Synthesis and biological evaluation of novel bisheterocycle-containing compounds as potential anti-influenza virus agents

Article abstract of DOI:10.1016/j.bmcl.2005.08.046

A series of novel 4,2-bisheterocycle tandem derivatives consisting of a methyloxazole and thiazole subunit were synthesized. Many compounds were found to inhibit human influenza A virus. Several analogues exhibited moderate biological activity and could s

Full text of DOI:10.1016/j.bmcl.2005.08.046

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5284–5287
Synthesis and biological evaluation of novel
bisheterocycle-containing compounds as potential
anti-influenza virus agents
Wen-Long Wang,a De-Yong Yao,b Min Gu,a Min-Zhi Fan,a Jing-Ya Li,a
Ya-Cheng Xingb and Fa-Jun Nana,*
aChinese National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes of Biological
Sciences, Graduate School of Chinese Academy of Sciences, Chinese Academy of Sciences,
189 Guo Shou Jing Road, Shanghai 201203, China
bCollege of Chemical Engineering, Qingdao University, 308 Ningxia Road, Qingdao, Shandong 266071, China
Received 22 June 2005; revised 8 August 2005; accepted 12 August 2005
Available online 23 September 2005
Abstract—A series of novel 4,2-bisheterocycle tandem derivatives consisting of a methyloxazole and thiazole subunit were synthe-
sized. Many compounds were found to inhibit human influenza A virus. Several analogues exhibited moderate biological activity
and could serve as leads for further optimizations for antivirus research.
ꢀ 2005 Elsevier Ltd. All rights reserved.
During the course of a search for bioactive metabolites
from marine microorganisms, a variety of chemically
interesting and biologically significant secondary metab-
olites containing heterocyclic tandem pairs consisting of
oxazole and thiazole subunits were reported.1 These
products display a wide range of biological activities,
such as antifungal, antileukemic, antimitotic, antibacte-
rial, and gene-regulating properties, and inspired strong
interest among many medicinal and synthetic chemists.2
It was also reported that the heterocyclic tandem pair
was possibly responsible for the biological activity,3
such as that seen with the three consecutive oxazoles
in Kabiramide C, which interfere with actin filament
dynamics by binding to subdomains 1 and 3 of G-actin
in an irreversible manner;4 and the antibiotic Bleomycin,
which interacts with DNA by binding of its bithiazole
moiety to the nucleic bases in a mode that is partially
intercalative, allowing penetration of the positively
charged moieties distal to the bithiazole into the major
groove.5 Therefore, heterocyclic tandem pairs may be
useful scaffolds in combinatorial libraries for new lead
discoveries. Leucamide A is a bioactive cyclic heptapep-
tide from the Australian marine sponge Leucetta micro-
raphis, containing a unique mixed 4,2-bisheterocycle
tandem pair consisting of a methyloxazole and a thia-
zole subunit, and which was found to be moderately
cytotoxic toward several tumor cell lines.6
Although Leucamide A itself does not show any antivi-
ral activity, the unique mixed 4,2-bisheterocycle tandem
pair in Leucamide A provides us with a useful scaffold
for searching for potentially therapeutic compounds,
especially antiviral agents, because the heterocyclic
tandem pair may provide bioactivity through specific
interactions with DNA/RNA. Following our first total
synthesis of Leucamide A,7 we constructed a library of
4,2-bisheterocycle tandem derivatives consisting of a
methyloxazole and a thiazole subunit. Here, we report
on the design and synthesis of a library of novel
compounds containing 4,2-bisheterocycle tandem pairs
and their inhibitory effects on influenza A virus in vitro
and a preliminary study of their structure–activity
relationships (Fig. 1).
Two series of compounds, 2a–2n and 3a–3e, were
designed with various substituents, as shown in
Figure 1. Synthesis of the compounds was as shown in
Scheme 1. Compound 4 was synthesized as a key interme-
diate for the combinatorial generation of 2a–2n and
Keywords: Synthesis; Bisheterocycle-containing compounds; Anti-
influenza virus.
*
Corresponding author. Tel./fax: +86 21 50800954; e-mail:
fjnan@mail.shcnc.ac.cn
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.046

W.-L. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5284–5287
5285
Figure 1. A focused library of 4,2-bisheterocycle tandem derivatives consisting of a methyloxazole and thiazole subunit.
Scheme 1. Synthesis of 4,2-bisheterocycle tandem derivatives 2a–2n and 3a–3e.
3a–3e. Therefore, the initial goal was to produce gram
quantities of 4 and 67 suitable for more in-depth in vitro
studies with the flexibility to produce analogues for explo-
ration of structure–activity relationships. Compounds
2a–2n were prepared as outlined in Scheme 1. The N-
Boc-protecting group of 4 was removed using trifluoro-
acetic acid (TFA) and the resulting amine 5 was treated
with acyl chloride to afford 2a–2e and 2l (Method A)8 or
reacted smoothly with the corresponding acid to give
2g–2k (Method B)8 or 2f (Method C).8 Compounds 3a–
3e were obtained by the method shown in Scheme 1. Com-
pound 6 was transformed into compound 7 by deprotec-
tion of the N-Boc group with CF3COOH–CH2Cl2,
followed by amidation with acyl chloride to afford 3a–
3c (Method A) or with the corresponding acid to give
3d–3e (Method B). Saponification of 2j and 2l with lithi-
um hydroxide in aqueous methanol gave the correspond-
ing carboxylic acids, 2n and 2m, respectively.
Acid derivatives (2m, 2n) were about twofold more ac-
tive than corresponding methyl ester derivatives (2f,
2k). These results suggested that the hydrophilic effects
of a carboxyl group might lead to enhancement of
activity, although no definitive explanations for these
observations have yet been obtained. Among com-
pounds 2a–2d bearing various R1 substituents on NH,
the compounds with the large side chain (cyclohexane-
carbonyl group) showed better inhibitory activity than
those with small ones, and these results indicated that
steric factors influence inhibitory activity. Increasing
side-chain size from hydrogen (5) and propionyl (2a) to
cyclobutanecarbonyl (2b) and large groups (2c, 2d) has
a clearly advantageous effect on antiviral activity. A
twofold increase in antiviral activity was observed for
2-methoxy-benzoyl derivatives (2f) compared to 3-fluo-
ro-benzoyl derivatives (2e), underscoring the importance
of having an electron-donating group on the phenyl.
A series of derivatives were evaluated for their ability to
inhibit influenza A virus H3N2 (A3 China/15/90) repli-
cation in Madin–Darby canine kidney (MDCK) cells.9
The results are summarized in Table 1. Several com-
pounds (2i, 2l–2n) showed moderate activity against
influenza A virus, with IC50 values of 29–37 lg/mL.
Among compounds 2g–2l, containing unsaturated ali-
phatic carbonyl substituents on the NH, the 4-methyl-
pent-3-enoyl derivative (2g) exhibited low activity
against influenza
A virus, with IC50 values of
192.45 lg/mL. With additional methylene spacers be-
tween the double bond and carbonyl group, the activity

5286
W.-L. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5284–5287
Table 1. Anti-influenza virus A activity and cytotoxicity of 4,2-
bisheterocycle tandem derivatives in MDCK cellsa
Table 1 (continued)
b
c
Compound
R1
R2
CC50
(lg/mL)
IC50
(lg/mL)
O
COOR2
O
O
O
N
H
O
3c
Me
577.35
77.04
N
N
F
N
COOR2
N
N
2a-2n
S
3a-3e
S
NHR1
NHR1
3d
3e
Me
Me
577.35
577.35
44.48
O
O
Compound
R1
R2
CC50
(lg/mL)
IC50
(lg/mL)
b
c
111.11
O
Me
Me
577.35
500
258.68
5
H
Me
577.35
258.69
2a
Leucamide A
Ribavirin
NA
>2000
NA
3.73
O
111.11
a Abbreviations and strains used: MDCK, Madin–Darby canine
kidney cells, influenza A H3N2 viruses (A3 China/15/90).
b Concentrations that cause microscopically detectable toxicity in
virus-infected cultures.
c Concentrations required to reduce virus-induced CPE in MDCK cells
by 50%.
2b
O
2c
2d
Me
Me
577.35
144.34
97.81
43.58
O
of 2i increased approximately sixfold. Deletion of two
terminal methyl groups from 2i, to give the correspond-
ing compound 2h, resulted in an eightfold decrease in
antiviral activity. Restricting the unsaturated alkyl chain
within a ring (2j, 2k) led to a threefold increase of anti-
viral activity when compared with the straight chain
analogue (2g). Compound 2l showed similar potency
when compared to compound 2i.
O
F
2e
Me
>1000
>86.23
OMe O
2f
2g
2h
2i
Me
Me
Me
Me
Me
Me
Me
333.33
>1000
577.35
577.35
231.12
333.33
231.12
47.72
192.45
258.69
37.03
68.71
64.15
37.03
O
O
In the case of the bisheterocycle tandem derivatives
bearing one more oxazole unit (3a–3e), 3b–3d were more
potent than the propionyl derivative (3a). These results
also indicated that introduction of a big hydrophobic
group might lead to enhancement of activity. Com-
pounds 3a–3e showed similar activity to their corre-
sponding analogues 2a, 2d, 2e, 2j, and 2g. These
results suggest that addition of an oxazole ring to the
bisheterocycle tandem pair made no difference to the
potency of the antiviral activity.
O
2j
O
O
The natural product Leucamide A was inactive against
influenza A virus, indicating that the restricted ring of
the 4,2-bisheterocycle tandem pair dramatically reduces
antiviral activity. Conversely, open chain 4,2-bishetero-
cycle tandem derivatives showed biological activities,
which may imply an advantage of a rather flexible side
chain, accommodating favorable conformation(s) for
bioactivity. In addition, all compounds showed no activ-
ity against influenza B virus. Most compounds showed
low cytotoxicity with the exception of 2d and 3b, which
nonetheless still exhibited a reasonable selectivity index
(CC50/IC50).
2k
2l
O
OMe O
2m
H
333.33
29.01
2n
3a
3b
H
480.75
577.35
144.34
29.01
160.25
57.78
O
These compounds containing
a 4,2-bisheterocycle
tandem pair were moderately effective against human
influenza A and may give us an opportunity to attain
novel potent anti-influenza agents to overcome drug-
resistant mutants. However, the molecular target of
these 4,2-bisheterocycle tandem derivatives is unknown
at present.
O
Me
Me
O

W.-L. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5284–5287
7. Wang, W.; Nan, F. J. Org. Chem. 2003, 68, 1636.
5287
In conclusion, by a discovery process inspired by a nat-
ural product, we have uncovered a novel and moderate
line of potential antiviral agents. These results suggest
that the 4,2-bisheterocycle tandem pair probably plays
a major role in the potent antiviral activity. These com-
pounds could certainly serve as leads for the develop-
ment of de novo antiviral agents. Further studies on
their structure–activity relationships, optimization of
these compounds, and identification of the molecular
targets of these compounds, as well as exploration for
new bisheterocycle tandem pairs, are actively underway
in our laboratory.
8. Typical procedure for preparation of compounds 2 and 3.
Method A: To a solution of amine (0.1 mmol) in 3 mL of a
1:1 EtOAc/H2O mixture was added excess NaHCO3
(160 mg, 1.9 mmol). The appropriate acid chloride
(0.15 mmol) was added at 0 ꢁC. The mixture was stirred
for 2–24 h with gradual warming to room temperature. The
reaction mixture was diluted with EtOAc (5 mL) and H2O
(2 mL). The aqueous phase was extracted with EtOAc. The
combined organic phases were then processed in the usual
way and chromatographed to yield the desired products.
Method B: To a stirred solution of appropriate acid
(0.1 mmol) in dry CH2Cl2 (2 mL) under N2 at 0 ꢁC were
added N-methylmorpholine (NMM) (0.13 mmol) and
isobutyl chloroformate (0.11 mmol). After 10 min, the
solution of amine in dry CH2Cl2 (2 mL) was added, and
then the solution was stirred for 1–24 h with gradual
warming to ambient temperature. The mixture was parti-
tioned between H2O (20 mL) and EtOAc (20 mL). The
phases were separated and aqueous phase was extracted
with EtOAc (4·10 mL). The combined organic phases were
then processed in the usual way to yield the desired
Acknowledgments
The Major State Hi-tech Research and Development
Program (Grant 2001AA234011), the Chinese Academy
of Sciences, and Shanghai Commission of Science and
Technology (Grant 02QB14013) are appreciated for
their financial support.
˚
products. Method C: To 5 (24 mg, 0.08 mmol) and 4 A
molecular sieves in DMF (1 mL) at À10 ꢁC were added
HOBT (27.0 mg, 0.2 mmol) and 2-methoxybenzoic acid
(13.0 mg, 0.088 mmol), and the resulting mixture was
stirred at À10 ꢁC for 20 min, then EDCI (16.0 mg,
0.088 mmol) was added and the mixture was stirred for
2 h with gradual warming to room temperature. The
reaction mixture was diluted with EtOAc (50 mL) and
H2O (20 mL). The aqueous phase was extracted with
EtOAc (4·20 mL). The combined organic phases were then
processed in the usual way and chromatographed to yield 2f
(29 mg, 86%).
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9. MDCK cells were grown as specified in EagleÕs minimum
essential medium with 10% heat-inactivated fetal bovine
serum (FBS) plus antibiotics (penicillin, 100 U/mL; strep-
tomycin, 100 U/mL). Influenza A H3N2 viruses (A3 China/
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old embryonated eggs. Virus titers were determined by
hemagglutinin titration, according to standard procedures.
Confluent MDCK monolayers were infected with Influenza
A viruses for 2 h at 37 ꢁC, after which the viral inoculum
was removed and cells were treated with different concen-
trations of compound. When CPE result of the viral control
group reached 4+, the result of compound treated group
was observed. The dilution that gives 50% cytopathic effect
was determined by the interpolating procedure of Reed and
Muench.