Design and synthesis of N-Aryl isothioureas as a novel class of gastric H+/K+-ATPase inhibitors

Article abstract of DOI:10.1002/ardp.201300276

To find new H⁺/K⁺-ATPase inhibitors for the treatment of peptic ulcer disease, a series of novel N-aryl isothiourea derivatives were synthesized and their structures were identified by ¹H NMR and GC-MS. The effects of these compounds on inhibiting gastric acid secretion were evaluated by the guinea pig stomach mucous membrane study with pantoprazole magnesium as a positive control. The results showed that, of the 37 N-aryl isothiourea compounds synthesized, 20 compounds have comparable or stronger gastric acid inhibitory activities than that of pantoprazole magnesium. The quantitative structure-activity relationships (QSARs) of the N-aryl isothiourea compounds were also studied by comparative molecular field analysis (CoMFA) computation, and the model structure that was supposed to give more powerful bioactivities was finally predicted. A series of novel N-aryl isothiourea derivatives were synthesized and evaluated for their effects of inhibiting gastric acid secretion using the guinea pig stomach mucous membrane study with pantoprazole magnesium as a positive control. Compounds 2c, 2e, and 2k have higher bioactivity. The quantitative structure-activity relationships also defined these structural requirements.

Full text of DOI:10.1002/ardp.201300276

Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
891
Full Paper
Design and Synthesis of N-Aryl Isothioureas as a Novel Class of
Gastric Hþ/Kþ-ATPase Inhibitors
Chao Ma, Anhui Wu, Yongqi Wu, Xuhong Ren, and Maosheng Cheng
Key Laboratory of Structure-Based Drugs Design and Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, P. R. China
To find new Hþ/Kþ-ATPase inhibitors for the treatment of peptic ulcer disease, a series of novel N-aryl
1
isothiourea derivatives were synthesized and their structures were identified by H NMR and GC-MS.
The effects of these compounds on inhibiting gastric acid secretion were evaluated by the guinea pig
stomach mucous membrane study with pantoprazole magnesium as a positive control. The results
showed that, of the 37 N-aryl isothiourea compounds synthesized, 20 compounds have comparable or
stronger gastric acid inhibitory activities than that of pantoprazole magnesium. The quantitative
structure–activity relationships (QSARs) of the N-aryl isothiourea compounds were also studied by
comparative molecular field analysis (CoMFA) computation, and the model structure that was
supposed to give more powerful bioactivities was finally predicted.
Keywords: Hþ/Kþ-ATPase / N-Aryl isothiourea / Synthesis
Received: July 22, 2013; Revised: August 28, 2013; Accepted: August 28, 2013
DOI 10.1002/ardp.201300276
Introduction
In our previous experiment, we found N-aryl isothiourea
compounds (Fig. 1, compounds 2a–2t) were converted into
mercaptans by hydrolyzing under acidic conditions. This
active species could bind to luminally accessible cysteines of
the gastric Hþ/Kþ-ATPase, resulting in disulfide formation and
acid secretion inhibition, and hence was supposed to possess
Hþ/Kþ-ATPase inhibiting activities with same mechanism as
benzimidazoles. It was reported that isothioureas have
pesticidal and antibacterial activities [6], and also can be
utilized as nitric oxide synthase inhibitor [7]. However, there
are no reports on their role in inhibiting the secretion of
gastric acid. To find out potent novel Hþ/Kþ-ATPase inhibitors
for peptic ulcer treatment, in this study, a series of new N-aryl
isothiourea derivatives were designed and synthesized. The
Peptic ulcer, a common and frequently encountered disease
with a morbidity of 15% in the whole population, is mainly
caused by the over-secretion of gastric acid [1]. Gastric Hþ/Kþ-
ATPase, which is located in the apical membrane of the
parietal cell and plays a major role in the last step of gastric
acid secretion, has therefore become the important target for
the development of drugs with activities in inhibiting the
secretion of gastric acid for peptic ulcer treatment. Current
clinically used Hþ/Kþ-ATPase inhibitors, such as omeprazole,
lansoprazole, and pantoprazole [2–4] (Fig. 1), are substituted
pyridylmethylsulfinyl benzimidazole prodrugs. Upon acid
catalysis, these prodrugs can be converted into the active
sulfenamide or sulfenic acid in the secretory canaliculus of
the stimulated parietal cell of the stomach and then bind to
luminally accessible cysteines of the gastric Hþ/Kþ-ATPase,
which result in disulfide formation and acid secretion
inhibition [5].
1
structures of new compounds were confirmed by H NMR,
GC-MS, and 13C NMR methods, and their gastric acid
inhibiting activities were evaluated by guinea pig stomach
mucous membrane study. The quantitative structure–
activity relationships (QSARs) of these compounds were also
studied by comparative molecular field analysis (CoMFA)
computation.
Correspondence: Prof. Maosheng Cheng, Key Laboratory of Structure-
Based Drugs Design and Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenhe District, Shenyang 110016, P. R. China.
E-mail: mscheng@syphu.edu.cn
Additional correspondence: Xuhong Ren
E-mail: xu_hong_ren@126.com
Fax: þ86 24 23995043
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
Figure 1. N-Aryl isothiourea compounds 2a–2t.
Results and discussion
easily achieved by standard procedure from substituted
2-methyl pyridine 3 [8] and substituted aniline 11 [9]. The
synthetic routes to 8a–8b and 13a–13m are shown in
Schemes 1 and 2, respectively. The synthetic route to target
compounds 2a–2t is shown in Scheme 3. The condensation of
intermediates 8a–8b and 13a–13m under acidic conditions to
Chemistry
Retrosynthetically, the title compounds 2a–2t can be
disconnected into two fragments, substituted 2-chloromethyl
pyridines 8a–8b and N-aryl thioureas 13a–13m, which can be
Scheme 1. Reagents and conditions: (a) H2O2, CH3COOH, reflux 24 h, 82%; (b) HNO3, H2SO4, 90°C, 24 h, 86%; (c) R’ONa, R’OH, 50°C,
12 h, 82%; (d) Ac2O, H2SO4, 110°C, 5 h and (e) 10%HCl, reflux 2 h, 69%; (f) SOCl2, CH2Cl2, room temperature, 2 h, 50%.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
N-Aryl Isothioureas as Gastric Hþ/Kþ-ATPase Inhibitors
893
Scheme 2. Reagents and conditions: (f) NH4SCN, acetone, reflux, 15 min; (g) substituted aniline, acetone, reflux, 30 min, 67–90%; (h) 5%
NaOH, 80–90°C, 3 h, 80–90%.
Scheme 3. Synthetic routes to the target compounds 2a–t.
construct the title compounds 2a–2t was studied in great
Table 1. Synthesis of 2a under different reaction conditions.
detail by using 2a (R1 ¼ CF3CH2, R2 ¼ 3-CH3, R3 ¼ 4-OC2H5) as
the model (Table 1). It was found that when the reaction was
Entry
Solvent
Time (h)
Temp. (°C)
Yield (%)
carried out in anhydrous ethanol at 50°C for 3 h, the
moderate yield of 61% can be achieved (Entry 6). No product
was obtained when the reaction was carried out at room
temperature (Entry 9). Additionally, due to easy decomposi-
tion of N-aryl thiourea 13 at high temperature, more than
60°C, the by-product yield increased evidently, which was
detected by thin-layer chromatography (TLC, petroleum ether/
acetic ether, 1:1). Finally, the optimal condition for the
condensation reaction, in anhydrous ethanol at 50°C for 3 h,
1
2
3
4
5
6
7
8
9
Iso-propanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
1
1
1
3
1
3
3
5
3
Reflux
Reflux
60–65
60–65
50–55
50–55
40–45
40–45
r.t.
17
21
32
34
42
61
33
33
(–)
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
was used and N-aryl isothiourea compounds 2a–2t were
synthesized.
For example, when the target compounds bear F, Br, or –CF3
substituent group on the benzene ring, they generally have
higher bioactivity, such as 2c, 2e, and 2k, with inhibitory
activity of 88, 91, and 95%, respectively. Especially, compound
2k, with CF3 group at the 3 position of the benzene ring, was
the most effective, with activity 1.5 times higher than that of
pantoprazol magnesium. However, the inhibitory activities of
N-aryl isothiourea compounds with CH3, OCH3, or OC2H5
groups at the 2 or 3 position of the benzene ring were
generally low, such as 2f, 2g, 2h, 2i, 2l, 2p, and 2q.
Furthermore, substituents of pyridine ring also contribute
Biological activity
The inhibitory activities of the target compounds against
gastric acid secretion were evaluated by the method described
in guinea pig stomach mucous membrane [10] study with
pantoprazole magnesium, a known clinically used Hþ/Kþ-
ATPase inhibitor, as a positive control. The activity was
represented as inhibition rate (%), and the results are
summarized in Table 2.
It was concluded that of 20 N-aryl isothiourea compounds
evaluated for inhibition of gastric acid secretion, 12
compounds showed higher or equal activity compared with
the positive control pantoprazol magnesium. The data given
in Table 3 indicates that the substituents on the phenyl ring
play an important role in inhibition of gastric acid secretion.
a lot to the inhibitory activity of N-aryl isothiourea
compounds. For instance, if 4-(20,20,20-trifluoro ethoxy) and
3-CH3 groups of pyridine ring were changed to 4-ethyoxy and
3-H, respectively, while keeping substituents on the phenyl
ring intact, the inhibitory activities of the N-aryl isothioureas
usually became lower. For further researching structure and
Table 2. Inhibition of the gastric acid secretion by the target compounds.
Compound
R1
R2
R3
Acid secretion inhibition (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CF3
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–CH2CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
H
H
H
H
H
H
H
4-OC2H5
4-OCH3
4-F
4-Cl
2-Br
4-CH3
2-CH3
2-CH3, 6-CH3
2-OC2H5
4-Br
69
75
88
83
91
56
54
48
51
87
95
34
70
72
64
48
35
86
82
73
2j
2k
2l
3-CF3
3-CH3
2-Cl
2m
2n
2o
2p
2q
2r
2s
2t
4-OCH3
4-OC2H5
2-CH3, 6-CH3
2-OC2H5
2-Br
4-F
4-Cl
Secretory volume of gastric acid ¼ [Hþ] ꢀ 0.05/(p ꢀ 1.252 ꢀ 0.25) mol cmꢁ2 hꢁ1
¼ 10ꢁpH ꢀ 4ꢀ10ꢁ3 mol cmꢁ2 hꢁ1
¼ 10ꢁpH ꢀ 4ꢀ 103 mmol cmꢁ2 hꢁ1
Inhibition rate (%) ¼ (A1 ꢁ A2)/A1 ꢀ 100%. A1: secretory volume of average elementary gastric acid; A2: secretory volume of gastric acid
after administration.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
N-Aryl Isothioureas as Gastric Hþ/Kþ-ATPase Inhibitors
895
Table 3. Inhibition of the gastric acid secretion by the target compounds.
Compound
R
Acid secretion inhibition (%)
Compound
R
Acid secretion inhibition (%)
3a
3b
3c
3d
3e
3f
4-OC2H5
4-OCH3
2-CH3, 6-CH3
2-CH3
2-Cl, 3-Cl
3-CF3
34.7
25
8.7
19.9
1.99
18.9
30.2
0.06
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-OC2H5
4-OCH3
4-CH3
2-CH3
4-Cl
2-Cl,3-Cl
2-Cl
2-Br
17.4
9.93
35.3
12.0
31.1
35.8
36.5
41.4
24.9
24.3
3g
3h
4-F
2-Br
3-CF3
Pantoprazol
activity relation of N-aryl isothiourea compounds, we also
synthesized unsubstitutional pyridine ring 3a–3h and quino-
line ring 4a–4i; their structures and activities data are shown
in Table 3.
calculations were performed on Silicon Graphics Inc (SGI)
Fuel workstation with IRIX 6.5 platform.
Subsequently, a dataset of 31 compounds was randomly
selected for training set. The regression analysis was carried
out using the partial least-squares (PLS) method. Briefly, cross-
validation analysis was performed. Furthermore, we intro-
duced all-orientation search (AOS) [11] to improve the q2
values. The conformation set corresponding to the highest
cross-validated r2(q2) value was then selected for further
non-cross-validation (conventional) analysis. For component
models with identical values, the component number
producing the smallest standard error of prediction (SEP)
was selected. The final linear regression between changes in
the steric and electrostatic fields and that in binding affinity
(pKi) of the training-set compounds was generated with the
optimum number of components equal to that yielding the
highest r2. PLS analysis results based on a least-squares fit are
listed in Table 4, which shows that all of the statistical indexes
are reasonably high. As listed in Table 4, a five-component
CoMFA model was obtained with cross-validated q2 of 0.415,
conventional r2 of 0.994, Fisher test value (F) of 698.744, and
standard errors of 0.052. The relative contributions to the
CoMFA model are 0.541 from the steric field and 0.439 from
the electrostatic field.
Quantitative structure–activity relationship (QSAR)
Three-dimensional quantitative structure–activity relation-
ship (3D-QSAR) studies were performed for 37 synthesized
N-aryl isothiourea compounds based on the structures and
activity data (Fig. 2). The CoMFA method in SYBYL6.91 (Tripos,
Ltd.) software package was used with the hope to find out
some clues in structural modifications for novel potent Hþ/Kþ-
ATPase inhibitors. A conformational database was created for
the 37-molecule dataset using the Omega conformer genera-
tion software (Omega, version 2.1.0, 2006; Openeye Scientific
Software, Santa Fe, NM, USA). The maximum number of
rotatable bonds was set large enough to accept all the
compounds. A maximum of 400 conformers were allowed for
each compound based on default RMSD cutoff (0.8 Å) and
energy window (25 kcal molꢁ1). 3D shape matching was
performed using the ROCS program (ROCS, version 2.1.1,
2006; Openeye Scientific Software, Santa Fe, NM, USA).
Compound 2k was used as the query for ROCS. All
The linear relationship shown in Fig. 3 indicates that the
fitting power is rationally potent and the predictive ability is
satisfactory.
The 3D “coefficient contours” produced by CoMFA are
shown in Fig. 3. Beneficial and detrimental steric interactions
are displayed in green and yellow contours, respectively,
while red and blue contours illustrate regions of desirable
negative and positive electrostatic interactions, respectively.
Some large regions of green contour around position 4 of
Figure 2. Alignment of N-aryl isothiourea analogs.
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896
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
Table 4. CoMFA results for the N-aryl isothioureas.
Field contribution
q2
Optimal components
r2
s
F
Steric
Electrostatic
Training set
Test set
0.451
6
0.994
0.844
0.052
0.199
698.744
0.541
0.439
groups and the presence of electron-withdrawing groups on
benzene ring of N-aryl isothioureas are usually beneficial to
their inhibitory activities.
Omega was designed for high-throughput generation of
conformational models to reproduce the bioactive conforma-
tion and Rapid Overlay of Chemical Structures (ROCS) was
used to perform large-scale structural comparison by using a
superposition method.
The QSARs of the N-aryl isothiourea compounds have also
been proposed on the basis of CoMFA computation. These
models have defined the structural requirements needed
for high inhibitory activities of the N-aryl isothioureas and
therefore offered some beneficial clues in structural mod-
ifications. The results from these studies may be helpful in
developing better Hþ/Kþ-ATPase inhibitors in future.
Figure 3. Correlation between the predicted activities (PA) of
N-aryl isothioureas analogs by CoMFA and experimental activities
(ꢁlogIC50, EA).
Conclusions
In summary, a series of novel N-aryl isothiourea derivatives
were synthesized. The inhibitory effects of these compounds
on gastric acid secretion were evaluated by the guinea pig
stomach mucous membrane study with pantoprazole mag-
nesium as a positive control. Biological results revealed that
20 compounds have comparable or stronger gastric acid
inhibitory activities than that of pantoprazole magnesium.
The data indicated that the target compounds bear F, Br, or
–CF3 substituent group on the benzene ring, and they
generally have higher bioactivity. Furthermore, 4-(20,20,20-
trifluoro ethoxy) and 3-CH3 as the substituents of pyridine
ring also contribute a lot to the inhibitory activity of N-aryl
isothiourea compounds. The QSARs have also defined the
structural requirements for high inhibitory activities of the
N-aryl isothioureas. This discovery offered some clues in our
future structural modifications.
Figure 4. CoMFA contour maps: (a) Steric contributions to the
gastric acid inhibitory activities of N-aryl isothioureas; (b) electro-
static contribution to the gastric acid inhibitory activities of N-aryl
isothioureas.
pyridine ring (Fig. 4) suggest that more bulky substituents
at this position will significantly improve the inhibitory
activities. The yellow polyhedron near position 3 of pyridine
ring and position 2, 5, and 6 of benzene ring indicates that
more steric bulk is unfavorable for inhibitory activities. The
blue contour near position 4 of pyridine ring (Fig. 4) suggests
that electron-donating substituents could increase the
activity, while the red polyhedrons near positions 3 and 4
of benzene ring indicate that electron-withdrawal substitu-
ents at these positions might play a favorable role in
inhibitory activities. Accordingly, N-aryl isothioureas with
bulky electron-donating groups at position 4 of pyridine ring
have larger inhibitory activities than those with small,
electron-withdrawing substituents. The addition of less bulky
Experimental
Chemistry
All commercially available reagents and solvents were used
without further purification. Analytical TLC was performed on
HSGF 254 (150–200 lm thickness, Yantai Huiyou Company,
China). Melting points of the compounds were measured in a
capillary tube on a BUCHI-450 melting point apparatus without
correction. 1H NMR and 13C NMR spectra were recorded in a
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
N-Aryl Isothioureas as Gastric Hþ/Kþ-ATPase Inhibitors
897
Bruker ARX 300 MHz. Chemical shift values were reported in
d (ppm), and coupling constants were reported in Hertz. Proton
coupling patterns were described as singlet (s), doublet (d), triplet
(t), quartet (q), multiplet (m), and broad (br). Low- and high-
resolution mass spectra GCMS were obtained by SHIMAU GCMS-
QP5050A spectrometer.
¼ 2.26 (3H, s, Py–CH3), 5.01 (2H, s, Py–CH2), 5.07 (1H, d, CF3CH2,
J ¼ 8.6 Hz), 5.12 (1H, d, CF3CH2, J ¼ 8.6 Hz), 7.42 (3H, t, Ar–H), 7.57
(2H, d, Ar–H,), 8.65 (1H, d, Py–H, J ¼ 6.0 Hz), 9.90 (2H, brs), 12.30
(1H, brs); GC-MS (m/z) (Mþ) calcd. for C16H15ClF3N3OS, 389.0576;
found: 389.06. (C9H9F3NOSHþ) 237.04, (C13H7N2Sþ) 222.74,
(C9H9F3NOþ) 204.06, (C7H6ClN2þ) 152.02, (C6H5ClNþ) 125.31.
Preparation of N-(4-ethoxybenzyl)-s-2-(3-methyl-4-
Preparation of N-(2-bromobenzyl)-s-2-(3-methyl-4-
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
dihydrochloride 2a
dihydrochloride 2e
To a solution of N-4-ethoxybenzyl thiourea 13a (0.71 g, 3.62 mmol)
in anhydrous ethanol 15 mL 2-chloromethyl-3-methyl-4-(20,20,20-
trifluoroethoxy)pyridine (1 g, 3.62 mol) was slowly added and the
resulting solution was stirred at 50°C for 3 h; the solvent was
concentrated and the crude product was recrystallized from
ethanol to give 2a (1.20 g, 61%) as white solid. Mp: 188–189°C;
1H NMR (DMSO-d6): d (ppm) ¼ 1.33 (3H, t, OCH2CH3, J ¼ 6.9 Hz),
2.24 (3H, s, Py–CH3), 4.06 (2H, dd, CH3CH2, J ¼ 6.8, 6.9 Hz), 4.88
(2H, s, Py–CH2), 5.06 (2H, d, CF3CH2, J ¼ 8.55 Hz), 7.03 (2H, d, Ar–H,
J ¼ 8.7 Hz), 7.25 (2H, d, Ar–H, J ¼ 8.8 Hz), 7.34 (1H, s, Ar–H), 8.52
(1H, s, Py–H), 10.03 (2H, brs), 11.98 (1H, brs); 13C NMR (DMSO-d6):
d (ppm) ¼ 10.3, 14.6, 18.6, 33.4, 56.0, 63.5, 64.6, 65.4, 108.3, 115.4,
121.7, 122.5, 125.4, 127.3, 145.3, 151.7, 158.2, 163.9; GC-MS (m/z)
(Mþ) calcd. for C19H21F3N2O2S, 399.1228; found: 399.10.
(C9H9F3NOSHþ) 237.04, (C9H9F3NOþ) 204.06, (C9H11N2Oþ)
162.95, (C14H8NSþ) 222.04, (C8H10NOþ) 136.08, (C7H7Nþ) 106.04.
In the same manner as described for 2a, 2e was prepared from 13e
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 62%. Mp: 184–185°C; 1H NMR (DMSO-d6): d (ppm) ¼ 2.30
(3H, s, Py–CH3), 5.08 (2H, s, Py–CH2), 5.12 (1H, d, CF3CH2,
J ¼ 8.5 Hz), 5.20 (1H, d, CF3CH2, J ¼ 8.5 Hz), 7.40 (1H, m, Ar–H), 7.51
(3H, m, Ar–H,), 7.80 (1H, d, Ar–H, J ¼ 7.8 Hz), 8.71 (1H, d, Py–H,
J ¼ 6.2 Hz), 9.84 (2H, brs); GC-MS (m/z) (Mþ þ 2) calcd. for
C16H15BrF3N3OS, 435.0071; found: 435.00. (C16H12BrF3N2OSþ)
416.08, (C13H10BrN3Sþ) 320.15, (C9H9F3 NOSHþ) 237.04,
(C13H7N2Sþ) 222.74, (C9H9F3NOþ) 204.06, (C7H3BrN2þ) 196.06,
(C6H5BrNþ) 168.38, (C7H7Nþ) 106.0, (C6H4Nþ) 90.03.
Preparation of N-(4-methylbenzyl)-s-2-(3-methyl-4-
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
dihydrochloride 2f
In the same manner as described for 2a, 2f was prepared from 13f
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 56%. Mp: 193–194°C; 1H NMR (DMSO-d6): d (ppm) ¼ 2.25
(3H, s, Ph–CH3), 2.34 (3H, s, Py–CH3), 4.95 (2H, s, Py–CH2), 5.04 (1H,
d, CF3CH2, J ¼ 8.4 Hz), 5.10 (1H, d, CF3CH2, J ¼ 8.4 Hz), 7.24 (2H, d,
Ar–H, J ¼ 8.1 Hz), 7.31 (2H, d, Ar–H, J ¼ 8.1 Hz), 7.40 (1H, s, Py–H),
8.59 (1H, s, Py–H), 9.98 (2H, brs), 12.10 (1H, brs); GC-MS (m/z) (Mþ)
calcd. for C17H18F3N3OS, 369.1123; found: 369.11. (C16H12F3N2OSþ)
336.86, (C15H13N2Sþ) 253.08, (C9H9F3NOSHþ) 237.04, (C13H7N2Sþ)
222.74, (C9H9F3NOþ) 204.06, (C8H9N2þ) 132.06.
Preparation of N-(4-methoxybenzyl)-s-2-(3-methyl-4-
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
dihydrochloride 2b
In the same manner as described for 2a, 2b was prepared from 13b
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 52%. Mp: 179–181°C; 1H NMR (DMSO-d6): d (ppm) ¼ 2.26
(3H, s, Py–CH3), 3.79 (3H, s, OCH3), 4.98 (2H, s, Py–CH2), 5.09 (1H, d,
CF3CH2, J ¼ 8.6 Hz), 5.25 (1H, d, CF3CH2, J ¼ 8.6 Hz), 7.04 (2H, d,
Ar–H, J ¼ 8.7 Hz), 7.28 (2H, d, Ar–H, J ¼ 8.7 Hz), 7.44 (1H, s, Ar–H),
8.62 (1H, s, Py–H), 10.03 (2H, brs), 11.98 (1H, brs); GC-MS (m/z) (Mþ)
calcd. for C17H18F3N3O2S, 385.1072; found: 385.11. (C15H13N2OSþ)
268.85, (C9H9F3NOSHþ) 237.04, (C9H9F3NOþ) 204.06, (C13H7N2Sþ)
222.74, (C8H9N2Oþ) 148.14, (C8H8NOþ) 133.06, (C7H7Nþ) 105.94.
Preparation of N-(2-methylbenzyl)-s-2-(3-methyl-4-
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
dihydrochloride 2g
In the same manner as described for 2a, 2g was prepared from 13g
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 50%. Mp: 192–194°C; 1H NMR (DMSO-d6): d (ppm) ¼ 2.20
(3H, s, Ph–CH3), 2.25 (3H, s, Py–CH3), 4.93 (2H, s, Py–CH2), 5.01 (1H,
d, CF3CH2, J ¼ 8.5 Hz), 5.05 (1H, d, CF3CH2, J ¼ 8.5 Hz), 7.34 (5H, m,
Ar–H), 8.52 (1H, s, Py–H), 9.98 (2H, brs), 11.98 (1H, brs); GC-MS (m/z)
(Mþ) calcd. for C17H18F3N3OS, 369.1123; found: 369.11.
(C9H9F3NOSHþ) 237.04, (C13H7N2Sþ) 222.74, (C9H9F3NOþ)
204.06, (C8H9N2þ) 132.06.
Preparation of N-(4-fluorobenzyl)-s-2-(3-methyl-4-(20,20,20-
trifluoroethoxy)pyridylmethyl) isothiourea dihydrochloride 2c
In the same manner as described for 2a, 2c was prepared from 13c
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 61% as white solid. Mp: 155–156°C; 1H NMR (DMSO-d6):
d (ppm) ¼ 2.24 (3H, s, Py–CH3), 4.89 (2H, s, Py–CH2), 5.02 (1H, d,
CF3CH2, J ¼ 8.6 Hz), 5.08 (1H, d, CF3CH2, J ¼ 8.6 Hz), 7.36 (2H, m,
Ar–H), 7.42 (3H, m, Ar–H), 8.52 (1H, d, Py–H, J ¼ 6.0 Hz), 9.88 (2H,
brs); GC-MS (m/z) (Mþ) calcd. for C16H15F4N3OS, 373.0872; found:
370.09. (C9H9F3NOSHþ) 237.04, (C13H7N2Sþ) 222.74, (C9H9F3NOþ)
204.06, (C7H6FN2Sþ) 168.38, (C7H5FNþ) 122.04, (C6H5FNþ) 109.86.
Preparation of N-(2,6 dimethylbenzyl)-s-2-(3-methyl-4-
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
dihydrochloride 2h
Preparation of N-(4-chlorobenzyl)-s-2-(3-methyl-4-
In the same manner as described for 2a, 2h was prepared from
13h and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyri-
dine, yield 49%. Mp: 181–182°C; 1H NMR (DMSO-d6): d (ppm) ¼ 2.17
(3H, s, Ph–CH3), 2.20 (3H, s, Ph–CH3), 2.29 (3H, s, Py–CH3), 4.81
(2H, s, Py–CH2), 5.07 (2H, m, CF3CH2), 7.26 (3H, m, Ar–H), 7.40 (1H,
d, Py–H, J ¼ 5.4 Hz), 8.44 (1H, d, Py–H, J ¼ 5.4Hz), 9.15 (1H, brs), 9.94
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
dihydrochloride 2d
In the same manner as described for 2a, 2d was prepared from
13d and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyri-
dine, yield 57%. Mp: 189–191°C; 1H NMR (DMSO-d6): d (ppm)
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
(1H, brs), 11.59 (1H, brs); GC-MS (m/z) (Mþ) calcd. for C18H20 F3N3OS,
383.1279; found: 383.13. (C17H17F3N3OSþ) 368.13, (C9H9F3NOSHþ)
237.04, (C13H7N2Sþ) 222.74, (C9H9F3NOþ) 204.06, (C9H11N2Sþ)
179.46, (C9H11N2þ) 145.24, (C8H8N2þ) 130.07.
dine, yield 56%. Mp: 185–186°C; 1H NMR (DMSO-d6): d (ppm)
¼ 2.26 (3H, s, Py–CH3), 4.93 (2H, s, Py–CH2), 5.03 (1H, d, CF3CH2,
J ¼ 6.8 Hz), 5.08 (1H, d, CF3CH2, J ¼ 6.8 Hz), 7.46 (4H, m, Ar–H), 7.65
(1H, m, Ar–H), 8.58 (1H, d, Py–H, J ¼ 5.4 Hz), 9.71 (2H, brs), 9.71
(2H, brs); GC-MS (m/z) (Mþ) calcd. for C16H15 ClF3N3OS 389.0576;
found: 389.10. (C16H15F3N3OSþ) 354.13, (C9H9F3NOSHþ) 237.04,
(C13H7N2Sþ) 222.74, (C9H9F3NOþ) 204.06, (C7H6ClN2þ) 152.02,
(C6H5ClNþ) 125.31.
Preparation of N-(2-ethoxybenzyl)-s-2-(3-methyl-4-(20,20,20-
trifluoroethoxy)pyridylmethyl) isothiourea dihydrochloride 2i
In the same manner as described for 2a, 2i was prepared from 13i
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 51%. Mp: 179–181°C; 1H NMR (DMSO-d6): d (ppm) ¼ 1.26
(3H, t, OCH2CH3), 2.25 (3H, s, Py–CH3), 4.07 (2H, m, OCH2CH3), 4.52
(2H, s, Py–CH2), 5.05 (1H, d, CF3CH2, J ¼ 8.6 Hz), 5.11 (1H, d,
CF3CH2, J ¼ 8.6 Hz), 7.33 (5H, m, Ar–H), 8.59 (1H, s, Py–H), 9.92 (2H,
brs), 11.6 (1H, brs); GC-MS (m/z) (Mþ) calcd. for C18H20F3N3OS,
399.1228; found: 399.12. (C9H9F3NOSHþ) 237.04, (C13H7N2Sþ)
222.74, (C9H9 F3NOþ) 204.06, (C9H11N2Oþ) 162.13, (C8H10NOþ)
134.07.
Preparation of N-(4-methoxylbenzyl)-s-2-(4-
ethoxypyridylmethyl) isothiourea dihydrochloride 2n
In the same manner as described for 2a, 2n was prepared from
13b and 2-chloromethyl-4-ethyoxypyridine, yield 32%. Mp: 175–
176°C; 1H NMR (DMSO-d6): d (ppm) ¼ 1.38 (3H, t, OCH2CH3,
J ¼ 6.8 Hz), 3.78 (3H, s, OCH3), 4.31 (1H, d, CH3CH2O, J ¼ 6.8 Hz),
4.36 (1H, d, CH3CH2O, J ¼ 6.8 Hz), 5.12 (2H, s, Py–CH2), 7.03 (2H, d,
Ar–H, J ¼ 8.6 Hz), 7.24 (2H, d, Ar–H, J ¼ 8.7 Hz), 7.45 (1H, d, Ar–H,
J ¼ 4.5 Hz), 7.79 (1H, s, Ar–H), 8.75 (1H, d, Py–H, J ¼ 6.6 Hz), 9.15
(1H, brs), 10.06 (1H, brs), 12.15 (1H, brs); 13C NMR (DMSO-d6):
d (ppm) ¼ 14.04, 31.78, 55.56, 66.40, 111.68, 113.11, 115.00,
127.13, 127.52, 144.88, 152.42, 159.00, 169.97; GC-MS (m/z) (Mþ)
calcd. for C16H19N3O2S 317.1198; found: 317.12. (C9H11N2OSþ)
195.25, (C8H10NOSHþ) 169.38, (C8H9N2Oþ) 148.31, (C8H8NOþ)
133.08, (C6H5NSþ) 123.41.
Preparation of N-(4-bromobenzyl)-s-2-(3-methyl-4-(20,20,20-
trifluoroethoxy)pyridylmethyl) isothiourea dihydrochloride 2j
In the same manner as described for 2a, 2j was prepared from 13j
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 60%. Mp: 190–192°C; 1H NMR (DMSO-d6): d (ppm) ¼ 2.27
(3H, s, Py–CH3), 4.99 (2H, s, Py–CH2), 5.04 (1H, d, CF3CH2,
J ¼ 12.9 Hz), 5.11 (1H, d, CF3CH2, J ¼ 12.9 Hz), 7.35 (2H, t, Ar–H),
7.45 (1H, d, Ar–H, J ¼ 8.4 Hz), 7.70 (2H, m, Ar–H), 8.64 (1H, s, Py–H),
9.89 (2H, brs), 11.98 (1H, brs); GC-MS (m/z) (Mþ) calcd. for
C16H15BrF3N3OS, 433.0071; found: 433.00. (C9H9F3NOSHþþ)
Preparation of N-(4-ethoxylbenzyl)-s-2-(4-
ethoxypyridylmethyl) isothiourea dihydrochloride 2o
In the same manner as described for 2a, 2o was prepared from 13c
and 2-chloromethyl-4-ethyoxypyridine, yield 34%. Mp: 161–162°C;
1H NMR (DMSO-d6): d (ppm) ¼ 1.37 (6H, m, 2OCH2CH3), 4.03 (2H,
dd, CH3CH2 J ¼ 6.8, 13.77 Hz), 4.35 (2H, dd, CH3CH2 J ¼ 6.8,
13.77 Hz), 5.10 (2H, s, Py–CH2), 7.02 (2H, d, Ar–H, J ¼ 8.7 Hz), 7.23
(2H, d, Ar–H, J ¼ 8.7 Hz), 7.44 (1H, t, Ar–H), 7.78 (1H, s, Ar–H), 8.74
(1H, s, Py–H), 10.06 (2H, brs), 12.15 (1H, brs); GC-MS (m/z) (Mþ) calcd.
for C17H21N3O2S 331.1354; found: 331.14. (C13H8N2Sþ) 225.04,
(C9H11N2OSþ) 196.25, (C8H10NOSHþ) 169.38, (C9H11N2Oþ) 163.14.
237.04, (C13H7N2Sþ) 222.74, (C9H9 F3NOþ) 204.06, (C7H3BrN2
196.25, (C6H5BrNþ) 168.38, (C7H6N2þ) 117.26.
)
Preparation of N-(3-trifluoromethylbenzyl)-s-2-(3-methyl-4-
(20,20,20-trifluoroethoxy)pyridylmethyl) isothiourea
dihydrochloride 2k
In the same manner as described for 2a, 2k was prepared from
13k and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyri-
dine, yield 41%. Mp: 122–124°C; 1H NMR (DMSO-d6): d (ppm)
¼ 2.27 (3H, s, Py–CH3), 5.09 (4H, m, Py–CH2, CF3CH2), 7.44 (1H, s,
Ar–H), 7.74 (4H, m, Ar–H), 8.63 (1H, s, Py–H), 9.5 (2H, brs); ESI-MS
(m/z) [MþH]þ calcd. for C17H15F6N3OS, 424.0840; found: 424.08.
(C16H9F6N2OSþ) 390.51, (C9H9F3NOSHþ) 238.07, (C13H7N2Sþ)
224.03, (C8H8N2Sþ) 164.14.
Preparation of N-(2,6 dimethylbenzyl)-s-2-(4-
ethoxypyridylmethyl) isothiourea dihydrochloride 2p
In the same manner as described for 2a, 2p was prepared from
13h and 2-chloromethyl-4-ethyoxypyridine, yield 32%. Mp: 166–
167°C; 1H NMR (DMSO-d6): d (ppm) ¼ 1.39 (3H, t, OCH2CH3,
J ¼ 6.8 Hz), 2.06 (6H, s, 2Ph–CH3), 4.31 (1H, d, CH3CH2, J ¼ 6.6 Hz),
4.36 (1H, d, CH3CH2, J ¼ 6.6 Hz), 5.15 (2H, s, Py–CH2), 7.23
(3H, m, Ar–H), 7.41 (1H, s, Ar–H), 7.76 (1H, d, Ar–H, J ¼ 6.5 Hz),
8.74 (1H, d, Py–H, J ¼ 6.3 Hz), 10.06 (2H, brs), 12.15 (1H, brs); GC-MS
(m/z) (Mþ) calcd. for C17H21N3OS 315.1405; found: 315.14.
(C16H18N3OSþ) 300.16, (C16H15N2OSþ) 282.09, (C15H12N2OSþ)
267.46, (C15H13N2Sþ) 253.25, (C14H10N2Sþ) 237.04, (C9H11N2OSHþ)
195.25, (C9H11N2Sþ) 179.46, (C8H10NO SHþ) 169.38, (C9H11Nþ)
146.31, (C7H5N2þ) 118.26.
Preparation of N-(3-methylbenzyl)-s-2-(3-methyl-4-(20,20,20-
trifluoroethoxy)pyridylmethyl) isothiourea dihydrochloride 2l
In the same manner as described for 2a, 2l was prepared from 13l
and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyridine,
yield 40%. Mp: 146–147°C; 1H NMR (DMSO-d6): d (ppm) ¼ 2.25
(3H, s, Ph–CH3), 2.34 (3H, s, Py–CH3), 4.91 (2H, s, Py–CH2), 5.05
(2H, s, CF3CH2), 7.15 (2H, d, Ar–H, J ¼ 8.7 Hz), 7.24 (1H, d, Ar–H,
J ¼ 7.8 Hz), 7.39 (2H, t, Ar–H), 8.55 (1H, s, Py–H), 9.92 (2H, brs),
12.15 (1H, brs); ESI-MS (m/z) (Mþ) calcd. for C17H18F3N3OS
369.1123; found: 370.01. (C17H15F3N2OSþ) 353.09, (C13H7N2Sþ)
224.03, (C9H9F3NOSHþ) 238.07.
Preparation of N-(2-ethoxylbenzyl)-s-2-(4-
ethoxypyridylmethyl) isothiourea dihydrochloride 2q
In the same manner as described for 2a, 2q was prepared from 13i
and 2-chloromethyl-4-ethyoxypyridine, yield 34%. Mp: 116–117°C;
1H NMR (DMSO-d6): d (ppm) ¼ 1.18 (3H, t, PhOCH2CH3, J ¼ 6.6 Hz),
1.37 (3H, t, PyOCH2CH3, J ¼ 6.7 Hz), 4.05 (2H, dd, PhOCH2CH3,
J ¼ 6.3, 13.2 Hz), 4.31 (2H, dd, PyOCH2CH3 J ¼ 6.7, 13.3 Hz), 5.04
(2H, s, Py–CH2), 7.01 (1H, t, Ar–H, J ¼ 7.5 Hz), 7.15 (1H, d, Ar–H,
Preparation of N-(2-chlorobenzyl)-s-2-(3-methyl-4-(20,20,20-
trifluoroethoxy)pyridylmethyl) isothiourea dihydrochloride 2m
In the same manner as described for 2a, 2m was prepared from
13m and 2-chloromethyl-3-methyl-4-(20,20,20-trifluoroethoxy)pyri-
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Arch. Pharm. Chem. Life Sci. 2013, 346, 891–900
N-Aryl Isothioureas as Gastric Hþ/Kþ-ATPase Inhibitors
899
J ¼ 8.4 Hz), 7.26 (1H, d, Ar–H, J ¼ 7.5 Hz), 7.37 (2H, d, Ar–H,
J ¼ 7.3 Hz), 7.62 (1H, s, Ar–H), 8.71 (1H, d, Py–H, J ¼ 6.2 Hz), 10.06
(2H, brs), 12.15 (1H, brs); GC-MS (m/z) (Mþ) calcd. for C17H21N3O2S
331.1354; found: 331.14. (C15H16N3OSþ) 285.09, (C9H11N2OSHþ)
195.25, (C8H10NOSHþ) 169.38, (C9H11N2Oþ) 163.13, (C8H10NOþ)
135.07, (C6H5NSþ) 123.41.
(0.5 mL), 5 mL absorption pipette, and so on. Animal: cavia
cobaya (Shen Yang Pharmaceutical University experimental
animal offered).
Method
Determination of cavia cobaya ex vivo gastric mucosa gastric
acid secretion: Recipe male cavia cobaya of 350–450 g body
weight, abrosia but refrain from water for 12 h, strike
cephalon to death. Recipe stomach, ablution chylostomach
with cold serosal fluid, porch greater curvature to clipper,
fixed the stomach at glass tube (end-cavea CSA 4.9 cm2),
membrana mucosa faced to lumens. Add 5 mL membrane
mucosa liquid to glass tube, to take the sample to immerse at
a bath tube with 30 mL serosal fluid, to make membrana
mucosa liquid and serosal fluid at the same level. Bath tube
constant temperature at 37 ꢂ 0.5°C, inject 0.95 O2 þ 0.05 CO2
to serosal fluid, pH 7.4, and inject 100% O2 to membrana
mucosa liquid. Renoveatur membrana mucosa liquid every
15 min, dislodge membrane liquid to survey pH with pH
meter. Pantoprazole magnesium and isothiourea chemical
compound is dissolved in DMSO and dilute with serosal
fluid, to recipe 0.2 mL sample to add to membrane mucosa
liquid in the bath tube, medicamentous solution has no effect
to pH of membrane mucosa liquid. After determine base
gastric acid secretary, add pantoprazole magnesium and
isothiourea compounds, final concentration in serosal fluid
is 5 ꢀ 10ꢁ5 mol Lꢁ1, contiguously detect 2.5 h; select to
isothiourea compounds with inhibiting activity, to retain
serosal fluid’s final at 5 ꢀ 10ꢁ6 mol Lꢁ1, contiguously detect.
Preparation of N-(2-bromobenzyl)-s-2-(4-
ethoxypyridylmethyl) isothiourea dihydrochloride 2r
In the same manner as described for 2a, 2r was prepared from 13e
and 2-chloromethyl-4-ethyoxypyridine, yield 42%. Mp: 185–186°C;
1H NMR (DMSO-d6): d (ppm) ¼ 1.37 (3H, t, OCH2CH3, J ¼ 6.6 Hz),
4.29 (2H, d, OCH2CH3, J ¼ 6.6 Hz), 4.85 (2H, s, Py–CH2), 7.33 (2H, d,
Ar–H, J ¼ 14.7 Hz), 7.40 (1H, d, Ar–H, J ¼ 7.2 Hz), 7.49 (2H, d, Ar–H,
J ¼ 13.2 Hz), 7.76 (1H, d, Ar–H, J ¼ 7.8 Hz), 8.62 (1H, d, Py–H,
J ¼ 5.8 Hz), 9.40 (2H, brs); GC-MS (m/z) (Mþþ2) calcd. for
C15H16BrN3OS 367.0197; found: 367.02. (C15H16N3OSþ) 286.09,
(C13H11N3Sþ) 241.05, (C7H6BrN2þ) 196.25, (C8H10NOSHþ) 169.38.
Preparation of N-(4-fluorobenzyl)-s-2-(4-
ethoxypyridylmethyl) isothiourea dihydrochloride 2s
In the same manner as described for 2a, 2s was prepared from 13a
and 2-chloromethyl-4-ethyoxypyridine, yield 51%. Mp: 177–179°C;
1H NMR (DMSO-d6): d (ppm) ¼ 1.38 (3H, t, OCH2CH3, J ¼ 6.9 Hz), 4.33
(2H, dd, OCH2CH3, J ¼ 6.9, 13.9 Hz), 5.09 (2H, s, Py–CH2), 7.38
(5H, m, Ar–H), 7.75 (1H, s, Ar–H), 8.74 (1H, d, Py–H, J ¼ 6.7 Hz), 9.98
(2H, brs), 12.38 (1H, brs); GC-MS (m/z) (Mþ) calcd. for C15H16FN3OS
305.0998; found: 305.10. (C13H8FN2Sþ) 243, (C9H11N2OSþ) 194.25,
(C8H10NOSHþ) 169.38, (C7H6FN2þ) 136.07, (C6H5NSþ) 123.41.
Preparation of N-(4-chlorobenzyl)-s-2-(4-
ethoxypyridylmethyl) isothiourea dihydrochloride 2t
In the same manner as described for 2a, 2t was prepared from 13d
and 2-chloromethyl-4-ethyoxypyridine, yield 45%. Mp: 181–182°C;
1H NMR (DMSO-d6): d (ppm) ¼ 1.38 (3H, t, OCH2CH3, J ¼ 6.9 Hz), 4.25
(2H, d, OCH2CH3, J ¼ 6.9 Hz), 4.82 (2H, s, Py–CH2), 7.23 (1H, s, Ar–H),
7.38 (2H, d, Ar–H, J ¼ 7.8 Hz), 7.57 (2H, d, Ar–H, J ¼ 7.5 Hz), 8.59 (1H,
d, Py–H, J ¼ 5.7 Hz), 9.98 (2H, brs), 12.38 (1H, brs); GC-MS (m/z) (Mþ)
calcd. for C15H16ClN3OS 321.0703; found: 321.07. (C13H8ClN2Sþþ)
This work was supported by Program for Innovative Research Team
of the Minisry of Education and Program for Liaoning Innovative
Research Team in University.
259.31, (C7H6ClN2Sþ) 186.14, (C8H10NOSHþ) 169.38, (C7H6ClN2
152.02.
)
The authors have declared no conflict of interest.
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