Synthesis and anti-CVB 3 evaluation of substituted 5-nitro-2-phenoxybenzonitriles

Article abstract of DOI:10.1016/j.bmcl.2008.07.099

The synthesis and SAR of a series of 60 substituted 2-phenoxy-5-nitrobenzonitriles (analogues of MDL-860) as inhibitors of enterovirus replication (in particular of coxsackievirus B3 (CVB 3)) are reported. Several of the analogues inhibited CVB 3 and othe

Full text of DOI:10.1016/j.bmcl.2008.07.099

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5123–5125
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-CVB 3 evaluation of substituted
5-nitro-2-phenoxybenzonitriles
b
a
a
a
Gerhard Pürstinger a, , Armando M. De Palma , Günther Zimmerhofer , Simone Huber , Sophie Ladurner ,
*
Johan Neyts b
a Institut für Pharmazie, Abteilung Pharmazeutische Chemie, Universität Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of a series of 60 substituted 2-phenoxy-5-nitrobenzonitriles (analogues of MDL-
860) as inhibitors of enterovirus replication (in particular of coxsackievirus B3 (CVB 3)) are reported. Sev-
eral of the analogues inhibited CVB 3 and other enteroviruses at low-micromolar concentrations.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 15 June 2008
Revised 23 July 2008
Accepted 25 July 2008
Available online 30 July 2008
Keywords:
5-Nitro-2-phenoxybenzonitriles
MDL-860
Enterovirus inhibitors
CVB 3
Antiviral
Enteroviruses are non-enveloped, single-stranded (+) RNA
viruses belonging to the picornavirus family. This large family har-
bours several pathogens that are implicated in a wide range of clin-
ical manifestations, affecting humans as well as animals.1 It is
estimated that enteroviruses cause each year 10–15 million (or
more) symptomatic infections.2 Although often mild and self-lim-
iting, enteroviruses may also be involved in more serious condi-
tions, which can be life-threatening, such as pancreatitis,
meningitis, encephalitis or myocarditis.2 Coxsackieviruses, and in
particular CVB 3, have often been associated with the development
of myocarditis, which may lead to sudden death in young adults or
progress to dilated cardiomyopathy.3–5
In the past, several compounds have been reported to be selec-
tive inhibitors of enteroviruses, some of which have entered clini-
cal trials.6 Pleconaril is the prototype of a series of broad-spectrum
picornavirus inhibitors, known as ‘WIN-compounds’.7 These com-
pounds block replication by inhibiting host cell attachment and/
or uncoating, as a result of binding into a pocket, located under-
neath the canyon on the virion surface.8 Despite its proven efficacy
in several studies,9,10 pleconaril was not approved by the FDA for
the treatment of the common cold (due to rhinovirus infections),11
but is still being studied on a compassionate base for the treatment
of life-threatening infections in children, such as meningitis or
encephalitis, but on the other hand it is also being developed at
Schering-Plough, under license of Viropharma, for the treatment
of rhinovirus infections in high-risk asthma and COPD (chronic
obstructive pulmonary disease) patients. Pleconaril is inactive
against the cardiotropic coxsackievirus B3 (Nancy strain).12,13
Although several research groups are still working on the synthesis
of antivirals structurally related to pleconaril,14–16 the search for
novel broad-spectrum inhibitors of enteroviruses replication re-
mains compulsory.
MDL-860 (2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile, 1)17–20
was previously reported to possess broad-spectrum in vitro activ-
ity against picornaviruses by inhibiting an early event in virus rep-
lication, after initial uncoating.19 This compound also elicited
in vivo efficacy in a model of coxsackievirus B3-induced myocardi-
tis.18 However, MDL-860 was never further developed. To gain in-
sight into the structural features that might contribute to a
potential improvement in antiviral activity, 60 analogues of this
lead compound were synthesized21 (Scheme 1) and evaluated22
for inhibition of coxsackievirus replication.
R1
R3
R1
R2
R3
O2N
O2N
a
R2
+
HO
Cl
O
CN
CN
* Corresponding author. Tel.: +43 512 507 5260; fax: +43 512 507 2940.
Scheme 1. Reagent and condition: (a) dry DMF, anhydrous K2CO3, room tempera-
E-mail address: Gerhard.Puerstinger@uibk.ac.at (G. Pürstinger).
ture, 24 h.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.099

5124
G. Pürstinger et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5123–5125
R1
R3
O2N
Cl
Cl
O2N
O2N
R
R2
O
O
O
CN
1 (MDL-860)
CN
CN
2 - 39
40 - 61
Figure 1. Structures of compounds 1 and 2–39.
Figure 2. Structures of compounds 40–61.
Table 1
Table 2
Anti-CVB 3 activities and cytotoxicities for di- and trisubstituted analogues 40–61
Anti-CVB 3 activities and cytotoxicities for compounds 1 and 2, and monosubstituted
analogues 3–39
R1, R2, R3
IC50
(l
M)
CC50 (lM)
S.I.b
a
a
Compound
R
IC50
(l
M)
CC50 (lM)
S.I.b
a
a
Compound
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
2,3-Cl2
2,4-Cl2
2,5-Cl2
3,5-Cl2
4.9 3.1
13 9.6
3.8 2.2
>81
39 9.0
7.3 4.3
3.7 2.7
3.7 2.6
>53
>324
>324
>324
81
>313
>334
>283
>283
53
>283
>346
>346
>330
>251
>346
>300
>334
>333
>324
146
>66.1
1
2
3
4
5
6
7
8
9
—
H
2-Cl
3-Cl
4-Cl
2-CH3
3-CH3
4-CH3
2-Br
3-Br
4-Br
2-OCH3
3-OCH3
4-OCH3
2-CN
3-CN
4-CN
6.5 4.9
8.7 1.5
6.9 3.1
4.9 0.9
5.0 4.1
57 15
6.4 4.6
6.2 2.2
>143
5.0 1.9
5.7 4.3
54 6.5
12 2.2
46 3.6
11 2.7
7.4 1.5
4.1 2.6
40 37
31 15
241 52
106 68
>352
>324
>416
>364
>364
>364
>393
>393
>393
>269
>313
>313
>370
>370
>370
>377
>377
>377
>354
>353
>392
>392
>352
>352
>354
>354
>351
>351
>336
>336
>273
>273
>335
>318
>349
>331
>314
>338
>353
>370
>49.8
>24.9
>85.3
n.a.
>47.8
>52.8
>74.3
>72.8
>6.9
>61.4
>63.4
1.9
>62.6
>54.9
>6.9
>30.8
>8.0
>34.3
>50.9
>92.0
>8.9
>11.4
>1.6
>3.7
n.a.
n.a.
>6.2
>10.1
>9.2
>106.4
>2.8
2-Cl-4-NO2
2-Cl-4-CN
2-Cl-4-Br
2-Br-4-Cl
2-NH2-4-Cl
3-Cl-4-Br
2-CH3-4-Cl
3-Me-4-Cl
3-Et-4-Cl
2,4-Br2
>8.0
>45.8
>76.5
>76.5
n.a.
>42.2
>31.5
6.7 0.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
11
2.0 1.6
>330
9
>173
n.a.
>40.3
>3.5
>115.4
>30.4
>33.3
n.a.
n.a.
>4.9
>4.0
6.2 0.1
99 71
2.6 0.2
11 2.2
10 9.0
>324
>146
59 27
72 20
3,4-Me2
3-Me-4-Br
3-Me-4-NO2
3-NO2-4-NH2
3,5-Me2
2,3,4-Cl3
2,4,5-Cl3
2,4,6-Cl3
3-Isopropyl
3-N(CH3)2
3-NH2
>291
>291
4-NH2
3-COOH
4-COOH
3-COCH3
4-COCH3
3-NO2
>352
a
Values are means of three independent experiments standard deviation.
In vitro selectivity index (CC50/IC50).
57 4.6
35 2.3
38 22
3.3 0.4
121 26
>336
5.7 1.9
5.7 3.1
>335
234 40
93 34
205 9.2
174 16
>338
b
4-NO2
3-NHCOCH3
4- NHCOCH3
3-I
better than that of the lead compound against CVB 3. In particular,
2,3-, 2,4- and 3,4-disubstitution with halogens (or in part with
other substituents such as methyl, cyano or nitro) were beneficial
for anti-CVB 3 activity. Most potent activities were measured for
the 4-chloro-3-methyl analogue 51 and for the 4-bromo-3-methyl
analogue 55. Interestingly, formal replacement of the methyl group
in compound 51 by an ethyl group resulted in an inactive analogue
(52). Also, replacement of the chlorine in compound 51 by a second
methyl group resulted in reduced anti-CVB 3 activity (54). Simi-
larly, 3,5-disubstitution with chlorine (43) or methyl (58), or tri-
substitution with chlorine (compounds 59–61) resulted in—more
or less—inactive analogues.
n.a.
>47.9
>47.9
n.a.
>1.4
>3.8
>1.6
>1.8
n.a.
n.a.
4-I
3-NH-CO-NH2
3-NH-CS-NH2
4-SCH3
4-SOCH3
4-SO2CH3
4-tert-Butyl
4-CONH2
4-CH2OH
>353
121 91
>3.1
a
Values are means of three independent experiments standard deviation.
In vitro selectivity index (CC50/IC50).
b
A selection of six compounds was evaluated against the other
five coxsackie B viruses (Table 3). All of the analogues, with the
exception of 51, which was inactive against CVB 4 and 6, had
broad-spectrum activity with analogues 3 and 40 being the most
potent ones.
First, the unsubstituted analogue 2 and a set of 38 monosubsti-
tuted MDL-860 analogues (3–39) (Fig. 1) were synthesized and
tested against CVB 3 (Table 1). Several compounds exhibited activ-
ities and selectivities comparable to that of the lead compound (1),
in particular when substituted with a halogen, a cyano group or a
4-nitro group. Introduction of polar and/or bulkier substituents
seemed to result in reduced antiviral activities. There also ap-
peared a preference—at least for some substituents—for positions
3 and 4.
Table 3
Activities (on Vero cells,
l
M)a of selected compounds against CVB 1, 2, 4, 5 and 6
Compound
CVB 1
CVB 2
CVB 4
CVB 5
CVB 6
Recently, it has been proposed that the anti-human rhinovirus 2
(HRV-2) activity of MDL-860 and its analogues is highly dependent
on hydrophobicity.23 This may possibly also be applied for the
activities against enteroviruses within this class of inhibitors.
Next, a set of 22 di- and trisubstituted MDL-860 analogues (40–
61) was prepared (Fig. 2, Table 2). Again, several of these com-
pounds had activities and selectivities comparable to or slightly
1
2
3
7
40
51
15 6.9
6.9 3.3
5.6 1.4
8.9 3.1
3.3 1.7
4.8 3.4
15 11
17 6.9
6.2 0.7
11 3.3
8.6 6.3
5.2 0.8
17 9.9
26 3.3
14 7.3
22 1.0
17 0.6
>346
18 9.4
10 4.8
3.1 2.1
13 6.1
4.8 2.2
2.1 0.5
12 5.2
19 4.2
8.1 0.5
22 4.4
10 4.2
>346
a
Values are means of three independent experiments standard deviation.

G. Pürstinger et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5123–5125
5125
Table 4
Activities (on MRC-5 cells,
picornaviruses
3. Archard, L. C.; Khan, M. A.; Soteriou, B. A.; Zhang, H.; Why, H. J.; Robinson, N.
M.; Richardson, P. J. Hum. Pathol. 1998, 29, 578.
4. Esfandiarei, M.; McManus, B. M. Annu. Rev. Pathol.: Mech. Dis. 2008, 3, 127.
5. Chapman, N. M.; Kim, K. S. Curr. Top. Microbiol. Immunol. 2008, 323, 275.
6. Barnard, D. L. Curr. Pharm. Des. 2006, 12, 1379.
7. Florea, N. R.; Maglio, D.; Nicolau, D. P. Pharmacotherapy 2003, 23, 339.
8. Pevear, D. C.; Fancher, M. J.; Felock, P. J.; Rossmann, M. G.; Miller, M. S.; Diana,
G.; Treasurywala, A. M.; McKinlay, M. A.; Dutko, F. J. J. Virol. 1989, 63, 2002.
9. Hayden, F. G.; Herrington, D. T.; Coats, T. L.; Kim, K.; Cooper, E. C.; Villano, S. A.;
Liu, S.; Hudson, S.; Pevear, D. C.; Collett, M.; McKinlay, M. Clin. Infect. Dis. 2003,
36, 1523.
10. Rotbart, H. A.; Webster, A. D. Clin. Infect. Dis. 2001, 32, 228.
11. Senior, K. Lancet Infect. Dis. 2002, 2, 264.
12. Groarke, J. M.; Pevear, D. C. J. Infect. Dis. 1999, 179, 1538.
13. Kandolf, R.; Hofschneider, P. H. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4818.
14. Shia, K.-S.; Li, W.-T.; Chang, C.-M.; Hsu, M.-C.; Chern, J.-H.; Leong, M. K.; Tseng,
S.-N.; Lee, C.-C.; Lee, Y.-C.; Chen, S.-J.; Peng, K.-C.; Tseng, H.-Y.; Chang, Y.-L.; Tai,
C.-L.; Shih, S.-R. J. Med. Chem. 2002, 45, 1644.
l
M)a of selected compounds against selected non-CVB
Compound
CVA 24
1.8 0.9
>240
23 7.7
>364
19 9.4
>350
Echo 9
Echo 11
Entero 68
1
2
3
5
7
27
40
42
51
>324
>416
35 18
>364
>393
>350
>323
>323
>346
6.7 3.7
>416
35 15
>364
>393
>350
12 9.0
>416
22
7
>364
76 28
>350
>309
>323
>323
>323
>346
>323
>323
>346
234 70
a
Values are means of three independent experiments standard deviation.
15. Krippner, G. Y.; Chalmers, D. K.; Stanislawski, P. C.; Tucker, S. P.; Watson, K. G.
Aust. J. Chem. 2004, 57, 553.
16. Makarov, V. A.; Riabova, O. B.; Granik, V. G.; Wutzler, P.; Schmidtke, M. J.
Antimicrob. Chemother. 2005, 55, 483.
17. Markley, L. D.; Tong, Y. C.; Dulworth, J. K.; Steward, D. L.; Goralski, C. T.;
Johnston, H.; Wood, S. G.; Vinogradoff, A. P.; Bargar, T. M. J. Med. Chem. 1986,
29, 427.
18. Padalko, E.; Verbeken, E.; De Clercq, E.; Neyts, J. J. Med. Virol. 2004, 72, 263.
19. Torney, H. L.; Dulworth, J. K.; Steward, D. L. Antimicrob. Agents Chemother. 1982,
22, 635.
Nine compounds were selected for evaluation against a set of 4
non-CVB enteroviruses (Table 4). This selection was based on the
activities against CVB 3 and on diversity with respect to the substi-
tution pattern of the phenoxy substructure. The lead compound
MDL-860 (1) proved active against CVA 24, echovirus 11 and
enterovirus 68, but was inactive against echovirus 9. Only the 2-
chloro analogue (3) proved active against all four non-CVB entero-
viruses, and five analogues (2, 5, 27, 40 and 42) were inactive
against any of the four viruses. Compound 40, therefore, seemed
to be a selective inhibitor of coxsackie B virus replication. Com-
pound 7 had moderate activity against CVA 24 and enterovirus
68, whereas the 3-methyl-4-chloro analogue 51 exhibited only
weak activity against CVA 24.
20. Powers, R. D.; Gwaltney, J. M.; Hayden, F. G. Antimicrob. Agents Chemother.
1982, 22, 639.
21. All compounds were characterized by 1H NMR spectra, MS spectra, combustion
analyses and melting points. All analogues except 33, 35 and 36 were prepared
by reaction of 2-chloro-5-nitrobenzonitrile with an appropriately substituted
phenol (Scheme 1). Compound 33 was synthesized by reaction of 20 with
potassium thiocyanate in a mixture of toluene and 6 N HCl (6 h at 90 °C).
Compounds 35 and 36 were prepared by oxidation of compound 34 with
metachloroperbenzoic acid (1 or 2.5 equiv, respectively) in dichloromethane at
ambient temperature. Synthesis of MDL-860 (1) as an example: To a mixture of
2-chloro-5-nitrobenzonitrile (300 mg), 3,4-dichlorophenol (268 mg, 1 equiv)
and anhydrous potassium carbonate (341 mg, 1.5 equiv) was added 3 mL of
dry DMF, and the resulting suspension was stirred for 24 h at ambient
temperature (tlc control: silica gel, eluent: light petroleum/ethyl acetate = 3:1
(v/v)). Then, water (50 mL) was slowly added and the resulting precipitate was
filtered, washed with water and dried. The crude product was purified by
recrystallization from a mixture of diisopropyl ether (20 mL) and ethyl acetate
(7 mL); off-white crystals; mp: 156–158 °C (155–156 °C);17 yield: 63%; 1H
NMR (200 MHz, DMSO-d6) d 8.88 (d, 1H, H6, J = 2.8 Hz), 8.43 (dd, 1H, H4, J = 9.3,
2.8 Hz), 7.84–7.78 (m, 2H, H20/50), 7.39 (dd, 1H, H60, J = 8.8, 2.8 Hz), 7.19 (d, 1H,
H3, J = 9.3 Hz); MS (CI): m/z calcd for C13H6Cl2N2O3 (M+) 308.0, found 307.9;
Anal. (C13H6Cl2N2O3): Calcd C, 50.51; H, 1.96; N, 9.06. Found C, 50.54; H, 2.09;
N, 8.97.
The exact mode of action of this class of compounds against
enteroviruses has yet to be figured out. Without knowing the tar-
get (and the structural differences of the target within the different
enteroviruses), it is very difficult to fully understand the obtained
structure–activity relationships (SARs).
In summary, several of the MDL-860 analogues are selective
inhibitors of CVB replication with activities that are comparable
to or slightly better than that of the lead compound. The SAR of this
class of compounds for CVB 3 does, however, not parallel the SAR
for other enteroviruses. At least one compound (3) was identified
that had broad-spectrum enterovirus activity and that was—unlike
the lead compound MDL-860—active against echovirus 9. Further
exploration of this class of compounds should allow obtaining fur-
ther insights into the SAR for inhibition of enterovirus replication.
Also studies are underway to identify the molecular target of this
class of compounds. Once this target has been identified, this infor-
mation may help to rationally design analogues with improved
antiviral activity.
22. Antiviral assay. The antiviral activity of the selected compounds was
determined using an MTS-based cytopathic effect (CPE) reduction assay and
was expressed as the 50% effective concentration (EC50), or the concentration
of compound that inhibits virus-induced cytopathic effect formation by 50%.
Cells, grown to confluency in 96-well plates, were infected with 100 CCID50 of
virus, one CCID50 being the 50% cell culture infective dose. After an adsorption
period of 2 h at 37 °C, virus was removed and serial dilutions of the compounds
were added. The cultures were further incubated at 37 °C for 3 days, until
complete CPE was observed in the infected and untreated virus control (VC).
After removal of the medium, 90 ll medium and 10 ll MTS/PMS (Promega,
Leiden, The Netherlands) were added to each well. After an incubation period
of 2 h at 37 °C, the optical density of each well was read at 498 nm in a
Acknowledgments
microplate reader. CPE values were calculated as follows:
%
CPE = 100*[ODCC ꢀ ODCVB3+Compound]/[ODCC ꢀ ODVC]. In these formulae, ODCC
corresponds to the optical density of the uninfected and untreated cell
cultures, ODVC represents the infected and untreated cell cultures and
We thank Mrs. M.-H. Stuyck for excellent technical assistance.
This work was supported by the VIZIER EU FP7—Integrated Project
(GrantLSHG-CT-2004–51196)andbyVIRGIL, theEuropeanNetwork
of Excellence on Antiviral Drug Resistance (Grant LSHM-CT-2004-
503359 from the Priority 1 ‘Life Sciences, Genomics and Biotechnol-
ogy’). Armando M. De Palma is a postdoctoral research fellow of the
FWO (Fonds voor Wetenschappelijk Onderzoek Vlaanderen).
ODCVB3+Compound are CVB3-infected cell cultures, treated with
a given
concentration of compound. Cytotoxic assay. The cytotoxicity of the
compounds was evaluated by the MTS-method and the 50% cytotoxic
concentration (CC50) was calculated. Briefly, the same experimental set-up
was used as for the antiviral assay, but for cytotoxicity determination,
uninfected cultures were incubated with serial dilution of compound for
three days at 37 °C. The cytotoxic activity was calculated using the following
formula: % CPE = 100*[ODCCꢀODCompound]/ODCC, where ODCC corresponds to the
optical density of the uninfected and untreated cell cultures and ODCompound
are uninfected cultures, treated with compound.
References and notes
1. Sawyer, M. H. Curr. Opin. Pediatr. 2001, 13, 65.
2. Rotbart, H. A. Antiviral Res. 2002, 53, 83.
23. Verma, R. P.; Hansch, C. Virology 2007, 359, 152.