Synthesis, in vitro evaluation, and docking studies of novel chromone derivatives as HIV-1 protease inhibitor

Article abstract of DOI:10.1016/j.molstruc.2011.06.035

Novel chromone derivatives with a benzopyran-4-one scaffold have been prepared by the one-pot cyclization reaction. The in vitro inhibitory activity of these new compounds towards HIV-1 protease have been evaluated using stop time HPLC method as the preliminary screening. The most potent compound, 7,8-dihydroxy-2-(3′-trifluoromethyl phenyl)-3-(3″- trifluoromethylbenzoyl)chromone (32), showed IC₅₀ = 0.34 μM. The molecular docking study supported results from experimental activity testing and also provided structure-activity relationship of this series.

Full text of DOI:10.1016/j.molstruc.2011.06.035

Journal of Molecular Structure 1001 (2011) 152–161
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, in vitro evaluation, and docking studies of novel chromone derivatives
as HIV-1 protease inhibitor
a
b
b
Jiraporn Ungwitayatorn a, , Chanpen Wiwat , Weerasak Samee , Patcharawee Nunthanavanit ,
⇑
Narumol Phosrithong c
a Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhya Road, Bangkok 10400, Thailand
b Faculty of Pharmacy, Srinakharinwirot University, Nakhon Nayok 26120, Thailand
c Faculty of Pharmacy, Siam University, 38 Petkasem Road, Bangkae, Bangkok 10160, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 April 2011
Received in revised form 10 May 2011
Accepted 24 June 2011
Available online 2 July 2011
Novel chromone derivatives with a benzopyran-4-one scaffold have been prepared by the one-pot cycli-
zation reaction. The in vitro inhibitory activity of these new compounds towards HIV-1 protease have
been evaluated using stop time HPLC method as the preliminary screening. The most potent compound,
7,8-dihydroxy-2-(30-trifluoromethyl phenyl)-3-(300-trifluoromethylbenzoyl)chromone (32), showed
IC50 = 0.34
lM. The molecular docking study supported results from experimental activity testing and
also provided structure–activity relationship of this series.
Keywords:
Ó 2011 Elsevier B.V. All rights reserved.
Chromone derivatives
HIV-1 protease inhibitor
Molecular docking
1. Introduction
One of the most intriguing small molecule, nonpeptidic HIV PR
inhibitors was warfarin (I) with IC50 = 30 mM (Fig. 1) [10]. A low
molecular weight template is very compelling. Focused screening
based on this result revealed the 4-hydroxy coumarin derivative,
phenprocoumon (II), exhibited improved enzymatic inhibitory
activity with Ki value of 1 mM [10]. Compounds such as 4-hydro-
xy-5,6-dihydropyrone series (III) have been reported as potent
HIV-1 PR inhibitor with IC50 in the nanomolar range [11,12]. In
our search for new series of potent HIV-1 PR inhibitors, we focused
on chromone derivatives whose general structure as shown in
Fig. 2.
HIV-1 protease (HIV-1 PR), a member of aspartic protease
superfamily, is an important target for the design of specific antivi-
ral agents dedicated to the treatment of HIV-1 infection and ac-
quired immunodeficiency syndrome (AIDS) [1–3]. It plays an
important role in the replication and maturation of infectious viral
particles since it is responsible for the processing of gag and gag-pol
polyprotein precursors into active viral structural proteins and rep-
licative enzymes such as reverse transcriptase, protease, and
integrase [4]. Inhibitors of the HIV-1 PR (amprenavir, atazanavir,
darunavir, indinavir, fosamprenavir, lopinavir, nelfinavir, ritonavir,
saquinavir and tipranavir) have been approved by the United
States Food and Drug Administration (US FDA) and are currently
in use in combination with reverse transcriptase inhibitors [5,6].
Despite combination chemotherapy, resistant variants have devel-
oped with reduced sensitivity to these inhibitors [7]. Therefore,
there is still need for new and improved drugs against HIV-1 PR
due to increasing viral resistance. Most of the HIV-1 PR inhibitors
reported are peptidomimetic, e.g., saquinavir, indinavir, ritonavir,
etc [8,9]. The major disadvantages of peptidomimetic compounds
are low bioavailability due to their high molecular weight, poor
solubility, and synthetic difficulties. Therefore, efforts have been
concentrated on developing nonpeptidic compounds.
2. Experimental
2.1. Physical measurements
All melting points were determined on an Electrothermal model
9100 capillary melting point apparatus and were uncorrected. 1H
NMR spectra were recorded on an ADVANCE 300 MHz Digital
NMR Spectrophotometer (Bruke Switzerland DPX-3000). Chemical
shifts were reported in ppm relative to the internal standard, tetra-
methylsilane (TMS). The NMR solvent used was deuterated
dimethylsulfoxide (DMSO, d = 2.54 ppm). FAB mass spectra were
determined on a MAT 90 (Finnigan) mass spectrometer and EI
mass spectra using INCOS 50. Elemental analyses were performed
by CHNS/O analyzer (Perkin Elmer PE2400 series II). Silica gel,
E. Merck (70–230 mesh), was used for column chromatography.
⇑
Corresponding author. Tel.: +66 2 644 8678; fax: +66 2 644 8695.
E-mail address: pyjuw@mahidol.ac.th (J. Ungwitayatorn).
0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.06.035

J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
153
O
OH
OH
O
OH
O
S
n
O
O
O
O
II
III
I
Fig. 1. Nonpeptidic HIV PR inhibitors.
(m, 3H, H30, H40, H50). MS m/z 268 (M)+. Anal. Calcd. for C16H12O4
(268.28 g/mol): C, 71.63; H, 4.52; O, 23.85. Found: C, 71.39; H,
4.27; O, 23.82.
2" 3"
O
O
C
5
A
8
4"
4
D
3
2
6
7
2.2.4. 7,8-Dihydroxy-2-pheny chromone (4)
C
5"
6"
2'
Yield 44.9%; yellow crystal; mp 191–192 °C. 1H NMR: 6.79 (d,
J = 2.56 Hz, 1H, H3), 6.94 (d, J = 8.72 Hz, 1H, H6), 7.39 (d,
J = 8.72 Hz, 1H, H5), 7.52–7.56 (m, 3H, H30, H40, H50), 8.01–8.08
(m, 2H, H20, H60). MS m/z 255 (M+H)+. Anal. Calcd. for C15H10O4
(254.25 g/mol): C, 70.86; H, 3.97; O, 25.17. Found: C, 70.59; H,
3.93; O, 25.18.
1'
3'
O
1
B
4'
6'
5'
chromone nucleus
Fig. 2. General structure of synthesized chromone derivatives.
2.2.5. 7-Hydroxy-2-methylchromone (5)
Yield 21.7%; yellow crystal; mp 205–206 °C. 1H NMR: 2.47 (s,
3H, CH3), 6.22 (s, 1H, H3), 6.49 (d, J = 2.37 Hz, 1H, H8), 6.72 (dd,
J = 8.62, 2.37 Hz, 1H, H6), 7.79 (d, J = 8.62 Hz, 1H, H5). MS m/z
176 (M)+. Anal. Calcd. for C10H8O3ꢁH2O (194.20 g/mol): C, 61.84;
H, 5.20; O, 32.96. Found: C, 61.98; H, 5.03; O, 32.99.
2.2. General procedure
To a stirred solution of phenolic ketone (1 mmol) in pyridine
(20 mL) was slowly added the appropriate acid chloride (1.5–2
equivalents) and then DBU (1,8-diazabicyclo[5,4,0] undec-7-ene)
(2 equivalents) in small portions. The reaction mixture was
refluxed at 120–140 °C for 24 h and pyridine was evaporated in
vacuo. The mixture was poured into 5 mL of concentrated HCl in
100 mL water and extracted with EtOAc (2 ꢀ 50 mL). The com-
bined organic layers were washed with water (2 ꢀ 50 ml), dried
over anhydrous sodium sulfate, and filtered. After evaporation,
the crude product was hydrolyzed by the mixture of dioxane
(2 mL), methanol (4 mL), water (1 mL) and 10% NaOH (2 mL), the
mixture was stirred at room temperature for 3–4 h. The mixture
was acidified with 4 N HCl and poured into 50 mL water, and then
extracted with EtOAc (2 ꢀ 50 mL). The combined organic layers
were washed with water (2 ꢀ 50 ml), dried over anhydrous sodium
sulfate, and filtered. After evaporation, the crude product was puri-
fied by column chromatography using mixture of EtOAc and hex-
ane as eluent.
2.2.6. 7-Hydroxy-2-benzylchromone (6)
Yield 42.2%; white crystal; mp 228–229 °C. 1H NMR: 2.15 (s, 2H,
CH2Ph), 6.17 (s, 1H, H3), 6.71 (d, J = 2.44 Hz, 1H, H8), 6.80 (dd,
J = 8.80, 2.44 Hz, 1H, H6), 7.20 (d, J = 8.12 Hz, 2H, H20, H60), 7.32–
7.44 (m, 3H, H30, H40, H50), 7.62 (d, J = 8.80 1H, H5). MS m/z 252
(M)+. Anal. Calcd. for C16H12O3 (252.28 g/mol): C, 76.17; H, 4.80;
O, 19.03. Found: C, 76.19; H, 4.71; O, 19.10.
2.2.7. 7-Hydroxy-2-benzyl-3-methylchromone (7)
Yield 27.3%; yellow crystal; mp 231–232 °C. 1H NMR: 1.05 (s,
3H, CH3), 2.26 (s, 2H, CH2Ph), 6.74 (s, 1H, H8), 6.81 (d, J = 8.83 Hz,
1H, H6), 7.18 (d, J = 7.14 Hz, 2H, H20, H60), 7.36–7.42 (m, 3H, H30,
H40, H50), 7.65 (d, J = 8.83 Hz, 1H, H5). MS m/z 266 (M)+. Anal. Calcd.
for C17H14O3 (266.31 g/mol): C, 76.67; H, 5.31; O, 18.02. Found: C,
76.31; H, 4.97; O, 18.72.
2.2.8. 7-Hydroxy-2-phenylchromone (8)
2.2.1. 8-Hydroxy-2-phenylchromone (1)
Yield 48.6%; yellow crystal; mp 246–247 °C. 1H NMR: 6.04 (s,
1H, H3), 6.33 (d, J = 2.25 Hz, 1H, H8), 6.48 (dd, J = 8.76, 2.25 Hz,
1H, H6), 7.34–7.46 (m, 5H, H20, H30, H40, H50, H60), 7.60 (d,
J = 8.76 Hz, 1H, H5). MS m/z 239 (M+H)+. Anal. Calcd. for
Yield 22.4%; white solid; mp 218–219 °C. 1H NMR: 6.21 (s, 1H,
H3), 6.85–6.88 (m, 2H, H6, H7), 7.57 (t, J = 7.51 Hz, 2H, H30, H50),
7.71 (t, J = 7.51 Hz, 1H, H400), 7.96 (d, J = 9.32 Hz, 1H, H5), 8.07 (d,
J = 7.44 Hz, 2H, H20, H60). MS m/z 238 (M)+. Anal. Calcd. for
C15H10O3 (238.25 g/mol): C, 75.61; H, 4.24; O, 20.15. Found: C,
C15H10O3 (238.25 g/mol): C, 75.61; H, 4.24; O, 20.15. Found: C,
75.60; H, 4.22; O, 20.18.
75.39; H, 4.34; O, 20.27.
2.2.9. 7-Hydroxy-3-methyl-2-phenylchromone (9)
2.2.2. 7,8-Dihydroxy-2-methylchromone (2)
Yield 29.5%; yellow crystal; mp 268–269 °C. 1H NMR: 1.94 (s,
3H, CH3), 6.81 (d, J = 2.18 Hz, 1H, H8), 6.89 (dd, J = 8.78, 2.18 Hz,
1H, H6), 7.52–7.54 (m, 3H, H30, H40, H50), 7.62–7.64 (m, 2H, H20,
H60), 7.88 (d, J = 8.78 Hz, 1H, H5). MS m/z 252 (M+H)+. Anal. Calcd.
for C16H12O3 (252.28 g/mol): C, 76.17; H, 4.80; O, 19.03. Found: C,
76.19; H, 4.76; O, 19.05.
Yield 19.2%; yellow crystal; mp 239–242 °C. 1H NMR: 2.43 (s,
3H, CH3), 6.49 (s, 1H, H3), 6.91 (d, J = 8.76 Hz, 1H, H6), 7.36 (d,
J = 8.76 Hz, 1H, H5). MS m/z 194 (M+2H)+. Anal. Calcd. for
C10H8O4ꢁ0.25H2O (196.68 g/mol): C, 61.06; H, 4.36; O, 34.58.
Found: C, 61.00; H, 4.27; O, 34.73.
2.2.3. 7,8-Dihydroxy-2-benzylchromone (3)
Yield 24.3%; yellow crystal; mp 259–260 °C. 1H NMR: 2.14 (s,
2H, CH2Ph), 6.15 (s, 1H, H3), 6.81 (d, J = 8.74 Hz, 1H, H6), 7.14 (d,
J = 8.74 Hz, 1H, H5), 7.20 (d, J = 8.24 Hz, 2H, H20, H60), 7.32–7.43
2.2.10. 7-Hydroxy-2-(40-nitrophenyl)chromone (10)
Yield 12.1%; yellow crystal; mp 298–299 °C. 1H NMR: 6.95 (dd,
J = 8.76, 2.17 Hz, 1H, H6), 7.02 (d, J = 2.17 Hz, 1H, H8), 7.10 (s, 1H,

154
J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
H3), 7.90 (d, J = 8.76 Hz, 1H, H5), 8.31–8.39 (m, 4H, H20, H30, H50,
H60). MS m/z 283 (M)+. Anal. Calcd. for C15H9NO5ꢁ0.25H2O
(287.49 g/mol): C, 62.67; H, 3.24; N, 4.87. Found: C, 62.87; H,
3.32; N, 4.75.
H20, H60). MS m/z 272 (M)+. Anal. Calcd. for C15H9FO4ꢁ0.50H2O
(280.73 g/mol): C, 64.18; H 3.41. Found: C, 64.31; H, 3.12.
2.2.19. 5,7-Dihydroxy-2-(30,40-difluorophenyl)chromone (19)
Yield 26.1%; yellow crystal; mp 359–360 °C. 1H NMR: 6.21 (d,
J = 2.03 Hz, 1H, H6), 6.55 (d, J = 2.03 Hz, 1H, H8), 7.04 (s, 1H, H3),
7.58–7.70 (m, 1H, H50), 7.93–8.00 (m, 1H, H20), 8.17–8.29 (m, 1H,
H60). MS m/z 290 (M)+. Anal. Calcd. for C15H8F2O4ꢁ0.25H2O
(294.72 g/mol): C, 61.13; H, 2.91. Found: C, 61.09; H, 2.88.
2.2.11. 7-Hydroxy-2-(30-trifluoromethylphenyl)chromone (11)
Yield 42.9%; yellow crystal; mp 258–260 °C. 1H NMR: 6.94 (dd,
J = 8.85, 2.43 Hz, 1H, H6), 7.06 (d, J = 2.43 Hz, 1H, H8), 7.09 (s, 1H,
H3), 7.80 (t, J = 7.96 Hz, 1H, H50), 7.88 (d, J = 8.85 Hz, 1H, H5),
7.95 (d, J = 7.96 Hz, 1H, H60), 8.33–8.40 (m, 2H, H20, H40). MS m/z
306 (M)+. Anal. Calcd. for C16H9F3O3 (306.24 g/mol): C, 62.75; H,
2.96. Found: C, 62.63; H, 3.09.
2.2.20. 5,7-Dihydroxy-2-(40-tert-butylphenyl)chromone (20)
Yield 35.4%; yellow crystal; mp 263–264 °C. 1H NMR: 1.35 (s,
9H, C(CH3)3), 6.21 (d, J = 2.04 Hz, 1H, H6), 6.50 (d, J = 2.04 Hz, 1H,
H8), 6.91 (s, 1H, H3), 7.59 (d, J = 8.50 Hz, 2H, H30, H50), 7.98 (d,
J = 8.50 Hz, 2H, H20, H60). MS m/z 310 (M)+. Anal. Calcd. for
2.2.12. 7-Hydroxy-2-(40-fluorophenyl)chromone (12)
Yield 41.6%; yellow crystal; mp 247–248 °C. 1H NMR: 6.87 (s,
1H, H3), 6.92 (dd, J = 8.76, 2.19 Hz, 1H, H6), 7.00 (d, J = 2.19 Hz,
1H, H8), 7.40 (t, J = 8.83 Hz, 2H, H30, H50), 7.88 (d, J = 8.76 Hz, 1H,
H5), 8.12 (dd, J = 8.83, 5.23 Hz, 2H, H20, H60). MS m/z 256 (M)+.
Anal. Calcd. for C15H9FO3ꢁ0.25H2O (260.73 g/mol): C, 69.10; H,
3.67. Found: C, 69.10; H, 3.54.
C
19H18O4ꢁ0.25H2O (314.74 g/mol): C, 72.54; H, 5.84. Found: C,
72.49; H, 5.44.
2.2.21. 5,7-Dihydroxy-2-(40-nitrophenyl)chromone (21)
Yield 26.5%; yellow crystal; mp 285–286 °C. 1H NMR: 6.22 (d,
J = 1.66 Hz, 1H, H6), 6.55 (d, J = 1.66 Hz, 1H, H8), 7.18 (s, 1H, H3),
8.30–8.43 (m, 4H, H20, H30, H50, H60). MS m/z 299 (M)+. Anal. Calcd.
for C15H9NO6ꢁ0.75H2O (312.74 g/mol): C, 57.61; H, 3.28; O, 34.63;
N, 4.48. Found: C, 57.38; H, 2.92; O, 35.0; N, 4.70.
2.2.13. 7-Hydroxy-2-(30,50-dinitrophenyl)chromone (13)
Yield 22.1%; yellow crystal; mp 347–348 °C. 1H NMR: 6.95 (dd,
J = 8.75, 2.16 Hz, 1H, H6), 7.10 (d, J = 2.16 Hz, 1H, H8), 7.32 (s, 1H,
H3), 7.90 (d, J = 8.75 Hz, 1H, H5), 8.95 (t, J = 1.95 Hz, 1H, H40),
9.14 (d, J = 1.95 Hz, 2H, H20, H60). MS m/z 328 (M)+. Anal. Calcd.
for C15H8N2O7 (328.23 g/mol): C, 54.89; H, 2.49; N, 8.53. Found:
C, 54.91; H, 2.27; N, 8.46.
2.2.22. 5,7-Dihydroxy-2-(30,50-dinitrophenyl)chromone (22)
Yield 11.0%; yellow crystal; mp 373–374 °C. 1H NMR: 6.26 (d,
J = 2.02 Hz, 1H, H6), 6.63 (d, J = 2.02 Hz, 1H, H8), 7.44 (s, 1H, H3),
8.97 (t, J = 2.06 Hz, 1H, H40), 9.16 (d, J = 2.06 Hz, 2H, H20, H60). MS
m/z 299 (M)+. Anal. Calcd. for C15H8N2O8 (344.23 g/mol): C,
52.34; H, 2.34; N, 8.14. Found: C, 52.20; H, 2.11; N, 7.83.
2.2.14. 7-Hydroxy-2-(30-chlorophenyl)chromone (14)
Yield 16.3%; yellow crystal; mp 268–269 °C. 1H NMR: 6.92 (dd,
J = 8.64, 2.08 Hz, 1H, H6), 6.99 (s, 1H, H3), 7.04 (d, J = 2.08 Hz, 1H,
H8), 7.58 (t, J = 7.33 Hz, 1H, H50), 7.65 (d, J = 7.33 Hz, 1H, H40),
7.89 (d, J = 8.64 Hz, 1H, H5), 8.03 (d, J = 7.33 Hz, 1H, H60), 8.14 (s,
1H, H20). MS m/z 272 (M)+. Anal. Calcd. for C15H9ClO3 (272.68 g/
mol): C, 66.07; H, 3.33. Found: C, 66.08; H, 3.22.
2.2.23. 5,7-Dihydroxy-2-(30-chlorophenyl)chromone (23)
Yield 58.1%; yellow crystal; mp 240–242 °C. 1H NMR: 6.21 (d,
J = 2.13 Hz, 1H, H6), 6.57 (d, J = 2.13 Hz, 1H, H8), 7.07 (s, 1H, H3),
7.59 (t, J = 8.02 Hz, 1H, H50), 7.68 (dt, J = 8.02, 1.72 Hz, 1H, H40),
8.05 (dt, J = 8.02, 1.72 Hz, 1H, H60), 8.13 (t, J = 1.72 Hz, 1H, H20).
MS m/z 299 (M)+. Anal. Calcd. for C15H9ClO4ꢁ0.50H2O (297.19 g/
mol): C, 60.62; H, 3.22. Found: C, 60.34; H, 2.89.
2.2.15. 7-Hydroxy-2-(30,40-dichlorophenyl)chromone (15)
Yield 20.3%; yellow crystal; mp 285–286 °C. 1H NMR: 6.91 (dd,
J = 8.48, 2.50 Hz, 1H, H6), 7.01 (s, 1H, H3), 7.03 (d, J = 2.50 Hz, 1H,
H8), 7.82 (d, J = 8.72 Hz, 1H, H50), 7.88 (d, J = 8.48 Hz, 1H, H5),
8.05 (dd, J = 8.72, 2.01 Hz, 1H, H60), 8.34 (d, J = 2.01 Hz, 1H, H20).
MS m/z 308 (M+2H)+. Anal. Calcd. for C15H8Cl2O3 (307.13 g/mol):
C, 58.66; H, 2.63. Found: C, 58.50; H, 2.55.
2.2.24. 5,7-Dihydroxy-2-(30,40-dichlorophenyl)chromone (24)
Yield 65.0%; yellow crystal; mp 334–335 °C. 1H NMR: 6.21 (d,
J = 1.98 Hz, 1H, H6), 6.58 (d, J = 1.98 Hz, 1H, H8), 7.10 (s, 1H, H3),
7.83 (d, J = 8.60 Hz, 1H, H50), 8.08 (dd, J = 8.60, 2.10 Hz, 1H, H60),
8.37 (d, J = 2.10 Hz, 1H, H20). MS m/z 299 (M)+. Anal. Calcd. for
2.2.16. 7-Hydroxy-2-(40-tert-butylphenyl)chromone (16)
Yield 44.7%; yellow crystal; mp 271–272 °C. 1H NMR: 1.35 (s,
9H, C(CH3)3), 6.83 (s, 1H, H3), 6.92 (dd, J = 8.64, 2.18 Hz, 1H, H6),
6.98 (d, J = 2.18 Hz, 1H, H8), 7.57 (d, J = 8.38 Hz, 2H, H30, H50),
7.88 (d, J = 8.64 Hz, 1H, H5), 7.97 (d, J = 8.38 Hz, 2H, H20, H60). MS
m/z 294 (M)+. Anal. Calcd. for C19H18O3ꢁ0.25H2O (298.85 g/mol):
C, 76.43; H, 6.19. Found: C, 76.77; H, 5.68.
C
15H8Cl2O4ꢁ0.50H2O (331.63 g/mol): C, 54.33; H, 2.50. Found: C,
54.42; H, 2.42.
2.2.25. 5,7-Dihydroxy-2-(40-methoxyphenyl)chromone (25)
Yield 43.2%; yellow crystal; mp 243–244 °C. 1H NMR: 3.90 (s,
3H, OCH3), 6.19 (d, J = 2.10 Hz, 1H, H6), 6.49 (d, J = 2.10 Hz, 1H,
H8), 6.86 (s, 1H, H3), 7.10 (d, J = 9.00 Hz, 2H, H30, H50), 8.02 (d,
J = 9.00 Hz, 2H, H20, H60). MS m/z 284 (M)+. Anal. Calcd. for
2.2.17. 5,7-Dihydroxy-2-(30-trifluoromethylphenyl)chromone (17)
Yield 70.1%; yellow crystal; mp 280–281 °C. 1H NMR: 6.21 (d,
J = 2.18 Hz, 1H, H6), 6.58 (d, J = 2.18 Hz, 1H, H8), 7.17 (s, 1H, H3),
7.81 (t, J = 7.95 Hz, 1H, H50), 7.98 (d, J = 7.95 Hz, 1H, H60), 8.33–
8.39 (m, 2H, H20, H40). MS m/z 322 (M)+. Anal. Calcd. for
C16H12O5 (284.26 g/mol): C, 67.60; H, 4.25. Found: C, 67.61; H,
4.05.
2.2.26. 5,7-Dihydroxy-2-(30-methoxyphenyl)chromone (26)
C16H9F3O4 (322.24 g/mol): C, 59.64; H, 2.82. Found: C, 59.65; H,
Yield 33.4%; yellow crystal; mp 242–243 °C. 1H NMR: 3.90 (s,
3H, OCH3), 6.21 (d, J = 2.10 Hz, 1H, H6), 6.53 (d, J = 2.10 Hz, 1H,
H8), 7.01 (s, 1H, H3), 7.18 (dd, J = 8.05, 2.20 Hz, 1H, H40), 7.48 (t,
J = 8.05 Hz, 1H, H50), 7.58 (dd, J = 2.20, 1.72 Hz, 1H, H20), 7.64 (d,
J = 8.05 Hz, 1H, H60). MS m/z 284 (M)+. Anal. Calcd. for C16H12O5
(284.26 g/mol): C, 67.60; H, 4.25. Found: C, 67.57; H, 3.86.
2.70.
2.2.18. 5,7-Dihydroxy-2-(40-fluorophenyl)chromone (18)
Yield 71.0%; yellow crystal; mp 270–271 °C. 1H NMR: 6.21 (d,
J = 2.02 Hz, 1H, H6), 6.52 (d, J = 2.02 Hz, 1H, H8), 6.98 (s, 1H, H3),
7.41 (t, J = 8.88 Hz, 2H, H30, H50), 8.14 (dd, J = 8.88, 5.25 Hz, 2H,

J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
155
2.2.27. 6-Hydroxy-2-(30-methoxyphenyl)chromone (27)
Calcd. for C24H18O7 (418.40 g/mol): C, 68.89; H, 4.34. Found: C,
68.38; H, 4.28.
Yield 12.0%; pink solid; mp 226–228 °C. 1H NMR: 3.84 (s, 3H,
OCH3), 6.98 (s, 1H, H3), 7.15 (d, J = 7.53, 1H, H40), 7.25 (dd,
J = 8.91, 2.75 Hz, 1H, H7), 7.31 (d, J = 2.75 Hz, 1H, H5), 7.47 (t,
J = 7.53 Hz, 1H, H50), 7.57 (s, 1H, H20), 7.64 (d, J = 7.53 Hz, 1H,
H60), 7.65 (d, J = 8.91 Hz, 1H, H8), 10.07 (s, 1H, C6–OH). MS m/z
268 (M)+. Anal. Calcd. for C16H12O4 (268.27 g/mol): C, 71.63; H,
4.51. Found: C, 71.45; H, 4.57.
2.2.35. 7,8-Dihydroxy-2-(40-fluorophenyl)-3-(400-
fluorobenzoyl)chromone (35)
Yield 10.0%; yellow crystal; mp 292–293 °C. 1H NMR: 7.00 (d,
J = 8.72 Hz, 1H H6), 7.22–7.34 (m, 4H, H30, H300, H50, H500), 7.39 (d,
J = 8.72 Hz, 1H, H5), 7.65 (dd, J = 8.86, 5.50 Hz, 2H, H200, H600) 7.99
(dd, J = 8.78, 5.40 Hz, 2H, H20, H60). MS m/z 417 (M+Na)+. Anal.
Calcd. for C22H12F2O5 (394.32 g/mol): C, 67.01; H, 3.07. Found: C,
67.06; H, 3.48.
2.2.28. 6-Hydroxy-2-(30-chlorophenyl)chromone (28)
Yield 28.1%; yellow crystal; mp 247–248 °C. 1H NMR: 7.03 (s,
1H, H3), 7.25 (dd, J = 8.92, 2.85 Hz, 1H, H7), 7.30 (d, J = 2.85 Hz,
1H, H5), 7.55–7.66 (m, 2H, H50, H60), 7.69 (d, J = 8.92 Hz, 1H, H8),
8.04 (dd, J = 7.74, 1.15 Hz, 1H, H40), 8.13 (s, 1H, H20), 10.10 (s, 1H,
C6–OH). MS m/z 272 (M)+. Anal. Calcd. for C15H9O3Cl (272.69 g/
mol): C, 66.07; H, 3.33. Found: C, 66.19; H, 3.19.
2.2.36. 7,8-Dihydroxy-2-(40-nitrophenyl)-3-(400-
nitrobenzoyl)chromone (36)
Yield 5%; yellow crystal; mp 327–328 °C. 1H NMR: 7.03 (d,
J = 8.57 Hz, 1H, H6), 7.41 (d, J = 8.57 Hz, 1H, H5), 7.88 (d,
J = 8.57 Hz, 2H, H200, H600), 8.20 (d, J = 8.57 Hz, 2H, H300, H500),
8.22–8.35 (m, 4H, H20, H30, H50, H60). MS m/z 448 (M)+. Anal. Calcd.
for C22H12N2O9 (448.34 g/mol): C, 58.94; H, 2.70; N, 6.25. Found: C,
58.95; H, 2.74; N, 5.98.
2.2.29. 6-Hydroxy-2-(40-fluorophenyl)chromone (29)
Yield 6.8%; pale yellow crystal; mp 261–262.5 °C. 1H NMR: 6.95
(s, 1H, H3), 7.25 (dd, J = 8.93, 2.67, 1H, H7), 7.30 (d, J = 2.67 Hz, 1H,
H5), 7.41 (t, J = 8.78 Hz, 2H, H30, H50), 7.64 (d, J = 8.93 Hz, 1H, H8),
8.13–8.17 (m, 2H, H20, H60), 10.04 (s, 1H, C6–OH). MS m/z 256 (M)+.
Anal. Calcd. for C15H9O3F (256 g/mol): C, 70.31; H, 3.51. Found: C,
70.30; H, 3.26.
2.2.37. 7,8-Dihydroxy-2-(40-methoxyphenyl)-3-(400-
methoxybenzoyl)chromone (37)
Yield 5.8%; pale yellow crystal; mp 253–254 °C. 1H NMR: 3.74
(s, 3H, 40-OCH3), 3.79 (s, 3H, 400-OCH3), 6.91–6.99 (m, 5H, H6, H50,
H30, H500, H300), 7.37 (d, J = 8.71 Hz, 1H, H5), 7.61 (d, J = 8.79, 2H,
H20, H60), 7.84 (d, J = 8.76 Hz, 2H, H200, H600). MS m/z 418 (M)+. Anal.
Calcd. for C24H18O7ꢁ0.75H2O (431.91 g/mol): C, 66.74; H, 4.20.
Found: C, 66.63; H, 4.36.
2.2.30. 6-Hydroxy-2-(30-trifluoromethylphenyl)chromone (30)
Yield 3.0%; yellow crystal; mp 259.1–260.3 °C. 1H NMR: d 7.15
(s, 1H, H3), 7.26 (dd, J = 8.96, 2.82, 1H, H7), 7.31 (d, J = 2.82 Hz,
1H, H5), 7.72 (d, J = 8.96 Hz, 1H, H8), 7.80 (t, J = 7.86 Hz, 1H, H50),
7.95 (d, J = 7.86 Hz, 1H, H60), 8.40 (m, 2H, H20, H40), 10.06 (s, 1H,
C6–OH). MS m/z 306 (M)+. Anal. Calcd. for C16H9O3F3 (306 g/mol):
C, 62.75; H, 2.94. Found: C, 63.26; H, 2.72.
2.2.38. 7-Hydroxy-2-(30,40-difluorophenyl)-3-(300,400-difluorobenzoyl)-
chromone (38)
Yield 7.0%; yellow crystal; mp 242–243 °C. 1H NMR: 6.98 (dd,
J = 8.30, 2.14 Hz, 1H, H6), 7.01 (d, J = 2.14 Hz, 1H, H8), 7.32–7.38
(m, 1H, H200), 7.48–7.60 (m, 2H, H50, H500), 7.72–7.79 (m, 1H,
H600), 7.80–7.87 (m, 1H, H20), 7.89 (d, J = 8.30 Hz, 1H, H5), 8.00–
8.08 (m, 1H, H60). MS m/z 437 (M+Na)+. Anal. Calcd. for
2.2.31. 6-Hydroxy-2-(40-tert-butylphenyl)chromone (31)
Yield 7.4%; white crystal; mp 233.5–235 °C. 1H NMR: d 1.31 (s,
9H, C(CH3)3), 6.89 (s, 1H, H3), 7.24 (dd, J = 8.97, 2.95 Hz, 1H, H7),
7.30 (d, J = 2.95 Hz, 1H, H5), 7.57 (d, J = 8.49 Hz, 2H, H30, H50),
7.63 (d, J = 8.97 Hz, 1H, H8), 7.98 (d, J = 8.49 Hz, 2H, H20, H60),
10.60 (s, 1H, C6–OH). MS m/z 294 (M)+. Anal. Calcd. for C19H18O3
(294.35 g/mol): C, 77.53; H, 6.16. Found: C, 78.13; H, 6.16.
C22H10F4O4 (414.40 g/mol): C, 63.78; H, 2.43. Found: C, 63.46; H,
2.70.
2.2.39. 7-Hydroxy-2-(30-trifluoromethylphenyl)-3-(300-trifluorometh-
ylbenzoyl)chromone (39)
2.2.32. 7,8-Dihydroxy-2-(30-trifluoromethylphenyl)-3-(300-
trifluoromethylbenzoyl)chromone (32)
Yield 10.9%; yellow crystal; mp 234–235 °C. 1H NMR: 7.01 (dd,
J = 8.43, 1.64 Hz, 1H, H6), 7.03 (d, J = 1.64 Hz, 1H, H8), 7.62–7.73
(m, 2H, H50, H500), 7.81–8.00 (m, 5H, H200, H400, H5, H40, H600), 8.18
(s, 1H, H20), 8.20 (d, J = 8.00 Hz, 1H, H60). MS m/z 501 (M+Na)+.
Anal. Calcd. for C24H12F6O4 (478.34 g/mol): C, 60.26; H, 2.53.
Found: C, 60.29; H, 2.33.
Yield 8.3%; yellow crystal; mp 208–209 °C. 1H NMR: 7.10 (d,
J = 8.68 Hz, 1H, H6), 7.41 (d, J = 8.68 Hz, 1H, H5), 7.62–7.75 (m,
2H, H50, H500), 7.82–7.87 (m, 2H, H40, H400), 7.97–8.00 (m, 2H,
H200, H600), 8.18 (s, 1H, H20), 8.22 (d, J = 8.04, 1H, H60). MS m/z 517
(M+Na)+. Anal. Calcd. for C24H12F6O5 (494.34 g/mol): C, 58.31; H,
2.45. Found: C, 58.28; H, 2.77.
2.2.40. 7-Hydroxy-2-(30-chlorophenyl)-3-(300-chlorobenzoyl)-
chromone (40)
2.2.33. 7,8-Dihydroxy-2-(30-chlorohenyl)-3-(300-chlorobenzoyl)
chromone (33)
Yield 23.1%; yellow crystal; mp 303–305 °C. 1H NMR: 6.98 (dd,
J = 8.13, 1.81 Hz, 1H, H6), 7.00 (d, J = 1.81, 1H, H8), 7.44–7.58 (m,
4H, H50, H500, H40, H60), 7.68 (s, 1H, H20), 7.70 (d, J = 1.67 Hz, 1H,
H400), 7.86–7.91 (m, 2H, H5, H600), 7.95 (d, J = 1.36 Hz, 1H, H200).
MS m/z 410 (M)+. Anal. Calcd. for C22H12O4Cl2 (411.24 g/mol): C,
64.25; H, 2.94. Found: C, 63.94; H, 2.81.
Yield 11.5%; yellow crystal; mp 256–257 °C. 1H NMR: 7.01 (d,
J = 8.71, 1H, H6), 7.39 (d, J = 8.71 Hz, 1H, H5), 7.45–7.58 (m, 4H,
H50, H40, H500, H400), 7.69 (d, J = 6.87 Hz, 1H, H60), 7.76 (s, 1H, H20),
7.88 (d, J = 7.69 Hz, 1H, H60), 7.96 (s, 1H, H20). MS m/z 426 (M)+.
Anal. Calcd. for C22H12O5Cl2ꢁ0.25H2O (431.74 g/mol): C, 61.20; H,
2.92. Found: C, 61.25; H, 2.82.
2.2.34. 7,8-Didroxy-2-(30-methoxyphenyl)-3-(300-
2.2.41. 7-Hydroxy-2-(30-methoxyphenyl)-3-(300-
methoxybenzoyl)chromone (34)
methoxybenzoyl)chromone (41)
Yield 15.0%; yellow crystal; mp 255–257 °C. 1H NMR: 3.66 (s,
3H, 30-OCH3), 3.85 (s, 3H, 300-OCH3), 7.01 (d, J = 8.62 Hz, 1H, H6),
7.05 (d, J = 9.17, 1H, H40), 7.18 (s, 1H, H20), 7.18–7.20 (m, 2H, H60,
H5), 7.34 (d, J = 7.79 Hz, 1H, H400), 7.38 (s, 1H, H200) 7.38–7.41 (m,
2H, H50, H500), 7.47 (d, J = 7.79 Hz, 1H, H600). MS m/z 418 (M)+. Anal.
Yield 14.1%; yellow crystal; mp 253–254 °C. 1H NMR: 3.65 (s,
3H, 300-OCH3), 3.75 (s, 3H, 30-OCH3), 6.97 (dd, J = 8.04, 2.29 Hz,
1H, H6), 6.99 (d, J = 2.29 Hz, H8), 7.04 (dd, J = 8.91, 1.98 Hz, 1H,
H400), 7.12–7.22 (m, 3H, H200, H40, H500), 7.32–7.41(m, 3H, H20, H50,
H600), 7.45–7.50 (m, 1H, H60), 7.89 (d, J = 8.04 Hz, 1H, H5). MS m/z

156
J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
402 (M)+. Anal. Calcd. for C24H18O6 (402.40 g/mol): C, 71.64; H,
4.51. Found: C, 72.46; H, 4.71.
Table 1
Composition of HPLC assay mixture.
Reagents
Negative control (
l
L)
Positive control (
lL)
Sample (lL)
2.2.42. 7-Hydroxy-2-(40,-fluorophenyl)-3-(400-
fluorobenzoyl)chromone (42)
Assay buffer
Substrate
50% DMSO
Inhibitor
Pepstatin A
Enzyme
20.4
1.6
1.6
–
–
1.4
20.4
1.6
–
–
1.6
1.4
20.4
1.6
–
1.6
–
Yield 8.5%; yellow crystal; mp 239-240 °C. 1H NMR: 6.95–7.02
(m, 2H, H6, H8), 7.25–7.37 (m, 4H, H30, H300, H50, H500), 7.63 (dd,
J = 8.84, 5.38 Hz, 2H, H200, H600), 7.89 (d, J = 9.39 Hz, 1H, H5), 8.00
(dd, J = 8.70, 5.48 Hz, 2H, H20, H60). MS m/z 401 (M+Na)+. Anal.
Calcd. for C22H12F2O4 (378.41 g/mol): C, 69.85; H, 3.20. Found: C,
69.92; H, 3.02.
1.4
Total volume
25.0
25.0
25.0
Table 2
Structures of chromone derivatives and % inhibition at 12.5
lg/mL concentration.
R5
O
R6
R7
R3
O
R2
R8
.
Compd
R2
R3
R5
R6
R7
R8
% Inhibition
1
2
3
4
5
6
7
8
9
Phenyl
CH3
Benzyl
Phenyl
CH3
Benzyl
Benzyl
Phenyl
H
H
H
H
H
H
CH3
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
64.34 1.01
92.02 0.24
93.30 0.07
88.17 1.09
56.00 1.74
50.26 4.73
18.97 5.79
75.04 1.72
57.29 5.06
63.52 1.63
29.27 1.10
37.52 7.03
26.32 4.49
27.36 3.84
47.63 2.11
78.89 5.71
74.80 0.26
88.13 2.33
80.25 3.25
89.29 3.47
27.20 0.82
10.48 2.52
73.62 0.58
85.26 1.20
88.68 2.27
97.48 1.72
75.29 2.13
47.48 0.94
54.56 4.68
66.68 0.32
31.98 2.46
93.16 1.74
50.59 1.39
53.13 2.95
84.94 1.54
92.24 1.70
35.23 4.07
88.98 1.44
74.98 3.86
54.17 0.81
24.67 4.31
50.79 0.97
74.47 2.76
34.06 9.89
88.53 1.75
53.39 3.22
88.39 0.11
43.19 4.07
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
Phenyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
40-(NO2)-Phenyl
30-(CF3)-Phenyl
40-(F)-Phenyl
30,50-(diNO2)-Phenyl
30-(Cl)-Phenyl
30,40-(diCl)-Phenyl
40-(t-butyl)-Phenyl
30-(CF3)-Phenyl
40-(F)-Phenyl
30,40-(diF)-Phenyl
40-(t-butyl)-Phenyl
40-(NO2)-Phenyl
30,50-(diNO2)-Phenyl
30-(Cl)-Phenyl
30,40-(diCl)-Phenyl
40-(OCH3)-Phenyl
30-(OCH3)-Phenyl
30-(OCH3)-Phenyl
30-(Cl)-Phenyl
40-(F)-Phenyl
30-(CF3)-Phenyl
40-(t-butyl)-Phenyl
30-(CF3)-Phenyl
30-(Cl)-Phenyl
30-(OCH3)-Phenyl
40-(F)-Phenyl
40-(NO2)-Phenyl
40-(OCH3)-Phenyl
30,40-(diF)-Phenyl
30-(CF3)-Phenyl
30-(Cl)-Phenyl
30-(OCH3)-Phenyl
40-(F)-Phenyl
40-(NO2)-Phenyl
40-(OCH3)-Phenyl
40-(t-butyl)-Phenyl
30-(OCH3)-Phenyl
40-(NO2)-Phenyl
40-(t-butyl)-Phenyl
H
H
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
300-(CF3)-benzoyl
300-(Cl)-benzoyl
300-(OCH3)-benzoyl
400-(F)-benzoyl
400-(NO2)-benzoyl
400-(OCH3)-benzoyl
300,400-(diF)-benzoyl
300-(CF3)-benzoyl
300-(Cl)-benzoyl
300-(OCH3)-benzoyl
400-(F)-benzoyl
400-(NO2)-benzoyl
400-(OCH3)-benzoyl
400-(t-butyl)-benzoyl
300-(OCH3)-benzoyl
400-(NO2)-benzoyl
400-(t-butyl)-benzoyl
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
OH
OH
H
H
H
H
OH
Pepstatin A (100
l
M)
91.07 1.53

J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
157
O
R
5
R5
O
O
O
R5
R8
O
O
O
R6
R7
R6
R7
R6
R7
CH3
R
R2
Cl
+
R2
OH
DBU, pyridine, reflux
= phenyl, substituted phenyl
R2
R8
R
2
R8
R , R , R , R = H, OH
5
6
7
8
R , R , R , R = H, OCOR
5
6
7
8
R = phenyl, substituted phenyl
10%NaOH, CH OH, dioxane
3
R5
R8
O
O
O
R5
O
O
R6
R7
R6
R7
R
+
R2
R2
R8
R , R , R , R = H, OH
5
6
7
8
O
O
O
CH3
R2
CH3
R2
Cl
O
DBU, pyridine, reflux
R2 = Ph, CH2Ph
R2
O
O
HO
OH
10%NaOH, CH OH, dioxane
3
O
CH3
HO
O
R2
(Chromones 7 and 9)
Scheme 1. Synthesis of chromone derivatives by one-pot cyclization reaction.
2.2.43. 7-Hydroxy-2-(40-nitrophenyl)-3-(400-nitrobenzoyl)chromone
(43)
Yield 5.1%; yellow crystal; mp 290–291 °C. 1H NMR: 6.98–7.07
(m, 2H, H6, H8), 7.83 (d, J = 8.78 Hz, 2H, H200, H600), 7.91 (d,
J = 9.05 Hz, 1H, H5), 8.21 (d, J = 8.78 Hz, 2H, H300, H500), 8.28 (d,
J = 8.72 Hz, 4H, H20, H30, H50, H60). MS m/z 455 (M+Na)+. Anal.
Calcd. for C22H12N2O8 (432.34 g/mol): C, 61.12; H, 2.80; N, 6.48.
Found: C, 61.11; H, 2.87; N, 6.87.
3.067
8.630
2.2.44. 7-Hydroxy-2-(40-methoxyphenyl)-3-(400-
methoxybenzoyl)chromone (44)
4.736
Yield 41.2%; yellow crystal; mp 300–301 °C. 1H NMR: 3.75 (s,
3H, 300-OCH3), 3.79 (s, 3H, 30-OCH3), 6.90–7.02 (m, 6H, H30, H300,
H50, H500, H6, H8), 7.57 (d, J = 8.98, 2H, H200, H600), 7.83 (d, J = 8.86,
2H, H20, H60), 7.87 (d, J = 8.50 Hz, 1H, H5). MS m/z 402 (M)+. Anal.
Calcd. for C24H18O6: (402.40 g/mol): C, 71.64; H, 4.51. Found: C,
71.62; H, 4.23.
2.2.45. 7-Hydroxy-2-(40-tert-butylphenyl)-3-(400-tert-
butylbenzoyl)chromone (45)
Yield 25.4%; white crystal; mp 271–273 °C. 1H NMR: 1.23 (s, 9H,
40-t-butyl), 1.27 (s, 9H, 400-t-butyl), 6.95 (dd, J = 7.95, 1.13, 1H, H6),
6.98 (d, J = 1.13 Hz, 1H, H8), 7.45 (d, J = 8.32 Hz, 2H, H30, H50), 7.49
(d, J = 8.48 Hz, 2H, H300, H500), 7.55 (d, J = 8.32 2H, H20, H60), 7.82 (d,
J = 8.48 2H, H200, H600), 7.86 (d, J = 7.95 Hz, 1H, H5). MS m/z 454
(M)+. Anal. Calcd. for C30H30O4 (454.56 g/mol): C, 79.27; H, 6.65.
Found: C, 79.09; H, 6.73.
0
5
10
Fig. 3. HPLC profile of a reaction mixture of HIV-1 PR and substrate after incubated
for 2 h at 37 °C.

158
J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
Fig. 4. Plots of the logarithm of concentrations (lg/mL) and % inhibition profiles and IC50 of chromones 2, 3, 26, 27, 30, 32, 36 and 45.
2.2.46. 5,7-Dihydroxy-2-(30-methoxyphenyl) 3-(300-
H, H20), 7.13 (dd, J = 8.24, 2.49 Hz, 1 H, H60), 7.21 (dd, J = 7.81,
methoxybenzoyl)chromone (46)
2.25 Hz, 1H, H400), 7.34 (t, J = 8.24 Hz, H, H50), 7.38 (t,
1
Yield 6.7%; yellow crystal; mp 167–168 °C. 1H NMR: 3.64 (s, 3H,
30-OCH3), 3.76 (s, 3H, 300-OCH3), 6.26 (d, J = 1.68 Hz, 1H, H6), 6.51
(d, J = 1.68, 1H, H8), 7.05 (dd, J = 8.24, 2.49 Hz, 1H, H40), 7.10 (s, 1
J = 7.81 Hz, 1 H, H500), 7.41 (s, 1H, H200), 7.54 (dd, J = 7.81, 2.25 Hz,
1H, H600). MS m/z 418 (M)+. Anal. Calcd. for C16H12O4ꢁ0.25H2O
(422.90 g/mol): C, 68.16; H, 4.41. Found: C, 68.16; H, 4.32.

J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
159
2.2.47. 5,7-Dihydroxy-2-(40-nitrophenyl)-3-(400-
nitrobenzoyl)chromone (47)
Graphics Inc., Mountain View, CA) using the sketch approach.
The fragment libraries in SYBYL database were used as building
blocks for the construction of larger ones. Each structure was
energy minimized using the standard Tripos force field (Powell
method and 0.05 kcal/mol Å energy gradient convergence criteria)
and electrostatic charge was assigned by the Gasteiger–Hückel
method. These conformations were used as starting conformations
to perform docking. The docking was performed using FlexiDock
option in SYBYL/Biopolymer program. The crystal structure of the
HIV-1 PR complexed with inhibitor (Aha001) was obtained from
the protein data bank (pdb 1AJX). The Aha001 structure was first
removed from the complex structure, then chromone inhibitor
was placed into the binding site. Water molecules and ions were
removed and hydrogen atoms were added at appropriate geome-
try. The charges were assigned by Kollman force field for protein
and Gasteiger–Hückel for ligands.
Yield 4.1%; yellow crystal; mp 288–289 °C. 1H NMR: 6.27 (d,
J = 1.75 Hz, 1H, H6), 6.54 (d, J = 1.75 Hz, 1H, H8), 7.82 (d,
J = 8.78 Hz, 2H, H200, H600), 8.22–8.38 (m, 6H, H20, H30, H300, H50,
H500, H60). MS m/z 448 (M)+. Anal. Calcd. for C22H12N2O9
(448.34 g/mol): C, 58.94; H, 2.70; N, 6.25. Found: C, 58.95; H,
2.74; N, 6.41.
2.2.48. 6-Hydroxy-2-(40-tert-butylphenyl)-3-(400-tert-
butylbenzoyl)chromone (48)
Yield 2.8%; pale orange solid; mp 232–234.0 °C. 1H NMR: 1.22
(s, 9H, 40-t-butyl), 1.68 (s, 9H, 400-t-butyl), 7.30 (broad d,
J = 2.95 Hz, 1H, H5), 7.31 (dd, J = 8.82, 2.95 Hz, 1H, H7), 7.45–7.54
(m, 6H, H30, H50, H300, H500, H20, H60), 7.66 (d, J = 8.82 Hz, 1H, H8),
7.83 (d, J = 8.32 Hz, 2H, H200, H600), 10.25 (s, 1H, C6–OH). MS m/z
454 (M)+. Anal. Calcd. for C30H30O4ꢁ0.5H2O (463.58 g/mol): C,
77.73; H, 6.74. Found: C, 77.74; H, 6.76.
3. Results and discussion
2.3. HIV-1 PR assay
3.1. Chemical synthesis
A stopped time HPLC assay [13] was performed by incubating
peptide substrate with the enzyme in small volumes (<0.1 mL). A
modified peptide, anthraniryl His-Lys-Ala-Arg-Val-Leu-(p-NO2-
Phe)-Glu-Ala-Nle-Ser-Amide, whose amino acid sequence corre-
sponds to the p24-p15 cleavage site of viral polyprotein was used
as a substrate. The following reagents were prepared for the assay:
anthranirylHis-Lys-Ala-Arg-Val-Leu-(p-NO2-Phe)-Glu-Ala-Nle-Ser-
Forty-eight chromone derivatives (Table 2) were prepared
according to Scheme 1. The one pot cyclization reaction was devel-
oped from the method of Riva et al. [14]. The phenolic ester inter-
mediate was initially formed from the reaction of phenolic ketone
and acid chloride. Abstraction of the acidic methylene proton by
DBU led to the formation of the carbanion. The carbanion nucleo-
phile attacked the ester carbonyl carbon and provided the b-dike-
tone intermediate. Cyclization of b-diketone followed by
dehydration resulted in the 2-substituted chromone ester. During
the cyclization process, the DBU could abstract more acidic hydro-
gen to generate another carbanion nucleophile which attacked the
carbonyl carbon of excess acid chloride. After dehydration process,
the 2,3-disubstitued chromone ester was obtained. Hydrolysis of
the chromone esters by the mixture of NaOH, methanol, water,
and dioxane finally yielded the chromone derivatives.
Amide (1
lg/L), substrate buffer (50 mM NaOAc, pH 4.9), inhibitors
(chromone derivatives, 12.5
l
g/mL), positive control (pepstatin A,
0.1 mM) dissolved in DMSO, assay buffer (50 mM NaOAc, pH 4.9,
200 mM NaCl, 5 mM dithiothreitol (DTT) and 10% (v/v) glycerol in
water and purified HIV-1 PR biosynthetic enzyme from E. coli
(0.005 lg/lL).
The composition of negative, positive control and sample reac-
tion mixtures for HPLC assay is shown in Table 1.
The reaction mixture was incubated at 37 °C for 2 h and then
terminated by addition of 2.5 lL of 10% trifluoroacetic acid. During
3.2. Evaluation of HIV-1 PR inhibitory activity
the incubation, the substrate was hydrolyzed at the site of Leu and
(p-NO2-Phe). After quenching with acid at various times (<60 min),
the sample was subjected to reverse phase HPLC. The resulting
peptidolytic products were thereby separated from the remaining
substrate using linear gradient of acetonitrile in 0.1% trifluoroacetic
acid (TFA) with spectrophotometric detection of the peptides at
280 nm. The percentage of inhibition was calculated from the ratio
of the substrate peak area to the hydrolysate peak area.
The ability of the chromone derivatives to inhibit the HIV-1 PR
was determined using HPLC assay for preliminary testing. The
principle of the assay is based on the catalytic cleavage of peptide
substrates bearing the chromogenic p-nitrophenylalanine. His-Lys-
Ala-Arg-Val-Leu-(p-NO2-Phe)-Glu-Ala-Nle-Ser-Amide peptide usu-
ally used as substrate for HIV-1 PR. Only the peptide bond between
the Leu and p-NO2-Phe residues is hydrolyzed [13]. The substrate
and Phe(NO2)-bearing hydrolysate could be monitored at wave-
length 280 nm and were eluted at 8.6 and 4.7 min, respectively
(Fig. 3). The results were reported as % inhibition as summarized
in Table 2. The inhibitory activity was in the range of 10–97%.
Chromone 26 exhibited highest activity with 97% inhibition at con-
Anegative control ꢂ Asample
%Inhibition ¼
ꢀ 100
Anegative control
ðA ¼ relative peak of the hydrolysateÞ
HPLC condition:
centration 12.5 lg/mL, while pepstatin A, a potent aspartyl prote-
Column: HypersilÒ BDS C18, particle size: 5
250 ꢀ 4.6 mm
lm,
Injection volume: 20 lL
Table 3
IC50 (lM) of the investigated chromone
derivatives.
Solvent: A. 0.1% trifluoroacetic acid in water, B. acetonitrile/
water (3:1)
Gradient: 22.5–40% of solvent B in 12 min
Flow rate: 1.5 mL/min
Compd
IC50 (lM)
2
3
6.89
5.22
UV detector: k 280 nm
26
27
30
32
36
45
2.53
2.4. Generation of the molecular structures and docking
23.35
26.92
0.34
0.65
11.50
The molecular structures of chromone derivatives were mod-
eled with SYBYL 6.9 molecular modeling program (Tripos Associ-
ates, Saint Louis, MO) on an Indigo Elan workstation (Silicon

160
J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
Fig. 5. (a) The hydrophobic interaction of chromone 32 in the active site of HIV-1 PR. (b) Schematic view of the binding conformation of chromone 32 in the enzyme active sites.
ase [15] showed 91% inhibition at 100
l
M in the same experiment.
active compound, 7,8-dihydroxy-2-(30-trifluoromethylphenyl)-3-
(300-trifluoromethylbenzoyl)chromone (32) showed IC50 = 0.34
which is more potent than the structurally related HIV-1 PR inhibi-
tors in 4-hydroxycoumarins series. The docking study revealed that
7,8-dihydroxyl groups and hydrophobic substituents both at posi-
tions 2 and 3 are required for better enzyme inhibition.
Some derivatives that exhibited medium and high % inhibition
were selected for further determination of IC50. IC50 values were
obtained from linear regression plots between log concentration
(log C) versus % inhibition (Fig. 4). The three most potent inhibitors
lM
were chromones 32, 36 and 26 with IC50 = 0.34, 0.65 and 2.53 lM,
respectively (Table 3).
Acknowledgements
3.3. Binding interaction study
The authors thank the High Performance Computer Center
(HPCC), National Electronics and Computer Technology Center
(NECTEC) of Thailand for providing SYBYL facilities.
The most active compound in this series, chromone 32 was
used as the representative molecule for discussion. As seen from
Fig. 5, the 3-trifluoromethyl benzoyl ring binds to its binding site,
S1 pocket (Val32, Ile500, Pro81, and Val82) via hydrophobic inter-
action with the hydrophobic amino acid residues. The 3-tri-
fluoro-methyl-phenyl ring also forms hydrophobic interaction
with the hydrophobic residues in S2 pocket (Leu23, Val32, Val82,
and Ile84). The 7-OH and 8-OH of the chromone form hydrogen
bonds with the carbonyl oxygens of Asp25 and Asp250. The docking
results supported the experimental activity testing that most of
the compounds which have both 7,8-dihydroxyl groups and hydro-
phobic substituents at positions 2 and 3 showed greater inhibition
(the only exception was chromone 26). Compounds with less than
20% inhibition such as chromones 7 and 22 are those whose struc-
tures are composed of only 7-hydroxy and hydrophobic substitu-
ent at position 2.
References
[1] C. Debouck, J.G. Gorniak, J.E. Strickler, T.D. Meek, B.W. Metcalf, M. Rosenberg,
Proc. Natl. Acad. Sci. USA 84 (1987) 8903.
[2] S. Seelmeier, H. Schmidt, V. Turk, K. von der Helm, Proc. Natl. Acad. Sci. USA 85
(1988) 6612.
[3] C.J. Flexner, N. Engl. J. Med. 338 (1998) 1281.
[4] N.E. Kohl, E.A. Emini, W.A. Schleif, L.J. Davis, J.C. Heimbach, R.A.F. Dixon, E.M.
Scolnick, I.S. Sigal, Proc. Natl. Acad. Sci. USA 85 (1988) 4686.
[5] Z. Temesgen, D. Warnke, M.J. Kasten, Expert. Opin. Pharmacother. 7 (2006)
1541.
[6] A.K. Patick, T.J. Boritzki, L.A. Bloom, Antimicrob. Agents Chemother. 1 (1997)
2159.
[7] D. Boden, M. Markowitz, Antimicrob. Agent Chemother. 42 (1998) 2775.
[8] D.J. Kempf, H.L. Shan, Curr. Pharm. Des. 2 (1996) 225.
[9] A. Brik, C. H Wong, Org. Biomol. Chem. 1 (2003) 5.
[10] S. Thaisrivongs, P.K. Tomich, K.D. Watenpaugh, K.-T. Chong, W.J. Howe, C.-P.
Yang, J.W. Strohbach, S.R. Turner, J.P. McGrath, M.J. Bohanon, J.C. Lynn, A.M.
Mulichak, P.A. Spinelli, R.R. Hinshaw, P.J. Pagano, J.B. Moon, M.J. Ruwart, K.F.
Wilkinson, B.D. Rush, G.L. Zipp, R.J. Dalga, F.J. Schwende, G.M. Howard, G.E.
Padbury, L.N. Toth, Z. Zhao, K.A. Koeplinger, T.J. Kakuk, S.L. Cole, R.M. Zaya, R.C.
Piper, P. Jeffrey, J. Med. Chem. 37 (1994) 3200.
4. Conclusion
We have synthesized a series of chromonederivatives as nonpep-
tidic HIV-1 PR inhibitor. The in vitro HIV-1 PR inhibitory activity test-
ing by HPLC assay was used as the preliminary screening. The most
[11] B.D. Tait, S. Hagen, S. Domagala, E.L. Ellsworth, C. Gajda, H.W. Hamilton, J.V.N.
Vara Prasad, D. Ferguson, N. Graham, D. Hupe, C. Nouhan, P.J. Tummino, C.

J. Ungwitayatorn et al. / Journal of Molecular Structure 1001 (2011) 152–161
161
Humblet, E.A. Lunney, A. Pavlovsky, J. Rubin, S.J. Gracheck, E.T. Baldwin, T.N.
Bhat, J.W. Erickson, S.V. Gulnik, B. Liu, J. Med. Chem. 40 (1997) 3781.
[12] S.R. Turner, J.W. Strohbach, R.A. Tommasi, P.A. Aristoff, P.D. Johnson, H.I.
Skulnick, L.A. Dolak, E.P. Seest, P.K. Tomich, M.J. Bohanon, M.-N. Horng, J.C.
Lynn, K.-T. Chong, R.R. Hinshaw, K.D. Watenpaugh, M.N. Janakiraman, S.
Thaisrivongs, J. Med. Chem. 41 (1998) 3467.
[13] C. Debouck, T.A. Tomaszec Jr., L.A. Ivanoff, J. Culp,
A
practical approach,
molecular biology, and drug discovery, in: J. Karn (Ed.), HIV, vol. 2, IRL Press,
New York, 1995, p. 73.
[14] C. Riva, C. De Toma, L. Donadel, C. Boi, R. Pennini, G. Motta, A. Leonardi,
Synthesis (February) (1997) 195.
[15] K. von der Helm, L. Gürtler, J. Eberle, F. Deinhardt, FEBS Lett. 247 (1989) 349.