, the purity of the compounds
was satisfactory after washing the precipitate with methanol thereby permitting omission of a
crystallisation step. Thus, the 4-hydroxycoumarins 5a-g were each reacted with POCl3 in
glacial acetic acid under reflux for at least 1 hour, after which the resulting precipitates were
filtered, washed with methanol and dried to give the respective products (6) in yields ranging
from 41% to 90%.
3

The 2,3-dihydroxysuccinodihydrazide 7, required for the final step in the preparation of the
title compounds 8a-g, was obtained via acid (HCl or AcOH)-catalysed reaction of racemic
diethyl tartrate with hydrazine hydrate in refluxing ethanol.21 The resulting precipitate was
recrystallised from methanol to give the desired dihydrazide 7 as a white solid, which was then
reacted with the 3-acetylated 4-hydroxycoumarins 6a-g in ethanol under reflux for at least 2
hours (Scheme 1).22 The precipitates formed in each reaction were filtered, washed with
methanol and dried to give the required bis[4-hydroxycoumarin-3-yl)ethylidene]-2,3-
dihydroxysuccinohydrazides 8a-g in high purity and in yields of 41% to 58%.
The targeted products 8a-g contain: i) the 4-hydroxycoumarin moiety present in the HIV-1 PR
inhibitor 1; ii) the hydroxyethylene peptide isosteres and overall C2 symmetry characteristic of
clinical HIV-1 PR inhibitors; and iii) the coumarin and hydrazine moieties present in the anti-
mycobacterial derivative 2 and the recently reported coumarin-3-carbohydrazide HIV-1 IN
inhibitors.13 Compounds 8a-g were thus screened for HIV-1 IN and PR inhibition potential,
anti-tuberculosis (M. tuberculosis) activity and cytotoxicity (against HeLa cells). The
opportunity was also taken to screen the compounds for anti-Plasmodium falciparum
(P.falciparum) and anti-trypanosome (T.b. brucei) activity. The results of the bioassays are
summarised in Table 1.
With the exception of the brominated derivative 8d, all of the title compounds (8) showed some
inhibition of HIV-1 IN – particularly, the parent system 8a with an IC50 value of 3.5 μM. It is
interesting to note that sequential introduction of a halogen substituent (R1 = F, Cl, Br) into the
unsubstituted aromatic rings in 8a (R1 = H) resulted in graded decreases in the HIV-1 IN
inhibition (8a: 59.4% > 8b: 35.4% > 8c: 27.6% > 8d: 0%) which appear to follow their
increasing bulk inversely and their electronegativity directly. The apparent advantage conferred
by the absence of additional substituents in the benzene ring is reflected in the HIV-1 inhibition
activity of other 4-hydroxycoumarin derivatives.11 Only one of the compounds (8e; 8.3%
inhibition) proved to be active, albeit very weakly, against HIV-1 PR at a concentration of 20
μM.
4

Table 1. Bioassay data for compounds 8a-g, showing % inhibition of HIV-1 IN and PR, %
viability of P. falciparum, T.b. brucei and HeLa cells at 20 µM and MIC90 values against M.
tuberculosis.
O
O
R2 OH
OH O
H
N
R1
N
N
N
R1
H
8a-g
O
OH
OH R2
O
O
Visual
MIC90
(μM)f g,
Calc.
MIC90
(μM)f,b
%
%
% P.
%
% HeLa
cell
%
Yield
Compd.
R1
R2
HIV-1 IN HIV-1 PR falciparum T.b.brucei
inhibitiona inhibitionb
viabilityc viabilityd
viabilitye
i
8a
8b
H
F
H
H
H
H
H
H
58
58
45
42
41
43
45
0.0
0.0
90.7
92.8
42.7
32.8
78.4
84.6
>125
>125
31.25
15.63
>125
>125
31.25
0.019f
>125
>125
54.25
18.91
>125
>125
29.46
0.007f
59.4
35.4
8c
Cl
27.6
0.0
0.0
93.8
44.6
100.0
100.0
85.3
8d
Br
0.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
8e
Me
MeO
26.4
8.3
8f
34.6
0.0
84.6
8g
5,6-Benzo
35.7
0.0
98.1
c
d
e
Control
100.0 a
99.8 b
_
_
_
Controls: achicoric acid; britonavir; cchloroquine: IC50 = 0.012 μM; dpentamidine: IC50 =0.039 μM; eemetine:
IC50 = 0.013 μM and frifampicin. gVisual MIC90 7D 7H9 GLU CAS Tx . hCalculated MIC90 7D 7H9 GLU
CAS Tx (μM). iIC50 = 3.5 μM.
The products 8a-g were also evaluated for anti-parasitic activity at a concentration of 20 μM
against both P.falciparum and T.b.brucei (which is responsible for sleeping sickness). The
products showed little, if any, significant activity in the P.falciparum screen; compounds 8a-c,
however, clearly exhibited anti-trypanosomal activity, decreasing residual parasite T.b. brucei
viabilities appreciably (8a: 43%; 8b: 33%; and 8c: 45%). When tested at a concentration of 20
μM against HeLa cells, none of the compounds 8a-g exhibited significant cytotoxicity. As
indicated earlier, coumarin-containing hydrazine derivatives (e.g., compound 2, Figure 1) have
been reported by Arshad et al.13 to exhibit minimum inhibitory concentrations (MIC) in the 15-
17 μM range against M.tuberculosis, and three of our compounds (8c, 8d and 8g) have also
shown promising anti-mycobacterial potential with relatively low visual MIC90 (31.25, 15.63
and 31.25 μM, respectively) and calculated MIC90 values (54.25, 18.91 and 29.46 μM,
respectively).
5

In silico docking studies were undertaken to assess the binding affinity of compounds 8a-g to
selected HIV-1 IN and PR, P. falciparum , T. Brucei and M.tuberculosis enzyme receptors, the
detailed results of which are summarised in the Supporting Information file. Clear correlations
between the in silico binding affinities and the in vitro data generally proved to be limited, but
several instances are noteworthy. Compound 8a, which exhibited the highest in vitro HIV-1
IN inhibition (Table 1) exhibited the best in silico binding affinity (-4.628 kcal/mol) in the non-
catalytic active-site of HIV-1 IN crystal structure PDB 4E1M (illustrated in Figure 2a) and the
second best binding affinity (-6.215 kcal/mol) in the HIV-1 IN crystal structure PDB 5FRN.
Interestingly, compound 8g, which exhibited the second-highest in vitro HIV-1 IN inhibition,
exhibited the best binding affinity (-6.794 kcal/mol) for 5FRN, but the lowest binding affinity
(-0.163 kcal/mol) for 4E1M; the binding of this ligand in the active site of 5FRN is illustrated
in Figure 2b. The strong in silico HIV-1 PR 1YT9 binding affinities for all of the ligands 8a-g
(between -6.744 and -9.420 kcal/mol, compared with -7.309 kcal/mol for the control, ritonavir)
are completely at odds with the in vitro data in Table 1! The corresponding in silico HIV-1 PR
IZP8 binding affinities for the ligands 8a-g are all significantly lower than that of ritonavir but
are at least consistent with their general, in vitro inactivity.
(a)
(b)
Figure 2. Potential hydrogen-bonding interactions between amino acid residues in: a) the
non-catalytic active-site of HIV-1 IN (PDB 4E1M) and compound 8a; and b) the active-site
of HIV-1 IN (PDB 5FRN) and compound 8g.
Although compounds 8a-g all exhibited very encouraging in silico binding affinities (relative
to chloroquine) with three different P.falciparum enzyme structures, none of them exhibited
6

significant in vitro anti-parasitic activity. The three compounds with the strongest in vitro anti-
trypanosomal activity (8a-c) exhibit the weakest in silico binding affinities with the T.b.brucei
enzyme structure! It is similarly difficult to draw any meaningful correlations between the in
vitro anti-microbacterial and the in silico enzyme-binding affinities of the title compounds.
In summary, it is apparent that the title compounds are readily accessible, exhibit insignificant
cytotoxicity and some of them show promising HIV-1 IN inhibition (IC50=3.5M), anti-
T.brucei activity (32% viability at 20 mM) and anti-mycobacterial potential (Visual MIC90 =
15.63 M). Experimental data for the synthesised compounds, NMR spectra for all new
compounds, the in silico docking protocols and the resulting binding affinities are provided in
the Supporting Information file. Bioassay protocols have been reported previously.23,24
Acknowledgements.
The authors thank Rhodes University and the South African Medical Research Council
(SAMRC) for generous financial support. This research project was funded by the South
African Medical Research Council (SAMRC) with funds from National Treasury under its
Economic Competitiveness and Support Package, and Rhodes University Sandisa Imbewu.
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Declarations of interest: none
8