Evaluation of novel N′-(3-hydroxybenzoyl)-2-oxo-2H-chromene-3-carbohydrazide derivatives as potential HIV-1 integrase inhibitors

Article abstract of DOI:10.1039/c8md00328a

In an attempt to identify potential new agents that are active against HIV-1 IN, a series of novel coumarin-3-carbohydrazide derivatives were designed and synthesised. The toxicity profiles of these compounds showed that they were non-toxic to human cells and they exhibited promising anti-HIV-1 IN activities with IC₅₀ values in nM range. Also, an accompanying molecular modeling study showed that the compounds bind to the active pocket of the enzyme.

Full text of DOI:10.1039/c8md00328a

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RESEARCH ARTICLE
Evaluation of novel N′-(3-hydroxybenzoyl)-2-oxo-
2H-chromene-3-carbohydrazide derivatives as
potential HIV-1 integrase inhibitors†
Cite this: DOI: 10.1039/c8md00328a
bc
Omobolanle J. Jesumoroti, a Faridoon, a Dumisani Mnkandhla,
ac
Michelle Isaacs,bc Heinrich C. Hoppe bc and Rosalyn Klein
*
In an attempt to identify potential new agents that are active against HIV-1 IN, a series of novel coumarin-
3-carbohydrazide derivatives were designed and synthesised. The toxicity profiles of these compounds
showed that they were non-toxic to human cells and they exhibited promising anti-HIV-1 IN activities with
IC50 values in nM range. Also, an accompanying molecular modeling study showed that the compounds
bind to the active pocket of the enzyme.
Received 3rd July 2018,
Accepted 1st November 2018
DOI: 10.1039/c8md00328a
rsc.li/medchemcomm
fication of scaffolds, these successful inhibitors belong to the
most well studied classes, and the two metal binding motif is
Introduction
Human immunodeficiency virus type 1 (HIV-1) is the causa-
tive agent for acquired immune deficiency syndrome
(AIDS).1,2 Although improvement in the management of HIV/
AIDS has been achieved through the use of antiretroviral
drugs, it remains one of the world's most serious health prob-
lems.3 Despite the progress made in development of a num-
ber of drugs for treating people living with HIV/AIDS, the use-
fulness of many of these drugs is limited by toxicity, side
effects and the development of HIV resistance.4–6 In develop-
ing countries, where the disease burden is highest, drugs
targeting protease and reverse transcriptase are most com-
monly used as components of highly active anti-retroviral
therapy (HAART),7 while integrase inhibitors are typically only
introduced as a third-line treatment. There is an ongoing
need for further development of anti-HIV drugs with reduced
toxicity, which can be introduced in first-line therapy.
HIV-1 IN has been demonstrated as an attractive target for
the development of new drugs since homologous enzymes
are lacking in the human host, and the relevant enzyme is es-
sential for a stable infection.8 Presently, the approved drugs
for inhibition of HIV-1 IN, namely Raltegravir (RAL) 1,9
elvitegravir10 (EVG), dolutegravir (DTG), and Bictegravir
(BIC)11,12 are all strand transfer inhibitors. Using Yan's classi-
conserved in each case.13 None of these are used as compo-
nents of HAART in developing countries, or even as second
line drugs, due to cost of manufacture.7
Inspired by the “scaffold-hopping” approach employed by
Fan and co-workers,14 and rather than building on the well-
studied but complex pyrimidine and quinolone scaffolds, we
decided to combine two of the less established scaffolds, cou-
marin derivatives combined with the hydrazide moiety. Com-
pound 2 was synthesised by Burke et al.15 and found to be ac-
tive in inhibiting 3′ processing rather than the strand
transfer step of IN. Compound 3 is an analogue of bis-salicyl
hydrazide,15 which has been described to inhibit IN through
metal chelation, but is only active in assays using Mn2+ and
not Mg2+, and lacked antiviral activity in cell-based assays.
Wild-type IN has been shown to use either Mn2+ or Mg2+.9
Structure activity relationship studies of salicylhydrazide in-
hibitors have showed that the salicyl moiety is essential for
activity.15,16 Therefore, we decided to incorporate the hydra-
zide group16,17 on the coumarin moiety18 to achieve a syner-
gistic effect in the anti-HIV-IN activity with possibility of re-
duced toxicity relative to the salicylhydrazides, and improved
synthetic efficiency relative to Raltegravir.19 In this study, we
designed 2-oxo-2H-chromene-3-carbohydrazide derivatives,
producing relatively simple potential lead compounds as
HIV-1 IN inhibitors.
a Department of Chemistry, Rhodes University, Grahamstown, 6140, South Africa.
E-mail: r.klein@ru.ac.za
We envisioned that 3-acyl-coumarin could mimic the
diketo-acid moiety of Raltegravir 1 (Fig. 1), and there was the
additional possibility of hydrogen bonding through the inter-
action of the oxygen in the pyran ring, as well as the other hy-
droxy, alkoxy and halogens with the amino acid residues
within the catalytic site of HIV-1 IN.18 The unique structure
of hydrazides has been proposed to inhibit cell proliferation
b Department of Biochemistry and Microbiology, Rhodes University, Grahamstown,
6140, South Africa
c Centre for Chemico- and Biomedicinal Research, Rhodes University, Grahams-
town, 6140, South Africa
† Electronic supplementary information (ESI) available: NMR and IR spectra and
mass spectral data. See DOI: 10.1039/c8md00328a
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Results and discussion
Derivatives of N′-[2-hydroxybenzoyl]-2-oxo-2H-chromene-3-
carbohydrazide, compounds 8a–t were synthesized
(Scheme 1) by reacting 2-oxo-2H-chromene-3-carbonyl chlo-
rides 4a–e with 2-hydroxybenzohydrazides 7a–e under basic
conditions. The acid chloride precursors 4a–e were success-
fully synthesized using previously reported Knoevenagel con-
densation20 of salicylic aldehydes 1a–e with diethyl malonate
to afford ethyl 2-oxo-2H-chromene-3-carboxylate derivatives
2a–e in good yields (73–92%). This was followed by alkaline
hydrolysis using NaOH to give 3a–e (75–89%), which were
reacted with oxalyl chloride to afford the key intermediates
4a–e which were used without further purification. Esterifica-
tion of substituted salicylic acids 5a–d using sulfuric acid in
methanol afforded the corresponding 2-hydroxy benzoate de-
rivatives 6a–d, hydrazinolysis of which afforded substituted
2-hydroxy benzohydrazides 7a–d. Finally, the target com-
Fig. 1 Raltegravir, its analogue (2) and hydrazide 3 showing the diketo
moiety.
pounds,
N′-[2-hydroxybenzoyl]-2-oxo-2H-chromene-3-
carbohydrazide derivatives 8a–t were obtained by stirring
7a–d with 4a–e in the presence of saturated Na2CO3
overnight.
through hydrophobic interaction and hydrogen bonding with
the target protein.17
Scheme 1 Reagents and conditions (i) diethyl malonate, piperidine, CH3COOH, ethanol, 80 °C, 12 h (ii) NaOH, ethanol, reflux (iii) oxalyl chloride,
reflux, 8 h. (iv) Conc. H2SO4, CH3OH, reflux, 24 h (v) 65% N2H4·H2O, 80–90 °C, overnight. (vi) Na2CO3, room temperature, overnight.
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Simple transformations of readily available starting mate-
rials were employed for the short, convergent synthesis of
compounds 8a–t from substituted salicylaldehydes and
salicylic acids. The methodology was, apparently, quite ro-
bust, facilitating the preparation of a fairly diverse library of
compounds. The yield of the final coupling step was variable,
depending on the substitution patterns on the two ring sys-
tems. Although the alkoxy substituents gave the best yields
(56–83%), the most efficient syntheses were between electron
rich salicyl hydrazides and coumarins bearing halides or hy-
drogen (8k–m and p–r). The simplicity of the method (six
steps, medium temperatures, ethanol as solvent) was consid-
ered advantageous.
Compounds 8a–t (Table 1) were evaluated for their in vitro
HIV-1 IN inhibition activity relative to chicoric acid (CA) as a
standard, according to the method described by McColl et al.21
Both chicoric acid and Raltegravir are strand transfer inhibitors,
which are effective in enzyme inhibition assays.13 CA is simply
the cheaper of the two. This method uses HIV-1 subtype C IN
bound to donor DNA immobilized in 96 well plates. Activity is
detected using absorbance of SureBlue Reserve at 620 nm, fol-
lowing oxidation. Entries 16–20 showed minimal inhibition
(less than 55%), and they were not examined further. It is nota-
ble that all of these compounds bear a methoxy group at posi-
tion 3 on the C-ring. By contrast, entries 1–5 (Table 1), with no
additional substitution on the C-ring, all showed inhibition
>85%. This agrees with the result found by Burke and co-
workers that the intact salicyl grouping is important for activ-
ity.15 Compounds 8a–o, which showed at least 70% inhibition
of extracellular HIV-1 IN, were examined further to determine
their IC50 values (calculated from dose response curves, in-
cluded in the ESI†). Among these compounds, two were particu-
larly noteworthy: 8b (IC50 = 14 nM), and 8c (IC50 = 13 nM), both
of which were found to be comparable to the standard, chicoric
acid (IC50 = 10 nM). These could thus serve as potential leads to
develop effective IN inhibitors.
From the table it is possible to discern three groups
according to IC50. The first group (8a–f, and h–j) show the
highest levels of inhibition, and IC50 values near or below
100 nM. Within this group the compounds with the highest
activity bore chloro- or bromo- substituents on ring A and
only a hydroxy on ring C. These compounds both have IC50
values comparable to CA. The lowest activity was observed for
those compounds bearing the methoxy groups on the C-ring
(8k–o and p–t). The compounds bearing chloro-substituents
on ring C showed some potential, particularly where this
was coupled with an electron rich coumaryl substituent (8i
and j).
In general, it was observed that the presence of chlorine
atom on either ring C (salicyl moiety) or the coumarin system
results in good anti-HIV-1 IN activity. The greatest impact is
the substitution on ring C, suggesting that this is most likely
to be the end of the molecule which binds to the metal ions
in the active site.
Table 1 Inhibitory potencies of coumarin hydrazides as measured in an HIV-I integrase assay
Compound
% inhibition
101.5
HIV-integrase IC50/nM (SE)a
10. (1)
Entry
Chicoric acid (+ve control)
A
C
1
2
3
4
5
6
7
8
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
H
6-Br
6-Cl
8-OMe
8-OEt
H
6-Br
6-Cl
8-OMe
8-OEt
H
6-Br
6-Cl
8-OMe
8-OEt
H
6-Br
6-Cl
H
H
H
H
H
5′Cl
5′Cl
5′Cl
90
94
95
89
85
91
70
79
86
88
83
88
86
70
60
41
54
44
32
24
70 (14)
14 (29)
13 (12)
47 (11)
104 (29)
33 (29)
175 (70)
41 (34)
23 (30)
31 (42)
5800 (1700)
163 (49)
605 (85)
901 (143)
592 (277)
—
9
5′Cl
5′Cl
10
11
12
13
14
15
16
17
18
19
20
5′OMe
5′OMe
5′OMe
5′OMe
5′OMe
3′OMe
3′OMe
3′OMe
3′OMe
3′OMe
—
—
—
—
8s
8t
8-OMe
8-OEt
a
IC50 values were determined from single replicates and are quoted with errors determined from the graphs (SE).
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Previous reports on the salicylhydrazide class of IN inhibi-
tors have shown that the majority of compounds in this class
are severely cytotoxic in cell-based assays, thus limiting their
therapeutic application.22 In order to investigate the thera-
peutic index of our compounds, the cytotoxicity of all 20 com-
pounds were measured against HeLa cells (Fig. 2). All the
compounds showed low levels of cytotoxicity with cell viabil-
ity remaining above 70% in most cases.
The docking of the representative carbohydrazide inhibi-
tors in this series (8i and 8c) to the active site of the mono-
meric subunit A of 1QS4 was performed using the Auto dock
4.2 programme.27 Compounds 8i and 8c bound well in the
active site of 1QS4 with hydrogen bonding through the inter-
action of the oxygen in the pyran ring, the methoxyl oxygen,
and the carbonyl oxygen with proximate active amino acid
residues (Glu152, Gln148, Gly149 Asn155, Thr66, His67,
Lys159 and Asp64) in the 1QS4 active site (Fig. 4). The close
proximity of the docked compounds to the binding site of
chicoric acid in the active site of the enzyme could explain
the inhibitory potency of these compounds when compared
to chicoric acid.
Fig. 4 highlighted an interesting comparison between the
two hydrazides chosen: the two ligands (8c and 8i) bind in
slightly different orientations, depending on the location of
the chloro substituent. In each case the ligand was centred
close to Asn155, with the ring bearing the chloro group clos-
est to Gln148. The result is that the particular binding mode
relative to the metal ions and the hydrophobic pocket
changes depending on the substituents.
The four best inhibitors in this series were 8b and c, and
8i and j, showing low or negligible cytocoxicity and excellent
enzyme inhibition. We chose the best of each pair to repre-
sent the different substitution on ring C. Thus, in order to ex-
plore the binding modes of compounds 8c (IC50 = 13 nM)
and 8i (IC50 = 23 nM) with the active site of the IN enzyme,
molecular modelling was employed. The 1QS4 crystal struc-
ture of HIV-1 IN was selected from the protein data bank.
Chicoric acid (L-CA, Fig. 3) was docked into the active site of
1QS4 because it binds to the same binding pocket as the
diketo acids (DKAs),23 including Raltegravir.24 However,
chicoric acid is larger than the DKAs, and thus has the poten-
tial to make significantly more contact with residues within
the active site. Therefore, L-CA represents an effective probe
of IN catalytic activity.25 In addition, several point mutations Experimental
of IN lead to resistance to DKAs, while reduced inhibition by
Materials and methods
L-CA is only observed with one.26 This suggests that binding
of L-CA might be more robust to mutations than various
DKAs, the option of several interactions with the active site
making up for the losses of one or two.
All solvents were chemically pure or pre-purified and freshly
distilled before use. Solvents were dried by standard proce-
dures prescribed in Vogel.20 The reagents were of analytical
grade as supplied by Sigma Aldrich or Fluka. Reactions were
monitored by thin layer chromatography (TLC) on Merck pre-
coated silica GF254 plates. Melting points (uncorrected) were
In our modeling studies it was observed that chicoric
acid docked near the binding site of the Mg2+, and hydro-
gen bonding interactions with Glu152, His67 and Gln148
were observed. The lowest energy binding mode of 8i over-
laid on chicoric acid (blue) in the active site of 1QS4
(Fig. 4) revealed that inhibitors 8i and chicoric acid fill the
binding pocket of 1QS4 in similar ways, with the coumaryl
end interacting with His67 and the salicyl end H-bonding
to Gln148.
1
determined using hot stage apparatus. H and 13C-NMR spec-
tra were recorded on a Bruker Avance 600 MHz spectrometer at
298 K, and were calibrated using the solvent signals; chemical
shifts are reported in ppm, and coupling constants are given in
Hertz (Hz). High-resolution mass spectra (HRMS): electrospray
ionisation accurate mass measurements were recorded in
Fig. 2 Cell toxicity assay (at 20 μM) of substituted N′-[2-hydroxybenzoyl]-2-oxo-2H-chromene-3-carbohydrazide derivatives (SD indicated with
shading on each bar). Shading of bars correlates with substitution on C-ring.
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Fig. 4 The binding mode of 8i and 8c (coloured by atom) in the active
site of 1QS4. Hydrogen bonds are shown as dashed lines.
recrystallized from ethanol to afford the corresponding cou-
marin hydrazides derivatives 8a–8t.
N ′ - ( 2 - H y d r o x y b e n z o y l ) - 2 - o x o - 2 H - c h r o m e n e - 3 -
carbohydrazide 8a28. Yellow solid. Yield (0.280 g, 69%), m.p.
>300 °C; [HRMS: m/z calculated for C17H13N2O5 (MH+)
325.0824. Found 325.0819]; νmax/cm−1 3391 and 3280 (2 × NH),
2700–3570 (br, OH), 1696 and 1608 (CO); δH (300 MHz,
DMSO-d6) 11.91 (1H, s, NH), 8.88 (1H, s, Ar–H), 8.00 (1H, dd, J
= 7.8 Hz, 1.4 Hz, Ar–H), 7.80–7.69 (2H, m, Ar–H), 7.52 (1H, d, J
= 8.3 Hz, Ar–H), 7.44 (1H, t, J = 7.5 Hz, Ar–H), 7.14–7.08 (1H,
m, Ar–H), 6.70–6.64 (2H, overlapping m, Ar–H); δC (75 MHz,
DMSO-d6) 164.4, 160.4 and 160.2 (CO), 153.6 (Ar–C), 153.4
(Ar–C), 145.9 (Ar–C), 137.8 (Ar–C), 133.5 (Ar–C), 130.0 (Ar–C),
129.9 (Ar–C), 128.2 (Ar–C), 125.1 (Ar–C), 120.8 (Ar–C), 119.5
(Ar–C), 118.9 (Ar–C), 116.5 (Ar–C) and 116.2 (Ar–C).
Fig.
3 Molecular docking and ligand interaction of 8i (A and B)
superimposed on chicoric acid (blue) and Mg2+ (black) in the active site
of 1QS4, showing similarities among active site interactions.
positive mode (or negative mode where specified) on a Waters
API Q-TOF Ultima spectrometer with an ESI† source.
6-Bromo-N′-(2-hydroxybenzoyl)-2-oxo-2H-chromene-3-
carbohydrazide 8b. Yellow solid (0.220 g, 44%), m.p. >300
°C; [HRMS (negative mode): m/z calculated for C17H10N2O5Br
(M − H) 400.9773. Found 400.9774]; νmax/cm−1 3038 and 3244
(2 × NH), 2835–3306 (br, OH), 1685 and 1545 (CO); δH (600
MHz, DMSO-d6) 15.15 (1H, s, NH), 11.91 (1H, s, NH), 8.83
(1H, s, Ar–H), 8.25 (1H, s, Ar–H), 7.85–7.76 (2H, overlapping
m, Ar–H), 7.48 (1H, d, J = 8.1 Hz, Ar–H), 7.09 (1H, s, Ar–H),
6.66–6.67 (2H, overlapping m, Ar–H); δC (150 MHz, DMSO-d6)
164.6, 160.2 and 160.0 (CO), 153.0 (Ar–C), 152.7 (Ar–C),
144.6 (Ar–C), 135.6 (Ar–C), 131.8 (Ar–C), 129.8 (Ar–C), 128.2
(Ar–C), 121.0 (Ar–C), 121.0 (Ar–C), 120.6, (Ar–C), 118.4 (Ar–C),
116.6 (Ar–C), 116.4 (Ar–C) and 116.1 (Ar–C).
Synthetic procedures
General procedure for synthesis of coumarin hydrazides
8a–8t28. To
a
stirred solution of the appropriate
2-hydroxybenzohydrazide (1.25 mmol) in 4 mL of water, was
added the appropriate solution of 2-oxo-2H-chromene-3-
carbonyl chlorides 4a (1.25 mmol) in 6 mL of toluene. There-
after, 6 mL of a saturated aqueous solution of Na2CO3 was
added to maintain pH ∼8, and the mixture was stirred for 4
h and left overnight. After the completion of the reaction as
monitored by TLC, the precipitate was filtered off, washed
with water and air-dried. The resultant crude material was
6-Chloro-N′-(2-hydroxybenzoyl)-2-oxo-2H-chromene-3-
carbohydrazide 8c. Yellow solid (0.210 g, 48%), m.p. >300 °C;
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[HRMS (negative mode): m/z calculated for C17H10N2O5Cl (M
− H) 357.0278. Found 357.0268]; νmax/cm−1 3250 and 3040 (2
× NH), 3300–2820 (br, OH) and 1685, 1545 (CO); δH (600
MHz, DMSO-d6) 15.20 (1H, s, NH), 11.93 (1H, s, NH), 8.85
(1H, s, Ar–H), 8.15 (1H, d, J = 2.5 Hz, Ar–H), 7.75 (2H, t, J =
9.0 Hz, Ar–H), 7.55 (1H, d, J = 8.8 Hz, Ar–H), 7.08 (1H, t, J =
7.6 Hz, Ar–H), 6.67–6.64 (2H, overlapping m, Ar–H); δC (150
MHz, DMSO-d6) 164.5, 160.1 and 160.0 (CO), 152.8 (Ar–C),
152.2 (Ar–C), 144.6 (Ar–C), 132.8 (Ar–C), 129.7 (Ar–C), 128.8
(Ar–C), 128.7 (Ar–C), 128.1 (Ar–C), 121.0 (Ar–C), 120.7 (Ar–C),
120.4 (Ar–C), 118.1 (Ar–C), 116.5 (Ar–C) and 116.1 (Ar–C).
N′-(2-Hydroxybenzoyl)-8-methoxy-2-oxo-2H-chromene-3-
carbohydrazide 8d. Yellow solid (0.24 g, 57%), m.p. >300 °C;
[HRMS: m/z calculated for C18H15N2O6 (MH+) 355.0930.
Found 355.0915]; νmax/cm−1 3290 (overlapping OH and NH),
3620–2740 (br, OH), and 1709 and 1608 (CO); δH (600 MHz,
DMSO-d6) 11.94 (1H, s, NH), 8.81 (1H, s, Ar–H), 7.79 (1H, d, J
= 7.4 Hz, Ar–H), 7.48 (1H, d, J = 7.1 Hz, Ar–H), 7.40–7.31 (2H,
m, Ar–H), 7.12 (1H, t, J = 7.0 Hz, Ar–H), 6.73–6.67 (2H,
overlapping m, Ar–H), 3.93 (3H, s, OCH3); δC (150 MHz,
DMSO-d6) 164.7, 160.4 and 160.3 (CO), 153.8 (Ar–C), 146.5
(Ar–C), 146.4 (Ar–C), 143.0 (Ar–C), 130.2 (Ar–C), 128.4 (Ar–C),
125.3 (Ar–C), 121.1 (Ar–C), 120.7 (Ar–C), 119.5 (2 × Ar–C),
116.8 (Ar–C), 116.5 (Ar–C), 115.7 (Ar–C) and 56.4 (OCH3).
N′-(2-Hydroxybenzoyl)-8-ethoxy-2-oxo-2H-chromene-3-
carbohydrazide 8e. Yellow solid (0.260 g, 60%), m.p. >300
°C; [HRMS: m/z calculated for C19H17N2O6 (MH+) 369.1087.
Found 369.1073]; νmax/cm−1 3471 and 3276 (2 × NH), 3610–
2770 (br, OH) and 1709 and 1608 (CO); δH (600 MHz,
DMSO-d6) 8.79 (1H, s, Ar–H), 7.80 (1H, d, J = 7.3 Hz, Ar–H),
7.46 (1H, d, J = 6.9 Hz, Ar–H), 7.37–7.29 (2H, m, Ar–H), 7.15
(1H, t, J = 7.2 Hz, Ar–H), 6.78–6.70 (2H, overlapping m, Ar–
H), 4.17 (2H, q, J = 6.6 Hz, –OCH2CH3), 1.41 (3H, t, J = 6.6
Hz,–OCH2CH3); δC (150 MHz, DMSO-d6) 164.5, 160.4 and
160.3 (CO), 154.4 (Ar–C), 146.9 (Ar–C), 145.8 (Ar–C), 143.2
(Ar–C), 130.8 (Ar–C), 128.7 (Ar–C), 125.5 (Ar–C), 121.2 (Ar–C),
120.2 (Ar–C), 119.6 (Ar–C), 119.3 (Ar–C), 117.2 (Ar–C), 116.7
(Ar–C) and 116.7 (Ar–C), 64.9 (OCH2) and 15.0 (CH3).
N′-(5-Chloro-2-hydroxybenzoyl)-2-oxo-2H-chromene-3-
carbohydrazide 8f. Yellow solid (0.194 g, 43%), m.p. >300 °C;
[HRMS: (negative mode) m/z calculated for C17H10N2O5Cl (M-
H) 357.0278. Found 357.0287]; νmax/cm−1 3392 and 3278 (2 ×
NH), 3610–2700 (br, OH), 1704 and 1612 (CO); δH (600
MHz, DMSO-d6) 11.82 (1H, s, NH), 8.88 (1H, s, Ar–H), 7.99
(1H, d, J = 7.5 Hz, Ar–H), 7.77–7.70 (2H, m, Ar–H), 7.51 (1H,
d, J = 8.3 Hz, Ar–H), 7.44 (1H, t, J = 7.4 Hz, Ar–H), 7.12 (1H,
d, J = 10.8 Hz, Ar–H), 6.72 (1H, d, J = 8.6 Hz, Ar–H); δC (150
MHz, DMSO-d6) 162.9, 160.5 and 159.5 (CO), 154.0 (Ar–C),
153.8 (Ar–C), 146.5 (Ar–C), 133.8 (Ar–C), 130.2 (Ar–C), 129.8
(Ar–C), 127.4 (Ar–C), 125.3 (Ar–C), 121.9 (Ar–C), 120.0 (Ar–C),
119.1 (Ar–C), 118.9 (Ar–C), 118.5 (Ar–C) and 116.3 (Ar–C).
6-Bromo-N′-(5-chloro-2-hydroxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8g. Yellow solid (0.210 g, 38%),
m.p. >300 °C; [HRMS: (negative mode) m/z calculated for
C17H9N2O5BrCl (M − H) 434.9383. Found 434.9381]; νmax/cm−1
3316 and 3043 (2 × NH), 3370–2990 (br, OH) and 1698 and
1547 (CO); δH (600 MHz, DMSO-d6) 15.40 (1H, s, NH),
11.83 (1H, s, NH), 8.83 (1H, s, Ar–H), 8.26 (1H, s, Ar–H), 7.85
(1H, d, J = 7.9 Hz, Ar–H), 7.70 (1H, s, Ar–H), 7.48 (1H, d, J =
8.7 Hz Ar–H), 7.09 (1H, d, J = 8.3 Hz, Ar–H), 6.69 (1H, J = 8.5
Hz, Ar–H); δC (150 MHz, DMSO-d6) 163.1, 160.0, 159.5
(CO), 153.3 (Ar–C), 152.7 (Ar–C), 144.9 (Ar–C), 135.8 (Ar–C),
131.9 (Ar–C), 129.4 (Ar–C), 127.3 (Ar–C), 122.2 (Ar–C), 120.8
(Ar–C), 120.4 (Ar–C), 119.8 (Ar–C), 118.4 (Ar–C), 118.2 (Ar–C)
and 116.7 (Ar–C).
6-Chloro-N′-(5-chloro-2-hydroxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8h. Yellow solid (0.244 g, 50%),
m.p. >300 °C; [HRMS: (negative mode) m/z calculated
C17H9N2O5Cl2 for (M − H) 390.9689. Found 390.9691]; νmax
/
cm−1 3291 and 3046 (2 × NH), 3600–2825 (br, OH), and 1709
and 1550 (CO); δH (600 MHz, DMSO-d6) 11.85 (1H, s, N–H),
8.84 (1H, s, Ar–H), 8.13 (1H, s, Ar–H), 7.76–7.68 (2H, m, Ar–
H), 7.55 (1H, d, J = 8.8 Hz, Ar–H), 7.10 (1H, d, J = 8.5 Hz, Ar–
H), 6.69 (1H, d, J = 8.6 Hz, Ar–H); δC (150 MHz, DMSO-d6)
163.1, 160.1 and 159.5 (CO), 153.3 (Ar–C), 152.4 (Ar–C),
145.0 (Ar–C), 133.1 (Ar–C), 129.5 (Ar–C), 130.0 (Ar–C), 128.9
(Ar–C), 127.3 (Ar–C), 122.3 (Ar–C), 120.4 (Ar–C), 120.4 (Ar–C),
119.9 (Ar–C), 118.3 (Ar–C) and 118.3 (Ar–C).
N′-(5-Chloro-2-hydroxybenzoyl)-8-methoxy-2-oxo-2H-
chromene-3-carbohydrazide 8i. Yellow solid (0.324 g, 67%),
m.p. >300 °C; [HRMS: (negative mode) m/z calculated for
C18H12N2O6Cl (M − H) 387.0384. Found 387.0377]; νmax/cm−1
3565 and 3230 (2 × NH), 3600–2825 (br, OH) and 1693 (ester
CO), 1608 and 1574 (amide CO); δH (300 MHz, DMSO-d6)
11.88 (1H, s, N–H), 8.84 (1H, s, Ar–H), 7.71 (1H, s, Ar–H),
7.52 (1H, d, J = 6.6 Hz, Ar–H), 7.38 (2H, t, J = 8.5 Hz, Ar–H),
7.10 (1H, d, J = 10.3 Hz, Ar–H), 6.70 (1H, d, J = 8.5 Hz, Ar–H),
3.94 (3H, s, OCH3); δC (75 MHz, DMSO-d6) 163.0, 160.2 and
159.6 (CO), 153.6 (Ar–C), 146.4 (Ar–C), 146.36 (Ar–C), 142.3
(Ar–C), 129.4 (Ar–C), 127.2 (Ar–C), 125.1 (Ar–C), 122.2 (Ar–C),
121.0 (Ar–C), 119.8 (Ar–C), 119.4 (Ar–C), 119.3 (Ar–C), 118.3
(Ar–C), 115.6 (Ar–C) and 56.3 (OCH3).
N′-(5-Chloro-2-hydroxybenzoyl)-8-ethoxy-2-oxo-2H-
chromene-3-carbohydrazide 8j. Yellow solid (0.29 g, 58%), m.
p. >300 °C; [HRMS: m/z calculated for C19H16N2O6Cl (MH+)
403.0697. Found 403.0700]; νmax/cm−1 3443 and 3261 (2 ×
NH), 3610–2700 (br, OH) and 1694 (ester CO), 1606, 1575
(amide CO); δH (600 MHz, DMSO-d6) 15.51 (1H, s, NH),
11.87 (1H, s, NH), 8.84 (1H, s, Ar–H), 7.70 (1H, s, Ar–H), 7.51
(1H, d, J = 6.8 Hz, Ar–H), 7.38 (1H, d, J = 7.5 Hz, Ar–H), 7.34
(1H, d, J = 7.7 Hz, Ar–H), 7.09 (1H, d, J = 7.7 Hz, Ar–H), 6.69
(1H, d, J = 8.20 Hz, Ar–H), 4.20 (2H, q, J = 6.8 Hz, OCH2),
1.43 (3H, t, J = 6.9 Hz, CH3); δC (150 MHz, DMSO-d6) 162.9,
160.2, and 159.6 (CO), 153.5 (Ar–C), 146.4 (Ar–C), 145.6
(Ar–C), 143.0 (Ar–C), 129.3 (Ar–C), 127.2 (Ar–C), 125.1 (Ar–C),
122.3 (Ar–C), 120.9 (Ar–C), 119.7 (Ar–C), 119.5 (Ar–C), 119.3
(Ar–C), 118.2 (Ar–C), 116.3 (Ar–C), 64.6 (OCH2) and 14.70
(CH3).
N′-(2-Hydroxy-5-methoxybenzoyl)-2-oxo-2H-chromene-3-
carbohydrazide 8k. Yellow solid (0.35 g, 79%), m.p. >300 °C;
[HRMS: m/z calculated for C18H15N2O6 (MH+) 355.0930.
Found 355.0927]; νmax/cm−1 3550–2700 (overlapping NH and
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OH) and 1704 (ester CO), 1609 and 1567 (amide CO) δH
(300 MHz, DMSO-d6) 11.97 (1H, s, NH), 8.79 (1H, s, Ar–H),
7.89 (1H, s, Ar–H), 7.70 (1H, s, Ar–H), 7.47–7.36 (3H,
overlapping m, Ar–H), 6.75–6.67 (2H, overlapping m, Ar–H),
3.66 (3H, s, OCH3); δC (75 MHz, DMSO-d6) 165.0, 161.3 and
155.0 (CO), 154.2 (Ar–C), 154.0 (Ar–C), 151.2 (Ar–C), 147.2
(Ar–C), 133.6 (Ar–C), 130.7 (Ar–C), 126.2 (Ar–C), 121.0 (Ar–C),
119.4 (Ar–C), 119.2.(Ar–C), 117.8 (Ar–C), 117.6 (Ar–C), 116.9
(Ar–C), 112.8 (Ar–C) and 56.2 (OCH3).
8.7, 3.2 Hz, Ar–H), 6.66 (1H, d, J = 8.7 Hz, Ar–H), 4.20 (2H, q,
J = 6.9 Hz, OCH2), 3.69 (3H, s, OCH3), 1.43 (3H, t, J = 6.9 Hz,
CH3); δC (150 MHz, DMSO-d6) 164.1, 160.1 and 153.9 (CO),
153.6 (Ar–C), 150.7 (Ar–C), 146.3 (Ar–C), 145.6 (Ar–C), 143.0
(Ar–C), 125.1 (Ar–C), 120.9 (Ar–C), 120.5, (Ar–C), 119.5 (Ar–C),
119.4 (Ar–C), 116.9 (Ar–C), 116.6 (Ar–C), 116.4 (Ar–C), 112.1
(Ar–C), 64.6 (OCH2), 55.4 (OCH3) and 14.7 (CH3).
N′-(2-Hydroxy-3-methoxybenzoyl)-2-oxo-2H-chromene-3-
carbohydrazide 8p. Yellow solid (0.35 g, 79%), m.p. >300 °C,
[HRMS: m/z calculated for C18H15N2O6 (MH+) 355.0930.
Found 355.0931]; νmax/cm−1 3270–3053 (2 × NH), 3570–2840
(br, OH) and 1700 (ester CO), 1609 and 1567 (amide CO);
δH (600 MHz, DMSO-d6) 15.50 (1H, s, OH), 11.88 (1H, s, N–
H), 8.89 (1H, s, Ar–H), 8.00 (1H, s, Ar–H), 7.73 (1H, s, Ar–H),
7.52 (1H, s, Ar–H), 7.44–7.39 (2H, overlapping, Ar–H), 6.79
(1H, s, Ar–H), 6.53 (1H, s, Ar–H), 3.73 (3H, s, OCH3); δC (150
MHz, DMSO-d6) 164.6, 160.4 and 153.6 (CO), 153.5 (Ar–C),
152.1 (Ar–C), 148.6 (Ar–C), 146.0 (Ar–C), 133.6 (Ar–C), 130.0
(Ar–C), 125.1 (Ar–C), 120.4 (Ar–C), 120.2 (Ar–C), 119.3 (Ar–C),
118.9 (Ar–C), 116.1 (Ar–C), 114.5 (Ar–C), 112.7 (Ar–C) and
55.7 (OCH3).
6-Bromo-N′-(2-hydroxy-3-methoxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8q. Yellow solid (0.41, 76%), m.
p. >300 °C; [HRMS: m/z for C18H14N2O6Br (MH+) 433.2100.
Found 433.0196]; νmax/cm−1 3464 and 3282 (2 × NH) and
3625–3625 (br, OH), 1703 (ester CO), 1616 and 1562 (amide
CO); δH (600 MHz, DMSO-d6) 15.53 (1H, s, OH), 11.89 (1H,
s, N–H), 8.85 (1H, s, Ar–H), 8.28 (1H, s, Ar–H), 7.86 (1H, d, J =
7.9 Hz, Ar–H), 7.49 (1H, d, J = 8.0 Hz, Ar–H), 7.39 (1H, d, J =
6.8 Hz, Ar–H), 6.79 (1H, d, J = 6.3 Hz, Ar–H), 6.53 (1H, s, Ar–
H), 3.72 (3H, s, CH3); δC (150 MHz, DMSO-d6) 164.7, 160.0
and 153.0 (CO), 152.6 (Ar–C), 151.7 (Ar–C), 148.4 (Ar–C),
144.6 (Ar–C), 135.6 (Ar–C), 131.8 (Ar–C), 129.0 (Ar–C), 128.3
(Ar–C), 120.8 (Ar–C), 120.5 (Ar–C), 118.4 (Ar–C), 116.6 (Ar–C),
114.7 (Ar–C), 112.8 (Ar–C) and 55.7 (OCH3).
6-Bromo-N′-(2-hydroxy-5-methoxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8l. Yellow solid (0.41, 76%), m.p.
>300 °C [HRMS: (negative mode) m/z calculated for
C18H12N2O6Br (M − H) 430.9879. Found 430.9870]; νmax/cm−1
3550–2700 (overlapping OH and NH) and 1703 (ester CO),
1611 (amide CO); δH (600 MHz, DMSO-d6) 11.87 (1H, s,
NH), 8.82 (1H, s, Ar–H), 8.26 (1H, s, Ar–H), 7.85 (1H, s, Ar–
H), 7.48 (1H, s, Ar–H), 7.36 (1H, s, Ar–H), 6.74 (1H, s, Ar–H),
6.65 (1H, s, Ar–H), 3.68 (3H, s, OCH3); δC (150 MHz, DMSO-
d6) 164.3, 160.0 and 153.6 (CO), 153.4 (Ar–C), 152.7 (Ar–C),
150.8 (Ar–C), 144.8 (Ar–C), 135.8 (Ar–C), 131.9 (Ar–C), 120.9
(Ar–C), 120.8 (Ar–C), 120.6 (Ar–C), 118.5 (Ar–C), 116.9 (Ar–C),
116.7 (Ar–C), 116.6 (Ar–C), 112.1 (Ar–C) and 55.5 (OCH3).
6-Chloro-N′-(2-hydroxy-5-methoxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8m. Yellow solid (0.40, 83%), m.
p. >300 °C; [HRMS: m/z calculated for C18H14N2O6Cl (MH+)
389.0540 found 389.0539]; νmax/cm−1 3510–2850 (overlapping
OH and NH), 1703 and 1609 (CO); δH (600 MHz, DMSO-d6)
11.91 (1H, s, N–H), 8.81 (1H, s, Ar–H), 8.10 (1H, s, Ar–H),
7.73 (1H, s, Ar–H), 7.57 (1H, s, Ar–H), 7.53 (1H, s, Ar–H), 6.73
(1H, s, Ar–H), 6.64 (1H, s, Ar–H) and 4.10 (3H, s, OCH3); δC
(150 MHz, DMSO-d6) 164.6, 160.3 and 153.8 (CO), 153.5
(Ar–C), 152.50 (Ar–C), 150.9 (Ar–C), 145.1.(Ar–C), 133.2 (Ar–C),
129.1 (Ar–C), 121.2 (Ar–C), 121.0 (Ar–C), 120.7 (Ar–C), 120.5
(Ar–C), 118.4 (Ar–C), 111.0 (Ar–C), 116.8 (Ar–C), 112.3 (Ar–C)
and 55.6 (OCH3).
N′-(2-hydroxy-5-methoxybenzoyl)-8-methoxy-2-oxo-2H-
chromene-3-carbohydrazide 8n. Yellow solid (0.299, 47%), m.
p. >300 °C; [HRMS: m/z calculated for C19H17N2O7 (MH+)
385.1036. Found 385.1040]; νmax/cm−1 3560–2770 (overlapping
OH and NH), 1701 (ester CO), 1609 and 1561 (amide
CO); δH (600 MHz, DMSO-d6) 11.81 (1H, s, N–H), 8.83 (1H,
s, Ar–H), 7.52 (1H, m, Ar–H), 7.34–7.41 (3H, m, Ar–H), 6.77
(1H, dd, J = 8.7, 3.3 Hz, Ar–H), 6.67 (1H, d, J = 8.7 Hz, Ar–H),
3.94 (3H, s, OCH3), 3.69 (3H, s, OCH3). δC (150 MHz, DMSO-
d6) 164.1, 160.0 and 154.1 (CO), 153.5 (Ar–C), 150.8 (Ar–C),
146.4 (Ar–C), 143.0 (Ar–C), 125.1 (Ar–C), 121.0 (Ar–C), 120.2 (2
× Ar–C), 119.4 (Ar–C), 117.1 (Ar–C), 116.7 (Ar–C), 115.6 (Ar–C),
112.1 (2 × Ar–C), 56.3 (OCH3) and 55.5 (OCH3).
8-Ethoxy-N′-(2-hydroxy-5-methoxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8o. Yellow solid (0.253 g, 51%),
m.p. >300 °C [HRMS: m/z calculated for C20H19N2O7 (MH+)
399.1184. Found 399.1144]; νmax/cm−1 3468 and 3259 (2 ×
NH), 3600–2780 (br, OH), 1688 (ester CO), 1607 and 1582
(amide CO); δH (600 MHz, DMSO-d6) 11.84 (1H, s, NH),
8.82 (1H, s, Ar–H), 7.49 (1H, d, J = 7.4 Hz, Ar–H), 7.39–7.37
(2H, m, Ar–H), 7.33 (1H, t, J = 7.9 Hz, Ar–H), 6.76 (1H, dd, J =
6-Chloro-N′-(2-hydroxy-3-methoxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8r. Yellow solid (0.40, 83%), m.p.
>300 °C; [HRMS: m/z calculated C18H14N2O6Cl for 389.0540
(MH+). Found 389.0537]; νmax/cm−1 3461, 3306 (2 × NH),
3600–2830 (br, OH) and 1710 (ester CO), 1613 and 1561
(amide CO); δH (600 MHz, DMSO-d6) 15.53 (1H, s, OH),
11.90 (1H, s, NH), 8.85 (1H, s, Ar–H), 8.15 (1H, s, Ar–H), 7.74
(1H, s, Ar–H), 7.55 (1H, d, J = 6.6 Hz, Ar–H), 7.39 (1H, d, J =
5.0 Hz, Ar–H), 6.79 (1H, s, Ar–H), 6.53 (1H, s, Ar–H), 3.72
(3H, s, OCH3); δC (150 MHz, DMSO-d6) 164.7, 160.0 and 153.0
(CO), 152.2 (Ar–C), 151.8 (Ar–C), 148.54 (Ar–C), 148.47 (Ar–
C), 144.7 (Ar–C), 132.9 (Ar–C), 128.84 (Ar–C), 128.76 (Ar–C),
120.5 (Ar–C), 120.4 (Ar–C), 120.3 (Ar–C), 118.1 (Ar–C), 114.7
(Ar–C), 112.7 (Ar–C) and 55.7 (OCH3).
N′-(2-Hydroxy-3-methoxybenzoyl)-8-methoxy-2-oxo-2H-
chromene-3-carbohydrazide 8s. Yellow solid (0.299 g, 47%),
m.p. >300 °C; [HRMS: m/z calculated for C19H17N2O7 (MH+)
385.1036. Found 385.1036]; νmax/cm−1 3390 and 3289 (2 ×
NH), 3625–2780 (br, OH) and 1701 (ester CO), 1609 and
1575 (amide CO); δH (600 MHz, DMSO-d6) 15.59 (1H, s,
OH), 11.89 (1H, s, NH), 8.84 (1H, s, Ar–H), 7.52 (1H, s, Ar–H),
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7.38 (3H, m, Ar–H), 6.80 (1H, s, Ar–H), 6.53 (1H, s, Ar–H),
3.94 (3H, s, OCH3), 3.72 (3H, s, OCH3); δC (150 MHz, DMSO-
d6) 164.6, 160.1 and 153.4 (CO), 152.1 (Ar–C), 148.6 (Ar–C),
146.3 (Ar–C), 146.2 (Ar–C), 142.9 (Ar–C), 125.1 (Ar–C), 121.0
(Ar–C), 120.4 (Ar–C), 120.3 (Ar–C), 119.4 (2 × Ar–C), 115.5 (Ar–
C), 114.5 (Ar–C), 112.7 (Ar–C), 56.2 (OCH3), 55.7 (OCH3).
8-Ethoxy-N′-(2-hydroxy-3-methoxybenzoyl)-2-oxo-2H-
chromene-3-carbohydrazide 8t. Yellow solid (0.253 g, 51%),
m.p. >300 °C; [HRMS: m/z calculated for C20H19N2O7 (MH+)
399.1192. Found 399.1189]; νmax/cm−1 3615 and 3284 (2 ×
NH), 3620–2830 (br, OH), 1699 (ester CO), 1610 and 1577
(amide CO); δH (600 MHz, DMSO-d6) 15.59 (1H, s, OH),
11.89 (1H, s, NH), 8.84 (1H, s, Ar–H), 7.51 (1H, s, Ar–H), 7.36
(3H, m, Ar–H), 6.79 (1H, s, Ar–H), 6.53 (1H, s, Ar–H), 4.20
(2H, s, OCH2), 3.72 (3H, s, OCH3), 1.43 (3H, s, OCH2CH3); δC
(150 MHz, DMSO-d6) 164.6, 160.2 and 153.3 (CO), 151.9
(Ar–C), 148.5 (Ar–C), 146.2 (Ar–C), 145.6 (Ar–C), 143.0 (Ar–C),
125.0 (Ar–C), 120.9 (Ar–C), 120.5 (Ar–C), 120.4 (Ar–C), 119.5
(Ar–C), 119.4 (Ar–C), 116.2 (Ar–C), 114.5 (Ar–C), 112.7 (Ar–C),
64.5 (OCH2), 55.7 (OCH3) and 14.7 (CH3).
cells, the cells were incubated with the compounds at a final
concentration of 25 μM for a period of 48 hours in 96-well
plates. The cells were seeded at 6.7 × 104 cells per well in
DMEM supplemented with 10% foetal bovine serum and
penicillin/streptomycin/amphotericin B and incubated at 37
°C in a 5% CO2 incubator for 24 hours, followed by com-
pound addition and an additional 48 hour incubation. The
numbers of cells surviving compound exposure were deter-
mined by adding 20 μL per well resazurin toxicology reagent
(Sigma-Aldrich) and measuring fluorescence (Exc 560 nm,
Em 590 nm) in a Spectramax M3 plate reader after an addi-
tional 2–4 hour incubation. Percentage cell viability in wells
exposed to test compounds was calculated relative to the av-
erage fluorescence readings in control wells containing no
compound.
Molecular Modelling
The structure and the molecular geometry of the synthesized
compounds were constructed using the ChemBio3D Ultra
Version 14 molecular modelling program (CambridgeSoft
Corporation, Cambridge, MA, USA). The X-ray crystal struc-
ture of the HIV IN enzymes was obtained from Protein Data
Bank (PDB code: 1QS4). The Discovery Studio Visualizer 3.5
(ref. 29) was used to prepare the protein for docking. All the
bound water molecules and the original ligand were removed
from the 1QS4 and polar hydrogen was added and non-polar
hydrogen was merged followed by the addition of Gasteiger
charges and assignment of AD4 atoms. Autodock 4.2 soft-
ware27 were employed in the preparation of the pdb and
pdbqt files of the ligand and the protein. The active site of
the 1QS4 was centered with the grid box dimensions −19.422
× 32.402 × 66.325 Å and a grid spacing of 0.5 Å. Mapping of
the protein active site and the surrounding residues were
done using Autogrid. The procedure for docking was
performed using the Lamarckian Genetic Algorithm, and the
geometry of the docked complex was view and analyzed using
Discovery Studio 4.0.22
Biological assays
In vitro IN assays. The HIV-1 IN strand transfer inhibition
assay was adapted from previously described methods.8
Briefly, 20 nM double-stranded biotinylated donor DNA (5′-
5biotin-TEG/ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3′
annealed to 5′-ACTGCTAGAGATTTTCCACACTGACTAAAAG-3′)
was immobilized in wells of streptavidin-coated 96-well
microtiter plates (R&D Systems, USA). Following incubation
at room temperature for 40 minutes and a stringent wash
step, 5 μg ml−1 purified recombinant HIV-1 subtype C IN in
IN buffer 1 (50 mM NaCl, 25 mM Hepes, 25 mM MnCl2, 5
mM β-mercaptoethanol, 50 μg ml−1 BSA, pH 7.5) was added
to individual wells. Test compounds and Raltegravir were
added to individual wells to a final concentration of 20 μM.
Recombinant HIV-1 subtype C IN was assembled onto the
pre-processed donor DNA through incubation for 45 minutes
at room temperature. Strand transfer reaction was initiated
through the addition of 10 nM (final concentration) double-
stranded FITC-labelled target DNA (5′-TGACCAAGGGCTAATT
CACT/36-FAM/-3′ annealed to 5′-AGTGAATTAGCCCTTGGTCA-/
36-FAM/-3′) in IN buffer 2 (same as buffer 1, except 25 mM
MnCl2 replaced with 2.5 mM MgCl2). After an incubation pe-
riod of 60 minutes at 37 °C, the plates were washed using
PBS containing 0.05% Tween 20 and 0.01% BSA, followed by
the addition of peroxidase-conjugated sheep anti-FITC anti-
body (Thermo Scientific, USA) diluted 1:1000 in the same
PBS buffer. Finally, the plates were washed and peroxidase
substrate (SureBlue Reserve™, KPL, USA) was added to allow
for detection at 620 nm using a Synergy MX (BioTek®) plate
reader. Absorbance values were converted to% enzyme activ-
ity relative to the readings obtained from control wells (en-
zyme without inhibitor).
Conclusions
The replacement of one salicylic moiety with a coumaryl moi-
ety has been shown here to significantly improve the cytotox-
icity of the resulting hydrazides. In addition, inhibition of
HIV-1 IN has been demonstrated, with nanomolar efficiency
for compounds with either hydrogen or chlorine on the
salicyl moiety and halogens or alkoxy groups on the couma-
rin. A docking study suggests that the orientation within the
active site positions the acyl hydrazide at the metal centres
and one of the aromatic rings directed toward the hydropho-
bic pocket.
In summary, we have successfully designed, synthesised
and evaluated a series of carbohydrazide derivatives as novel
HIV-1 inhibitors. However, the structure of these compounds
could be optimised in the development of new drug candi-
dates for IN inhibition. Such optimization may include the
incorporation of fluoro groups (common to other approved
Cell toxicity assays. To determine the activity of the test
compounds against human cervix adenocarcinoma (HeLa)
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inhibitors), as well as the exploration of specific modes of
toxicity.
12 K. Rosa and M. Hoffman, FDA Approves Bictegravir/FTC/TAF
Combination HIV Therapy, 2018.
13 Y. Li, S. Xuan, Y. Feng and A. Yan, Targeting HIV-1 integrase
with strand transfer inhibitors, 2015, pp. 435–449.
14 X. Fan, F. Zhang, R. I. Al-Safi, L. Zeng, Y. Shabaik, B.
Debnath, T. W. Sanchez, S. Odde, N. Neamati and Y. Long
Design of HIV-1 integrase inhibitors targeting the catalytic
domain as well as its interaction with LEDGF/p75: A scaffold
hopping approach using salicylate and catechol groups, 2011,
pp. 4935–4952.
15 H. Zhao, N. Neamati, S. Sunder, H. Hong, S. Wang, G.
Milne, Y. Pommier and T. J. Burke, J. Med. Chem., 1997, 40,
937–941.
16 N. Neamati, S. Sunder and Y. Pommier, Drug Discovery
Today, 1997, 2, 487–498.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This research project was funded by the South African Medi-
cal Research Council (MRC) with funds from the National
Treasury under its Economic Competitiveness and Support
Package. O. J. Jesumoroti is supported by the Organization for
Women in Science in the Developing World.
Notes and references
17 F. Tian, F. Jiang, X. Han, C. Xiang, Y. Ge, J. Li, Y. Zhang,
R. Li, X. Ding and Y. Liu, J. Phys. Chem. B, 2010, 114,
14842.
1 J. d'Angelo, J. F. Mouscadet, D. Desmaële, F. Zouhiri and H.
Leh, Pathol. Biol., 2001, 49, 237–246.
2 F. Barre-Sinoussi, J. C. Chermann, F. Rey, M. T. Nugeyre, S.
Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. Vezinet-
Brun, C. Rouzioux, W. Rozenbaum and L. Montagnier,
Science, 1983, 220, 868–871.
3 A. Pani, A. G. Loi, M. Mura, T. Marceddu, P. La Colla and
M. E. Marongiu, Curr. Drug Targets: Infect. Disord., 2002, 2,
17–32.
4 G. Pais, X. Zhang, C. Marchand, N. Neamati, K. Cowansage,
E. S. Svarovskaia, V. K. Pathak, Y. Tang, M. Nicklaus, Y.
Pommier and T. J. Burke, J. Med. Chem., 2002, 45,
3184–3194.
5 Y. Mehellou and E. De Clercq, J. Med. Chem., 2010, 53, 521.
6 K. B. Ingale and M. S. Bhatia, Antiviral Chem. Chemother.,
2011, 22, 95–105.
18 J. H. Park, S. U. Lee, S. H. Kim, S. Y. Shin, J. Y. Lee, C. Shin,
K. H. Yoo and Y. S. Lee, Arch. Pharmacal Res., 2008, 31, 1–5.
19 V. Summa, A. Petrocchi, F. Bonelli, B. Crescenzi, M. Donghi,
M. Ferrara, F. Fiore, C. Gardelli, O. Gonzalez Paz, D. J.
Hazuda, P. Jones, O. Kinzel, R. Laufer, E. Monteagudo, E.
Muraglia, E. Nizi, F. Orvieto, P. Pace, G. Pescatore, R.
Scarpelli, K. Stillmock, M. V. Witmer and M. Rowley, J. Med.
Chem., 2008, 51, 5843–5855.
20 D. E. Polyansky and D. C. Neckers, J. Phys. Chem. A,
2005, 109, 2793–2800.
21 D. J. McColl and X. Chen, Antiviral Res., 2010, 85, 101–118.
22 N. Neamati, H. Hong, J. M. Owen, S. Sunder, H. E. Winslow,
J. L. Christensen, H. Zhao, T. J. Burke, G. Milne and Y.
Pommier, J. Med. Chem., 1998, 41, 3202–3209.
7 G. Meintjes, M. A. Moorhouse, S. Carmona, N. Davies, S.
Dlamini, C. Van Vuuren, T. Manzini, M. Mathe, Y. Moosa, J.
Nash, J. Nel, Y. Pakade, J. Woods, G. Van Zyl, F. Conradie
and F. Venter, South. Afr. J. HIV Med., 2017, 18, e24.
8 Y. Goldgur, F. Dyda, A. B. Hickman, T. M. Jenkins, R. Craigie
and D. R. Davies, Proc. Natl. Acad. Sci. U. S. A., 1998, 95,
9150–9154.
9 J. A. Grobler, K. Stillmock, B. Hu, M. Witmer, P. Felock, A. S.
Espeseth, A. Wolfe, M. Egbertson, M. Bourgeois, J. Melamed,
J. S. Wai, S. Young, J. Vacca and D. J. Hazuda, Proc. Natl.
Acad. Sci. U. S. A., 2002, 99, 6661–6666.
10 B. J. Dong and J. Cocohoba, Clin. Ther., 2008, 30, 1747–1765.
11 M. Tsiang, G. S. Jones, J. Goldsmith, A. Mulato, D. Hansen,
E. Kan, L. Tsai, R. A. Bam, G. Stepan, K. M. Stray, A.
Niedziela-Majka, S. R. Yant, H. Yu, G. Kukolj, T. Cihlar, S. E.
Lazerwith, K. L. White and H. Jin, Antimicrob. Agents
Chemother., 2016, 60, 7086.
23 M. C. Lee, J. Deng, J. M. Briggs and Y. Duan Large-Scale
Conformational Dynamics of the HIV-1 Integrase Core Domain
and Its Catalytic Loop Mutants, 2005, pp. 3133–3146.
24 A. L. Perryman, S. Forli, G. M. Morris, C. Burt, Y. Cheng,
M. J. Palmer, K. Whitby, J. A. McCammon, C. Phillips and
A. J. Olson, J. Mol. Biol., 2010, 397, 600–615.
25 E. F. Healy, J. Sanders, P. J. King and W. E. Robinson, J. Mol.
Graphics Modell., 2009, 27, 584–589.
26 T. T. Charvat, D. J. Lee, W. E. Robinson and A. R.
Chamberlin, Bioorg. Med. Chem., 2006, 14, 4552–4567.
27 O. Trott and A. J. Olson, J. Comput. Chem., 2010, 31, 455.
28 A. D. Beldovskaya, G. A. Dushenko, N. I. Vikrishchuk, L. D.
Popov, Y. V. Revinskii, I. E. Mikhailov and V. I. Minkin,
Russ. J. Org. Chem., 2013, 49, 1861.
29 B. Dassault Systèmes, Discovery Studio Modeling
Environment, Release 3.5, 2016, Dassault Systèmes, San
Diego, 2015.
This journal is © The Royal Society of Chemistry 2018
Med. Chem. Commun.