Investigation of the pharmacophore space of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) NTPase/helicase by dihydroxychromone derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.07.009

Aryl diketoacids have been identified as the first SARS-CoV NTPase/helicase inhibitors with a distinct pharmacophore featuring an arylmethyl group attached to a diketoacid. In order to search for the pharmacophore space around the diketoacid core, three c

Full text of DOI:10.1016/j.bmcl.2009.07.009

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4538–4541
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Investigation of the pharmacophore space of Severe Acute Respiratory
Syndrome coronavirus (SARS-CoV) NTPase/helicase by dihydroxychromone
derivatives
a,
Chaewoon Lee a, , Jin Moo Lee b, , Na-Ra Lee b, Dong-Eun Kim a, Yong-Joo Jeong b, , Youhoon Chong
*
*
a Department of Bioscience & Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea
b Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Aryl diketoacids have been identified as the first SARS-CoV NTPase/helicase inhibitors with a distinct
pharmacophore featuring an arylmethyl group attached to a diketoacid. In order to search for the phar-
macophore space around the diketoacid core, three classes of dihydroxychromone derivatives were pre-
pared. Based on SAR study, an extended feature of the pharmacophore model of SARS-CoV NTPase/
helicase was proposed which is constituted of a diketoacid core, a hydrophobic arylmethyl substituent,
and a free catechol unit.
Received 4 June 2009
Revised 1 July 2009
Accepted 3 July 2009
Available online 9 July 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
SARS (Severe Acute Respiratory Syndrome)
NTPase/helicase
Dihydroxychromone
Pharmacophore
In 2002, SARS (Severe Acute Respiratory Syndrome) caused by
coronavirus (SARS-CoV) quickly spread to nearly 30 countries lead-
ing to infection over 8000 people, and almost 800 deaths world-
wide.1 Although new cases of the infection have not been
reported since 2004, SARS remains as a global health threat due
to its high mortality and lack of therapeutic agents.2
with proven stability and safety,5 share the similar structural motif
to the diketoacid of an ADK: two phenolic hydroxyl groups and a
carbonyl oxygen serve together as an excellent mimic for the dike-
toacid functionality (Fig. 1). In addition, compared with an ADK
with substituents attached only on one side of the diketoacid, var-
ious functionalities can be introduced to both sides of the
dihydroxychromone core to allow extended investigation of the
pharmacophore space. Thus, we designed novel dihydroxychro-
mone derivatives with an arylmethyl functionality shown to play
a critical role in inhibitory activity of ADKs3 as well as a catechol
moiety known to be responsible for various biological activities
of flavonols6 on either side (2 and 3, Fig. 2) or both sides (4,
Fig. 2) of the dihydroxychromone scaffold. Herein, we present a
In our previous study,3 we have identified aryl diketoacids
(ADKs) as novel anti-SARS agents with selective inhibition
(IC50 = 5.4–13.6 lM) against duplex DNA-unwinding activity of
SARS-CoV NTPase/helicase without significant impact on the ATP-
ase activity. It is of particular interest that, among the SARS-CoV
NTPase/helicase inhibitors reported to date,3,4 ADK is the only
example with distinct structure–activity relationship to provide
an initial feature of the pharmacophore model composed of a dike-
toacid core with an appropriately positioned arylmethyl substitu-
ent (1, Fig. 1).
In this study, as a part of our ongoing efforts to delineate a com-
plete pharmacophore model via structural variation on ADK, we
designed several dihydroxychromone derivatives as a bioisostere
of ADK in which the diketoacid moiety of ADK with poor drug-like
property is replaced with a dihydroxychromone scaffold (Fig. 1).
Dihydroxychromones, a class of naturally-occurring flavonoids
* Corresponding authors. Tel.: +82 2 910 5454; fax: +82 2 910 4415 (Y.-J.J.); tel.:
+82 2 2049 6100; fax: +82 2 454 8217 (Y.C.).
E-mail addresses: jeongyj@kookmin.ac.kr (Y.-J. Jeong), chongy@konkuk.ac.kr (Y.
Chong).
These two authors contributed equally to this work.
Figure 1. Comparison of ADK and dihydroxychromone.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.009

C. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4538–4541
4539
Figure 2. Design of dihydroxychromone derivatives with mono- (2 and 3) and di-substituents (4) on the dihydroxychromone scaffold.
Scheme 1. Syntheses of 2-[N-(arylmethyl)methyl]-5,6-dihydroxy chromone derivatives (2a: R = 4-Cl, 2b: R = 3-Cl, 2c: R = 3-CN). Reagents and conditions: BnBr, K2CO3,
acetone, 60 °C; (b) AcCl, Pyr, 60 °C; (c) LiHMDS, THF, À78 °C; (d) SeO2, bromobenzene, 160 °C; (e) (R-Ph)CH2NH2, AcOH, MeOH, NaBH3CN, rt; (f) BBR3, CH2Cl2, rt.
novel pharmacophore model of SARS-CoV NTPase/helicase inhibi-
tors via syntheses and biological evaluation of three classes of
dihydroxychromone derivatives.
prepared by degradation of pentabenzylated quercetin9 (65%
yield, Scheme 2). Esterification of phenol 10 with piperonyloyl
chloride gave the corresponding ester. Cyclization of the ester
in the presence of K2CO3 and phase transfer catalyst,10 followed
by hydrogenolysis provided the flavonol 3b (72% yield for two
steps).
2-[N-(Arylmethylamino)methyl]-5,6-dihydroxychromones (2,
Fig. 2) superimposable to ADK were prepared starting from aceto-
phenone 5 (Scheme 1).7 Regioselective benzylation of 58 followed
by acetylation provided the fully protected acetophenone 7 (71%
yield for two steps), which underwent cyclization (48% yield) and
subsequent oxidation to give the aldehyde 9 in 91% yield. The de-
sired dihydroxychromone derivatives 2a–2c were prepared from
the intermediate 9 by reductive amination followed by deprotec-
tion with BBr3 in CH2Cl2 (31–38% yield).
Free or protected catechol was introduced at the other side
of the dihydrochromone to give flavonol derivatives (3, Scheme
2). The free catechol derivative [quercetin (3a)] was obtained
from commercial source and the 3,6-dihydroxychromone deriv-
ative with protected catechol functionality at 2 position (3b)
was synthesized starting from the phenol 10 which was
Synthesis of dihydroxychromones with substituents on both
sides (4) was accomplished by selective alkylation on 7-O position
of the flavonol (3a and 3b) (Scheme 3). Thus, peracetylation of 3a
(or 3b) followed by selective deacetylation of 7-OAc with thiophe-
nol and imidazole in NMP at 0 °C gave the 7-O-mono deprotected
flavonol 12a (or 12b).11 Treatment of 12a (or 12b) with substituted
benzyl bromide in acetone in the presence of K2CO3 at room tem-
perature, followed by methanolysis provided the desired com-
pounds 4a–4f in 25–29% yields.
The synthesized dihydroxychromone derivatives12 were tested
for their inhibitory activities against ATPase and duplex DNA-
unwinding activities of the helicase by phosphate release assay13

4540
C. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4538–4541
Scheme 2. Syntheses of 3b from commercially available 3a (quercetin). Reagents and conditions: (a) piperonyloly chloride, Pyr; (b) K2CO3, TBAB, toulene, 90 °C; (c) H2, Pd/C,
MeOH/CH2Cl2.
Scheme 3. Syntheses of dihydrochromone derivatives with substituents on both sides of the core diketoacid mimic. Reagents and conditions: Ac2O, Pyr; (b) PhSH, imidazole,
NMP, 0 °C, rt; (c) (R3-Ph)CH2Br, K2CO3, acetone, rt; (d) NH3/MeOH, rt.
and FRET-based assay,14 respectively. Cloning and purification of
the SARS-CoV helicase were performed as previously described.15
Like ADK analogues,3 dihydroxychromone derivatives showed
no significant inhibition against helicase ATPase activity. Only mar-
Interestingly, substitution of arylmethyl functionality on 7-O
position of 3b remarkably increased the activity of the resulting
compounds (4d–4f, Table 1). The inhibitory activity of quercetin
(3a) was also improved upon introduction of arylmethyl substitu-
ent at 7-O position (4a–4c, Table 1). It is noteworthy that flavonol
derivatives with free catechol moieties (4a–4c) are usually two to
three times more active than the protected catechol counterparts
(4d–4f). The synergistic effect of the two substituents attached to
the opposite side of the dihydrochromone core suggests the pres-
ence of two distinct binding sites on the target enzyme: a hydro-
ginal inhibition of the ATPase activity (20.9 and 25.4 lM) was ob-
served with two disubstituted dihydroxychromone derivatives (4a
and 4c, Table 1). Unexpectedly, compounds 2a–2c, superimposable
to the ADK scaffold inhibited neither ATPase nor duplex DNA-
unwinding activity of SARS-CoV ATPase/helicase. Presumably, the
high flexibility of (N-arylmethyl)methyl substituent of 2a–2c com-
pared with the ADK counterpart resulted in loss of inhibitory
activity.
phobic arylmethyl binding site and
a catechol binding site
capable of hydrogen bonding interaction.
A flavonol with protected catechol group (3b) also failed to
show any inhibition against ATPase/helicase. However, a flavonol
with free catechol (quercetin, 3a) selectively inhibited the duplex
On the basis of this study, we hypothesize an extended pharma-
cophore model (Fig. 3) of SARS-CoV NTPase/helicase inhibitors
composed of three key components including a diketoacid core, a
hydrophobic site and a free catechol moiety.
DNA-unwinding activity in micromolar range (IC50 = 8.1 lM, Table
1) to indicate the possible role of the free catechol moiety in the
binding interaction with the target enzyme presumably as a hydro-
gen bond donor.
In summary, in order to investigate the pharmacophore space
around the diketoacid core of SARS-CoV NTPase/helicase inhibitors,
three classes of dihydroxychromone derivatives were prepared in

C. Lee et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4538–4541
4541
Table 1
which two different substituents, arylmethyl and catechol, are at-
tached on opposite ends. The synthesized dihydroxychromones
showed selective inhibition against duplex DNA-unwinding activ-
ity of SARS-CoV NTPase/helicase. Moreover, the inhibitory activity
was enhanced by combination of the two spatially separated sub-
stituents, which indicates two different binding sites in the target
enzyme. Taken together, an extended feature of the pharmaco-
phore model was proposed which is constituted of a diketoacid
core, a hydrophobic arylmethyl substituent, and a free catechol
unit. Further structure–activity study around the proposed phar-
macophore model is warranted for discovery of more potent inhib-
itors of SARS-CoV NTPase/helicase.
IC50 values of flavonol derivatives against SARS-CoV helicase ATPase activity and
duplex DNA-unwinding activity
Acknowledgments
This work was supported by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare & Family
Affairs, Republic of Korea (A08-4628-AA2023-08N1-00010A), a
grant from ORP 11-30-68 (NIAS), and a grant from Biogreen 21
(Korea Ministry of Agriculture and Forestry). Y.-J. Jeong was sup-
ported by the Korea Research Foundation Grant funded by the Kor-
ean Government (KRF-2008-313-C00531) and the research
program 2009 of Kookmin University in Korea.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.009.
References and notes
1. (a) Peiris, J. S.; Lai, S. T.; Poon, L. L.; Guan, Y.; Yam, L. Y.; Lim, W.; Nicholls, J.;
Yee, W. K.; Yan, W. W.; Cheung, M. T.; Cheng, V. C.; Chan, K. H.; Tsang, D. N.;
Yung, R. W.; Ng, T. K. Lancet 2003, 361, 1319; (b) Drosten, C.; Gunther, S.;
Preiser, W.; van der Werf, S.; Brodt, H. R.; Becker, S.; Rabenau, H.; Panning, M.;
Kolesnikova, L.; Fouchier, R. A.; Berger, A.; Burguiere, A. M.; Cinatl, J.; Eickmann,
M.; Escriou, N. N. Engl. J. Med. 2003, 348, 1967.
2. http://www.who.int/csr/sars/en.
3. Lee, C.; Lee, J. M.; Lee, N. R.; Jin, B. S.; Jang, K. J.; Kim, D. E.; Jeong, Y. J.; Chong, Y.
Bioorg. Med. Chem. Lett. 2009, 19, 1636.
4. (a) Kliger, Y.; Levanon, E. Y.; Gerber, D. Drug Discovery Today 2005, 10, 345; (b)
Yang, N.; Tanner, J. A.; Wang, Z.; Huang, J. D.; Zheng, B. J.; Zhu, N.; Sun, H. Chem.
Commun. 2007, 4413; (c) Kesel, A. J. Anti-Infective Agents Med. Chem. 2006,
5, 161.
5. Spedding, G.; Ratty, A.; Middleton, E., Jr. Antiviral Res. 1989, 12, 99.
6. (a) Morel, I.; Lescoat, G.; Cogrel, P.; Sergent, O.; Pasdeloup, N.; Brissot, P.;
Cillard, P.; Cillard, J. Biochem. Pharmacol. 1993, 4, 13; (b) Van Acker, S. A. B. E.;
Van den Berg, D. J.; Tromp, M. N. J. L.; Griffoen, D. H.; van Bennekom, W. P.; van
der Vijjgh, W. J. F.; Bast, A. Free Radical Biol. Med. 1996, 20, 331; (c) Chiang, L. C.;
Chiang, W.; Liu, M. C.; Lin, C. C. J. Antimicrob. Chmother. 2003, 52, 194; (d)
Formica, J. V.; Regelson, W. Food Chem. Toxicol. 1995, 33, 1061.
7. Prakash, O.; Pundeer, R.; Kaur, H. Synthesis 2003, 18, 2768.
8. Wollenweber, E.; Iinuma, M.; Tanaka, T.; Mizuno, M. Phytochemistry 1990, 29,
633.
Compds
IC50
(l
M)
ATPase
Duplex DNA-unwinding
2a
2b
2c
3a
3b
4a
4b
4c
4d
4e
4f
>50
>50
>50
>50
>50
20.9 0.5
>50
25.4 1.5
>50
>50
>50
>50
>50
8.1 0.3
>50
4.1 0.3
5.2 0.4
2.7 0.1
9.3 0.4
15.4 0.8
8.1 0.3
42.9 5.4
9. Hauteville, M.; Chadenson, M.; Chopin, J. Bull. Soc. Chim. Fr. 1979, 11, 124.
10. Caldwell, S. T.; Petersson, H. M.; Farrugia, L. J.; Mullen, W.; Crozier, A.; Hartley,
R. C. Tetrahedron 2006, 62, 7257.
11. Li, M.; Han, X.; Yu, B. J. Org. Chem. 2003, 68, 6842.
12. See Supplementary data for experimental and characterization data for the
final compounds (2a–2c, 3b, and 4a–4f) as well as previously unreported
intermediates.
13. (a) Baykov, A. A.; Evtushenko, O. A.; Avaeva, S. M. Anal. Biochem. 1988, 171, 266;
(b) Wardell, A. D.; Errington, W.; Ciaramella, G.; Merson, J.; McGarvey, M. J. J.
Gen. Virol. 1999, 80, 701; (c) Martin, G. R.; Yvette, M. N.; Chrisotomos, P.;
Laurence, H. P.; Paul, W.; Wynne, A. Anal. Biochem. 2004, 327, 176.
14. Jang, K. J.; Lee, N. R.; Yeo, W. S.; Jeong, Y. J.; Kim, D. E. Biochem. Biophys. Res.
Commun. 2008, 366, 738.
15. Yang, N.; Tanner, J. A.; Wang, Z.; Huang, J. D.; Zheng, B. J.; Zhu, N.; Zun, H. Chem.
Commun. 2007, 4413.
Figure 3. The proposed pharmacophore model of SARS-CoV helicase inhibitors.