Towards the synthesis of coumarin derivatives as potential dual-action HIV-1 protease and reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.tetlet.2010.09.121

3-(Chloromethyl)coumarins, obtained via acid-catalysed cyclisation of salicylaldehyde-derived Baylis-Hillman adducts, have been treated with propargylamine; reaction of the resulting 3-alkynylmethylcoumarins with azidothymidine (AZT) in the presence of a

Full text of DOI:10.1016/j.tetlet.2010.09.121

Tetrahedron Letters 51 (2010) 6325–6328
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Tetrahedron Letters
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Towards the synthesis of coumarin derivatives as potential dual-action
HIV-1 protease and reverse transcriptase inhibitors
Temitope O. Olomola a, Rosalyn Klein a, Kevin A. Lobb a, Yasien Sayed b, Perry T. Kaye a,
⇑
a Department of Chemistry and Centre for Chemico- and Biomedicinal Research, Rhodes University, Grahamstown 6140, South Africa
b Protein Structure–Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2010
Revised 15 September 2010
Accepted 24 September 2010
Available online 18 October 2010
3-(Chloromethyl)coumarins, obtained via acid-catalysed cyclisation of salicylaldehyde-derived Baylis–
Hillman adducts, have been treated with propargylamine; reaction of the resulting 3-alkynylmethyl-
coumarins with azidothymidine (AZT) in the presence of a Cu(I) catalyst has afforded a series of
cycloaddition products for evaluation, in their own right, as potential dual-action HIV-1 protease and
non-nucleoside reverse transcriptase inhibitors, and as scaffolds for further structural elaboration.
Ó 2010 Elsevier Ltd. All rights reserved.
The development of novel therapeutics for the treatment of
Acquired Immunodeficiency syndrome (AIDS) remains a major
challenge. The genome of human immunodeficiency virus type 1
(HIV-1) encodes 15 distinct proteins,1 including essential enzymes,
such as reverse transcriptase (RT) and protease (PR), which have
been identified as therapeutic targets. Problems associated with
the development of drug-resistant viral variants2–4 have led to
the introduction of highly active antiretroviral therapy (HAART),
typically involving concomitant treatment with a mixture of nucle-
oside and non-nucleoside HIV-1 RT inhibitors and an HIV-1 PR
inhibitor.
As part of our research programme directed at the design,
synthesis and evaluation of novel compounds as potential HIV-1
protease inhibitors, particular attention has been focused on the
preparation of coumarin derivatives (2H-chromen-2-ones or
2H-1-benzopyran-2-ones), including ritonavir analogues (1).5 Many
coumarin-containing compounds are known to exhibit medicinally
useful properties including anti-HIV, anti-cancer, anti-fungal, anti-
bacterial and anti-coagulant activities.6–11 Phenprocoumon (2), for
example, is a competitive HIV-1 PR inhibitor and has served as a lead
compound in the design of non-peptidic inhibitors,12–17 while
calanolide A has shown promise as an HIV-1 non-nucleoside RT
inhibitor (NNRTI).4 Azidothymidine (AZT), on the other hand, has
found clinical use as a nucleoside analogue RT inhibitor. In continu-
ation of research directed at: (i) exploring applications of Baylis–
Hillman methodology in the construction of complex molecular
targets and (ii) the development of novel HIV-1 PR inhibitors,5,12
we now report the use of Baylis–Hillman-derived 3-(chloro-
methyl)coumarins to prepare a range of compounds, which contain
both coumarin and triazolothymidine moieties, as potential dual-
action HIV-1 PR and RT inhibitors, and as scaffolds for further struc-
tural elaboration.
Ph
O
O
OH
O
OH
H
N
R
N
H
R
O
O
O
Ph
2
1
DABCO-catalysed Baylis–Hillman reactions of the salicylaldehy-
des (3a–e; Scheme 1) with t-butyl acrylate (4) gave the corre-
sponding adducts 5a–e, cyclisation of which with HCl–AcOH
afforded the 3-(chloromethyl)coumarins 6a–e.18 These compounds
(6a–e) have been shown to undergo preferential SN attack, by
nitrogen nucleophiles, at the exocyclic 3-methylene centre19 rather
than attack at C-4 involving an SN (or conjugate addition–elimina-
tion) mechanism. Reaction of each of the 3-(chloromethyl)couma-
rins 6a–e with propargylamine (7) in THF also followed the direct
SN pathway to give the corresponding substitution products 8a–e
in yields ranging from 52% to 80% (Table 1). These alkynylated
products were then subjected to Cu(I)-catalysed cycloaddition
with azidothymidine (AZT; 9) in the presence copper(II) sulfate
and ascorbic acid—the latter to effect the reduction of Cu(II) to
Cu(I). The resulting triazole derivatives 10a–e, which were isolated
in yields ranging from 64% to 76%. (Table 1) contain both a couma-
rin and an AZT moiety. Interestingly, the 1,2,3-triazole moiety has
been shown to be an effective replacement for the peptide group in
HIV-1 protease inhibitors, facilitating binding by hydrogen-bond-
ing to structural water.20
A preliminary Saturation Transfer Difference (STD) NMR screen-
ing experiment, using a mixture of compounds 10a–e as potential
ligands, indicated that some of these compounds do, in fact, bind to
HIV-1 subtype C PR (the subtype endemic to Sub-Saharan Africa).
⇑
Corresponding author. Tel.: +27 46 6038268; fax: +27 46 6225109.
E-mail address: P.Kaye@ru.ac.za (P.T. Kaye).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.09.121

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T. O. Olomola et al. / Tetrahedron Letters 51 (2010) 6325–6328
R1
CHO
OH
OH
O
R1
R1
OBut
Cl
3a-e
i
ii
R2
OH
O
O
O
R2
R2
OBut
4
5a-e
6a-e
H2N
iii
1
2
R
R
7
a
b
c
d
e
H
Br H
H OMe
H OEt
Cl H
H
R1
N
H
O
O
R1
R2
OH
8a-e
iv
N
O
N
N
O
R2
H
N
N3
N
NH
O
N
HO
O
O
O
N
H
O
O
10a-e
9
Scheme 1. Reagents and conditions: (i) DABCO, CHCl3, rt; (ii) HCl, AcOH, reflux; (iii) propargylamine (7), THF; (vi) sodium ascorbate, Cu2SO4ꢀ5H2O, H2O–THF.
Table 1
Yields obtained for the transformations 6 ? 8 ? 10 (Scheme 1)
Substrate
R1
R2
Yield of 8 (%)
Yield of 10 (%)
6a
6b
6c
6d
6e
H
Br
H
H
Cl
H
H
OMe
OEt
H
80
52
65
70
70
65
76
75
70
64
Computer modelling studies were also used to explore the in silico
docking of these compounds in the HIV-1 PR and NNRT enzyme
receptor sites.
Figure 2. Overlay of the docked conformations of compound 10a (in black) and
ritonavir (in grey) in the HIV-1 PR active site, the latter ligand as it is found in the
crystal structure of HIV-1 (PDB 1HXW).21
Docking of the parent system 10a into the receptor site of HIV-1
PR21 revealed potential hydrogen-bonding interactions between
proximate protein residues and both the coumarin and the AZT
moieties (Fig. 1). Solvent (H2O)-mediated hydrogen-bonding be-
tween the triazole moiety and the receptor-site residue Gly-27
would serve to enhance binding. Moreover, as is evident in Figure
2, there is close correspondence between the docked conformation
of compound 10a and the X-ray crystal structure21 of the known
inhibitor, ritonavir, in the active site of HIV-1 PR. While the ligands
10a–e lack a suitably located lipophilic group to occupy the P1
pocket22—a feature of most active PR inhibitors—the synthetic
methodology reported herein is currently being extended to per-
mit early attachment of such a group.
The non-nucleoside binding pocket of HIV-1 RT appears to be
quite forgiving in the variety of structural motifs it can accommo-
date.23 Docking of ligands 10a–e into this pocket reveals that: (i)
they exhibit potential hydrogen-bonding interactions with amino
acid residues that line the pocket (Fig. 3) and (ii) the coumarin moi-
ety occupies the same cavity as efavirenz24—observations which
Gly27
O
N
O
OH
O
Lys101
H
N
H
Thr1139
Glu1138
H
N
O
N
H
N
H
N
N
N
N
N
O
O
O
O
N
H
N
H
O
O
O
O
Asp29
Arg8
Arg8
Lys101
Asp29
Asp30
Lys172
Figure 1. Schematic representation of potential hydrogen-bonding interactions
(<4 Å) between compound 10a and residues in the receptor cavity of HIV-1 protease
(PDB 1HXW)21 as determined using AUTODOCK 4.2.
Figure 3. Schematic representation of hydrogen-bonding interactions (<4 Å)
between AZT-coumarin product 10a and residues in the non-nucleoside pocket of
HIV-1 reverse transcriptase (PDB 1IKW)24 as determined using AUTODOCK 4.2.

T. O. Olomola et al. / Tetrahedron Letters 51 (2010) 6325–6328
6327
21. Kempf, D. J.; Marsh, K. C.; Denissen, J. F.; Mcdonald, E.; Vasavanonda, S.;
Flentge, C. A.; Green, B. E.; Fino, L.; Parks, C. H.; Kong, X.-P.; Wideburg, N. E.;
Saldivar, A.; Ruiz, L.; Kati, W. M.; Sham, H. L.; Robins, T.; Stewart, K. D.; Hsu, A.;
Plattner, J. J.; Leonard, J. M.; Norbeck, D. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
2484–2488.
22. Wlodawer, A.; Vondrasek, J. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 249–
284.
23. Ren, J.; Nichols, C.; Bird, L. E.; Fujiwara, T.; Sugimoto, H.; Stuart, D. I.; Stammers,
D. K. J. Biol. Chem. 2000, 275, 14316–14320.
24. Lindberg, J.; Sigurdsson, S.; Lowgren, S.; Andersson, H. O.; Sahlberg, C.; Noreen,
R.; Fridborg, K.; Zhang, H.; Unge, T. Eur. J. Biochem. 2002, 269, 1670–1677.
25. Furman, P. A.; Fyfe, J. A.; St. Clair, M. H.; Weinhold, K.; Rideout, J. L.; Freeman, G.
A.; Lehrman, S. N.; Bolognesi, D. P.; Broder, S.; Mitsuya, H.; Barry, D. W. Proc.
Natl. Acad. Sci. U.S.A. 1986, 83, 8333–8337.
26. Schneider, B.; Biondi, R.; Sarfati, R.; Agou, F.; Guerreiro, C.; Deville-Bonne, D.;
Veron, M. Mol. Pharmacol. 2000, 57, 948–953.
27. De Clercq, E. Int. J. Antimicrob. Agents 2009, 33, 307–320.
28. General procedures for the synthesis and characterisation of compounds 8a–e and
10a–e are as follows.
indicate the possibility of their acting as non-nucleoside RT inhib-
itors (NNRTIs). The nucleoside analogue (NRTI) pro-drug AZT 9,
however, is incorporated, following in vivo triphosphorylation,25
into the transcribed DNA being generated in the RT active site. It
has been suggested that the azido group in AZT 9 may interfere ste-
rically with the phosphorylation process,26 and it is possible that
similar steric constraints may preclude the ligands 10a–e from act-
ing as NRTI pro-drugs. However, prior in vitro phosphorylation [as
effected in the nucleotide analogue (NtRTI) tenofovir27] might well
permit phosphorylated derivatives of compounds 10a–e to act at
the RT active site. Ongoing studies are focusing on: functional elab-
oration of the coumarin-AZT scaffold in compounds of type 10; en-
zyme–inhibition assays of the synthetic ligands; and use of the
resulting SAR data in the development of novel, dual-action HIV-
1 PR and RT inhibitors.
3-[(2-Propynylamino)methyl]coumarin (8a). Propargylamine (7) (0.6 mL,
In conclusion, the 3-(chloromethyl)coumarins 6a–e, obtained
via Baylis–Hillman methodology, have been successfully reacted
with propargylamine (7) to afford compounds 8a–e, which have
been used, in turn, as substrates for ‘Click Chemistry’ cycloaddition
with azidothymidine (AZT).28 This four-step sequence, using read-
ily available reactants, provides convenient access to the structur-
ally complex coumarin-AZT conjugates 10a–e, STD NMR binding-
and in silico modelling studies of which encourage exploration of
their potential as dual-action HIV-1 PR/RT inhibitors.
8.8 mmol) was added to
a solution of 3-(chloro-methyl)coumarin (5a)
(0.80 g, 4.1 mmol) in dry THF (5 mL). After stirring at room temperature for
48 h, the reaction mixture was concentrated in vacuo and purified using flash
chromatography [on silica gel; elution with hexane/EtOAc (3:2)] to afford 3-
[(2-propynylamino)methyl]coumarin (8a) as an off-white solid (0.7 g, 80%), mp
107–108 °C [HRMS: m/z calculated for C13H12NO2 (MH+): 214.0868. Found:
214.0860]; m
max/cmꢁ1 (neat) 1694 (C@O); dH (400 MHz, CDCl3) 1.78 (1H, s, NH),
2.24 (1H, t, J = 2.4 Hz, C„CH), 3.48 (2H, d, J = 2.4 Hz, CH2–C„CH), 3.82 (2H, s,
CH2NH), 7.26 (1H, m, ArH), 7.32 (1H, d, J = 8.1 Hz, ArH), 7.48 (2H, m, ArH) and
7.73 (1H, s, 4-H); dC (100 MHz, CDCl3) 38.1 and 48.2 (CH2N), 72.4 and 82.0
(C„CH), 117.0, 119.6, 124.9, 127.3, 128.1, 131.6, 139.9 and 153.7 (Ar–C) and
161.8 (C@O).
6-Bromo-3-[(2-propynylamino)methyl]coumarin (8b) (0.55 g, 52%) as an off-
white solid, mp 136–137 °C [HRMS: m/z calculated for C13H11BrNO2 (MH+)
Acknowledgements
291.9973. Found: 291.9966];
m
max/cmꢁ1 (neat) 1712 (C@O); dH (400 MHz,
CDCl3) 1.76 (1H, br s, NH), 2.23 (1H, s, C„CH), 3.46 (2H, s, CH2–C„CH), 3.80
(2H, s, CH2NH), 7.18 (1H, d, J = 8.7 Hz, ArH), 7.54 (1H, d, J = 8.8 Hz, ArH), 7.58
(1H, s, ArH) and 7.64 (1H, s, 4-H); dC (100 MHz, CDCl3) 38.1 and 48.0 (CH2N),
72.5 and 81.9 (C„CH), 117.4, 118.6, 121.2, 128.8, 130.3, 134.2, 138.3 and 152.5
(Ar–C) and 161.0 (C@O).
The authors thank Rhodes University for a bursary (to T.O.O.),
the South African Medical Research Council (MRC) and Rhodes Uni-
versity for generous financial support, Aspen Pharmacare for the
supply of azidothymidine used for this work and the referee for
helpful comments.
8-Methoxy-3-[(2-propynylamino)methyl]coumarin (8c) (0.70 g, 65%) as a pale
yellow solid, mp 89–91 °C [HRMS: m/z calculated for
C
14H14NO3 (MH+)
244.0974. Found: 244.0973];
m
max/cmꢁ1 (neat) 1702 (C@O); dH (400 MHz,
CDCl3) 1.83 (1H, br s, NH), 2.21 (1H, s, C„CH), 3.45 (2H, s, CH2–C„CH), 3.78
(2H, s, CH2NH), 3.91 (3H, s, OCH3), 7.01 (2H, d, J = 7.9 Hz, ArH), 7.15 (1H, t,
J = 7.9 Hz, ArH) and 7.67 (1H, s, 4-H); dC (100 MHz, CDCl3) 38.0 and 48.2
(CH2N), 56.7 (OCH3), 72.4 and 82.0 (C„CH), 113.5, 119.5, 120.3, 124.7, 127.5,
139.9, 143.3 and 147.5 (Ar–C) and 161.2 (C@O).
References and notes
1. Frankel, A. D.; Young, J. A. T. Annu. Rev. Biochem. 1998, 67, 1–25.
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Kovacs, J. A.; Falloon, J.; Walker, R. E.; Masur, H.; Haneiwich, S. E.; O’Neill, D. G.;
Decker, M. R.; Metcalf, J. A.; Deloria, M. A.; Laskin, O. L.; Salzmant, N.; Lane, H. C.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5608–5612.
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1–9.
5. Kaye, P. T.; Musa, M. A.; Nchinda, A. T.; Nocanda, X. W. Synth. Commun. 2004,
34, 2575–2589.
6. Spino, C.; Dodier, M.; Sotheeswaran, S. Bioorg. Med. Chem. Lett. 1998, 8, 3475–
3478.
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D.; Koshimizu, K.; Ohigashi, H. Bioorg. Med. Chem. Lett. 2000, 10, 59–62.
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Furukawa, H. Cancer Lett. 2001, 169, 15–19.
10. Yamaguchi, T.; Fukuda, T.; Ishibashi, F.; Iwao, M. Tetrahedron Lett. 2006, 47,
3755–3757.
11. Yamamoto, Y.; Kurazono, M. Bioorg. Med. Chem. Lett. 2007, 17, 1626–1628.
12. Rashamuse, T. J.; Musa, M. A.; Klein, R.; Kaye, P. T. J. Chem. Res. 2009, 5, 302–
305.
13. Bourinbaiar, A. S.; Tan, X.; Nagorny, R. AID 1993, 7, 129–130.
14. Tummino, P. J.; Ferguson, D.; Hupe, L.; Hupe, D. Biochem. Biophys. Res. Commun.
1994, 200, 1658–1664.
15. Tummino, P. J.; Ferguson, D.; Hupe, D. Biochem. Biophys. Res. Commun. 1994,
201, 290–294.
8-Methoxy-3-[(2-propynylamino)methyl]coumarin (8d) (0.75 g, 70%) as a pale
yellow solid, mp 120–121 °C [HRMS: m/z calculated for C15H16NO3 (MH+)
258.1130. Found: 258.1124];
m
max/cmꢁ1 (neat) 1699 (C@O); dH (400 MHz,
CDCl3) 1.46 (3H, t, J = 7.0 Hz, OCH2CH3), 1.85 (1H, br s, NH), 2.23 (1H, t,
J = 2.4 Hz, C„CH), 3.46 (2H, d, J = 2.4 Hz, CH2–C„CH), 3.79 (2H, s, CH2NH), 4.15
(2H, q, J = 7.0 Hz, OCH2CH3), 7.01 (2H, 2 ꢂ overlapping d, ArH), 7.15 (1H, dd,
ArH) and 7.68 (1H, s, 4-H); dC (100 MHz, CDCl3) 15.2 (CH3), 38.0 and 48.2
(CH2N), 65.4 (CH2O), 72.4 and 82.0 (C„CH), 114.8, 119.5, 120.4, 124.7, 127.4,
140.1, 143.6 and 146.8 (Ar–C) and 161.4 (C@O).
6-Chloro-3-[(prop-2-ynylamino)methyl]coumarin (8e) (0.76 g, 70%) as a pale
yellow solid, mp 116–117 °C [HRMS: m/z calculated for C13H11ClNO2 (MH+)
248.0478. Found: 248.0460];
m
max/cmꢁ1 (neat) 1702 (C@O); dH (400 MHz,
CDCl3) 1.76 (1H, br s, NH), 2.27 (1H, t, J = 2.4 Hz, C„CH), 3.50 (2H, d, J = 2.4 Hz,
CH2–C„CH), 3.84 (2H, s, CH2NH), 7.28 (1H, d, J = 8.5 Hz, ArH), 7.45 (2H, m,
ArH) and 7.69 (1H, s, 4-H); dC (100 MHz, CDCl3) 38.1 and 48.0 (CH2N), 72.6 and
81.8 (C„CH), 118.4, 120.7, 127.3, 128.7, 130.1, 131.5, 138.5 and 152.0 (Ar–C)
and 161.2 (C@O).
4-{[(2H-1-Benzopyran-2-one-3-yl)methylamino]methyl}-1-[(2S,3R,5R)-5-(5-
methyl-2,4-dioxo-1,2,3,4-tetrahydro-pyrimidin-1-yl)-2-
(hydroxymethyl)tetrahydrofuran-3-yl]-1H-1,2,3-triazole
(10a).
30-Azido-30-
deoxythymidine (9) (0.61 g, 2.3 mmol) was dissolved in H2O/THF (1:1;
12 mL) and 3-[(2-propynylamino)methyl]coumarin (8a) (0.49 g, 2.3 mmol),
sodium ascorbate (96 mg, 0.49 mmol) and Cu2SO4ꢀ5H2O (17 mg, 68
lmol)
were added to the solution. After stirring for 24 h at room temperature, the
mixture was extracted with CH2Cl2 (2 ꢂ 100 mL) and washed sequentially with
H2O (50 mL) and brine (30 mL). The organic layers were combined, dried over
anhydrous MgSO4, filtered and concentrated in vacuo. The crude material was
purified by flash chromatography [on silica gel; elution with EtOAc and then
with MeOH/EtOAc (2:3)] to afford the coumarin-AZT conjugate (10a) (0.72 g,
16. Vara Prasad, J. V. N.; Para, K. S.; Lunney, E. A.; Ortwine, D. F.; Dunbar, J. B., Jr.;
Ferguson, D.; Tummino, P. J.; Hupe, D.; Tait, B. D.; Domagala, J. M.; Humblet, C.;
Bhat, T. N.; Liu, B.; Guerin, D. M. A.; Baldwin, E. T.; Erickson, J. W.; Sawyer, T. K.
J. Am. Chem. Soc. 1994, 116, 6989–6990.
65%) as
C
a
light brown solid, mp 125–127 °C [HRMS: m/z calculated for
23H25N6O6 (MH+) 481.1836. Found: 481.1823];
m
max/cmꢁ1 (neat) 3291 (OH)
17. Lunney, E. A.; Hagen, S. E.; Domagala, J. M.; Humblet, C.; Kosinski, J.; Tait, B. D.;
Warmus, J. S.; Wilson, M.; Ferguson, D.; Hupe, D.; Tummino, P. J.; Baldwin, E. T.;
Bhat, T. N.; Liu, B.; Erickson, J. W. J. Med. Chem. 1994, 37, 2664–2677.
18. Kaye, P. T.; Musa, M. A.; Nocanda, X. W. Synthesis 2003, 531–534.
19. Kaye, P. T.; Musa, M. A. Synth. Commun. 2004, 34, 3409–3417.
20. Brik, A.; Alexandratos, J.; Lin, Y.-C.; Elder, J. H.; Olson, A. J.; Wlodawer, A.;
Goodsell, D. S.; Wong, C.-H. ChemBioChem 2005, 6, 1167–1169.
and 1684 (C@O); dH (400 MHz, methanol-d4) 1.90 (3H, s, CH3), 2.70 and 2.85
(2H, m, CH2CHN), 3.72 and 3.95 (4H, s, 2 ꢂ NCH2), 3.76 (1H, dd, J = 3.2, 12.3 Hz,
CHaOH), 3.89 (1H, dd, J = 2.9, 12.2 Hz, CHbOH), 4.30–4.34 (1H, m, OCHCHN),
5.37–5.42 (1H, m, OCHCH2OH), 6.47 (1H, t, J = 6.5 Hz, OCHN), 7.32–7.35 (2H, m,
ArH), 7.54–7.59 (1H, m, ArH), 7.63 (1H, dd, J = 1.3, 7.9 Hz, ArH), 7.90 (1H, s,
ArH), 7.93 (1H, s, ArH) and 8.05 (1H, s, ArH); dC (100 MHz, methanol-d4) 10.0
(CH3), 36.5 (CH2CHN), 41.8 and 46.4 (CH2N), 58.5 (CHN), 59.6 (CH2O), 83.9

6328
T. O. Olomola et al. / Tetrahedron Letters 51 (2010) 6325–6328
(HOCH2CHO), 84.2 (NCHO), 109.2, 114.8, 118.2, 121.6, 123.3, 125.0, 126.8,
8.2 Hz, OCHCHN), 5.37–5.43 (1H, m, OCHCH2OH), 5.46 (1H, t, J = 4.60 Hz, OH),
6.41 (1H, t, J = 6.6 Hz, OCHN), 7.43 (1H, d, J = 8.8 Hz, ArH), 7.59 (1H, dd, J = 2.4,
8.8 Hz, ArH), 7.86 (2H, d, J = 2.6 Hz, ArH), 7.98 (1H, s, ArH), 8.23 (1H, s, ArH) and
11.34 (1H, br s, NHC@O); dC (100 MHz, DMSO-d6) 13.1 (CH3), 38.0 (CH2CHN),
44.4 and 48.0 (CH2N), 60.0 (CHN), 61.6 (CH2O), 84.8 (HOCH2CHO), 85.4 (NCHO),
110.5, 118.8, 121.5, 123.5, 128.0, 129.1, 129.4, 131.5, 137.1, 138.3, 147.2, 151.3,
151.9, 160.9 and 164.6 (Ar–C and C@O).
130.1, 135.7, 139.3, 144.9, 150.0, 152.1, 160.6 and 164.2 (Ar–C and C@O).
Coumarin-AZT conjugate (10b) (0.51 g, 76%) as an off-white solid, mp 129–
130 °C [HRMS: m/z calculated for
C
23H24BrN6O6 (MH+) 559.0941. Found:
559.0922];
m
max/cmꢁ1 (neat) 3367 (OH) and 1674 (C@O); dH (400 MHz, DMSO-
d6) 1.82 (3H, s, CH3), 2.60–2.72 (2H, m, CH2CHN), 3.59–3.72 (4H, overlapping s
and dd, CH2OH and NCH2CC@O), 3.84 (2H, s, NCH2CN), 4.16–4.22 (1H, m,
OCHCHN), 5.33–5.39 (1H, m, OCHCH2OH), 6.42 (1H, t, J = 6.5 Hz, OCHN), 7.38
(1H, d, J = 8.8 Hz, ArH), 7.72 (1H, d, J = 8.8 Hz, ArH), 7.83 (1H, s, ArH), 7.98 (2H,
overlapping s, ArH), 8.21 (1H, s, ArH) and 11.38 (1H, br s, NHC@O); dC
(100 MHz, DMSO-d6) 13.2 (CH3), 38.0 (CH2CHN), 44.4 and 48.0 (CH2N), 60.0
(CHN), 61.6 (CH2O), 84.8 (HOCH2CHO), 85.4 (NCHO), 110.5, 117.0, 119.1, 122.0,
123.4, 129.5, 131.0, 134.2, 137.1, 138.1, 147.3, 151.4, 152.4, 160.9 and 164.7
(Ar–C and C@O).
Saturation Transfer Difference (STD) NMR studies using HIV-1 subtype
C
protease.29 The over-expression and purification of the wild-type PR has been
described previously.30 Briefly, plasmid DNA encoding the PR was transformed
into Escherichia coli BL21 (DE3) pLysS cells. The protease was over-expressed
and purified from inclusion bodies. The inclusion bodies were dissolved in 8 M
urea and refolded by extensive dialysis into protease storage buffer (10 mM
sodium acetate, 1 mM NaCl and 1 mM DTT, pH 5.0). The protease was
estimated to be P99% pure with an apparent oligomeric molecular mass of
22 kDa. The protein concentration was determined spectrophotometrically
using an extinction coefficient E1% of 11.8 at 280 nm. A buffered, aqueous
solution of HIV-1 subtype C protease (0.35 mL) was freeze-dried and then re-
constituted in an equal volume of D2O. The five ligands 10a–e (2 mg of each)
were dissolved together in D2O (0.25 mL) and the resulting solution was added
to the protein solution. Magnetic resonance saturation of the protein was
achieved using a train of four Guassian bell-shaped pulses separated by a 1 ms
delay at a power level of 40 dB. The frequency of the saturating on-resonance
pulse was at 1.64 ppm and the off-resonance pulse at 20 ppm. No spin-lock
filter was used.
Coumarin-AZT conjugate (10c) (0.45 g, 75%) as a yellow solid, mp 126–127 °C
[HRMS: m/z calculated for C24H27N6O7 (MH+) 511.1941. Found: 511.1940];
m
max/cmꢁ1 (neat) 3291 (OH) and 1671 (C@O); dH (400 MHz, DMSO-d6) 1.81
(3H, s, CH3), 2.58–2.75 (2H, m, CH2CHN), 3.59–3.71 (4H, overlapping s and dd,
CH2OH and NCH2CC@O), 3.83 (2H, s, NCH2CN), 3.90 (3H, s, OMe), 4.16–4.21
(1H, m, OCHCHN), 5.27–5.37 (2H, m, OCHCH2OH and OH), 6.41 (1H, t,
J = 5.6 Hz, OCHN), 7.23–7.32 (3H, m, ArH), 7.81 (1H, s, ArH), 7.95 (1H, s, ArH),
8.18 (1H, s, ArH) and 11.36 (1H, br s, NHC@O); dC (100 MHz, DMSO-d6) 13.1
(CH3), 38.0 (CH2CHN), 44.4 and 48.0 (CH2N), 56.9 (OCH3), 59.9 (CHN), 61.6
(CH2O), 84.7 (HOCH2CHO), 85.4 (NCHO), 110.5, 114.2, 120.1, 120.6, 123.3,
125.3, 128.3, 137.1, 139.7, 142.7, 147.3, 147.4, 151.3, 161.1 and 164.7 (Ar–C
and C@O).
Computer modelling studies. The structures of the coumarin derivative 10a and
the protein were prepared using Discovery Studio Visualiser.31 The protease
and reverse transcriptase structures were obtained using the HIV-1-PR and -RT
coordinates taken from the RCSB Protein Data Base (PDB entry code 1HXW21
and 1IKW,24 respectively); all water molecules were removed from the original
PDB file, hydrogen atoms were added and each atom assigned an AUTODOCK Type
using AUTODOCK Tools (ADT). The AUTODOCK 4.2 programme32 was used to explore
the binding mode of compound 10a when docked in the protease binding site.
For docking calculations, Gasteiger partial charges33 were assigned to the
coumarin derivatives and non-polar hydrogen atoms were merged. All torsions
were allowed to rotate during docking.
Coumarin-AZT conjugate (10d) (0.44 g, 70%) as a yellow solid, mp 114–116 °C
[HRMS: m/z calculated for C25H29N6O7 (MH+) 525.2079. Found: 525.2077];
m
max/cmꢁ1 (neat) 3306 (OH) and 1679 (C@O); dH (400 MHz, DMSO-d6) 1.40 (3H,
t, J = 6.9 Hz, OCH2CH3), 1.81 (3H, s, CH3), 2.58–2.73 (2H, m, CH2CHN), 3.60–3.71
(4H, overlapping s and dd, CH2OH and NCH2CC@O), 3.83 (2H, s, NCH2CN), 4.14–
4.20 (3H, overlapping m and q, OCHCHN and OCH2CH3), 5.31–5.38 (2H, m,
OCHCH2OH and OH), 6.41 (1H, t, J = 6.5 Hz, OCHN), 7.23–7.28 (3H, m, ArH),
7.82 (1H, s, ArH), 7.94 (1H, s, ArH), 8.19 (1H, s, ArH) and 11.43 (1H, br s,
NHC@O); dC (100 MHz, DMSO-d6) 13.1 and 15.5 (2 ꢂ CH3), 38.0 (CH2CHN), 44.5
and 48.0 (CH2N), 59.9 (CHN), 61.6 and 65.2 (2 ꢂ CH2O), 84.8 (HOCH2CHO), 85.4
(NCHO), 110.5, 115.1, 120.1, 120.6, 123.3, 125.3, 128.2, 137.1, 139.8, 142.8,
146.5, 147.4, 151.3, 161.2 and 164.6 (Ar–C and C@O).
29. Mayer, M.; Meyer, B. Angew Chem., Int. Ed. 1999, 38, 1784–1788.
30. Velazquez-Campoy, A.; Todd, M. J.; Vega, S.; Freire, E. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 6062–6067.
31. Discovery Visual Studio, Release 2.0, San Diego, Accelrys Software Inc., 2007.
32. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639–1662.
Coumarin-AZT conjugate (10e) (0.40 g, 64%) as an off-white solid, mp 103–
105 °C [HRMS: m/z calculated for
C
23H24ClN6O6 (MH+) 515.1446. Found:
515.1435];
m
max/cmꢁ1 (neat) 3260 (OH) and 1679 (C@O); dH (400 MHz, DMSO-
d6) 1.80 (3H, s, CH3), 2.58–2.74 (2H, m, CH2CHN), 3.60–3.71 (4H, overlapping s
and dd, CH2OH and NCH2CC@O), 3.82 (2H, s, NCH2CN), 4.16 (1H, dd, J = 3.5,
33. Gasteiger, J.; Marsili, M. Tetrahedron 1980, 36, 3219–3228.