Novel furocoumarins as potential HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2014.07.008

A series of seven novel, rationally designed N-substituted 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamides have been prepared as potential HIV-1 integrase (IN) inhibitors via a five-step pathway commencing with resorcinol and diethyl 2-acetylglutarate, and the HIV-1 IN inhibition potential of these compounds has been examined relative to raltegravir, a known HIV-1 IN inhibitor.

Full text of DOI:10.1016/j.bioorg.2014.07.008

Accepted Manuscript
Novel furocoumarins as potential HIV-1 integrase inhibitors
Temitope O. Olomola, Salerwe Mosebi, Rosalyn Klein, Telisha Traut-
Johnstone, Judy Coates, Raymond Hewer, Perry T. Kaye
PII:
S0045-2068(14)00061-3
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.07.008
YBIOO 1744
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
23 May 2014
Please cite this article as: T.O. Olomola, S. Mosebi, R. Klein, T. Traut-Johnstone, J. Coates, R. Hewer, P.T. Kaye,
Novel furocoumarins as potential HIV-1 integrase inhibitors, Bioorganic Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.bioorg.2014.07.008
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Novel Furocoumarins as Potential HIV-1 Integrase Inhibitors
Temitope O. Olomola,a Salerwe Mosebi,b Rosalyn Klein,a Telisha Traut-Johnstone,b
Judy Coates,b Raymond Hewer b and Perry T. Kaye a*
aDepartment of Chemistry and Centre for Chemico- and Biomedicinal Research,
Rhodes University, Grahamstown, 6140, South Africa.
bBiomed, Advanced Materials Division, Mintek, Randburg, 2125, South Africa.
ABSTRACT. A series of seven novel, rationally designed N-substituted 3-{3,5-
dimethylfuro[3,2-g]coumarin-6-yl}propanamides have been prepared as potential HIV-1
integrase (IN) inhibitors via a five-step pathway commencing with resorcinol and diethyl 2-
acetylglutarate, and the HIV-1 IN inhibition potential of these compounds has been examined
relative to raltegravir, a known HIV-1 IN inhibitor.
Key Words: Furocoumarins, HIV-1 integrase, strand-transfer inhibitors, rational design
1. Introduction.
Earlier research on the development of effective anti-HIV agents has focussed on
inhibitors of the critical HIV-1 enzymes, reverse transcriptase (RT) and protease (PR).1,2 The
emerging challenges of drug resistance have been addressed using combinations of RT and
PR inhibitors in what is known as ‘Highly Active Anti-Retroviral Therapy (HAART)’ −an
approach which has enjoyed considerable success.3 Nevertheless, there is an urgent need to
develop new and more effective therapeutics and the drug targets have been extended to
include HIV-1 integrase (IN) inhibitors,4 which either block the strand transfer of viral DNA
into the host genome or disrupt the 3′-processing of the viral DNA LTR units.5 Since the
approval of raltegravir 16 for clinical use by the FDA in 2007, other potential scaffolds have
been explored, inter alia the β-diketo acid (DKA) moiety,7 which is present in compound 28
and which is capable of coordinating metal ions in the enzyme active site.
O
HN
N
O
F
O
N
H
N
OH
O
1
2
O
O
OH
O
raltegravir
_______________
*To whom correspondence should be addressed. Prof. P.T. Kaye, E-mail: P.Kaye@ru.ac.za;
Tel. +27 46 6038268.Department of Chemistry, Rhodes University, Grahamstown, 6140, South Africa.

2. Results and Discussion.
Computer modelling studies suggested that the endocyclic oxygens in furocoumarin
derivatives, such as compound 3a (Figure 1), could also coordinate metal ions in the HIV-1
IN enzyme active site and inhibit the integration of viral DNA. The coumarin moiety is found
in various medicinally active natural products, including umbilliferone (7-hydroxycoumarin)9
and warfarin.10. In our own research on applications of Baylis-Hillman chemistry, we have
reported several approaches to coumarins and their elaboration to novel analogues11 of the
clinically established HIV-1 PR inhibitor, ritonavir, and, more recently, the synthesis and
evaluation of coumarin-AZT conjugates as potential dual-action HIV-1 PR/RT inhibitors.12,13
In our approach to the synthesis of the furocoumarins 3a-g as potential HIV-1 IN inhibitors,
however, we decided to follow the five-step pathway outlined in Scheme 1.
3 x H-bonds
to Lys159 (IN)
solvent accessible sites
CH3
O
O
H3C
N
N
H
potential
DNA interaction
O
O
H-bond to Asn155 (IN)
Mg2+ interaction
sites
H-bond to
viral DNA (A18)
Figure 1. Computer-modelled 2-D interactions of compound 3a with the HIV-1 IN active site
obtained using Tripos Sybyl molecular modelling software.
Resorcinol 4 was reacted overnight with diethyl 2-acetylglutarate 5 at 0 °C in an
ethanolic solution of HCl, generated in situ by the cautious addition of acetyl chloride to dry
ethanol (CAUTION! exothermic reaction), and the known coumarin derivative 614 was
isolated in 64% yield (Scheme 1). Following a literature procedure,15 alkylation of the
phenolic hydroxyl group in compound 6 was achieved by treatment with highly lachrymatory
chloroacetone in the presence of freshly calcined K2CO3; the intermediate 7 was obtained,
after recrystallisation, in 73% yield. The critical furanocoumarin scaffold 8 was obtained in
good yield (79% after recrystallisation) by heating the ether 7 under basic conditions.15 This
mechanistically interesting cyclisation is considered16 to proceed via initial opening of the
coumarin ring to afford a phenoxide ion which, in turn, activates the para-position to
intramolecular cyclisation and formation of the furan ring −processes presumably
accompanied by saponification of the ester group; subsequent protonation and acid-catalysed
dehydration then regenerates the coumarin moiety
With the common scaffold, 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanoic acid
8, in hand, access to the targeted furanocoumarin carboxamides 3a-g was achieved in two
steps, viz., activation of the carboxylic acid group by treatment with N-hydroxysuccinimide
and N,N’-diisopropylcarbodiimide (DIC) to generate the succinimido intermediates 9
(Scheme 1), followed by in situ reaction with each of the amines 10a-g. The resulting, novel
carboxamides 3a-g were obtained in moderate to good yield (63 - 83% after recrystallisation;

Table 1) and fully characterised. Chromatographed samples were tested for their inhibition of
the HIV-1 IN strand-transfer process using the method adapted from Hazuda et al.17
O
O
HCl,
HO
OH
OEt
O
OEt
OEt
EtOH
K2CO3
O
4
O
O
O
O
HO
O
O
+
7
6
EtO
O
Cl
i) NaOH
ii) H2SO4
O
5
O
RNH2
OH
N
O
O
O
a
b
c
O
H2N
8
O
N
OH
O
H2N
O
O
N
NH2
N
d
O
O
O
H2N
N
O
9
RNH2
10a-g
e
f
H2N
H2N
O
NHR
O
O
O
g
H2N
3a-g
Scheme 1. Synthetic route to furocoumarin analogues 3a-g.
Table 1. Yields and % HIV-1 IN strand-transfer enzyme inhibition activities for
O
compounds 3a-g at 10 μM.
NHR
O
O
O
Compound
R
% Yield
% Inhibitiona
3a
(pyridine-2-yl)methyl
(furan-2-yl)methyl
5-(diethylamino)pent-2-yl
(pyridine-3-yl)methyl
benzyl
78
80
76
81
82
63
83
-
16.61 (3.11)
5.21 (7.94)
4.98 (5.96)
10.31 (6.62)
11.12 (7.48)
8.48 (7.44)
15.92 (7.66)
92.77 (0.61)
3b
3c
3d
3e
3f
3g
tert-butyl
propyl
Raltegravir
-
a Standard deviation in parentheses.

Percentage enzyme inhibition values for compounds 3a-g and raltegravir (a known
HIV-1 IN inhibitor) are reported in Table 1. While some of the synthetic ligands exhibited
negligible inhibition activities at a concentration of 10 μM, a number of compounds exhibited
stastically significant inhibition (p > 0.05) at this concentration [viz., 3a (16.61%); 3d
(10.31); 3e (11.12%); and 3g (15.92%)] and may warrant further investigation in the
development of possible lead compounds. While compounds 3a and 3d contain pyridyl
moieties with hydrogen-bonding potential, compounds 3e and 3g contain non-polar moieties
(benzyl and propyl, respectively). In silico docking studies of these specific compounds is
expected to guide future developments.
3. Conclusions.
The structure of a potential HIV-1 IN inhibitor has been identified from an
examination of its virtual interactions with a model of the HIV-1 IN active site based on
available sub-domain X-ray crystal structures and NMR structures. This compound and a
series of six analogues have been successfully synthesised and HIV-1 IN inhibition studies
reveal that some of the ligands exhibit statistically significant inhibition at 10 μM
concentrations. Ongoing research in this area at Rhodes is being focussed on the development
of cinnamate ester-AZT and DKA analogue-AZT conjugates as potential dual-action HIV-1
integrase/reverse transcriptase inhibitors.
4. Experimental.
4.1. General.
NMR spectra were recorded on a Bruker AMX 400 spectrometer at 303 K in CDCl3
or DMSO-d6 and calibrated using solvent signals [δ : 7.26 ppm for residual CHCl3 and 2.50
H
ppm for residual DMSO; δ : 77.0 ppm (CDCl3) and 39.5 (DMSO-d6)]. Melting points were
C
measured using a hot stage apparatus and are uncorrected. IR spectra were recorded on a
Perkin Elmer Spectrum 100 FT-IR spectrometer with a diamond window, and compounds
were analysed neat. High-resolution mass spectra (HRMS) were recorded on a Waters API
Q-TOF Ultima spectrometer (University of Stellenbosch, Stellenbosch, South Africa). All
computational protein manipulations were performed on a dual-core desktop PC with a Red
Hat Enterprise Linux Version 5.0 operating system. All protein visualisations, manipulations
and docking runs were performed using the commercial software package SybylTM version
8.0, licensed from TriposTM (Tripos Inc., St. Louis, MO, USA, 2008).
4.2. Computer modelling.
A three-dimensional model of the HIV-1 IN active site was prepared based on sub-
domain X-ray crystal structures and NMR structures available in the Protein Data Bank
(PDB1QS4, PDB1B9F, PDB1EX4, PDB1K6Y, PDB2B4J, PDB1MM8 and PDB1TQR). A
virtual, drug-like compound database of 731 compounds was prepared through filtering of the
ZINC commercial database according to Lipinski’s Rule of 5. Ten different orientations of
each compound were docked into the prepared HIV-1 IN active site using the SurflexDockTM
application. Compound 3a was identified as a potential inhibitor based on the virtual
interactions with the enzyme active site predicted by the docking studies.

4.3. Chemistry.
4.3.1. Synthesis of intermediates 6-8.
The intermediates were obtained using reported methods: ethyl 3-(7-hydroxy-4-
methylcoumarin-3-yl)propanoate 6 as a pale yellow solid (44.2 g, 64%), m.p. 126-127 °C
(lit.14 124 °C); ethyl 3-[7-(2-oxopropoxy)-4-methylcoumarin-3-yl]propanoate 7 as white
crystals (28.1 g, 73%), m.p. 122-123 °C (lit.15 115-116 °C); and 3-{3,5-dimethylfuro[3,2-
g]coumarin-6-yl}propanoic acid 8 as a yellow solid (16.9 g, 79%), m.p. 220-221 °C (lit.15
215-216 °C). The general procedure for the synthesis of the novel furocoumarin
carboxamides 3a-g is illustrated by the following example; analytical data for the other
compounds are detailed below.
4.3.2. Synthesis and analytical data for the furocoumarin ligands 3a-g.
4.3.2.1. General procedure: Synthesis of 3-{3,5-Dimethylfuro[3,2-g]coumarin-6-yl}-N-
[(pyridin-2-yl)methyl]-propanamide 3a.
3-{3,5-Dimethylfuro[3,2-g]coumarin-6-yl}propanoic acid 8 (1.0 g, 3.5 mmol) was
dissolved in dry dioxane (50 mL) in a 2-necked round-bottomed flask and N-
hydroxysuccinimide (0.44 g, 3.8 mmol) was added. The solution was stirred under N2 for 20
min.; N,N′-diisopropylcarbodiimide (0.6 mL, 4 mmol) was then added through a septum and
the solution stirred vigorously for 2 h. 2-Picolylamine 10a (0.4 mL, 4 mmol) was then
introduced through the septum and the mixture stirred for 4 h at r.t. under N2. Ice-cooled
water was then added to the mixture and the resulting precipitate was filtered off.
Recrystallisation from 2-propanol gave 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}-N-
[(pyridin-2-yl)methyl]propanamide 3a as a yellow solid (1.03 g, 78%), m.p. 212-213 °C
[HRMS: m/z calculated for C22H21N2O4 (MH+) 377.1501. Found 377.1498]; δ (400 MHz;
H
DMSO-d6) 2.26 (3H, s, 3-CH3), 2.41 (2H, t, J = 7.5 Hz, CH2Ar), 2.45 (3H, s, 5-CH3), 2.85
(2H, t, J = 7.5 Hz, CH2CO), 4.32 (2H, d, J = 5.7 Hz, NCH2), 7.18 (2H, m, ArH), 7.57 (2H, m,
ArH), 7.83 (1H, s, ArH), 7.90 (1H, s, ArH), 8.42 (1H, d, J = 4.3, ArH) and 8.51 (1H, t, J =
5.7 Hz, NH); δ (100 MHz; DMSO-d6) 7.6 and 15.2 (CH3), 23.7, 33.9 and 44.2 (CH2), 98.8,
C
115.6, 116.0, 116.3, 120.9, 122.0, 122.4, 125.9, 136.5, 143.6, 147.9, 148.8, 149.7, 155.2 and
158.6 (Ar-C), 160.7 and 171.5 (C=O).
4.3.2.2. Analytical data for the furocoumarin ligands 3b-g.
4.3.2.2.1. 3-{3,5-Dimethylfuro[3,2-g]coumarin-6-yl}-N-[(furan-2-yl)methyl]propanamide 3b,
a yellow solid (1.02 g, 80%), m.p. 215-217 °C [HRMS: m/z calculated for C21H20NO5 (MH+)
366.1342. Found 366.1331]; δ (400 MHz; CDCl3) 2.28 (3H, s, 3-CH3), 2.53 (5H,
H
overlapping s and t, 5-CH3 and CH2Ar), 3.02 (2H, t, J = 7.4 Hz, CH2CO), 4.39 (2H, d, J = 5.5
Hz, NCH2), 6.14 (1H, d, J = 2.1 Hz, ArH), 6.20 (1H, m, ArH), 6.24 (1H, br s, NH), 7.17
(1H, s, ArH), 7.29 (1H, s, ArH), 7.43 (1H, s, ArH) and 7.66 (1H, s, ArH); δ (100 MHz;
C
CDCl3) 7.9 and 15.5 (CH3), 24.1, 34.9 and 36.4 (CH2), 99.2, 107.3, 110.2, 114.9, 115.6,
116.6, 122.4, 126.3, 142.0, 142.9, 148.3, 150.2, 151.2 and 155.9 (Ar-C), 162.0 and 171.8
(C=O).
4.3.2.2.2. N-[5-(Diethylamino)pent-2-yl]-3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propan-
amide 3c, a yellow solid (1.02 g, 76%), m.p. 59-60 °C [HRMS: m/z calculated for
C25H35N2O4 (MH+) 427.2597. Found 427.2611]; δ (400 MHz; CDCl3) 0.93 (6H, t, J = 6.5
H
Hz, 2 × CH2CH3), 1.04 (3H, d, J = 5.9 Hz, CHCH3), 1.37 (4H, m, CHCH2CH2), 2.25 [5H, m

(overlapping s and t), 3-CH3 and CH2Ar], 2.39-2.48 (6H, series of signals, 3 × NCH2), 2.54
(3H, s, 5-CH3), 2.99 (2H, t, J = 6.5 Hz, CH2CO), 3.90 (1H, br s, NCH), 6.22 (1H, d, J = 6.7
Hz, NH), 7.29 (1H, s, ArH), 7.41 (1H, s, ArH) and 7.65 (1H, s, ArH); δ (100 MHz; CDCl3)
C
7.8, 11.2, 15.4 and 20.6 (CH3), 23.3, 24.3, 34.6, 35.3, 46.6 and 52.6 (CH2), 45.0 (NCH), 99.2,
114.8, 115.6, 116.6, 122.7, 126.3, 142.9, 148.1, 150.1 and 155.9 (Ar-C), 162.0 and 171.3
(C=O).
4.3.2.2.3. 3-{3,5-Dimethylfuro[3,2-g]coumarin-6-yl}-N-[(pyridin-3-yl)methyl]propanamide
3d, an off-white solid (1.07 g, 81%), m.p. 234-236 °C [HRMS: m/z calculated for
C22H21N2O4 (MH+) 377.1501. Found 377.1513]; δ (400 MHz; DMSO-d6) 2.25 (3H, s, 3-
H
CH3), 2.37 (2H, t, J = 7.5 Hz, CH2Ar), 2.43 (3H, s, 5-CH3), 2.83 (2H, t, J = 7.5 Hz, CH2CO),
4.25 (2H, d, J = 5.7 Hz, NCH2), 7.17 (1H, m, ArH), 7.50-7.51 (2H, overlapping s and m,
ArH), 7.80 (1H, s, ArH), 7.86 (1H, s, ArH), 8.37 (1H, d, J = 3.9, ArH) and 8.43 (2H,
overlapping s and m, ArH and NH); δ (100 MHz; DMSO-d6) 7.5 and 15.1 (CH3), 23.7, 33.9
C
and 39.9 (CH2), 98.7, 115.5, 115.9, 116.2, 122.3, 123.3, 125.8, 134.9, 135.0, 143.5, 147.8,
147.9, 148.7, 149.7 and 155.2 (Ar-C), 160.6 and 171.4 (C=O).
4.3.2.2.4 N-Benzyl-3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamide 3e, an off-white
solid (1.07 g, 82%), m.p. 230-232 °C [HRMS: m/z calculated for C23H22NO4 (MH+)
376.1549. Found 376.1565]; δ (400 MHz; DMSO-d6) 2.26 (3H, s, 3-CH3), 2.37 (2H, t, J =
H
7.5 Hz, CH2Ar), 2.45 (3H, s, 5-CH3), 2.84 (2H, t, J = 7.5 Hz, CH2CO), 4.21 (2H, s, NCH2),
7.15 (5H, s, ArH), 7.58 (1H, s, ArH), 7.85 (1H, s, ArH), 7.92 (1H, s, ArH) and 8.42 (1H, br s,
NH); δ (100 MHz; DMSO-d6) 7.6 and 15.2 (CH3), 23.8, 34.0 and 42.1 (CH2), 98.8, 115.7,
C
116.1, 116.4, 122.5, 125.9, 126.7, 127.2, 128.2, 139.5, 143.7, 148.0, 149.8 and 155.2 (Ar-C),
160.7 and 171.3 (C=O).
4.3.2.2.5. N-tert-Butyl-3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamide 3f, a white
solid (0.75 g, 63%), m.p. 84-85 °C [HRMS: m/z calculated for C20H24NO4 (MH+) 342.1705.
Found 342.1698]; δ (400 MHz; CDCl3) 1.28 [9H, s, C(CH3)3], 2.26 (3H, s, 3-CH3), 2.39
H
(2H, t, J = 7.5 Hz, CH2Ar), 2.54 (3H, s, 5-CH3), 2.97 (2H, t, J = 7.5 Hz, CH2CO), 5.67 (1H,
br s, NH), 7.28 (1H, s, ArH), 7.41 (1H, s, ArH) and 7.65 (1H, s, ArH); δ (100 MHz; CDCl3)
C
7.8, 15.5 and 28.6 (CH3), 24.2 and 36.0 (CH2), 51.0 [C(CH3)3], 99.1, 114.8, 115.6, 116.6,
122.8, 126.3, 142.8, 148.0, 150.1 and 155.8 (Ar-C), 162.0 and 171.3 (C=O).
4.3.2.2.6. 3-{3,5-Dimethylfuro[3,2-g]coumarin-6-yl}-N-propylpropanamide 3g, a yellow
solid (0.95 g, 83%), m.p. 211-213 °C [HRMS: m/z calculated for C19H22NO4 (MH+)
328.1549. Found 328.1561]; δ (400 MHz; DMSO-d6) 0.75 (3H, t, J = 7.2 Hz, CH3), 1.33
H
(2H, m, CH2CH3), 2.24-2.29 (5H, overlapping s and t, 3-CH3 and CH2Ar), 2.47 (3H, s, 5-
CH3), 2.79 (2H, t, J = 7.2 Hz, CH2CO), 2.95 (2H, m, NCH2), 7.55 (1H, s, ArH), 7.82 (1H, s,
ArH) 7.88 (1H, m, NH) and 7.92 (1H, s, ArH); δ (100 MHz; DMSO-d6) 7.6, 11.4 and 15.3
C
(CH3), 23.9, 34.1, 39.5 and 40.4 (CH2), 51.0 (CH2CH3), 98.8, 115.7, 116.0, 122.6, 125.9,
143.7, 147.9, 149.8 and 155.2 (Ar-C), 160.8 and 171.2 (C=O).
4.4. Biological evaluation.
The HIV-1 IN strand transfer inhibition assay was adapted from previously described
methods.17 Briefly, 0.15 µM double-stranded biotinylated donor DNA (5’-biotin-
GTGTGGAAAATCTCTAGCA-3’ and 5’-ACTGCTAGAGATTTTCCACAC-3’) was added
to the wells of streptavidin-coated 96-well microtiter plates (R&D Systems, USA). Following
incubation at room temperature for 60 minutes and a stringent wash step, 1 µM purified

recombinant HIV-1 subtype B IN (in the presence of MgCl2 and MnCl2) was assembled onto
the pre-processed donor DNA through incubation for 30 minutes at 22 °C. Following a wash
step, compounds 3a-g and Raltegravir were titrated into individual wells at a final concentration
of 10 µM. The microtiter plates were incubated for 30 minutes at 37 °C, washed and the
strand transfer reaction was initiated through the addition of 0.25 µM double-stranded FITC-
labelled
target
DNA
(5’-TGACCAAGGGCTAATTCACT-FITC-3’
and
5’-
AGTGAATTAGCCCTTGGTCA-FITC-3’) in Hepes buffer containing MgCl2 and MnCl2.
After an incubation period of 60 minutes at 37 °C, the plates were washed as before and an
alkaline phosphatase (AP) – conjugated anti-FITC secondary antibody (Sigma, USA) was
added. Finally, the plates were washed and substrate (BluePhos, KPL, USA) was added to
allow for detection at 620 nm using a microplate reader (xMarkTM, Bio-Rad, USA). All
inhibition values are the average of triplicate experiments.
6. Acknowledgments
We thank MINTEK and Rhodes University for generous financial support and permission to
publish these findings. T.O.O. gratefully acknowledges Obafemi Awolowo University, Ile-
Ife, Nigeria for study leave.
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Novel Furocoumarins as Potential HIV-1 Integrase Inhibitors
Temitope O. Olomola,a Salerwe Mosebi,b Rosalyn Klein,a Telisha Traut-Johnstone,b
Judy Coates,b Raymond Hewer b and Perry T. Kaye a*
Graphical abstract
3 x H-bonds
to Lys159 (IN)
solvent accessible sites
CH3
O
O
H3C
N
N
H
potential
DNA interaction
O
O
H-bond to Asn155 (IN)
Mg2+ interaction
sites
H-bond to
viral DNA (A18)

Novel Furocoumarins as Potential HIV-1 Integrase Inhibitors
Temitope O. Olomola,a Salerwe Mosebi,b Rosalyn Klein,a Telisha Traut-Johnstone,b
Judy Coates,b Raymond Hewer b and Perry T. Kaye a*
Highlights.
• An HIV-1 IN active site model was constructed using sub-domain X-ray and NMR
structures.
• The model was used to identify a furocoumarin system as a potential HIV-1 IN
inhibitor.
• The potential HIV-1 IN inhibitor and six structural analogues have been synthesised.
• Some of the furocoumarin ligands exhibit low but statistically significant inhibition at
10 μM.