Discovery, total synthesis, HRV 3C-protease inhibitory activity, and structure-activity relationships of 2-Methoxystypandrone and its analogues

Article abstract of DOI:10.1016/S0960-894X(01)00648-5

2-Methoxystypandrone, a naphthoquinone, was isolated from a Chinese herb Polygonum cuspidatum by bioassay guided fractionation using HRV 3C-protease assay. It showed an IC₅₀ value of 4.6 μM and is moderately selective. A new 10-step, total synthesis of 2-methoxystypandrone was accomplished in 45% overall yield using a Diels-Alder approach. Several analogues of this compound were prepared. Isolation, synthesis and HRV 3C-protease structure-activity relationships of these compounds have been described.

Full text of DOI:10.1016/S0960-894X(01)00648-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3143–3146
Discovery, Total Synthesis, HRV 3C-Protease Inhibitory Activity,
and Structure–Activity Relationships of2-Methoxystypandrone
and Its Analogues
a,
b
a
b
Sheo B. Singh, * Pia L. Graham, Robert A. Reamer and Michael G. Cordingley
aMerck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bMerck Research Laboratories, West Point, PA 19486, USA
Received 28 August 2001; accepted 24 September 2001
Abstract—2-Methoxystypandrone, a naphthoquinone, was isolated from a Chinese herb Polygonum cuspidatum by bioassay guided
fractionation using HRV 3C-protease assay. It showed an IC50 value of 4.6 mM and is moderately selective. A new 10-step, total
synthesis of 2-methoxystypandrone was accomplished in 45% overall yield using a Diels–Alder approach. Several analogues of this
compound were prepared. Isolation, synthesis and HRV 3C-protease structure–activity relationships of these compounds have been
described. # 2001 Elsevier Science Ltd. All rights reserved.
Human rhinoviruses (HRVs) are members of the picor-
navirus family of viruses and are the single most sig-
nificant causative agents of the common cold. More
than 100 serotypes of these viruses are known and they
typically infect upper respiratory tract in humans and
target nasal epithelial cells. A 220 kDa polypeptide is
translated by the positive-sense viral RNA genome
upon entry into the host cell. This polypeptide is pro-
teolytically processed by virus-specific proteases (HRV
revealed potent HRV 3C-protease activity in the
extractsof Chine es herb, Polygonum cuspidatum. The
bioassay guided fractionation of the extract led to the
isolation of three compounds identified as 2-methoxy-
stypandrone (2), emodin (3), and methoxyemodin (4).
The compound 2 showed IC50 value of 4.6 mM in HRV
3C-protease assay. The present paper describes the dis-
covery, structure, a new total synthesis, HRV 3C-pro-
tease inhibitory activity, and SAR of 2-
methoxystypandrone and related compounds.
1
2
A and HRV 3C) to afford structural and functional
2
proteinsthat are required for replication of HRV.
HRV 3C-protease, a cysteine protease, is responsible for
the polypeptide cleavage between Q-G amino acidsand
playsa critical role in the replication cycle of HRVsand
thuscon ts itutesa potential therapeutic target for the
2
,3
control of HRVsand common cold.
the inhibitorsof thisenzyme isin pha es II clinical
AG-7088, one of
development for the treatment against the infections of
4
HRVs.
Isolation and Structure
Screening of natural product extractsagain st HRV 3C-
protease using a small peptide containing Q-G scissile
bond as a substrate led to the discovery of thysanone
(1) asa novel inhibitor from an extract of a fungu,s
Thysanophora penicilloides. Continued screening
Sephadex LH 20 chromatography of a 1:1 CH OH–
3
CH Cl extract (100 mg) of P. cuspidatum followed by
2
2
5
chromatographies on silica gel and reversed-phase C-8
HPLC yielded 2 (1 mg), 3 (5 mg), and 4 (2 mg). The
structure of 2 waselucidated as2-methoxy st ypandrone
by comparison of spectral data (UV, IR, and 1H
6
NMR). The structures of 3 and 4 were determined by
comparison with authentic samples.
*Corresponding author. Tel.:+1-732-594-3222; fax:+1-732-594-6880;
e-mail: sheo_singh@merck.com
0
960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00648-5

3144
S. B. Singh et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3143–3146
Synthesis of2-Methoxystypandrone
turned out to be challenging but wasefficiently accom-
plished by using a mixture of dioxane–MeOH–H O–
2
The quantity of 2-methoxystypandrone isolated from
the plant extract was very small and resupply of the
compound for further biological evaluationshad to be
accomplished by a total synthesis. A 14-step total 9%
overall yielding synthesis of this compound was repor-
H SO (3:6:2:0.8), which gave deprotected product 10 in
2
99% yield after chromatography. Standard acetylation
of compound 10 furnished compound 11 in 93%
4
yield. Friesrearrangement of compound
ꢀ
11 with
BF Et O at 60 C exclusively produced ortho-acetylated
.
3
2
6
b
ted by Hughesand Sargent.
However, we decided to
product 12 in 85% yield (vide infra), which wasacetyl-
ated to give acetate 13 in 95% yield. Oxidation of
compound 13 with ceric ammonium nitrate (CAN) in a
design a new synthesis that would be more amenable for
analogue synthesis for SAR. In our synthetic approach
we took advantage of the regio-selective Diels–Alder
9
mixture of CH CN–H O yielded 78% of para-quinone
3
2
7
reactions of suitably substituted chloro-benzoquinone
14, which upon saponification furnished 2 which was
1
and appropriately substituted O-silyloxy butadiene fol-
lowed by oxidation for the construction of the naph-
thoquinone. A 10-step 45% overall yielding synthesis
identical (TLC, HPLC, H NMR) to natural product
2-methoxystypandrone.
(
Scheme 1) of 2 follows.
Fries Rearrangement
Thus, Diels–Alder reaction of 1-trimethylsilyloxy-1, 3-
8
.
BF Et O catalyzed Friesrearrangement of compound
11 produced stable BF -adduct 15 (Scheme 2) which was
2
butadiene (5) with 2-methoxy-5-chloro-benzoquinone
(6) in refluxing benzene followed by Jonesoxidation
3
2
afforded naphthoquinone (7) in 88% yield. MOM pro-
tection of the phenolic group produced compound 8 in
99% yield, which wasreduced with os dium dithionite
and alkylated with dimethyl sulfate to give trimethyl
ether (9) in 97% yield. Deprotection of the MOM ether
purified by silica gel chromatography and was char-
1
0
acterized by spectral analysis. It showed a molecular
ion at m/z 338 (with m/z 337, which was20% of m/z 338
1
1
peak). The B NMR spectrum showed one signal for
the boron nucleusat d 0.20 (relative to external refer-
.
19
ence of BF Et O at d 0.0). The F NMR spectrum of
3
2
15 showed two signals for equivalent fluorine atoms at
d À147.52 and d À147.59 (relative to internal C F at
6
6
d À163.0) in a 1:4 ratio consistent with the natural
1
0
11
abundance ratiosof B and B, respectively. In addi-
1
3
tion, the broad band decoupled C NMR spectrum
of 15 showed two triplets at d 193.9 (J=1.7 Hz) and
d 171.1 (J=2.2 Hz) due to covalent bonding of the
boron atom to CH CO and the aromatic oxygen. The
3
hydrolysis of the adduct in water was slower (45% con-
version in 30 min and 100% after overnight as observed
1
by H NMR analysis in CD Cl and addition of D O)
2
2
2
and wasfa st in MeOH.
Scheme 2.
Sources ofAnalogues
Compound 16 (Table 1) wasprepared by t sa ndard
acetylation of compound 7. Compounds 17 and 18 were
prepared by a method described by Jung et al.1 Com-
pound 19 was isolated as an occasional by-product of
the Jonesoxidation reaction during preparation of 7. 5-
Hydroxy-7-methyl-1,4-naphthoquinone (20) waspre-
pared by analogouscycloaddition reactionsof 5 with 2-
chloroquinone followed by oxidation. Compounds 21–
26 were acquired from Aldrich Chemical Company.
Xanthomegnin (27) wasobtained from our internal
collections.
1
Scheme 1. Reagentsand condition s: (i) Benzene, reflux, 24 h; (ii) Jones
ꢀ
oxidation; rt, 20 min; (iii) MOMCl, DIPEA, CH
iv) (a) Sodium dithionite, dioxane–H
dimethyl sulfate, rt, 2 h; (v) dioxane–MeOH–H
2
Cl
2
, 0 C to rt, 16 h;
(
2
O, 1:1, rt, 15 min; (b) KOH,
2
O–H
ꢀ
2
SO
4
, 3:6:2:0.8,
3.
2
rt, 16 h; (vi) Ac
Ac O, C N, rt, 20 h; (ix) CAN (excess), CH
x) KOH, MeOH–H O, 6:1, 0.5 min, 6 N HCl.
2
O, C
5
H
5
N, rt, 20 h; (vii) BF Et
O, 60 C, 35 min; (viii)
2
5
H
5
3 2
CN–H O, 3:1, 30 min;
(
2

S. B. Singh et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3143–3146
Table 1. List of compounds and biological activities
3145
Compd
Structure
HRV3C (IC50, mM) Papain (IC50, mM) Compd
Structure
HRV3C (IC50, mM) Papain (IC50, mM)
1
4
7
NT
19
10.5
NT
2
3
4
7
4.6
>150
>150
2.4
23
20
21
22
23
24
25
26
6.4
26
NT
NT
NT
NT
33
NT
>150
21
NT
76% (29 mM)
67% (120 mM)
24% (91 mM)
45% (18 mM)
14
16
17
8.1
8.2
40
NT
NT
1.4
124
46.5
18
>150
NT
27
6
NT
NT, not tested.
Structure–Activity Relationship
three substituents (cf. 23) led to the elimination of the
selectivity for HRV 3C-protease. The transposition of
the methoxy group from C-2 to C-3 caused significant
reduction in the activity (14 vs 17). Complete loss of the
activity wasob es rved when the methoxy group was
replaced with a hydroxy group (21 vs 22) and (14, 17 vs
18). The ortho-quinoneswere generally more potent
than corresponding para-quinones( 23 vs 24). The size
of the quinoneshaslittle effect on the protea se activity.
For example, complex tricyclic naphthoquinones us ch
asthy as none ( 1) and xanthomegnin (27) showed IC50
valuesof 47 and 6 mM, respectively. Substituted 1,4-
anthraquinones us ch asemodin ( 3) and methylemodin
(4) were inactive. In contrast the simple 9,10-phena-
thraquinone wasthe mots active compound of the
series and showed an IC50 value of 1.4 mM with a high
degree of selectivity. Chrysenequinone (26) showed a
modest activity (IC50 46.5 mM). The activity profiles
of 1,4-quinones(cf. 2) and 1,2-qunones(cf. 25) were
different. The former compound showed a time
dependent inhibition which wasnot affected by pre-
incubation reaction with DTT whereasthe latter is
2
-Methoxystypandrone (2) wasevaluated in HRV 3C-
2
a
protease HPLC assay. It showed an IC50 value of
.6 mM and the inhibition wastime dependent. In order
4
to measure the selectivity of this compound against
other cysteine proteases, it was evaluated against
papain. It showed 5-fold selectivity for HRV 3C-pro-
tease over papain (Table 1). Naphthoquinone without
acetyl substitution (cf. 7) was2-fold more potent than
the corresponding acetylated compound 2. Acetylation
of the phenolic group caused 2-fold reduction (cf. 14)
compared to the parent compound 2 and over 16-fold
reduction (cf. 16) compared to compound 7. Elimina-
tion of either phenolic (19) or methoxy (cf. 20) groups
from 7 caused 5- or 3-fold diminution of the HRV 3C-
protease activity. Elimination of both the methyl and
the hydroxy groupsof 7 caused more significant reduc-
tion in HRV 3C-protease activity (cf. 21) but the elim-
ination of methoxy group (cf. 23), the third substituent,
did not have any deleteriouseffect on the potency (26 vs
2
1 mM). However, it appearsthat the elimination of all

3146
S. B. Singh et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3143–3146
a time dependent inhibitor but inhibition ispotentiated
by reaction with DTT.
2. (a) Cordingley, M. G.; Register, R. B.; Callahan, P. L.;
Garsky, V. M.; Colonno, R. J. J. Virol. 1989, 63, 5037. (b)
Cordingley, M. G.; Callahan, P. L.; Garsky, V. M.; Sardana,
V. V.; Colonno, R. J. J. Biol. Chem. 1990, 265, 9062.
3. For a recent review, see: Dragovich, P. S. Exp. Opin. Ther.
Pat. 2001, 11, 177.
2
-Methoxystypandrone and other active compounds
reported in this letter contain reactive sites, which is
consistent with most of the inhibitors of HRV 3C-
protease reported in the literature that also possess
the Michael addition site including clinical candidate
4
5
. Graul, A.; Castaner, J. Drugs Future 2000, 25, 9.
. (a) Singh, S. B.; Cordingley, M. G.; Ball, R. G.; Smith, J. L.;
Dombrowski, A. W.; Goetz, M. A. Tetrahedron Lett. 1991, 32,
279. (b) For synthesis and absolute stereochemistry, see
Donner, C. D.; Gill, M. Tetrahedron Lett. 1999, 40, 3921.
. (a) Kimura, Y.; Kozawa, M.; Baba, K.; Hata, K. Planta
3
,4
AG-7088. The mode of action of these inhibitors are
through irreversible inactivation of the enzyme.
5
6
In summary, we have described the discovery, efficient
synthesis and HRV 3C-protease activity of 2-methoxy-
stypandrone and analogues. In addition, we also
described and compared the activities of ortho- versus
para-quinones. It appears that the mode of action of the
two classes of compounds is different.
Med. 1983, 48, 164. (b) Hughes, A. B.; Sargent, M. V. J.
Chem. Soc., Perkin Trans. 1 1989, 449.
7
2
4
8
5
9
. (a) Boisvert, L.; Brassard, P. Tetrahedron Lett. 1983, 24,
453. (b) Boisvert, L.; Brassard, P. J. Org. Chem. 1988, 53,
052.
. Ishida, A.; Mukaiyama, T. Bull. Chem. Soc. Japan 1977,
0, 1161.
. Experimental details. Aqueous solution of CAN (300 mg
in 0.4 mL H
solution of 13 (50 mg in 0.4 mL CH CN). After 3 h stirring at
2
O) wasadded drop wi es (in 1 h) to a CH
3
CN
Acknowledgements
3
rt the reaction mixture wasdiluted with 10 mL H
with CH Cl and chromatographed on silica gel column.
Elution with 49:1 CH Cl –MeOH afforded 35 mg of 14 asa
chromatographically homogeneousyellow powder.
0. 15: HREIMS (m/z) 338.1143 (calcd
C H O BF :338.1136), H NMR (400 MHz, CD Cl ) d, 2.60
2
O, extracted
The authorsare thankful to Mr. Greg Koch for pro-
viding papain activity data, Ms. Debbie Zink for mass
spectral analysis, Drs. Michael Goetz and George
Albers-Schonberg for their generous support.
2
2
2
2
1
for
1
1
6
17
5
2
2
2
(
3H, d, J=1.9 Hz), 2.79 (3H, s), 3.82 (3H, s), 4.02 (3H, s), 4.03
3H, s), 6.58 (1H, s), 7.26 (1H, m), C NMR (100 MHz,
1
3
(
CD Cl ) 25.1, 28.3, 56.7, 57.1, 61.6, 95.6, 109.3, 114.4, 117.3,
References and Notes
2
2
1
. (a) Gwaltney, J. M., Jr. In Viral Infection of Man: Epide-
134.4, 136.1, 136.7, 158.2, 160.7, 171.1 (t, J=2.2 Hz), 193.9 (t,
1
miology and Control, 2nd ed.; Evans, E. A., Ed.; Plenum: New
York, 1982; p 491. (b) Couch, R. B. In Fields Virology, 3rd
ed.; Fields, B. N., Knipe, D. M., Howley, P. M., Chanock, R.
M., Melnick, J. L., Monath, T. P., Roizman, B., Straus, S. E.,
Eds.; Lippincott-Raven: Philadelphia, 1996; Vol. 1, p 713.
2 2
J=1.8 Hz). 12: H NMR (250 MHz, CD Cl ) d, 2.34 (3H, d,
J=0.8 Hz), 2.56 (3H, s), 3.84 (3H, s), 3.97 (3H, s), 4.05 (3H, s),
6.60 (1H, s), 7.31 (1H, t, J=0.8 Hz), 9.78 (1H, br s).
11. Jung, M. E.; Lowe, J. A., III; Lyster, M. A.; Node, M.;
Pfluger, R. W.; Brown, R. W. Tetrahedron 1984, 40, 4751.