Thiourea inhibitors of herpes viruses. Part 1: Bis-(aryl)thiourea inhibitors of CMV

Article abstract of DOI:10.1016/S0960-894X(03)00586-9

Bis-(aryl)thioureas were found to be potent and selective inhibitors of cytomegalovirus (CMV) in cultured HFF cells. Of these, the thiazole analogue 38 was investigated as a potential development candidate.

Full text of DOI:10.1016/S0960-894X(03)00586-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2929–2932
Thiourea Inhibitors of Herpes Viruses. Part 1: Bis-(aryl)thiourea
Inhibitors of CMV
Jonathan D. Bloom,* Martin J. DiGrandi, Russell G. Dushin, Kevin J. Curran,
Adma A. Ross, Emily B. Norton, Eugene Terefenko, Thomas R. Jones,
Boris Feld and Stanley A. Langy
Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
Received 15 January 2003; accepted 24 April 2003
Abstract—Bis-(aryl)thioureas were found to be potent and selective inhibitors of cytomegalovirus (CMV) in cultured HFF cells. Of
these, the thiazole analogue 38 was investigated as a potential development candidate.
# 2003 Elsevier Ltd. All rights reserved.
High-throughput screening against a panel ofviruses in
Human cytomegalovirus (CMV) is a ubiquitous patho-
gen belonging to the herpes family of viruses.1 Trans-
mission can be perinatal or through body fluids.
Seroprevalence rates in adults range from 50–90%,
depending on the socioeconomic population examined.1
CMV infection in immunocompetent adults is usually
asymptomatic. However, as an opportunistic pathogen,
CMV causes clinically significant disease in the absence
ofnormal immunity. In this regard, relevant clinical
populations include infected infants, and immunocom-
promised adults. Although in recent years HAART2
therapies have significantly restored immune function in
AIDS patients, there remains a need for safer, more
effective CMV therapies for infected neonates and
transplant patients as well as AIDS patients in whom
HAART therapy has failed.
cultured cells revealed thiourea 1 (Fig. 2) as a weak
inhibitor ofHerpes simplex virus (HSV). An early
attempt to maximize this activity led to the preparation
ofphenyl analogue 2, which unexpectedly displayed
inhibitory activity against CMV in cultured human
foreskin fibroblast (HFF) cells. Inhibitory activities of
analogues against CMV and HSV quickly diverged,
leading to parallel series.
Thioureas 3 could be prepared (Scheme 1) by reacting
appropriately substituted anilines 7 with isothiocyanates
6 in acetonitrile or THF. Alternatively, 3 could also be
prepared by sequential treatment ofanilines 7 with
Current therapies for CMV (Fig. 1) include ganciclovir,3
cidofovir4 and foscarnet.5 These drugs typically produce
significant side effects including neutropenia and
nephrotoxicity as well as exhibit poor bioavailability,
although valganciclovir6 (a valine ester prodrug ofgan-
cyclovir) has shown improved bioavalability. Recently,
fomivirsen, a 21-nucleotide anti-sense therapy has been
approved for intravitreal injection.7 A review ofnon-
Figure 1. Current CMV therapies.
8
nucleoside inhibitors ofCMV has recently appeared.
*Corresponding author. Fax:+1-845-602-5561; e-mail: bloomj@
wyeth.com
yCurrent address: Ribapharm Inc., 3300 Hyland Ave., Costa Mesa,
CA 92626, USA.
Figure 2. Initial HSV and CMV inhibitors.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00586-9

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J. D. Bloom et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2929–2932
gave the 4-carboxy-1,2,3-thiadiazole methyl ester, which
could be converted to the acid chloride 14 as described in
Scheme 2. 4-Carboxy-1,2,3-thiadiazole was purchased.
The preparation ofanalogues in which the central phe-
nylenediamine ring was substituted (Scheme 4) was
accomplished by acylation ofthe commercially avail-
able substituted nitroanilines 15 to yield amides 16.
Reduction ofthe nitro group with palladium and
cyclohexene13 in refluxing ethanol or with iron and
ammonium chloride14 in ethanol gave the substituted
anilines 17. Conversion to the desired thiourea was
identical to the procedure described in Scheme 1.
Scheme 1. (i) RC(O)Cl, TEA, CH2Cl2; (ii) TFA; (iii) 1,10-thiocar-
bonyldiimidazole; (iv) hot CH3CN.
1,10-thiocarbonyldi-(1,2,4)-triazole,9 followed by addi-
tion ofanilines 5. This latter approach was advantageous
when employing less nucleophilic anilines. Requisite iso-
thiocyanates 6 were prepared in an efficient three-step
process beginning with the acylation ofmono-BOC phe-
nylenediamine 4, deprotection with TFA and treatment of
the resulting free anilines 5 with 1,10-thiocarbonyldiimi-
dazole. Anilines 7 reported herein are commercially
available, with the exception of3-chloro-4-trifluoro-
All compounds in the program were assayed15 against
the herpes viruses CMV, HSV and varicella zoster
(VZV). Additionally, screening against non-herpes
viruses such as respiratory syncytial virus (RSV) and the
MTS cellular toxicity assay16 were performed to distin-
guish between specific anti-viral activity and non-spe-
cific host cell toxicity. All ofthe compounds reported
herein (Fig. 3) have RSV and MTS IC50 values of >10
mg/mL. These compounds are considered to be non-
cytotoxic and RSV and MTS data are not included in
the tables.
methylaniline, which was prepared in two steps by
10
reaction oftrifluoromethylcopper
with 3-chloro-4-
iodonitrobenzene, followed by reduction of the nitro
group.
The SAR resulting from variation of the acyl group is
shown in Table 1. Replacement ofthe methyl group in 1
with larger alkyls such as ethyl (18) did not improve
activity versus HSV. However, introduction ofa phenyl
group (2) led to a 10-fold reduction in the IC50 against
both HSV and CMV. Replacement ofphenyl by a het-
eroaromatic group such as 2-furoyl (19) further
increased the potency against CMV 20-fold, to 0.2 mg/
mL, but HSV activity was lost. At this point, the HSV
The preparation ofnon-commercially available car-
boxylic acid chlorides is shown in Scheme 211 and
Scheme 3.12 Condensation ofethyl isocyanoacetate,
8
with N,N-dimethylformamide dimethyl acetal gave ethyl
3-dimethylamino-2-isocyano acrylate, 9. Treatment of 9
with hydrogen sulfide and TEA yielded 4-carboethoxy
thiazole, 10 which was converted to the acid chloride, 11
by basic hydrolysis followed by exposure to oxalyl
chloride.
Substituted 1,2,3-thiadiazole 4-carboxylates (Scheme 3)12
were prepared by reaction ofthe appropriately sub-
stituted b-keto ester 12 with p-toluenesulfonyl azide to
form the intermediate diazo ketone 13. Subsequent
treatment with Lawesson’s reagent in refluxing toluene
Scheme 4. (i) Excess RC(O)Cl, THF, reflux; (ii) Pd/C/cyclohexene,
EtOH; or Fe/NH4Cl, EtOH.
Figure 3. Generic structure ofCMV inhibitors 18–38.
Table 1. Variation ofthe acyl group
Scheme 2. (i) Me2NCH(OMe)2; (ii) H2S, TEA; (iii) NaOH; (iv)
(COCl)2.
Compd
X
Y
R
CMVa HSVa VZVa
1
18
2
2,4-(OMe)2-5-Cl
2,4-(OMe)2-5-Cl
2,4-(OMe)2-5-Cl
2,4-(OMe)2-5-Cl
H
H
H
H
Me
Et
Ph
2-Furb
45
NT
4
3.0
3.0
0.3
>10
>10
>10
NT
19
0.2
>10
NT=not tested; MTS>10 mg/mL for all compounds.
aIC50, mg/mL.
Scheme 3. (i) TsN3, TEA (ii) Lawesson’s reagent; (iii) NaOH; (iv)
(COCl)2.
b2-Fur=2-furoyl.

J. D. Bloom et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2929–2932
Table 4. Heteroaryl R groups
2931
and CMV programs diverged, and all SARs reported
herein involved our efforts directed toward finding
potent CMV inhibitors. A paper describing the SARs of
the HSV series will be forthcoming.
Compd
X
Y
R
CMVa,b
33
34
35
36
37
38
3-Cl–4-CF3
3-Cl–4-CF3
3,5-(CF3)2
3,5-(CF3)2
3-Cl–4-CF3
3,5-(CF3)2
H
H
H
H
H
H
2-Pyridyl
4-(1,3-Oxazoyl)
4-(1,3-Oxazoyl)
4-(1,2,3-Thiadiazolyl)
4-(1,3-Thiazolyl)
4-(1,3-Thiazolyl)
>10
0.02
0.03
0.02
0.006
0.008
With R held constant as 2-furoyl, we explored the SAR
ofRing A ( Table 2). Alkyl and electron withdrawing
groups, especially meta and para to the thiourea nitro-
gen, showed increased potency over non-substituted
analogues (20). Disubstituted analogues (21–23 and 25)
were approximately as potent as monosubstituted (24
and 26). The most potent furan-containing inhibitor
was the 3-trifluoromethyl-4-chloro analogue 22, which
had an IC50 of0.03 mg/mL.
aIC50, mg/mL.
bVZV and HSV>10 mg/mL for all analogues in table.
Substitution on Ring B (Table 3) diminished or elimi-
nated activity altogether in every case. This was true for
analogues bearing electron withdrawing groups (30 and
31) and electron-donating groups (27 and 32). The only
analogue to retain good activity was 28 (Y=2-Me),
although it was 3-fold less potent than the correspond-
ing unsubstituted analogue 19.
Figure 4. Advanced lead candidate.
compound might be problematic. Although the com-
pounds in series were stable under conditions ofthe in
vitro assay, this unexpected instability (attributed to
hydrolysis ofthe thiourea group) prompted us to aban-
don this series and focus on modified structures with
improved stability characteristics. This will be the sub-
ject ofa future paper.
Heterocycles other than furan were synthesized and this
resulted in the most potent analogues in the series
(Table 4). We had previously demonstrated a large
advantage in activity ofa five-membered ring (ufran)
over a six-membered ring (benzene) for R (2 vs 19) and
this trend continued for other aromatic groups. The 2-
pyridyl analogue 33 had an IC50 of >10 mg/mL. How-
ever, a number offive-membered heterocycles showed
activity that was equipotent (1,3-oxazoles 34 and 35) or
superior (thiadiazole 36 and thiazole 37) to the corre-
sponding 2-furoyl analogue. Thiazoles 37 and 38 were
the most active compounds in the series with IC50s of
0.006 and 0.008 mg/mL, respectively.
HTS identified a weak inhibitor ofHSV. Chemical
synthesis improved this activity, but also revealed activity
against CMV, another herpes virus. During the course of
this program the activity versus CMV was improved
7500-fold, yielding an extremely potent (0.006 mg/mL)
inhibitor ofCMV in cultured HFF cells without loss of
selectivity. Unsuitable physical properties precluded fur-
ther development ofany compounds in this series.
Thiazole 3817 (Fig. 4) was found to be unstable under
forcing conditions, especially in acidic media at elevated
temperatures, suggesting that the in vivo stability ofthe
References and Notes
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Table 2. Ring A SAR
Compd
X
Y
R
CMVa
HSVa
VZVa
2. Palella, F. J.; Delaney, K. M.; Moorman, A. C.; Loveless,
M. O.; Fuhrer, J.; Satten, G. A.; Aschman, D. J.; Holmberg,
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20
21
22
23
24
25
26
H
H
H
H
H
H
H
H
2-Furoyl
2-Furoyl
2-Furoyl
2-Furoyl
2-Furoyl
2-Furoyl
2-Furoyl
0.30
0.09
0.03
0.06
0.06
0.04
0.06
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
7
3-Cl–4-Me
3-CF3–4-Cl
3-Cl–4-F
3-CF3
3,5-(CF3)2
3-CH3
>10
aIC50, mg/mL.
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Table 3. Ring B SAR
Compd
X
Y
R
CMVa HSVa VZVa
27
28
29
30
31
32
2,4-(OMe)2–5-Cl
2,4-(OMe)2–5-Cl
2,4-(OMe)2–5-Cl
2,4-(OMe)2–5-Cl
2,4-(OMe)2–5-Cl
3-OMe
2-Furb >10
2-Fur 0.6
2-Fur >10 >10 >10
2-Fur >10 >10 >10
8 >10
>10 >10
2-Me
3-Me
2-CN
2-CF3
2-Fur
2.0
2.5
>10
4
2,4-(OMe)2–5-Cl 3,6-(OMe)2 2-Fur >10 >10
aIC50, mg/mL.
b2-Fur, 2-Furoyl

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J. D. Bloom et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2929–2932
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15. To calculate IC50s, the compounds were serially diluted
and tested in duplicate. The Sigmoidal Hill Slope (0–100)
17. Physical data for 38: calcd For C19H12F6N4OS2: C 46.53;
H 2.47; N 11.42. Found: C 46.31; H 2.42; N 11.34. EI-MS
[M+H]+=491. 1H NMR (300 MHz, DMSO-d6) d 7.41 (d,
2H), 7.77 (b s, 1H), 7.86 (d, 2H), 8.27 (s, 2H), 8.52 (d, 1H),
9.29 (d, 1H), 10.15 (s, 1H), 10.28 (s, 1H), 10.44 (s, 1H).