A novel class of potent influenza virus inhibitors: Polysubstituted acylthiourea and its fused heterocycle derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.09.033

A series of polysubstituted and fused heterocycle derivatives of acylthiourea was prepared and the biological activity against influenza virus was evaluated. Of the analogues that demonstrated IC₅₀s < 0.1 μM, acylthiourea derivatives 16 and 50 were further investigated as candidates with the most potential for future development. The SAR of these compounds are discussed and they represent a novel class of highly potent and selective inhibitors of influenza virus.

Full text of DOI:10.1016/j.bmcl.2005.09.033

Bioorganic & Medicinal Chemistry Letters 16 (2006) 162–166
A novel class of potent influenza virus inhibitors: Polysubstituted
acylthiourea and its fused heterocycle derivatives
Chuanwen Sun,a,* Hai Huang,b Meiqing Feng,b Xunlong Shi,b
Xiaodong Zhanga and Pei Zhoub,*
aBio-X Life Science Research Center, Department of Life Science and Technology,
Shanghai Jiaotong University, Shanghai 200030, China
bDepartment of Biosynthetic Drugs, School of Pharmacy, Fudan University, Shanghai 200032, China
Received 8 July 2005; revised 6 September 2005; accepted 12 September 2005
Available online 10 October 2005
Abstract—A series of polysubstituted and fused heterocycle derivatives of acylthiourea was prepared and the biological activity
against influenza virus was evaluated. Of the analogues that demonstrated IC50s < 0.1 lM, acylthiourea derivatives 16 and 50 were
further investigated as candidates with the most potential for future development. The SAR of these compounds are discussed and
they represent a novel class of highly potent and selective inhibitors of influenza virus.
Ó 2005 Published by Elsevier Ltd.
Influenza virus is the contagious etiologic agent that
causes an acute respiratory infection followed by sec-
ondary bacterial infections, hence it has always been a
major threat to human health worldwide. Each year in
the USA alone, influenza epidemics cause morbidity
and mortality. More than 200,000 patients are admitted
to hospitals because of influenza and there are approxi-
mately 36,000 influenza-related deaths.
evaluations of polysubstituted and fused heterocycle
derivatives of acylthioureas for their anti-viral activity.
In our early attempts to screen for activity against differ-
ent viruses, acylthiourea 1 (Fig. 1) was revealed as a
weak inhibitor of influenza virus. As part of the pro-
gram to maximize this activity and to identify more
effective inhibitors of influenza virus, we decided to com-
bine these two parts into one molecule and investigate
the resulting anti-viral activity. A new class of polysub-
stituted acylthioureas and their fused heterocycle deriv-
atives (designated PAFHs, series I–XII) were prepared.
A highly specific anti-influenza virus activity in cell
culture was discovered and evaluated. Of these
acylthiourea derivatives 16 and 50 inhibited virus in
cultured MDCK cells at concentrations of 0.08 and
0.09 lM, respectively. These compounds were further
investigated for their potential for future development.
At present, effective therapy for influenza virus is limited
due to newly discovered resistance1 in mutant strains. In
addition, current anti-viral agents, such as amantadine
and rimantadine, provide only limited protection due
to a narrow spectrum of activity.2 Thus, research in
the design and screening for more effective anti-viral
drugs for the chemotherapy of influenza virus infection
is a high priority.
Recently, thiourea derivatives have been reported to be
effective against HIV3,4 and to have bactericidal action.5
Additionally, nitrogen heterocycles are known as versa-
tile functional groups in active compounds such as fun-
gicides.6 Few studies have been done concerning
Compounds were prepared using the routes shown in
Schemes 1–3. The intermediates N0-(4,6-disubstituted
pyrimidine-2-yl)-thiourea II and N0-(tert-butylamino-
F
O
F
O
S
O
Me
Keywords: Influenza virus; Inhibitor; Acylthioureas; Heterocycle;
Antiviral activity.
N
N
O
H
H
*
Corresponding authors. Tel.: +86 021 54237431; fax: +86 021
64225149; e-mail addresses: sunwillin@sjtu.edu.cn; pzhou@shmu.
edu.cn
Figure 1. Initial influenza inhibitor, 1.
0960-894X/$ - see front matter Ó 2005 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2005.09.033

C. Sun et al. / Bioorg. Med. Chem. Lett. 16 (2006) 162–166
163
Scheme 1. Reagents: (a) SOCl2; (b) HCl; (c) KSCN/CH3CN; (d) Et3N; (e) Br2.
Scheme 2. Reagents: (a) SOCl2; (b) HCl; (c) KSCN/CH3CN; (d) Et3N.
carbonyl)-thiourea IX (Scheme 2) were obtained by
treatment with potassium thiocyanate (KSCN) and a
hot aqueous solution of 2-amino-4,6-disubstituted
pyrimidine or tert-butylurea, respectively. N0-(4,6-Di-
substituted pyrimidine-2-yl)-N-(5-aryl-2-furoyl) thio-
urea III (Scheme 1) was prepared by the reaction of 5-
aryl-2-furoic acid with SOCl2 to form the intermediate
5-aryl-2-furoyl chloride. Subsequent treatment of the
intermediate II gave the desired acylthiourea derivative.
The preparation of N0-(4,6-disubstituted pyrimidine-2-
acylthioureas III and VI were converted in a straightfor-
ward manner to thiadiazolo[2,3-a]pyrimidines IV and
VII by oxidizing cyclization with the addition of drops
of bromine as oxidant. Note that the intermediate
substituted nicotinyl chloride V, substituted formyl
chloride VIII were synthesized by treating the same acyl-
ating agent SOCl2 with substituted nicotinic acid and
substituted formic acid,7,8 respectively.
The target adduct X was synthesized by the reaction of
intermediates VIII with IX (Scheme 2) in a 1:1 molar ra-
tio in acetonitrile, and the resulting precipitate was col-
lected by filtration and recrystallized from CH3CN to
yl)-N-substituted-b-pyridinecarbonyl
(Scheme 1) was nearly identical to the procedure for
thiourea
VI
the acylthiourea derivation III described above. Then

164
C. Sun et al. / Bioorg. Med. Chem. Lett. 16 (2006) 162–166
Scheme 3. Reagents: (a) NH3(H2O); (b) (COCl)2; (c) 2-amino-4,6-disubstituted pyrimine; (d) H2NCONHC(CH3)3.
give the final compound X. Treatment of substituted
acylisocyanates with 2-amino-4,6-substituted pyrimi-
dines and tert-butylurea, respectively, yielded acylurea
derivatives XI and XII (Scheme 3).
electron-donating groups such as Me, OMe, OEt, and
OH in this portion of the molecule (Table 1; 15–18).
Substitution by electron-withdrawing groups, such as
Cl, (19–21) led to poor activity. For instance, the acyl-
thiazole 16 was the most active compound in the series
with an IC50 of 0.08 lM. However, the 4-nitrophenyl
substitution did not offer any improvement over 2-chlor-
ophenyl substituted analogues. A number of corre-
sponding 4-nitrophenyl derivatives (22–24) had no
appreciable activity against influenza virus when com-
pared with their 2-chlorophenyl counterparts (15 and
16). Interestingly, the 4-nitrophenyl derivative 25 was
an exception, which was found to inhibit the influenza
virus in vitro at 0.36 lM and showed activity compara-
ble to that of 17. Nonetheless, substitutions on the
pyrimidine ring profoundly affect the ability to inhibit-
ing influenza virus in vitro.
All compounds were assayed11 against the influenza A3/
beijing/30/95(H1N1) virus, which were amplified and
titrated in MDCK cells. Additionally, general cytotoxic-
ity was measured using the MTS cellular toxicity as-
say9,10 to distinguish between specific anti-viral activity
and non-specific host cell toxicity. All the compounds
reported herein have MTS IC50 values of >300 lM
and are considered to be non-cytotoxic (these data are
not included in the tables).
The SAR resulting from variation for the X and Y sub-
stituents in the pyrimidine ring are shown in Table 1.
Different substituents at the 4- or 6-position in the
pyrimidine ring had greatest influence on activity. In this
series of 5-aryl-2-furoyl thioureas bearing a substituted
pyrimidine ring, there was a large preference for
With the right substituent W held constant as the tert-
butylaminocarbonyl group, we also explored the SAR
of substitutions at Q (Table 2). Introduction of a specific
functional cycloalkyl, such as chrysanthemoyl (33 and
34), resulted in a significant increase in potency.
Table 1. SAR of polysubstituted pyrimidinyl acylthiourea analogues
X
Table 2. tert-Butyl acylthiourea analogue SAR
N
O
S
O
S
O
R
Me
Me
O
N
Y
N
H
N
H
Me
Q
N
H
N
H
N
H
15-25
26-34
a
d
Compound
R
X
Y
IC50
Compound
Q
IC50
15
16
17
18
19
20
21
22
23
24
25
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
OEt
OEt
OH
Me
1.65
0.08
0.32
1.77
14.5
>20
>20
>20
>20
11.3
0.36
26
27
5-(2-Cl-Ph)-2-Furyl
5-(4-NO2-Ph)-2-Furyl
Ph
1.42
1.30
1.79
1.83
1.67
1.43
1.35
0.51
0.26
OEt
Me
28
29
OMe
Cl
Cl
OMe
Cl
OMe
(2,4-Cl2-Ph)-OCH2–
2,6-F2-Ph
30
OEt
Me
31
32aS-(+)
33a Cis-(ꢀ)
34a Trans-(ꢀ)
2-Me-1-(4-Cl-Ph)-Pr
*CFPCb
*DCPCc
OMe
OMe
OEt
OEt
OH
4-NO2
4-NO2
4-NO2
4-NO2
OMe
OEt
Me
a Compounds 32–34 are pure enantiomers as indicated.
Me
b *CFPC, 3-(2-chloro-3,3,3-trifluropropenyl)-2,2-dimethyl cyclopropyl.
c *DCPC, 3-(2,2-dichloro ethenyl)-2,2-dimethyl cyclopropyl.
d IC50 of influenza (H1N1) virus, lM.
MTS IC50 > 300 lM for all compounds.
a IC50 of influenza (H1N1) virus, lM.

C. Sun et al. / Bioorg. Med. Chem. Lett. 16 (2006) 162–166
165
Replacement of 5-aryl-2-furyl by groups, such as phenyl
or methoxyl (28 and 29), resulted in a slight decrease in
the potency (Table 2), while other substituents (30 and
31) showed activity that was equipotent to that of ana-
logue 26. With regard to the substituent W, introduction
of the tert-butylaminocarbonyl group at this position
afforded improved activity. Replacement of polysubsti-
tuted pyrimidine by tert-butylaminocarbonyl (26 and
27) further increased the potency 8- to 10-fold over ana-
logue 19 and 24. Analogue 34 was the most potent com-
pound in this series and inhibited influenza virus in vitro
at a concentration of 0.26 lM.
ring (39–43) and other pyridyl analogues (45 and 46)
was also examined. They did not achieve good efficiency
against influenza virus. Additionally, substitution of
chloro- at the 2- or 6-position in the pyridine ring was
not a major determinant in activity against influenza
virus (39 vs 42) in this series.
There was little correlation between the formation
of thiadiazolo[2,3-a]pyrimidine ring and potency
(Table 4). The former had a relatively small effect on
potency against influenza virus (47 vs 25). However,
compounds 49 and 50 in this series inhibited the virus
with an IC50 of 0.35 and 0.09 lM, respectively. Com-
pound 49 showed improved potency over the corre-
sponding un-ringed analogues such as 15. The high
potency of 50 bearing the same diethyl groups as 16 is
further support of a preference for electron-donating
groups, such as ethoxy, in pyrimidine derivatives.
It was noted that these tert-butyl acylthiourea analogues
have improved water solubility while retaining good
lipophilicity. Although we can speculate that the de-
crease in logP values may contribute partially to the in-
crease in anti-viral potency, the role of substituents such
as tert-butylaminocarbonyl in improving the activity
against influenza virus is unclear and this will be part
of the subject of a forthcoming paper.
The necessity for a thiourea functionality (Table 5) was
also investigated. The corresponding ureas (54–60) were
found to be less effective than their thiourea counter-
parts. Replacement of the thiourea bridge with urea
diminished or eliminated activity altogether in most
Heterocycles other than furan were synthesized and
resulted in lowered potency (Table 3). This demonstrat-
ed a large advantage in activity of the existence of a five-
membered ring (furan) over corresponding analogues
without the furan ring in most instances, and this trend
continued for other series of compounds. The com-
pounds containing furan ring (35 and 36) were 15-fold
more potent than the corresponding 6-chlorophenyl
substituted pyridyl analogues (37 and 38), which had
an IC50 of >20 lM. However, the only analogue with
no furan ring that retained good activity was the test
compound 44, although it was 2-fold less potent than
the corresponding furyl substituted analogue 36. This
result suggests that a furan ring at this position provides
significantly improved activity and should be included in
the rational design of new influenza virus inhibitors.
Variation of the X and Y substituents in the pyrimidine
Table 4. Effect of thiadiazolo[2,3-a]pyrimidines ring on inhibition of
influenzavirus
X
O
Q
S
N
N
N
N
Y
47-53
a
Compound
Q
X
Y
IC50
47
48
49
50
51
52
53
5-(4-NO2-Ph)-2-Furyl
5-(4-NO2-Ph)-2-Furyl
5-(2-Cl-Ph)-2-Furyl
6-Cl-3-py
Me
OEt
OH
OEt
Me
0.67
5.25
0.35
0.09
9.32
2.41
OEt
OEt
OEt
Cl
6-Cl-3-py
2-Cl-3-py
3-py
OMe
OMe
OMe
Table 3. SAR of heteroaryl Q groups
OMe
OMe
>20
X
a IC50 of influenza (H1N1) virus, lM.
O
S
N
Y
N
Q
N
H
N
H
Table 5. Thiourea versus urea comparison
37-46
O
O
a
W
Compound
Q
X
Y
IC50
Q
N
N
H
H
35
36
37
38
39
40
41
42
43
44
45
46
5-(4-NO2-Ph)-2-Furyl
5-(2-Cl-Ph)-2-Furyl
6-Cl-3-py
OMe
OMe
OMe
OMe
OEt
Me
Cl
1.22
1.29
54-60
Me
Cl
>20
a
Compound
Q
W
IC50
>20
6-Cl-3-py
6-Cl-3-py
>20
>20
54
55
56
57
58
59
60
Ph
2,6-F2-Ph
CONH(t-Bu)
CONH(t-Bu)
OEt
OH
Me
OEt
Cl
14.1
15.5
6-Cl-3-py
2-Cl-3-py
Me
Me
8.58
7.19
>20
>20
2.59
>20
18.5
2-Me-1-(4-Cl-Ph)-Pr CONH(t-Bu)
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
4,6-(Me)2-2-Pymb
4,6-(OMe)2-2-Pym
4-Cl-6-(OMe)-2-Pym
4-Cl-6-Me-2-Pym
>20
>20
15.9
>20
2-Cl-3-py
2-Cl-3-py
OEt
OEt
2-Cl-3-py
3-py
5,6-Cl2-3-py
OMe
OMe
OMe
Cl
OMe
OMe
a IC50 of influenza (H1N1) virus, lM.
b Pym, pyrimidinyl.
a IC50 of influenza (H1N1) virus, lM.

166
C. Sun et al. / Bioorg. Med. Chem. Lett. 16 (2006) 162–166
Acknowledgments
C2H5O
O
S
N
We thank Professor James C. Reynolds for revising the
manuscript. We also gratefully acknowledge research
specialist Wei Li for valuable advice.
O
OC2H5
N
H
N
N
H
Cl
Cl
16
OC2H5
O
S
N
References and notes
N
N
50
OC2H5
N
1. Hayden, F. G.; Belshe, R. B.; Glover, R. D. New Engl.
J. Med. 1989, 321, 1696.
2. Erik, De Clercq J. Clin. Virol. 2001, 22, 73.
3. Venkatachalam, T. K.; Mao, C. Bioorg. Med. Chem. 2004,
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331.
5. Sarkis, G. Y.; Faisal, E. D. J. Heterocycl. Chem. 1985, 22,
137.
Figure 2. Advanced potential development candidates 16 and 50.
cases. For instance, 55 and 56 had a 10-fold reduction in
activity compared to 31 and 32. In this respect, it follows
that the thiourea bridge is an important contribution to
inhibitory activity against influenza virus.
6. Bessard, Y.; Crettaz, R. Tetrahedron 2000, 56, 4739.
7. Krutosikova, A.; Kovac, J.; Sykora, V. Collect. Czech.
Chem. Commun. 1994, 39, 1892.
8. Oliveto, E. P. Chem. Heterocycl. Compd. 1962, 14, 178.
9. Barltrop, J. A.; Owen, T. C. Bioorg. Med. Chem. Lett.
1991, 1, 611.
Although some of the PAFHs have a relative low solubil-
ity (<10 mg/mL at room temperature), their high lipophil-
icity is expected to lead to an efficient penetration of the
PAFHs through cellular membranes and biological barri-
ers. This property may be advantageous for this novel
class of influenza inhibitors, as it may facilitate the uptake
of the lipophilic PAFHs into cells or biological compart-
ments where the virus accumulates.
10. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
11. The MDCK cells were grown in 96-well microtiter plates
with influenza virus at 50 plaque-forming units (PFU)/
well. Each compound was serially diluted and tested to
calculate its IC50 (Tables 1–5) and all values are means of
at least three independent experiments on different days.
12. Physical data: N0-(4,6-diethoxylpyrimidin-2-yl)-N-[5-(2-
chlorophenyl)-2-furoyl]thiourea (compound 16). Yield:
86%, mp: 175–177 °C; IR (KBr plate, cmꢀ1): 1632
(C@O), 3195 (N@H), 1235 (C@S); 1H NMR (DMSO-
d6): dHppm 7.24–7.86 (m, 6H, Ar-H), 6.15 (s, 1H,
pyrimidine-5-H), 12.08 (s, 1H, N0-H), 12.46 (s, 1H, N0-
H), 1.22 (t, 6H, CH3), 4.08 (q, 4H, OCH2). Anal.
Calcd for C20H19ClN4O4S: C, 53.75; H, 4.26; N, 12.54.
Found: C, 53.64; H, 4.35; N, 12.41.
In this paper, we have described the synthesis of a ser-
ies of polysubstituted and fused heterocycle derivatives
of acylthioureas. Evaluation of their in vitro anti-viral
activity revealed compounds with high potent cellular
activity against wild-type influenza virus in cultured
MDCK cells. Analogues 16 and 5012 (Fig. 2) inhibited
the influenza virus with an IC50 of 0.08 and 0.09 lM,
respectively. These novel inhibitors were investigated
as candidate compounds with the most potential for
future drug development. This research has lead to a
better understanding of SAR of influenza virus inhib-
itors and thereby provides some insight into the ra-
tional design of new anti-flu agents. Further studies
are ongoing to assess the inhibitory activity against
resistant influenza virus strains as well as searching
for the specific influenza target(s) and mechanism-
based drugs.
5,7-Diethoxyl-2-(6-chloro-3-pyridinecarbonylimino)-2H-
1,2,4-thiadiazolo[2,3-a]pyrimidine (compound 50). Yield:
72%, mp: 176–178 °C; IR (KBr plate, cmꢀ1): 1690 (C@O),
1
1630 (C@N); H NMR (DMSO-d6): dHppm 7.60–8.95 (m,
3H, pyridine-H), 5.65 (s, 1H, pyrimidine-5-H), 1.16 (t, 3H,
CH3), 4.18 (q, 2H, OCH2). Anal. Calcd for
C20H17ClN4O4S: C, 47.43; H, 3.69; N, 18.45. Found: C,
47.51; H, 3.60; N, 18.36.