Synthesis and HIV protease inhibitory activity of new 4-hydroxy-2-pyrone derivatives

Full text of DOI:10.1002/1521-4184(200010)333:10<319::AID-ARDP319>3.0.CO;2-A

Communications to the Editor
Synthesis and HIV Protease Inhibitory Activity of New
4-Hydroxy-2-pyrone Derivatives
a)
a)
a)
a)
b)
b)
a)
Yong Sup Lee* , Sun Nam Kim , Yong Sil Lee , Jae Yeol Lee , Chong-Kyo Lee , Hae Soo Kim , and Hokoon Park
a) Division of Life Sciences, Korea Institute of Science & Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Korea
b) Pharmaceutical Screening Center, Korea Research Institute of Chemical Technology, P.O. Box 107, Yusong, Taejeon 305-600, Korea
Key Words: HIV; protease inhibitors; nonpeptide; 4-hydroxy-2-pyrone; sulfonamide
Human immunodeficiency virus (HIV) is the causative chain on HIV protease inhibitory activity, based on the C -
2
[7]
agent of acquired immunodeficiency syndrome (AIDS). Sev- symmetric structure of the HIV protease . Herein, we report
eral enzymes are important to the life cycle of this virus. on the synthesis and HIV protease inhibitory activities of new
Therefore, reverse transcriptase, integrase, and protease are 4-hydroxy-2-pyrone derivatives 3, which have a sulfonamide
considered to be promising targets for the development of group at the phenyl ring at C-6β position of the pyrone ring.
anti-AIDS drugs. One therapeutic approach of intense study
over the past few years has been inhibition of HIV protease, taining 4-hydroxypyrones is depicted in Scheme 2. Acid cata-
Preparation of a representative series of sulfonamide-con-
[1]
which plays a key role in viral mutation
.
lyzed condensation of 4-hydroxy-6-methyl-2-pyrone (4) and
[6]
A number of potent HIV protease inhibitors has been iden- α-cyclopropylbenzyl alcohol gave the substituted 2-pyrone
tified , but these compounds are peptide-derived structures, 5 in 66% yield. Treatment of 5 with 2.2 equiv. of lithium
[2]
and in many cases their clinical development is hindered by diisopropylamide followed by ethyl iodide resulted in an
poor phamacokinetics, including low oral bioavailability and alkylation at the C-6α position to provide 6 in 66% yield.
[3]
rapid excretion . Recently, Pharmacia & Upjohn company
Repeated alkylation of 6 at the C-6α position with m-ni-
discovered that nonpeptide HIV protease inhibitors of the trobenzyl bromide gave racemic 7 in 34% yield. Reduction
4-hydroxy-2-pyrone class as exemplified by compound 1 of the nitro group in 7 was accomplished using catalytic
have high inhibitory activity against HIV protease with good hydrogenation on palladium charcoal to provide amine 8,
[4]
pharmacokinetic characters . Enzymatic and antiviral ac- which could be coupled with a wide variety of arylsulfonyl
tivity of 4-hydroxy-2-pyrone class could be improved by chlorides in the presence of pyridine to afford the final
introduction of sulfonamide functionality at the meta position sulfonamides 3 as a racemic mixture. Compound 3l was
[5]
of phenyl side chain, as exemplified by compound 2
.
prepared in 82% from 3k by catalytic hydrogenation of the
nitro group. To examine the effect of arylsulfonamide func-
tionality on the activity, unsubstituted 4-hydroxy-2-pyrone 9
was also prepared in 56% yield from 6 by alkylation with
[8]
benzyl bromide
.
The resulting 4-hydroxy-2-pyrone derivatives 3, which
have a meta-sulfonamide group of phenyl ring at C-6β, have
[9]
been tested as HIV protease inhibitors as shown in Table 1
.
The inhibitory activity of unsubstituted derivative 9 was
included for comparison. The benzenesulfonamide-contain-
ing derivatives (3a–3g) were found to be less active than the
parent compound 9. The activities within this series generally
increased when an electron-withdrawing group was substi-
tuted in the benzene ring compared to unsubstituted phenyl
derivative (3a) and compounds (3) containing electron-do-
nating group with the exception of 3c. On the other hand,
pyridinesulfonamide-containing derivatives (3h–3l) were
more potent than the parent compound 9 with the exception
of 3h. Unfortunately, most compounds showed high cyto-
Scheme 1
Although many efforts to improve the HIV protease inhibi- toxicity (CC = 3.8–70.0 µM) with the exception of 3f and
50
tory activity by the modification of the C-3 side chain of could therefore not inhibit HIV replication in the cell culture
4-hydroxy-2-pyrone have been undertaken, studies on struc- assay under nontoxic concentration (data not shown). Com-
ture-activity relationship of C-6 side chain have remained pound 3f did not exhibit noticeable antiviral activity even
[6]
relatively rare . Thus, we wished to investigate the effects though it has the least cytotoxicity (CC = 170.4 µM) among
50
of a sulfonamide functionality of the phenyl ring at C-6 side the compounds. However, compound 3j showed less cyto-
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/1010/0319 $17.50 +.50/0

320
Lee and co-workers
Scheme 2
toxicity (CC = 71.4 µM) while exhibiting moderate in vitro
50
antiviral activity (EC = 23.6 µM) from a cell culture assay
50
[10]
using HIV-I
infected MT4 cells
.
IIIB
In conclusion, 4-hydroxy-2-pyrone derivatives, which con-
tain a pyridinesulfonamide functionality at C-6 side chain
showed higher HIV protease inhibitory activities than unsub-
stituted and benzenesulfonamide-containing 4-hydroxy-2-
pyrone derivatives. Among synthesized compounds, 3j
showed moderate antiviral activity under the nontoxic con-
centration. For more extensive SAR studies, we are undertak-
ing syntheses of 4-hydroxy-2-pyrone derivatives containing
an arylsulfonamide at the ortho- or para position of phenyl
ring at the C-6 side chain.
Acknowledgement
We are grateful for the support of this work by the Ministry of Science and
Technology, Korea (2N20110).
Arch. Pharm. Pharm. Med. Chem. 333, 319–322 (2000)

HIV Protease Inhibitory Activity
321
(CDCl3+CD3OD) δ 7.61–6.79 (m, 13H, Ph), 5.67 (s, 1H, pyrone),
3.79 (s, 3H, OCH3), 3.39 (d, 1H, J = 10.3 Hz, CH-Ph), 2.80 (m, 2H,
PhCH2), 2.48 (m, 1H, CH-pyrone), 1.89–1.54 (m, 3H, propyl and
cyclopropyl), 0.86 (t, 3H, J = 7.4 Hz, 3H), 0.68 (m, 1H, cyclopropyl),
0.49 (m, 1H, cyclopropyl), 0.24 (m, 2H, cyclopropyl). 3d: (CDCl3)
δ 7.78–6.83 (m, 13H, Ph), 5.66 (s, 1H, pyrone), 3.67 (d, 1H, J = 9.1 Hz,
PhCH), 2.79 (m, 2H, PhCH2), 2.46 (m, 1H, CH-pyrone), 1.67–1.53 (m,
3H, propyl and cyclopropyl), 0.85 (t, 3H, J = 7.3 Hz, CH3), 0.71 (m,
1H, cyclopropyl), 0.55 (m, 1H, cyclopropyl), 0.39 (m, 1H, cyclo-
propyl), 0.25 (m, 1H, cyclopropyl).
References
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3e: (CDCl3+CD3OD) δ 8.21–6.78 (m, 13H, Ph), 5.65 (s, 1H, pyrone),
3.39 (d, 1H, J = 9.8 Hz, PhCH), 2.83 (m, 2H, PhCH2), 2.50 (m, 1H,
CH-pyrone), 1.87–1.53 (m, 3H, propyl and cyclopropyl), 0.87 (t, 3H, J
= 7.3 Hz, CH3), 0.69 (m, 1H, cyclopropyl), 0.48 (m, 1H, cyclopropyl),
0.24 (m, 2H, cyclopropyl). 3f: (CDCl3+CD3OD) δ 7.59–6.73 (m, 13H,
Ph), 5.70 (s, 1H, pyrone), 3.38 (d, 1H, J = 9.6 Hz, PhCH), 2.80–
2.71 (m, 2H, PhCH2), 2.34 (m, 1H, CH-pyrone), 2.13 (s, 3H, CH3CO),
1.92–1.49 (m, 3H, propyl and cyclopropyl), 0.83 (t, 3H, J = 7.3 Hz,
CH3), 0.68 (m, 1H, cyclopropyl), 0.48 (m, 1H, cyclopropyl), 0.25 (m,
2H, cyclopropyl). 3g: (CDCl3+CD3OD) δ 8.07–6.80 (m, 13H, Ph),
5.63 (s, 1H, pyrone), 3.40 (m, 1H, PhCH), 2.89–2.77 (m, 2H, PhCH2),
2.50 (m, 1H, CH-pyrone), 1.89–1.54 (m, 3H, propyl and cyclopropyl),
0.85 (t, 3H, J = 7.3 Hz, CH3), 0.68 (m, 1H, cyclopropyl), 0.49 (m, 1H,
cyclopropyl), 0.25 (m, 2H, cyclopropyl). 3h: (CDCl3) δ 8.64 (d, 1H, J
= 5.4 Hz, pyridine), 7.82–6.79 (m, 12H, pyridine and Ph) 5.66 (s, 1H,
pyrone), 3.66 (d, 1H, J = 9.4 Hz, PhCH), 2.77 (m, 2H, PhCH2), 2.42 (m,
1H, CH-pyrone), 1.69–1.62 (m, 3H, propyl and cyclopropyl), 0.83 (m,
3H, CH3), 0.73 (m, 1H, cyclopropyl), 0.55 (m, 1H, cyclopropyl),
0.39 (m, 1H, cyclopropyl), 0.24 (m, 1H, cyclopropyl). 3i: (CD3OD)
δ 8.72 (s, 1H, pyridine), 7.94–6.62 (m, 11H, pyridine and Ph), 5.59 (1s,
1H, pyrone), 3.12 (1H, overlapped with solvent, PhCH), 2.62 (m, 2H,
PhCH2), 2.36 (m, 1H, CH-pyrone), 1.69–1.43 (m, 3H, propyl and cy-
clopropyl), 0.69 (t, 3H, J = 7.4 Hz, CH3), 0.49 (m, 1H, cyclopropyl),
0.26 (m, 1H, cyclopropyl), 0.04–0.01 (m, 2H, cyclopropyl). 3j:
(CDCl3) δ 8.91 (s, 1H, pyridine), 8.03–6.83 (m, 11H, pyridine and Ph),
5.64 (s, 1H, pyrone), 3.67 (d, 1H, J = 9.2 Hz, PhCH), 2.80 (m, 2H,
PhCH2), 2.45 (m, 1H, CH-pyrone), 1.69–1.47 (m, 3H, propyl and cy-
clopropyl), 0.83 (t, 3H, J = 7.4 Hz, CH3), 0.69 (m, 1H, cyclopropyl),
0.54 (m, 1H, cyclopropyl), 0.37 (m, 1H, cyclopropyl), 0.24 (m, 1H,
cyclopropyl). 3k: (CDCl3+CD3OD) δ 9.37 (m, 1H, pyridine), 8.49–
6.78 (m, 11H, pyridine and Ph), 5.64 (s, 1H, pyrone), 3.37 (d, 1H, J
=10.0 Hz, PhCH), 2.83–2.75 (m, 2H, PhCH2), 2.43 (m, 1H, CH-py-
rone), 1.88–1.50 (m, 3H, propyl and cyclopropyl), 0.84 (t, 3H, J = 7.3
Hz, CH3), 0.68 (m, 1H, cyclopropyl), 0.45 (m, 1H, cyclopropyl), 0.25–
0.15 (m, 2H, cyclopropyl). 3l: (CD3OD) δ 7.92 (s, 1H, pyridine), 7.52–
6.73 (m, 11H, J = 9.0 Hz), 5.74 (s, 1H, pyrone), 3.32 (1H, overlapped
with solvent, PhCH), 2.81–2.73 (m, 2H, PhCH2), 2.46 (m, 1H, CH-py-
rone), 1.95–1.48 (m, 3H, propyl and cyclopropyl), 0.86 (m, 3H, CH3),
0.69 (m, 1H, cyclopropyl), 0.44 (m, 1H, cyclopropyl), 0.19–0.18 (m,
2H, cyclopropyl).
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1989, 246, 1149–1152. b) M. Clare, Perspect. Drug Dis. Des. 1993, 1,
49–68.
[8] All new compounds are characterized by 300 MHz 1H NMR spectros-
copy. 5: (DMSO-d6) δ 7.36 (d, 2H, J = 7.3 Hz, Ph), 7.23 (dd, 2H, J =
7.3, 6.9 Hz, Ph), 7.13 (d, 1H, J = 6.9 Hz, Ph), 6.00 (s, 1H, pyrone),
3.23 (d, 1H, J = 10.4 Hz, PhCH-), 2.13 (s, 3H, CH3), 1.85 (m, 1H,
cyclopropyl), 0.63–0.17 (m, 4H, cyclopropyl). 6: (CDCl3) δ 7.50 (d,
2H, J = 7.5 Hz, Ph), 7.30 (dd, 2H, J=7.5, 7.2 Hz, Ph), 7.22 (d, 1H, J =
7.2 Hz, Ph), 5.93 (s, 1H, pyrone), 3.62 (d, 1H, J = 9.6 Hz, PhCH-),
2.37 (t, 2H, J = 7.5 Hz, propyl), 1.75–1.59 (m, 3H, propyl & cyclo-
propyl), 0.95 (t, 3H, J = 7.4 Hz, propyl), 0.71–0.28 (m, 4H, cyclo-
propyl). 7: (CDCl3) δ 8.10 (m, 1H, Ph), 7.96 (s, 1H, Ph), 7.49–7.27 (m,
7H), 6.87 (br, 1H, OH), 5.62 (s, 1H, pyrone), 3.81 (m, 1H, PhCH-),
3.07–2.98 (m, 2H, m-NO2-Ph-CH2), 2.55 (m, 1H, CH-pyrone),
1.70 (m, 2H, propyl), 1.29 (m, 1H, cyclopropyl), 0.92 (t, 3H, J = 7.4
Hz, CH3), 0.75 (m, 1H, cyclopropyl), 0.66 (m, 1H, cyclopropyl),
0.47 (m, 1H, cyclopropyl), 0.28 (m, 1H, cyclopropyl).
9: (CDCl3) δ 7.50–7.07 (m, 10H, Ph), 6.51 (br, 1H, OH), 5.58 (s, 1H,
pyrone), 3.86 (d, 1H, J = 8.9 Hz, PhCH), 2.96–2.83 (m, 2H, PhCH2),
2.52 (m, 1H, CH-pyrone), 1.72–1.40 (m, 3H, propyl and cyclopropyl),
0.88 (m, 3H, CH3), 0.75–0.68 (m, 1H, cyclopropyl), 0.62–0.49 (m, 2H,
cyclopropyl), 0.31–0.25 (m, 1H, cyclopropyl).
8: (CDCl3) δ 7.43–7.21 (m, 6H, Ph and OH), 6.95 (t, 1H, J = 7.7 Hz,
Ph), 6.43 (m, 2H, Ph), 6.33 (s, 1H, Ph), 5.52 (s, 1H, pyrone), 3.81 (m,
1H, PhCH), 2.78 (m, 1H, CH-pyrone), 2.65 (m, 1H, m-NH2Ph-CH),
2.43 (m, 1H, m-NH2Ph-CH), 1.65–1.50 (m, 4H, NH2 and propyl),
1.19 (m, 1H, cyclopropyl), 0.79 (t, 3H, J = 7.4 Hz, CH3), 0.67–0.65 (m,
1H, cyclopropyl), 0.54–0.43 (m, 2H, cyclopropyl), 0.23–0.18 (m, 1H,
cyclopropyl). 3a: (CDCl3+CD3OD) δ 7.68–6.79 (m, 14H, Ph),
5.66 (1s, 1H, pyrone), 3.39 (d, 1H, J = 9.8 Hz, PhCH), 2.80 (m, 2H,
PhCH2), 2.48 (m, 1H, CH-pyrone), 1.85–1.54 (m, 3H, propyl and
cyclopropyl), 0.86 (t, 3H, J = 7.3 Hz, CH3), 0.67 (m, 1H, cyclopropyl),
0.49–0.47 (m, 1H, cyclopropyl), 0.24 (m, 2H, cyclopropyl). 3b:
(CDCl3+CD3OD) δ 7.51–6.78 (m, 13H, Ph), 5.68 (1s, 1H, pyrone),
3.37 (d, 1H, J = 10.1 Hz, PhCH), 2.80 (m, 2H, PhCH2), 2.45 (m, 1H,
CH-pyrone), 2.35 (s, 3H, CH3-Ph), 1.91–1.51 (m, 3H, propyl and cy-
clopropyl), 0.86 (t, 3H, J = 7.3 Hz, CH3), 0.68 (m, 1H, cyclopropyl),
0.49 (m, 1H, cyclopropyl), 0.24 (m, 2H, cyclopropyl). 3c:
[9] HIV protease inhibitory activity was measured as follows: HIV pro-
tease and the fluorogenic substrate, Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-
Arg-NH2 were purchased from Bachem Bioscience. The compounds
were serially diluted by 10 fold with assay buffer (50 mM sodium
acetate, pH 5.2, 200 mM NaCl, 5 mM dithiothreitol, 10% glycerol).
Reaction mixture consisted of 9 µl of 10× concentrated compounds, 6.5
µl of 1 mg/ml substrate dissolved in 100% DMSO, 72.5 µl assay buffer
and 2 µl of HIV protease. The reaction was initiated by addition of 2 µl
of HIV protease (0.7 µg/µl) and incubated at room temperature. The
activity was measured by monitoring the increase in fluorescence
intensity at the emission maximum of 460 nm (excitation wavelength
was 345 nm) using fluorescence microplate reader (Fluoroskan Ascent,
Labsystems).
Arch. Pharm. Pharm. Med. Chem. 333, 319–322 (2000)

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Lee and co-workers
[10] Evaluation of Antiviral activity was measured as follows: Standard
virus-induced CPE (cytopathic effect) inhibition assay which employed
3-(4,5-dimethyl thiazoly-2)-2,5-diphenyl tetrazolium bromide (MTT)
to measure inhibition of virus-mediated cell death was used with slight
modification. MT-4 cells on log phase were pelleted and infected with
HIV-1IIIB virus at an M.O.I. (multiplicity of infection) of 100 CCID50
(50% cell culture inhibitory dose) per well. The cells were immediately
resuspended with RPMI 1640/10% FBS at the concentration of 1.2 ×
105 cells/ml. 100 µl of the resuspended cells were dropped to the wells
of 96 well plate containing 100 µl of 2 × concentrated compounds in
duplicate. After 5 days incubation at 37 °C, the cells were observed
microscopically and quantified by MTT assay. The liquid was aspirated
until 50 µl of liquid and all cells remained, and 20 µl of 7.5 mg/ml MTT
solution was added. The plate were further incubated for 1 h, and 100 µl
of acidified isopropanol was added and shaken on a microplate shaker
until the formazan form completely dissolved. The absorbance at
540 nm by using 690 nm as reference wavelength was measured with
microplate reader (Vmax, Molecular Devices). The antiviral effective
concentration was expressed as the EC50 or concentration of the com-
pound required to inhibit virus-induced CPE by 50%. To measure the
effect of compounds on the host cell growth, mock-infected cells were
applied to the compound containing wells of the same plates in dupli-
cate. The cytotoxic concentration was expressed as the CC50 or concen-
tration of the compound required to inhibit cell growth by 50%.
Received: June 19, 2000 [FP499]
Arch. Pharm. Pharm. Med. Chem. 333, 319–322 (2000)