Inhibition of mast cell leukotriene release by thiourea derivatives

Article abstract of DOI:10.1016/S0960-894X(02)00992-7

Mast cell derived leukotrienes (LT's) play a vital role in pathophysiology of allergy and asthma. We synthesized various analogues of indolyl, naphthyl and phenylethyl substituted halopyridyl, thiazolyl and benzothiazolyl thioureas and examined their in vitro effects on the high affinity IgE receptor/FcERI-mediated mast cell leukotriene release. Of the 22 naphthylethyl thiourea compounds tested, there were seven active compounds and N-[1-(1-naphthyl)ethyl]-N′-[2-(ethyl-4-acetylthiazolyl)]thiourea (17 and 16) (IC₅₀=0.002 μM) and N-[1-(1R)-naphthylethyl]-N′-[2-(5-methylpyridyl)]thiourea (5) (IC₅₀=0.005 μM) were identified as the lead compounds. Among the 11 indolylethyl thiourea compounds tested, there were seven active compounds and the halopyridyl compounds N-[2-(3-indolylethyl)]-N′-[2-(5-chloropyridyl)]thiourea and N-[2-(3-indolylethyl)]-N′-[2-(5-bromopyridyl)]thiourea were the most active agents and inhibited the LTC₄ release with low micromolar IC₅₀ values of 4.9 μM and 6.1 μM, respectively. The hydroxylphenyl substituted compounds N-[2-(4-hydroxyphenyl)ethyl]-N′-[2-(5-chloropyridyl)]thiourea (IC₅₀=12.6 μM), N-[2-(4-hydroxyphenyl)ethyl]-N′-[2-(5-bromopyridyl)]thiourea (IC₅₀=16.8 μM) and N-[2-(4-hydroxyphenyl)ethyl]-N′-[2-(pyridyl)]thiourea (IC₅₀=8.5 μM) were the most active pyridyl thiourea agents. Notably, the introduction of electron withdrawing or donating groups had a marked impact on the biological activity of these thiourea derivatives and the Hammett sigma values of their substituents were identified as predictors of their potency. In contrast, experimentally determined partition coefficient values did not correlate with the biological activity of the thiourea compounds which demonstrates that their liphophilicity is not an important factor controlling their mast cell inhibitory effects.

Full text of DOI:10.1016/S0960-894X(02)00992-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 485–488
Inhibition of Mast Cell Leukotriene Release by Thiourea
Derivatives
Taracad K. Venkatachalam,a Sanjive Qazi,b Peter Samuelc and Fatih M. Uckund,*
aDepartment of Chemistry, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
bDepartment of Immunology, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
cDepartment of Virology, Parker Hughes Institute, 2699 Patton Road, Roseville, MN 55113, USA
dDrug Discovery Program, Parker Hughes Cancer Center, 2848 Patton Road, Roseville, MN 55113, USA
Received 19April 2002; accepted 9October 2002
Abstract—Mast cell derived leukotrienes (LT’s) play a vital role in pathophysiology of allergy and asthma. We synthesized various
analogues of indolyl, naphthyl and phenylethyl substituted halopyridyl, thiazolyl and benzothiazolyl thioureas and examined their
in vitro effects on the high affinity IgE receptor/FcERI-mediated mast cell leukotriene release. Of the 22 naphthylethyl thiourea
compounds tested, there were seven active compounds and N-[1-(1-naphthyl)ethyl]-N0-[2-(ethyl-4-acetylthiazolyl)]thiourea (17 and
16) (IC50=0.002 mM) and N-[1-(1R)-naphthylethyl]-N0-[2-(5-methylpyridyl)]thiourea (5) (IC50=0.005 mM) were identified as the
lead compounds. Among the 11 indolylethyl thiourea compounds tested, there were seven active compounds and the halopyridyl
compounds N-[2-(3-indolylethyl)]-N0-[2-(5-chloropyridyl)]thiourea and N-[2-(3-indolylethyl)]-N0-[2-(5-bromopyridyl)]thiourea were
the most active agents and inhibited the LTC4 release with low micromolar IC50 values of 4.9 mM and 6.1 mM, respectively. The
hydroxylphenyl substituted compounds N-[2-(4-hydroxyphenyl)ethyl]-N0-[2-(5-chloropyridyl)]thiourea (IC50=12.6 mM), N-[2-(4-
hydroxyphenyl)ethyl]-N0-[2-(5-bromopyridyl)]thiourea (IC50=16.8 mM) and N-[2-(4-hydroxyphenyl)ethyl]-N0-[2-(pyridyl)]thiourea
(IC50=8.5 mM) were the most active pyridyl thiourea agents. Notably, the introduction of electron withdrawing or donating groups
had a marked impact on the biological activity of these thiourea derivatives and the Hammett sigma values of their substituents
were identified as predictors of their potency. In contrast, experimentally determined partition coefficient values did not correlate
with the biological activity of the thiourea compounds which demonstrates that their liphophilicity is not an important factor
controlling their mast cell inhibitory effects.
# 2002 Elsevier Science Ltd. All rights reserved.
Mast cells participate in the pathophysiology of allergy
and asthma through the release of chemical mediators,
including the pro-inflammatory leukotrienes (LTs) after
crosslinking of their high affinity surface IgE receptors/
FceRI.1ꢀ4 Repeated stimulation of mast cells in patients
with allergic asthma may cause sustained synthesis and
release of LTs contributing to the significant and per-
sistent bronchoconstriction and inflammatory airway
response during episodes of exacerbation. In recent
years, several strategies aimed at inhibiting the synthesis
and release of leukotrienes (e.g., use of 5-LO inhibitors)
or blocking their action at the receptor level (e.g., use of
specific LTD4 antagonists) have been explored as treat-
ment modalities for asthma.5ꢀ8 The purpose of the pre-
sent study was to examine members of our thiourea
compound library9ꢀ12 for mast cell inhibitory activity in
an attempt to identify thiourea compounds capable of
inhibiting FceRI-mediated LTC4 release from mast cells.
Naphthyl-, indolyl- and phenyl-substituted halo pyridyl,
thiazolyl and benzothiazolyl thiourea compounds were
synthesized according to Scheme 1.9 The chiral thiourea
derivatives were synthesized starting from their respec-
tive chiral amines and their chirality was confirmed
using optical rotation values. The structures of the
compounds were altered by changing the substituents
on the pyridyl, thiazolyl and benzothiazolyl rings. Halo
and methyl substitutions were chosen to determine the
effects of electron withdrawing and donating groups,
respectively, on the mast cell inhibitrory activity.
Table 1 shows four different classes of thiourea com-
pounds that were tested for biological activity. The total
number of active compounds and the total number of
*Corresponding author. Tel.: +1-651-796-5450; fax: +1-651-796-
5493; e-mail: fatih_uckun@ih.org
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00992-7

486
T. K. Venkatachalam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 485–488
Scheme 1.
Table 1.
pounds 5 and 7 were more potent than the mast cell inhib-
itory control dimethoxyquinazoline compound WHI-
P97.13 The most potent compound (5) in this series is a
eutomer while its enantiomer (6) is a distomer. Table 2(B)
shows the biological activity of chiral naphthyl thioureas
when the pyridyl ring is replaced with a thiazolyl or ben-
zothiazolyl moiety. The thiazolyl substituted compounds
with methyl groups inhibited LTC4 release from IgE/
antigen-stimulated mast cells at micromolar concentra-
tions. Notably, the ester analogues 16 and 17 exhibited
potent activity with low nanomolar IC50 values in the
nanomolar range.‘R’ and ‘S’ isomers of these thiazolyl-
substituted compounds were equally potent implying
that stereochemistry does not significantly affect their
biological activity. On the other hand, compounds 15
and 18 were found to be eutomers and compounds 14
and 19 were distomers. By comparison, benzothiazolyl-
substituted compounds showed only marginal activity.
Introduction of methyl or methoxy substituents on the
benzothiazolyl moiety eliminated the biological activity of
the parent compound. Of the 22 naphthylethyl thiourea
compounds tested, N-[1-(1-naphthylethyl]-N0-[2-(ethyl-4-
acetylthiazolyl)]thiourea (16 and 17) (IC50=0.002 mM)
and N-[1-(1R)-naphthylethyl]-N0-[2-(5-methylpyridyl)]thiourea
(5) (IC50=0.005 mM) were identified as the lead com-
pounds. Active inhibitors of LTC4 release were also
identified among indolylethyl thiourea compounds.
Thioureas Compounds
tested
Total number of
active compounds
Potent compounds
<0.005 mM activity
Class 1
Class 2
Class 3
Class 4
11
11
11
295
2
5
7
0
1
2
0
Class 1: chiral naphthyl ethyl pyridyl thioureas; Class 2: chiral naph-
thyl ethyl thiazolyl, benzothiazolyl thioureas; Class 3: indolyl ethyl
pyridyl, thiazolyl and benzothiazolyl thioureas; Class 4: phenylethyl
pyridyl, thiazolyl and benzothiazolyl thioureas.
Table 2. (A) Structure–activity of naphthyl pyridyl thioureas
Compd
Isomer
X
Activity in mM
1
2
R
S
5-Cl
5-Br
5-Br
5-Cl
5-Me
5-Me
>100
>100
>100
>100
0.005
>100
1.6
3
S
4
5
6
R
R
S
7
8
9
10
S
S
R
S
6-Me
4,6-diMe>100
4,6-diMe>100
H
H
—
>100
>100
6.8a
11
WHI-P97
R
—
Table 1 class 3 compounds show the biological activity
observed for indolyl ethyl-substituted thioureas. In the
pyridyl substituted compounds, halo substitution pro-
duced active compounds. The most active compound in
the series was the chloropyridyl substituted thiourea
compound. Thiazolyl-substituted compounds were also
active albeit with less potency. Interestingly, the ester
analogue of thiazolyl substituted compound showed
higher potency. Benzothiazolyl compounds in this series
were inactive.
(B) Structure–activity of naphthyl pyridyl thioureas
Compd
Isomer
Y
Z
Activity in mM
12
13
14
15
16
17
18
19
20
21
22
S
R
S
R
S
R
R
S
H
H
NA
NA
>100
>100
83.2
Me
Me
NA
NA
Table 1 class 4 compounds show the biological activity
obtained for 29differently substituted phenylethyl
thioureas. Only the hydroxy substituted compounds
exhibited mast cell inhibitory activity. Substituents such
as methyl, fluoro, bromo and chloro yielded inactive
compounds, irrespective of the position of substituent in
the phenyl ring. We also examined the activity of thia-
zolyl and benzothiazolyl substituted phenylethyl
thiourea compounds and found that none of the com-
pounds in the series showed potent biological activity.
Substitutions in the thiazolyl ring or the benzothiazolyl
ring did not improve their potency.
25.9
CH2COOEt
CH2COOEt
NA
NA
NA
H
0.002
0.002
18.9
NA
NA
NA
NA
H
>100
>100
>100
>100
S
R
R
4-Me
4-Me
6-OMe
potent compounds are shown in each class. Table 2(A)
shows the biological activity observed for chiral naph-
thyl thioureas. Except for the methyl substituted com-
pounds 5 (IC50=0.005 mM) and 7 (IC50=1.6 mM), none
of these compounds was active (IC50>100 mM). Com-
We also investigated the role played by various elec-
tron donating and electron withdrawing groups on

T. K. Venkatachalam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 485–488
487
leukotriene C4 release. A significant correlation was
Stimulation of Mast Cells and Mediator Assays
found for the indolyl ethyl substituted compounds.
The regression between Hammett and the IC50
values showed that introducing electron with-
drawing groups such as Br and Cl resulted in
more potent compounds that showed a statistically
RBL-2H3 cells were sensitized with a monoclonal
anti-dinitrophenyl (DNP) IgE antibody (0.24 mg/
mL) for 1 h at 37 ꢂC in a 48-well tissue culture
plate. Unbound IgE was removed by washing the
cells with phosphate buffered saline, pH 7.4. After
washing the BMMC were re-suspended in RPMI-
hepes buffer whereas PIPES-buffered saline contain-
ing 1 mM calcium chloride was added to the mono-
layers of the RBL-2H3 cells. In order to study the
biologic effects of the test compounds, sensitized mast
cells were further incubated with the test compounds at
the indicated concentrations (or vehicle alone) for 1 h.
The cells were then challenged with 20 ng/mL DNP-
BSA for 30 min at 37 ꢂC. The plates were centrifuged at
200g for 10 min at 4 ꢂC. Supernatants were removed
and saved. LTC4 levels were also determined in cell free
supernatants.13,14
significant relationship between
(R2=67%,
slope=ꢀ1.55ꢁ0.3,
df=13, p=0.0003).
s
and activity
t-ratio=ꢀ5.24,
In summary, among the three classes of thiourea
compounds studied for mast cell inhibitory activ-
ity, naphthyl substituted thiazolyl thioureas exhib-
ited the most potent activity followed by indolyl
ethyl and phenylethyl thiourea compounds. The
mast cell inhibitory activity shown by both ‘R’
and ‘S’ isomers of naphthyl thiourea compounds
demonstrate that a few derivatives were found to be
eutomers and distomers. In the case of indolyl ethyl
substituted thiourea series, the halopyridyl moiety was
found to be more beneficial than both thiazolyl and
benzothiazolyl units in the structure. Halo substitutions
on the pyridyl ring showed improved potency. In the
case of phenyl ethyl substituted thioureas, the hydroxy
substituent on the phenyl ring showed improved
potency compared to other substituents. In addition we
found that pyridyl ring in the structure was an essential
feature of these phenyl ethyl thiourea compounds.
Substituted halopyridyl, indolyl and naphthyl
thiourea compounds represent a new chemical class
of anti-allergic agents inhibiting IgE/FcRI receptor
mediated mast cell LTC4 release. The lead com-
Physicochemical properties of selected thiourea
compounds.
N-[1-(1R)-(1-Naphthylethyl)]-N-[2-(5-methylpyridyl)]-
thiourea (5). Mp 186–187 ꢂC; H NMR (DMSO-d6) d
1
12.22 (d, 1H, J=8.1 Hz), 10.59(s, 1H), 8.19(d, 1H,
J=8.1 Hz), 7.92 (d, 2H, J=8.4 Hz), 7.83 (d, 1H, J=7.8
Hz), 7.56–7.46 (m, 5H), 7.06 (d, 1H, J=8.4 Hz), 6.32 (t,
1H), 2.11 (s, 3H), 1.65 (d, 3H, J=6.9Hz); 13C NMR
(DMSO-d6) d 178.5, 152.0, 144.9, 139.9, 139.1, 133.6,
130.6, 128.9, 127.9, 127.0, 126.6, 125.8*, 123.4, 122.9,
112.4, 50.5, 21.6, 17.4; IR 3440, 3232, 3027, 2969, 1600,
1562, 1492, 1195, 798 cmꢀ1; UV (MeOH) 203, 222, 250,
pounds
N-[1-(1-naphthylethyl]-N0-[2-(ethyl-4-acetyl-
thiazolyl)]thiourea and N-[1-(1R)-naphthylethyl]-N0-[2-
(5-methylpyridyl)]thiourea are nanomolar inhibitors of
mast cell LTC4 release.
272,
294
nm;
MALDI-TOF
m/z
322.8
(C19H19N3S2+2H+). [a]ꢀ31.0; 5 HPLC Rt: 14.01
min;% purity 99.0; elemental analysis: calcd: C, 70.99;
H, 5.96; N, 13.07. Found: C, 71.04; H, 6.04; N, 13.17.
N-[1-(1S)-(1-Naphthylethyl)]-N0-[2-(ethyl-4-acetylthiazo-
lyl)]thiourea (16). Mp 107–109 ꢂC; 1H NMR (DMSO-d6)
d 11.54 (s, 1H), 10.10 (s, 1H), 8.15 (t, 1H), 7.96 (t, 1H),
7.86 (t, 1H), 7.61–7.50 (m, 4H), 6.89(s, 1H), 6.21 (s,
1H), 4.05–4.00 (m, 2H), 3.64 (s, 2H), 1.62 (d, 3H, J=3.9
Hz), 1.16–1.09(m, 3H); 13C NMR (DMSO-d6) d 177.2,
170.3, 161.5, 143.9, 139.3, 134.1, 131.0, 129.4, 128.4,
127.2, 126.5, 126.2, 123.7, 123.3, 109.9, 61.1, 50.5,
31.2, 21.9, 14.9; IR 3444, 3185, 3047, 2927, 1577,
1554, 1519, 1230 cmꢀ1; MALDI-TOF m/z 401.0
(C20H21N3O2S2+2H+); [a]: +22.8; HPLC Rt: 6.91
min;% purity 100.0, elemental analysis: calcd: C, 60.13;
H, 5.30; N, 10.52. Found: C, 60.06; H, 5.30; N, 10.50.
Statistical Analysis
The IC50 values were correlated with Hamett using a
linear regression model (JMP software, SAS institute
Inc, Cary, NC). Slope parameters were estimated and
the t-ratios compared to zero slope were calculated to
assess the statistical significance. p values of less than
0.05 were deemed significant.
Stimulation of Mast Cells
RBL-2H3 rat mast cells were sensitized with mono-
clonal anti DNP IgE antibody (0.24 mg/mL) for 1 h at
37ꢂ C in a 48-well tissue culture plate. Unbound IgE
was removed by washing the cells with PIPES-buffered
saline. After washing, PIPES-buffered saline containing
1 mM calcium chloride was added to the monolayers of
the RBL-2H3 cells. The cells were challenged with 20
ng/mL DNP-BSA for 30 min at 37 ꢂC. The plate was
centrifuged at 200g for 10 min at 4 ꢂC, Supernatants
were removed and saved. To study the effect of test
drugs, RBL-2H3 rat mast cells were incubated with the
drugs at the indicated concentrations or vehicle for 30
min prior to antigen challenge.
N-[1-(1R)-(1-Naphthylethyl)]-N0-[2-(ethyl-4-acetylthiazo-
lyl)]thiourea (17). Mp 105–107 ꢂC; 1H NMR (DMSO-d6)
d 11.54 (s, 1H), 10.09(s, 1H), 8.14 (d, 1H, J=7.8 Hz),
7.95 (d, 1H, J=7.8 Hz), 7.86 (t, 1H), 7.62–7.50 (m, 4H),
6.89(s, 1H), 6.20 (t, 1H), 4.02 (q, 2H), 3.63 (s, 2H), 1.62
(d, 3H, J=5.4 Hz), 1.12 (t, 3H); 13C NMR (DMSO-d6)
d 176.5, 169.7, 160.8, 143.3, 138.7, 133.5, 130.4, 128.8,
127.8, 126.5, 125.9, 125.6, 123.1, 122.7, 109.4, 60.5, 49.9,
36.6, 21.3, 14.3; IR 3463, 3166, 2981, 1743, 1573, 1535,
1508, 1380, 1211, 1164, 794 cmꢀ1; MALDI-TOF m/z
401.2 (C20H21N3O2S2+2H+); [a]ꢀ25.5; HPLC Rt: 9.89

488
T. K. Venkatachalam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 485–488
min;% purity 99.9, elemental analysis: calcd: C, 60.13;
H, 5.30; N, 10.52. Found: C, 60.40; H, 5.36; N, 10.53.
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Acknowledgements
The authors thank R. Malaviya for technical assistance
and performing the LTC4 release assays.
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