Synthesis and in vitro antifungal activities of new 3-substituted benzopyrone derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.09.072

A series of new benzopyrone compounds were designed and synthesized and their antifungal activities in vitro were evaluated. The results showed that the benzopyrone derivatives with short terminal alkyl chain exhibited potent antifungal activity, which represent a novel class of promising leads for the development of novel non-azole antifungal agents. Compound 5j is the most potent one with MIC₈₀ value 1.5 μg/mL against Trichophyton rubrum. Flexible molecular docking was used to analyze the structure-activity relationships (SARs) of the compounds. The designed compounds interact with CA-CYP51 through hydrophobic and van der Waals interactions. Copyright

Full text of DOI:10.1016/j.bmcl.2010.09.072

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7106–7109
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro antifungal activities of new 3-substituted
benzopyrone derivatives
Zhiliang Lv, Chunquan Sheng, Yikai Zhang, Tiantian Wang, Jilu Feng, Hailing Sun, Hanyu Zhong,
⇑
Mingfeng Zhang, Huan Chen, Ke Li
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2010
Revised 30 August 2010
Accepted 14 September 2010
Available online 17 September 2010
A series of new benzopyrone compounds were designed and synthesized and their antifungal activities
in vitro were evaluated. The results showed that the benzopyrone derivatives with short terminal alkyl
chain exhibited potent antifungal activity, which represent a novel class of promising leads for the devel-
opment of novel non-azole antifungal agents. Compound 5j is the most potent one with MIC80 value
1.5 lg/mL against Trichophyton rubrum. Flexible molecular docking was used to analyze the structure–
activity relationships (SARs) of the compounds. The designed compounds interact with CA-CYP51
through hydrophobic and van der Waals interactions.
Keywords:
Antifungal activity
Benzopyrone derivative
Synthesis
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Lanosterol 14a-demethylase (CYP51)
Molecular docking
Over the past decades, opportunistic infection caused by fungus
has displayed dramatically rising tendency, especially among the
patients undergoing anticancer chemotherapy or organ transplants
and patients with AIDS.1,2 Clinically, antifungal agents can be
classified as azoles (Fig. 1), polyenes, echinocandins and allyl-
amines.3 Azoles, possessing high therapeutic index, were broadly
used as the first-line drugs in clinic for the treatment of the fungal
infections. Unfortunately, the extensive use and prolonged therapy
with azole antifungal agents have led to severe resistance, which
significantly limited the utilization of them.4 Therefore, the devel-
opment of novel antifungal agents has been constantly required
clinically.
heme of a lot of host cytochrome P450 enzymes, particularly mam-
malian CYP3A4.7–10 Previous studies have indicated that azole
function group is not only an important pharmacophore but also
a key toxicophore for hepatotoxicity. We believe that it is impor-
tant to design novel non-azole antifungal compounds with good
selectivity to the binding site of fungal CYP51 and without coordi-
nation binding to the heme iron atom of CYP51.
In our previous study, we have reported a series of non-azole
antifungal lead compounds containing the benzopyran scaffold
(Fig. 2).11 The binding study showed that these benzopyran deriv-
atives could interact with CYP51 without binding with heme. Flex-
ible molecular docking model revealed that the long alkyl side
chain interacted with the hydrophobic S3 subsite in the active site,
which represented a narrow and hydrophobic cleft. The 7-phenolic
hydroxyl group formed an hydrogen bond with the side chain
hydroxyl group of catalytic residue T311 in the S1 subsite, which
represents the hydrophilic hydrogen bond binding site. The benz-
opyran scaffold was located in S2 subsite, which is representing
the core hydrophobic area.11,12 In order to extend the structure–
activity relationships (SARs) of the above antifungal leads, a series
of benzopyrone compounds were designed. We first replaced the
benzopyran scaffold by benzopyrone in order to investigate the
effect of core scaffold on the antifungal activity. The methoxyl
group was retained and long lipophilic alkyl and aromatic side
chains were introduced into position 3 of benzopyrone ring, which
can interact with the hydrophobic S3 subsite. As a result, a series of
benzopyrone derivatives with long alkyl and aromatic ester side
chains were synthesized. The design rationale was supported by
Lanosterol 14
lyzes the oxidative removal of the 14
to give
a
-demethylase (CYP51), the enzyme that cata-
-methyl group of lanosterol
-
14,15desaturated intermediates in ergosterol biosynthesis,
a
D
is a primary target for the development of antifungal drugs.5 Azoles
inhibit CYP51 by a mechanism in which the heterocyclic nitrogen
atom (N-3 of imidazole and N-4 of triazole) binds to the heme iron
atom in the substrate binding site of the enzyme. The plasma
membrane is made more vulnerable to be damaged by the accu-
mulation of precursor 14a-methylated sterols, which results in
an inhibition of the growth of fungi.6 However, CYP51 is a member
of the cytochrome P450 superfamily, which exists not only in fungi
but also in mammals. Cases of fatal hepatotoxicity have been
reported because of the ability of azoles to coordinate with the
⇑
Corresponding author. Tel.: +86 21 81871237.
E-mail address: proflike@sina.com (K. Li).
0960-894X/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.072

Z. Lv et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7106–7109
7107
Figure 1. Chemical structures of azole antifungal agents.
Figure 2. Lead compound and 5j.
their antifungal activities in vitro as well as the docking model
with CYP51.
The chemical synthesis of the benzopyrone derivatives was out-
lined in Scheme 1. As a key intermediate of our designed com-
defined by the National Committee for Clinical Laboratory Stan-
dards (NCCLS).19 Candida albicans and Cryptococcus neoformans
are ATCC standard strains, and the others are clinic isolates. Fluco-
nazole (FLZ) was used as the positive control. The MIC values are
listed in Table 1. The results indicated that half of the synthesized
compounds exhibited moderate antifungal activities against all the
five tested human pathogenic fungus. Compounds 4, 5a–5f, and
5m–5o didn’t show antifungal activity or just displayed marginal
activity against some of the tested pathogenic fungus (such as
5b, 5q, and 5r). Compounds 5g–5l exhibited broad-spectrum anti-
fungal activities. However, their antifungal activities were lower
than that of the positive control FCZ. Among the synthesized com-
pounds, compound 5j was the most potent one and exhibited mod-
erate antifungal activity against the five tested fungi. The MIC80
pounds, compound
3 was synthesized through a reported
procedure via four steps from paenol.13,14 Coupling of intermediate
3 with propynol by the Sonogashira reaction15–17 afforded the key
intermediate 4 with good yield. Intermediate 4 reacted with
substituted acyl chlorides in dry DMF to afford the target
compounds 5a–5r. Their chemical structures were confirmed by
IR, ESI-MS, and NMR spectroscopic analysis18 and the purity of
all the new compounds were determined by HPLC with two quite
different systems (see Supplementary data).
In vitro antifungal activity was measured by means of the min-
imal inhibitory concentrations (MIC) using the serial dilution
method in 96-well microtest plates according to the methods
value of compound 5j against Trichophyton rubrum was 1.5
lg/
mL, which was 30-fold more potent than that of compound 4. From
Scheme 1. Reagents and conditions: (a) Na, ethyl formate, ice bath; (b) HCl, HOAc, reflux; (c) piperidine, MeOH, reflux; (d) DCM, I2, rt; (e) CuI, PdCl2(Ph3P)2, DMF, rt; (f) DMAP,
DMF, substituted acyl chlorides.

7108
Z. Lv et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7106–7109
Table 1
In vitro antifungal activity of the target compoundsa
Compounds
R
C. alb.
C. neo.
C. par.
T. rub.
M. gyp.
4
—
>100
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
25
>100
>100
50
>100
>100
50
>100
>100
>100
50
12.5
25
25
5a
5b
5c
5d
5e
5f
5g
5h
5i
4-Nitriophenyl
3,5-Dinitrophenyl
Phenyl
>100
>100
>100
>100
6.25
12.5
3.125
1.5
Benzyl
4-Chlorobenzyl
2-Chlorobenzyl
Ethyl
Propyl
Isopropyl
12.5
50
25
50
50
25
50
12.5
25
5j
Butyl
25
5k
5l
Isobutyl
n-Pentyl
Cyclohexyl
n-Hexyl
n-Heptyl
Isoheptyl
2,2,3,3-Tetramethylcyclopropyl
Methyl
>100
>100
>100
>100
>100
>100
>100
>100
0.25
50
50
50
6.25
25
25
>100
>100
>100
>100
50
>100
>100
1
12.5
>100
>100
>100
12.5
12.5
25
5m
5n
5o
5p
5q
5r
>100
>100
>100
25
>100
>100
32
>100
>100
>100
12.5
>100
>100
0.25
FCZ
—
0.5
a
Abbreviations: C. alb.: Candida albicans; C. neo.: Cryptococcus neoformans; C. par.: Candida parapsilosis; T. rub.: Trichophyton rubrum; M. gyp.: Microsporum gypseum.
the results of antifungal activity, the preliminary SARs were
obtained. Compound 4 did not show any antifungal activity. The
introduction of substituted phenyl and benzyl groups on the termi-
nal hydroxyl, marginal activity was found. For example, com-
pounds 5b and 5f were active at 50 lg/mL against T. rubrum and
Microsporum gypseum. The introduction of alkyl groups on the hy-
droxyl resulted in obvious improvement of the activity, which sug-
gested that the alkyl group was necessary for its binding affinity
with CYP51. Moreover, the length of the alkyl group attached with
the terminal hydroxyl played an important role in the antifungal
activity. Compounds 5g–5l containing the alkyl group within five
carbon atoms exhibited the best activity among the target com-
pounds. Compound 5j with a 4 carbon alkyl substituent showed
the best antifungal activity among the synthesized compounds.
On the contrary, the loss of antifungal activity was observed for
the compounds with the alkyl groups which was longer than five
carbon atoms (such as the compounds 5m–5r). For compound
5p, the propyl branch of the isoheptyl could contribute to the anti-
fungal activity a lot. For the substituted benzopyrone scaffolds rep-
resent a novel non-azole antifungal agent, it is necessary for us to
make further modification in order to obtain more information
about SARs.
The binding studies were carried out by the flexible docking
using the affinity module within the Insight II software package.20
Figure 3 shows the docking conformation of compound 5j in the
active site of CYP51 of C. albicans (CA-CYP51). Unlike azoles, the
benzopyrone ring of compound 5j did not coordinate with the
heme group. Instead, its conformation is parallel to the heme group
and stack with it. In addition, the benzopyrone heterocycle ring
was located in S2 hydrophobic pocket interacting with the sur-
rounding residues lined with Leu376, Val509, and Ile379. The alkyl
side chain of the compound was oriented into the S3 pocket and
formed hydrophobic and van der Waals interactions with Phe126,
Ala114, Phe145, and Lys143. Due to the steric limitation of this
pocket, benzyl, phenyl and long alkyl substituent groups would
form stereo-collision with the active site. The docking conforma-
tion could explain activity tendency of this series of compounds,
though the methoxyl group did not form H bonds with the residue
of the subsites effectively. In the future study, various alkyl side
chains will be introduced into position 3 of benzopyrone and some
hydrophilic groups will be employed to substitute the methoxyl
group in order to obtain more SAR information.
Figure 3. The docking conformation of compound 5j in the active site of CA-CYP51.
In summary, a series of new benzopyrone derivatives were de-
signed and synthesized. In vitro antifungal activity assay indicated
that the target compounds had potent antifungal activity against
the five tested human pathogenic funguses. The results indicated
that the alkyl chain with 2–5 carbon atoms exhibited better effec-
tive than the others. The mode of action of benzopyrone molecules
showed that the affinity of the lead molecules for CA-CYP51 was
mainly attributed to their non-bond interactions with the residues
of CYP51 without coordinating with the heme. The novel structure
reported here represented a new scaffold type for the development
of novel non-azole antifungal agents.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 30973640) and Shanghai Rising-Star
Program (Grant No. 09QA1407000).

Z. Lv et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7106–7109
7109
13. Lv, Z.; Sheng, C.; Wang, T.; Zhang, Y.; Liu, J.; Feng, J.; Sun, H.; Zhong, H.; Niu, C.;
Li, K. J. Med. Chem. 2009, 53, 660.
Supplementary data
14. Dong, H.; Li, K.; Zheng, C.; Liu, J.; Lü, Z.; Li, T.; Liu, C. Acta Chim. Sinica 2009, 67,
819.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.072.
15. Miller, M. W.; Johnson, C. R. J. Org. Chem. 1997, 62, 1582.
16. Elangovan, A.; Wang, Y.-H.; Ho, T.-I. Org. Lett. 2003, 5, 1841.
17. Garcia, D.; Cuadro, A. M.; Alvarez-Builla, J.; Vaquero, J. J. Org. Lett. 2004, 6, 4175.
18. Representative analytical data for compound 4: 1H NMR (300 MHz, CDCl3) d:
3.914 (3H, s, CH3–O), 4.518 (2H, d, J = 6 Hz, CH2–O), 6.843 (1H, d, J = 2.4 Hz, Ar-
H), 6.981 (1H, dd, J1 = 2.4 Hz, J2 = 9 Hz, Ar-H), 8.099 (1H, s, C@CH–O), 8.145 (1H,
d, J = 9 Hz, Ar-H), 10.302 (1H, d, J = 6 Hz, OH); 13C NMR (300 MHz, CDCl3) d:
52.08, 55.89, 89.40, 100.44, 110.40, 115.03, 117.39, 127.68, 157.68, 158.36,
164.36, 174.58; LC–MS, m/z: 231.1 (M+1)+. Compound 5b: 1H NMR (300 MHz,
CDCl3) d: 3.919 (s, 3H, CH3–O), 5.314 (2H, s, CH2–O), 6.854 (1H, d, J = 2.4 Hz, Ar-
H), 6.988 (1H, dd, J1 = 2.4 Hz, J2 = 9 Hz, Ar-H), 8.128 (1H, s, C@CH–O), 8.158 (1H,
d, J = 1.8 Hz, Ar-H), 9.217 (2H, d, J = 1.8 Hz, Ar-H), 9.247 (1H, d, J = 1.8 Hz, Ar-H);
13C NMR (300 MHz, CDCl3) d: 54.98, 55.92, 78.67, 87.28, 100.49, 109.83,
115.22, 117.25, 122.67, 127.25, 129.65, 133.17, 148.67, 157.67, 158.82, 161.90,
164.50, 174.44; LC–MS, m/z: 425.21 (M+1)+. Compound 5j: 1H NMR (300 MHz,
CDCl3) d: 0.899 (3H, t, J = 7.5 Hz, CH3CH2), 1.328 (2H, m, CH3CH2CH2), 1.593
(2H, m, CH2CH2), 2.354 (2H, t, J = 7.5 Hz, COCH2CH2), 3.911 (3H, s, CH3–O),
4.935 (2H, s, CH2–O), 6.839 (1H, d, J = 2.4 Hz, Ar-H), 6.977 (1H, dd, J1 = 2.4 Hz,
J2 = 9 Hz, Ar-H), 8.107 (1H, s, C@CH–O), 8.142 (1H, d, J = 9 Hz, Ar-H); LC–MS, m/
z: 315.12 (M+1)+.
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