Substituted N-phenylisothioureas: Potent inhibitors of human nitric oxide synthase with neuronal isoform selectivity

Article abstract of DOI:10.1021/jm960785c

S-Ethyl N-phenylisothiourea (4) has been found to be a potent inhibitor of both the human constitutive and inducible isoforms of nitric oxide synthase. A series of substituted N-phenylisothiourea analogues was synthesized to investigate the structure-activity relationship of this class of inhibitor. Each analogue was evaluated for human isoform selectivity. One analogue, S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (39), exhibited 115-fold and 29-fold selectivity for the neuronal isoform versus the inducible and endothelial derived constitutive isoforms, respectively. Studies have shown the substituted N-phenylisothiourea 39 binds competitively with L,-arginine.

Full text of DOI:10.1021/jm960785c

J . Med. Chem. 1997, 40, 1901-1905
1901
Su bstitu ted N-P h en ylisoth iou r ea s: P oten t In h ibitor s of Hu m a n Nitr ic Oxid e
Syn th a se w ith Neu r on a l Isofor m Selectivity
Barry G. Shearer,*,† Shuliang Lee,† J effrey A. Oplinger,† Lloyd W. Frick,‡ Edward P. Garvey,§ and
Eric S. Furfine§
Glaxo Wellcome Research and Development, Five Moore Drive, Research Triangle Park, North Carolina 27709
Received November 12, 1996X
S-Ethyl N-phenylisothiourea (4) has been found to be a potent inhibitor of both the human
constitutive and inducible isoforms of nitric oxide synthase. A series of substituted N-
phenylisothiourea analogues was synthesized to investigate the structure-activity relationship
of this class of inhibitor. Each analogue was evaluated for human isoform selectivity. One
analogue, S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (39), exhibited 115-fold and 29-fold
selectivity for the neuronal isoform versus the inducible and endothelial derived constitutive
isoforms, respectively. Studies have shown the substituted N-phenylisothiourea 39 binds
competitively with L-arginine.
In tr od u ction
Nitric oxide (NO) has been recognized as an important
mediator of diverse physiological processes including
blood pressure homeostasis, platelet aggregation, neu-
rotransmission, and immunological defense mecha-
nisms.1 The endogenous synthesis of NO is derived
from the oxidation of L-arginine to L-citrulline. The
L-arginine-nitric oxide pathway is mediated by a family
of enzymes known as nitric oxide synthase (NOS).2
Structurally distinct NOS enzymes have been identified
which are generally classified as constitutive (cNOS) or
inducible (iNOS) isoforms based on mechanism of
regulation.3 Two distinct constitutive NOS isoforms
have been distinguished in the vascular endothelium
(eNOS) and in the brain (nNOS). All NOS isoforms are
heme-containing enzymes sharing similiar cofactor
requirements for NADPH, FAD, FMN, and tetrahydro-
biopterin for activity. The constitutive NOSs, however,
are tightly regulated by Ca2+ concentrations and are
responsible for the low, transient bursts of NO genera-
tion involved in cellular communication processes. In
contrast, iNOS is regulated transcriptionally and is
expressed in response to various cytokines and endo-
toxin. The activation of iNOS results in prolonged
elevated levels of NO production.
The distinct roles demonstrated by NO in normal
physiology are attributed to the different NOS isoforms.
The uncontrolled production of NO, however, has been
implicated in numerous disease states4 including septic
shock,5,6 inflammatory arthritis,7 neurodegenerative
diseases,8 chronic ileitus,9 and insulin-dependent dia-
betes mellitus.10 Thus, selective NOS isoform inhibition
to regulate NO synthesis has potential therapeutic
benefits.
F igu r e 1. Arginine-based NOS inhibitors.
F igu r e 2. Structure of S-ethyl N-phenylisothiourea.
biological processes. The majority of described inhibi-
tors are modified arginine analogues. One such ana-
logue, NG-methyl-L-arginine (L-NMA, 1), has been shown
to normalize blood pressure in animal models of cyto-
kine-induced and septic shock.11,12 Recently, we and the
laboratories of Narayanan and Griffith independently
reported the amino acid derived S-methyl and S-ethyl
isothiourea derivatives 2 and 3 (Figure 1) as a new class
of potent NOS inhibitor.13,14
Prior to these reports, we discovered a class of
structurally unrelated isothiourea analogues to be
potent NOS inhibitors. We found simple S-alkyl isothio-
ureas to be potent NOS inhibitors during our initial
search for isoform selective NOS inhibitors.15 Nakane
and co-workers have also reported independent detailed
studies identifying S-ethyl isothiourea as a potent and
selective inhibitor of iNOS.16 Further structural inves-
tigation of our compound series identified S-ethyl N-
phenylisothiourea (4) (Figure 2) as a potent NOS
inhibitor exhibiting selectivity for the neuronal iso-
form.17 This selectivity differs from the iNOS selectivity
observed for simple S-alkyl isothioureas. We initiated
synthetic efforts to investigate the potential of this class
of nonamino acid based inhibitor.18 This communication
reports the synthesis of a limited series of substituted
N-phenylisothiourea analogues and their ability to
inhibit purified human NOS isoforms.
Despite the immense interest in nitric oxide, disclo-
sures of novel NOS inhibitors have been limited. In-
hibitors with high isoform selectivity are required to
further define the role of each NOS isoform in various
* Address correspondence to: Barry G. Shearer, Department of
Medicinal Chemistry, Glaxo Wellcome Research and Development,
Five Moore Drive, Research Triangle Park, NC 27709. Tel: (919) 483-
9860. FAX: (919) 315-0430. E-mail: shearer∼bg@glaxo.com.
† Department of Medicinal Chemistry.
‡ Division of Bioanalysis and Drug Metabolism.
§ Department of Molecular Biochemistry.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00785-6 CCC: $14.00 © 1997 American Chemical Society

1902 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Shearer et al.
Sch em e 1a
a
(a) PhCONCS, acetone, room temperature; (b) NaOH, THF-H2O, reflux; (c) R′I, acetone, reflux; (d) (i) NaHCO3, Et2O-H2O; (ii) HCl,
Et2O.
Sch em e 2a
similiar to SAR evidence reported for simple alkyl
isothioureas.15 Second, N-methylation of either isothio-
urea nitrogen as in derivatives 13 and 16 (Scheme 2)
abolishes NOS inhibition. The role of the isothiourea
NH groups in hydrogen-bonding interactions may be
crucial for NOS inhibition. Steric and conformational
considerations, however, cannot be dismissed. Ad-
ditionally, the N-phenyl ring is a key component for
potent inhibition. Heteroaromatic replacement of the
phenyl group with a pyridyl ring results in a significant
decrease in NOS inhibition as evidenced by analogues
47-49. Despite their reduced potency, these com-
pounds retain selectivity for the neuronal isoform.
Further SAR investigation of the phenyl ring led to
enhanced nNOS selectivity. The improvement in se-
lectivity was achieved by selectively decreasing both
iNOS and eNOS inhibition potency without markedly
effecting nNOS activity. Para substitution of the aro-
matic ring has the greatest influence upon selectivity.
The inducible enzyme appears to be the most sensitive
isoform to N-phenylisothioureas structurally modified
at the p-phenyl position. The unsubstituted N-phenyl-
isothiourea analogue 4 is a potent inhibitor of all three
isoforms (Ki < 1 µM) displaying highest activity toward
nNOS. In general, those analogues possessing a para
electron-withdrawing group (EWG) exhibit a significant
decrease in iNOS inhibition. This trend is supported
by the weak iNOS inhibition (Ki >35 µM) displayed by
the p-OCF3, p-CO2Et, p-CF3, and p-NO2 derivatives 25,
29, 39, and 45, respectively. The inhibition of eNOS
also decreased (Ki > 1 µM) but to a lesser extent than
observed for iNOS. In contrast, the inhibition of nNOS
by these para-EWG-substituted S-ethyl N-phenylisothio-
ureas remained submicromolar with the exception of
analogue 29.
a
(a) PhCONCS, THF, 0 °C to room temperature; (b) K2CO3,
EtOH-H2O, reflux; (c) EtI acetone, reflux; (d) CH3NCS, EtOH,
reflux.
Ch em istr y
The synthesis of our general series is outlined in
Scheme 1. Many of the N-phenylthioureas employed
in this study were available from commercial sources.
The remaining thioureas were readily accessible from
substituted anilines 5. Treatment of substituted anilines
5 with benzoyl isothiocyanate and subsequent hydroly-
sis of the benzoyl group provided the requisite thioureas
7.19 Sulfur alkylation of the substituted thioureas 7
with alkyl iodides efficiently afforded the targeted
substituted N-phenylisothioureas 8. Crystallization of
the isothiourea hydroiodide salts, however, often proved
to be difficult. Purification was simplified by conversion
to the corresponding hydrochloride salts 9 as illustrated
in Scheme 1. Additionally, the N,S-disubstituted N-
phenylisothiourea analogues 13 and 16 were synthe-
sized in a similiar manner as depicted in Scheme 2.
Incorporation of electron-releasing groups (ERGs)
para to the isothiourea functionality also influences
inhibition selectivity as exemplified by comparing com-
pounds 24, 26-28, and 32-35 with unsubstituted
analogue 4. These modified derivatives display a de-
crease in nNOS inhibition similiar in magnitude to the
para-EWG-substituted derivatives. The magnitude of
decrease in eNOS and iNOS inhibition for these ana-
logues, however, was significantly lower relative to the
observed inhibitions of the para-EWG-substituted de-
rivatives. As a result, the effect of the para-ERGs upon
selectivity in this reported series was less pronounced
than those observed for the analogues possessing EWGs.
Resu lts a n d Discu ssion
The ability of substituted N-phenylisothioureas to
inhibit human NOS isoforms was determined by moni-
toring the conversion of L-[14C]arginine to L-[14C]citrul-
line as previously described.15,20-23 Table 1 summarizes
these results. Several general structure-activity rela-
tionship (SAR) observations regarding these analogues
can be derived from evaluation of the results presented
in Table 1. First, inhibition potency is directly related
to the steric bulk of the S-alkyl substituent. Optimal
potency is observed for the S-ethyl isothioureas. The
analogous S-methyl isothioureas 20, 38, and 44 display
a decrease in inhibition potency, and compounds 40-
43 show inhibition rapidly diminishes with larger or
branched alkyl substitution on sulfur. This trend is
Steric factors associated with p-alkyl substituents also
affect inhibition potency. Replacement of the small
p-methyl substituent in compound 32 with the larger
branched isopropyl or cyclohexyl groups to give ana-

Substituted N-Phenylisothioureas
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1903
Ta ble 1. Substituted N-Phenylisothioureas and Their Inhibition of Human NOSa
Ki, (µM)
selectivityc
compdb
R
R′
X
mp, °C
100
87-90
95-96
172-174
154-157
75-78
75-77
127-128
96-97
iNOS
eNOS
nNOS
iNOS/ nNOS
7.3
19
17
5.3
eNOS/ nNOS
4d
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
H
Et
Et
Et
Et
I
I
0.87
4.7
2.9
2.4
42
4.2
1.4
3.2
4.9
55
0.40
2.0
1.8
1.8
2.6
0.9
1.6
3.1
2.7
17
0.12
0.25
0.17
0.45
0.57
0.14
0.17
0.56
0.29
0.70
0.19
0.21
0.34
1.6
58
11
0.18
1.1
0.33
1.5
3.3
8.0
11
4.0
4.6
6.4
9.4
5.5
9.3
24
2-Br
2-Cl
3-Cl
4-Cl
4-Cl
Cl
Cl
I
Cl
Cl
I
I
Cl
I
I
I
I
I
Me
Et
74
30
8.2
5.7
16
79
16
31
7.4
39
2-OCH3
3-OCH3
4-OCH3
4-OCF3
4-OPh
4-OCH2Ph
4-OH
4-CO2Et
4-CO2H
3-CO2H
4-CH3
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
140-143
172-174
135
3.1
6.6
2.5
63
4.0
2.8
2.7
12
21
13
7.9
7.5
80-85
214-215
149-152
138-141
66-68
0%e
44
14%e
14
I
4.0
11
9.1
76
19
16
1.3
5.6
5.9
23
9.3
13
26
6.7
29
Cl
Cl
Cl
I
Cl
Cl
I
Cl
Cl
Cl
Cl
Cl
I
1.9
10
25
28
22
3.2
0%e
37
1.0
6.5
7.7
14
18
28
2-iPr
4-iPr
113-116
174-175
75-77
4-c-C6H11
2-CF3
1.4
1.1
4.6
0.32
64
3-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-NO2
102
2.9
Me
Et
125-127
126-127
169-170
67-70
151
71-72
31
9.4
0%e
0%e
0%e
0%e
17
9.1
9.0
17
115
nPr
iPr
Bu
CH2Ph
Me
Et
4%e
0%e
0%e
0%e
0%e
54
29
23
23
22
29
0%e
11%e
3.3
195-198
161-162
138-141
127-128
96-97
5.2
14
6.4
3.5
7.0
23
4-NO2
I
I
I
Cl
f
0.66
1.4
4.8
1.0
0.8
82
21
4.8
23
28
4-N(CH3)2
Ar ) 2-pyridyl
Ar ) 3-pyridyl
Ar ) 4-pyridyl
Et
Et
Et
Et
7.0
16
138-141
a
Inhibition constants were obtained by measuring percent inhibition with at least three concentrations of inhibitor as described in ref
b
c
15. Values had a standard deviation of e10% (n g 3). All compounds gave satisfactory analyses for C, H, N, S, X ((0.4%). Defined as
the ratio of Ki iNOS or Ki eNOS to Ki nNOS. Purchased from Aldrich Chemical Co. e Percent inhibition using 25 µM of test compound.
d
f Not isolated as a HX salt.
logues 34 and 35 resulted in reduced inhibition of all
three isoforms.
For a given substituent, para substitution imparts the
greatest selectivity as shown by comparison of the
trifluoromethyl analogues 36, 37, and 39, methoxy
derivatives 22-24, and isopropyl analogues 33 and 34.
The p-CF3 analogue 39 proved to be the the most
selective compound. The selectivity of this inhibitor for
nNOS versus iNOS and eNOS is 115-fold and 29-fold,
respectively.
The nNOS binding of the p-CF3 analogue 39 was
competitive with L-arginine as illustrated in Figure 3.
The equation V ) Vmax[S]/{[S] + Km(1 + [I]/Ki)} was
fitted to the data to give Km ) 2 µM and Ki ) 0.12 µM.
This result suggests that the enzyme-binding interac-
tions of compound 39 and structurally related N-
phenylisothioureas occur within the L-arginine binding
F igu r e 3. Competitive inhibition of nNOS by compound 39.
Velocity of nNOS catalyzed conversion of L-arginine to L-
citrulline in the presence of 0, 150, and 300 nM concentrations
of 39 was determined as described in ref 22. The solid lines
site.
The potent and selective analogue 39 was selected for
in vivo evaluation. The pharmacokinetic properties and
the ability of isothiourea 39 to inhibit nNOS in intact
represent the fit of velocity ) Vmax[arginine]/{[arginine] + Km-
cells within brain tissue were investigated as a prelimi-
(1 + [39]/Ki)} where Vmax ) 19000 ( 3000 pmol/min, Km ) 2.0
( 0.6 µM, and Ki ) 0.12 ( 0.03 µM.
nary predictor of potential in vivo efficacy. When

1904 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Shearer et al.
CH2Cl2. The combined organic layers were washed with brine
and dried over MgSO4. Removal of solvent at reduced pressure
and recrystallization from ethyl acetate-hexane afforded 3.77
g (70%) of the desired thiourea 7 (R ) 4-OCF3) as a white
administered intravenously to mice at 25 mg/kg, com-
pound 39 was found to readily penetrate the blood-
brain barrier, immediately reaching concentrations in
the brain equal to the plasma concentration. The ratio
of brain to plasma concentration essentially remained
equivalent throughout the experiment. This compound,
however, was rapidly cleared (5.6 (L/kg)/h) in a biphasic
manner (t1/2R ) 4 min and t1/2â ) 31 min), and several
unidentified plasma metabolites were observed im-
mediately after dosing. Compound 39 disappointingly
failed to inhibit nNOS activity in rat brain slices
employing previously described assay methods.14 Other
potent isothiourea inhibitors of purified NOS enzymes
have been shown to exhibit significantly decreased inhi-
bition potency in whole cell assays due to poor cellular
uptake.15 The efficiency of substituted N-phenylisothio-
ureas to penetrate into specific cells is unknown and
may be a contributing factor to the result observed for
compound 39. Further detailed studies are required.
On the basis of the above results, the in vivo evaluation
of these N-phenylisothioureas was not pursued.
In summary, substituted N-phenylisothioureas are
potent, competitive inhibitors of human NOS displaying
selectivity for the neuronal isoform. Preliminary SAR
evidence indicates that the constitutive isoforms nNOS
and eNOS are more tolerable than iNOS toward struc-
tural modifications to this class of inhibitor. As a result,
greater nNOS versus iNOS selectivity than nNOS
versus eNOS selectivity was achieved. The further
development of these and related agents for in vivo
therapeutic evaluation is in progress.
1
solid: mp 136-137 °C; 200 MHz H NMR (DMSO-d6) δ 7.33
(2H, d, J ) 9.0 Hz), 7.57 (2H, d, J ) 9.0 Hz), 9.81 (1H, bs).
Anal. (C8H17N2OSF3) C, H, N, S.
Gen er a l P r oced u r e for th e P r ep a r a tion of Su bstitu ted
N-P h en ylisoth iou r ea Hyd r oiod id es 8. The procedure for
S-eth yl-N-(4-p h en oxyp h en yl)isoth iou r ea (26) is represen-
tative. To a stirred solution of 1-(4-phenoxyphenyl)thiourea
(7, R ) 4-OPh) (2.00 g, 8.19 mmol) in 20 mL of acetone was
added iodoethane (7.61 g, 48.7 mmol). The mixture was heated
to reflux for 3 h, cooled to room temperature, and concentrated
at reduced pressure, giving a viscous oil. Crystallization from
acetone-pentane afforded 2.00 g (61%) of the desired N-
phenylisothiourea hydroiodide 26 as a beige solid. mp 140-
143 °C; 200 MHz 1H NMR (DMSO-d6) δ 1.34 (3H, t, J ) 7.4
Hz), 3.29 (2H, q, J ) 7.4 Hz), 7.17 (5H, m), 7.42 (4H, m). Anal.
(C15H16N2OS‚HI) C, H, N, S, I.
Gen er a l P r oced u r e for th e P r ep a r a tion of Su bstitu ted
N-P h en ylisoth iou r ea Hyd r och lor id es 9. The procedure for
S-eth yl-N-[4-(tr iflu or om eth oxy)p h en yl]isoth iou r ea (25)
is representative. To a stirred solution of 1-[4-(trifluoromethox-
y)phenyl]thiourea (7, R ) 4-OCF3) (3.00 g, 12.7 mmol) in 100
mL of acetone was added iodoethane (5.85 g, 37.5 mmol). The
mixture was heated to reflux and left overnight. After being
cooled to room temperature, the mixture was concentrated at
reduced pressure, giving an oil. This oil was dissolved in water
and washed with pentane. The aqueous layer was poured into
saturated aqueous NaHCO3 (75 mL) and extracted with Et2O.
The organic layer was dried over MgSO4, concentrated to a
volume of approximately 200 mL, and treated with 1 N HCl
in Et2O (15.0 mL, 15.0 mmol). After being stirred for 20 min,
the mixture was concentrated at reduced pressure and placed
in vacuo overnight to give a gummy foam. Trituration with
pentane provided 3.35 g (88%) of the desired N-phenylisothio-
urea hydrochloride 25 as a white solid: mp 96-97 °C; 200 MHz
1H NMR (D2O) δ 1.38 (3H, t, J ) 7.4 Hz), 3.20 (2H, q, J ) 7.4
Hz), 7.44 (4H, s). Anal. (C10H11N2OSF3‚HCl) C, H, N, S, Cl.
1-Ben zoyl-3-m eth yl-3-p h en ylth iou r ea (11). To a stirred,
cooled (0 °C) solution of N-methylaniline (10.7 g, 100 mmol)
in 60 mL of THF was added benzoyl isothiocyanate (19.6 g,
120 mmol). The mixture was allowed to warm to room tem-
perature while stirring overnight and concentrated at reduced
pressure giving a solid. Recrystallization from ethyl acetate-
hexane afforded 22.7 g (84%) of 11 as a white solid: mp 136-
137 °C; 200 MHz 1H NMR (DMSO-d6) δ 3.69 (3H, s), 7.19-
7.66 (11H, m), 10.8 (1H, bs). Anal. (C15H14N2OS) C, H, N, S.
1-Meth yl-1-p h en ylth iou r ea (12). To a stirred solution of
1-benzoyl-3-methyl-3-phenylthiourea (11) (10.0 g, 37.0 mmol)
in EtOH (120 mL) was added 1.23 M aqueous K2CO3 (60 mL,
73.8 mmol). The resulting yellow mixture was heated to
reflux. After 24 h, the mixture was cooled to room tempera-
ture, concentrated at reduced pressure, and extracted with
EtOAc. The organic layer was dried over MgSO4. Solvent was
removed at reduced pressure and the crude product was
recrystallized from ethyl acetate-hexane to afford 600 mg
(10%) of pure 12 as a white solid: mp 98-103 °C; 200 MHz
1H NMR (DMSO-d6) δ 3.47 (3H, s), 7.26-7.68 (5H, m). Anal.
(C8H10N2S) C, H, N, S.
Exp er im en ta l Section
Solvents and reagents were reagent grade and used without
further purification. Melting points were determined on a
Thomas-Hoover capillary melting point apparatus and are
uncorrected. All 1H NMR spectra were recorded on Varian
Unity 300 MHz or Varian Gemini 200 MHz spectrometers.
Chemical shifts (δ) are reported downfield from tetramethyl-
silane (Me4Si) in parts per million (ppm) of the applied field.
Peak multiplicities are abbreviated: singlet, s; broad singlet,
bs; doublet, d; triplet, t; quartet, q; multiplet, m. Coupling
constants (J values) are reported in hertz. Analytical thin-
layer chromatography (TLC) was used to monitor reactions.
Plates (2.5 × 10 cm) precoated with silica gel GHLF of 0.25-
mm thickness, supplied by Analtech, were used. Combustion
microanalyses were performed by Atlantic Microlab, Inc.,
Norcross, GA.
Gen er a l P r oced u r e for th e P r ep a r a tion of Su bstitu ted
1-Ben zoyl-3-p h en ylth iou r ea s 6. The procedure for 1-ben -
zoyl-3-[4-(t r iflu or om et h oxy)p h en yl]t h iou r ea (6,
R )
4-OCF 3) is representative. To a stirred solution of 4-(trifluo-
romethoxy)aniline (5.24 g, 29.6 mmol) in 100 mL of acetone
was added benzoyl isothiocyanate (5.46 g, 33.5 mmol). After
being stirred overnight, the mixture was concentrated at
reduced pressure, giving a yellow solid. Recrystallization from
ethyl acetate-hexane afforded 9.29 g (92%) of the desired
substituted phenylthiourea as a pale yellow solid: mp 124-
125 °C; 200 MHz 1H NMR (DMSO-d6) δ 7.39-7.73 (5H, m),
7.83 (2H, d, J ) 9.0 Hz), 8.01 (2H, d, J ) 7.2 Hz), 11.67 (1H,
s,), 12.60 (1H, s). Anal. (C15H11N2O2SF3) C, H, N, S.
S-Eth yl-1-m eth yl-1-ph en ylisoth iou r ea Hydr oiodide (13).
To a stirred solution of 1-methyl-1-phenylthiourea (12) (1.89
g, 11.4 mmol) in 20 mL of acetone was added iodoethane (5.90
g, 34.1 mmol). The mixture was heated to reflux for 4 h, cooled
to room temperature, and concentrated at reduced pressure,
giving a viscous oil. Crystallization from acetone-pentane
afforded 3.10 g (86%) of the desired N-phenylisothiourea
hydroiodide 13 as a white solid: mp 135-137 °C; 200 MHz
1H NMR (DMSO-d6) δ 1.24 (3H, t, J ) 7.4 Hz), 3.16 (2H, q, J
) 7.4 Hz), 3.49 (3H, s), 7.54 (5H, m), 9.24 (1H, bs). Anal.
(C10H14N2S‚HI) C, H, N, S, I.
Gen er a l P r oced u r e for th e P r ep a r a tion of Su bstitu ted
N-P h en ylth iou r ea s 7. The procedure for N-[4-(tr iflu o-
r om eth oxy)p h en yl]th iou r ea (7, R ) 4-OCF 3) is represen-
tative. To a stirred solution of 1-benzoyl-3-[4-(trifluoromethoxy)-
phenyl]thiourea (6, R ) 4-OCF3) (7.80 g, 22.9 mmol) in 100
mL of THF was added 2.0 N aqueous NaOH (25.0 mL, 50.0
mmol). The mixture was heated to reflux for 3 h, cooled to
room temperature, and concentrated at reduced pressure. The
residue was suspended in water and extracted repeatedly with
1-Meth yl-3-p h en ylth iou r ea (15). To a stirred solution of
aniline (5.11 g, 54.9 mmol) in 100 mL of EtOH was added
methyl isothiocyanate (4.70 g, 64.3 mmol). The mixture was
heated to reflux overnight, cooled to room temperature, and

Substituted N-Phenylisothioureas
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1905
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Class of Potent Nitric Oxide Synthase Inhibitors. J . Med. Chem.
1994, 37, 885-887.
(14) Furfine, E. S.; Harmon, M. F.; Paith, J . E.; Knowles, R. G.; Salter,
M.; Kiff, R. J .; Duffy, C.; Hazelwood, R.; Oplinger, J . A.; Garvey,
E. P. Potent and Selective Inhibition of Human Nitric Oxide
Synthases. Selective Inhibition of Neuronal Nitric Oxide Syn-
thase By S-Methyl-L-Thiocitrulline and S-Ethyl-L-Thiocitrulline
J . Biol. Chem. 1994, 269, 26677-26683.
(15) Garvey, E. P.; Oplinger, J . A.; Tanoury, G. J .; Sherman, P. A.;
Fowler, M.; Marshall, S.; Harmon, M. F.; Paith, J . E.; Furfine,
E. S. Potent and Selective Inhibition of Human Nitric Oxide
Synthases. J . Biol. Chem. 1994, 269, 26669-26676.
(16) Nakane, M.; Klinghofer, V.; Kuk, J . E.; Donnelly, J . L.; Budzik,
G. P.; Pollock, J . S.; Basha, F.; Carter, G. W. Novel Potent and
Selective Inhibitors of Inducible Nitric Oxide Synthase. Mol.
Pharmacol. 1995, 47, 831-834.
concentrated at reduced pressure, giving a dark yellow solid.
Recrystallization from EtOH afforded 4.32 g (47%) of the
desired thiourea 15 as a white solid. The mother liquor was
concentrated at reduced pressure and recrystallized from ethyl
acetate-hexane to provide an additional 3.70 g (41%) of 15 as
1
a white solid: 200 MHz H NMR (DMSO-d6) δ 2.93 (3H, bs),
7.14 (1H, m), 7.36 (4H, m), 7.71 (1H, bs), 9.54 (1H, bs).
S-Eth yl-1-m eth yl-3-ph en ylisoth iou r ea Hydr oiodide (16).
Compound 16 was prepared from 1-methyl-3-phenylthiourea
(15) (1.60 g, 9.62 mmol) and iodoethane (1.76 g, 11.3 mmol)
as described for compound 13 to afford 2.77 g (89%) of a white
solid: mp 134 °C; 300 MHz 1H NMR (D2O) δ 1.28 (3H, bs),
3.06 (5H, bs), 7.34 (2H, m), 7.48 (3H, m). Anal. (C10H14N2S‚
HI) C, H, N, S, I.
Ack n ow led gm en t. The authors thank Mark Salter
(Stevenage, U.K.) for rat brain slice data.
(17) The weak inhibition of rat iNOS by S-methyl N-phenylisothio-
urea was reported during the course of this work: Southan, G.
J .; Szabo, C.; Thiemermann, C. Isothioureas: Potent Inhibitors
of Nitric Oxide Synthases with Variable Isoform Selectivity. Br.
J . Pharmacol. 1995, 111, 510-516.
(18) The following patents describing other classes of substitututed
N-phenyl derived NOS inhibitors appeared after completion of
this work: Cowart, M.; Kerwin, J . F. 2-Nitroaryl and 2-Cy-
anoaryl Compounds as Regulators of Nitric Oxide Synthase. WO
9412163. MacDonald, J . E.; Gentile, R. J .; Murray, R. J .
Guanidine Derivatives Useful in Therapy. WO 9421621. Gentile,
R. J .; Murray, R. J .; MacDonald, J . E. Amidine Derivatives with
Nitric Oxide Synthetase Activities. WO 9505363.
(19) Rasmussen, C. R.; Villani, F. J .; Weaner, L. E.; Reynolds, B. E.;
Hood, A. R.; Hecker, L. R.; Nortey, S. O.; Hanslin, A.; Costanzo,
M. J .; Powell, E. T.; Milinari, A. J . Improved Procedures for the
Preparation of Cycloalkyl-, Arylalkyl-, and Arylthioureas. Syn-
thesis 1988, 456-459.
(20) Human iNOS was purified from the human colorectal adeno-
carcinoma cell line DLD-1 as described in the following: Sher-
man, P. A.; Laubach, V. E.; Reep, B. R.; Wood, E. R. Purification
and cDNA Sequence of an Inducible Nitric Oxide Synthase from
a Human Tumor Cell Line. Biochemistry 1993, 32, 11600-
11605.
(21) Human eNOS was purified from human placental as described
in the following: Garvey, E. P.; Tuttle, J . V.; Covington, K.;
Merrill, B. M.; Wood, E. R.; Baylis, S. A.; Charles, I.G. Purifica-
tion and Characterization of the Constitutive Nitric Oxide
Synthase from Human Placenta. Arch. Biochem. Biophys. 1994,
311, 235-241.
(22) Human nNOS was purified as described for bovine brain nNOS
as reported in the following: Furfine, E. S.; Harmon, M. F.;
Paith, J . E.; Garvey, E. P. Selective Inhibition of Constitutive
Nitric Oxide Synthase by L-NG-Nitroarginine. Biochemistry
1993, 32, 8512-8517.
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