Synthesis and biological evaluation of cyanoguanidine derivatives of loratadine

Article abstract of DOI:10.1016/j.bmcl.2012.08.041

Cyanoguanidine derivatives of loratadine (3a-i) were synthesized and screened for antitumor and anti-inflammatory activity. The most promising compound 3c (R = n-C₈H₁₇) possessed at least twofold higher in vitro cytotoxicity than 5-f

Full text of DOI:10.1016/j.bmcl.2012.08.041

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6076–6080
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and biological evaluation of cyanoguanidine derivatives of loratadine
Wukun Liu a,b, Jinpei Zhou a, , Tong Zhang a, Huibin Zhang a, Haiyang Zhu a, Yanhua Cheng a,
⇑
Ronald Gust b,c,
⇑
a Department of Medicinal Chemistry, China Pharmaceutical University, Tongjia Xiang 24, 210009 Nanjing, PR China
b Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Str, 2+4, 14195 Berlin, Germany
c Institute of Pharmacy, University of Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyanoguanidine derivatives of loratadine (3a–i) were synthesized and screened for antitumor and anti-
inflammatory activity. The most promising compound 3c (R = n-C8H17) possessed at least twofold higher
in vitro cytotoxicity than 5-fluorouracil against mammary (MCF-7 and MDA-MB 231) as well as colon
(HT-29) carcinoma cells. The mode of action, however, is so far unclear. The participation of the COX-
1/2 enzymes on the cytotoxicity, however, is very unlikely. Nevertheless all compounds showed stronger
in vivo anti-inflammatory activity than ibuprofen in the xylene-induced ear swelling assay in mice.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 6 August 2012
Accepted 10 August 2012
Available online 16 August 2012
Keywords:
Cyanoguanidine derivatives
Benzo[5,6]cyclohepta[1,2-b]pyridine
Antitumor
Anti-inflammation
Loratadine
Cancer, a group of malignant diseases is responsible for tremen-
dous health costs associated with high levels of mortality and mor-
bidity and remains the second leading cause of death in the world.1
Although chemotherapy is the mainstay of cancer therapy, the use
of available chemotherapeutics is often limited due to undesirable
side effects and the development of resistance during the therapy.
Therefore, the successful treatment of cancer still remains a chal-
lenge in the 21st century, and clearly underscores the need of novel
chemotherapeutic agents.
Benzo[5,6]cyclohepta[1,2-b]pyridine derivatives (for examples
see Fig. 1) exhibited various biological effects and had therefore at-
tracted considerable pharmaceutical interest.2–14 Recently, it was
demonstrated that the second-generation H1 histamine antagonist
loratadine (Fig. 1) induces cell cycle arrest of tumor cells in G2/M
phase.6 Then further investigations documented its potential as a
chemotherapeutic agent as well as a modifier of radiation respon-
siveness in the treatment of cancer and might warrant further clin-
ical evaluation.7 In preclinical studies lonafarnib (Fig. 1), a farnesyl
protein transferase inhibitor showed selective anticancer activity
in a broad range of solid and hematologic tumor cell lines, includ-
ing those with wild-type Ras. This cellular Ras plays an important
role in cellular proliferation mediated by growth factor receptor.
Moreover, lonafarnib showed activity against human lung, pros-
tate, pancreas, colon, bladder and glioblastom also in in vivo stud-
ies.8–11
In our group, we started a project to optimize loratadine as che-
motherapeutic agent for the treatment of inflammatory cancerous
diseases (e.g. the mammary carcinoma). In the first part of this
study we designed a series of thiourea derivatives containing the
benzo[5,6]cyclohepta[1,2-b]pyridine moiety of loratadine. These
compounds were as active as 5-fluorouracil (5-FU) in vitro against
tumor cells and caused stronger anti-inflammatory effects than
ibuprofen in vivo.12
In the next step we combined the benzo[5,6]cyclohepta[1,2-
b]pyridine core with a cyanoguanidine moiety which is a highly
efficient pharmacophore.15–23 The influence of alkyl chains and
substituted aromatic rings at the cyanoguanidine on the in vitro
cytotoxicity and the anti-inflammatory potency were studied.
The synthetic routes to get the target compounds 3a–i are out-
lined in Schemes 1 and 2. The synthon desloratadine (1), synthe-
sized according to previously published methods12–14 was
reacted with substituted methyl N’-cyanocarbamimidothioates
2a–e in toluene under reflux for 6.5 h yielding 3a–e (Scheme 1).24
Compounds 2a–e were previously obtained from substituted
amines and dimethyl cyanocarbonimidodithioate.
Unfortunately, this synthetic route failed in the synthesis of 3f–
i. So we used the N-cyano-1-piperidinecarboximidothioic acid
methyl ester 425 as intermediate. Reaction with substituted amines
in toluene under reflux for 9 h gave the corresponding compounds
3f–i (Scheme 2).26
⇑
Corresponding authors. Tel.: +86 25 83471532; fax: +86 25 83271480 (J.Z.); tel.:
+43 512 507 58200; fax: +43 512 507 58299 (R.G.).
All new compounds (3a–i) as well as loratadine, desloratadine,
and the established antitumor drug 5-FU were screened for
E-mail addresses: jpzhou668@163.com (J. Zhou), ronald.gust@uibk.ac.at (R.
Gust).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.041

W. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6076–6080
6077
Cl
Cl
Cl
Br
Cl
N
N
N
N
Br
O
N
H
N
N
N
N
NH2
O
O
O
N
Desloratadine
Loratadine
Rupatadine
Lonafarnib
Figure 1. Representatives of benzo[5,6]cyclohepta[1,2-b]pyridine derivatives.
Table 1
Cytotoxicity against MCF-7, MDA-MB 231 and HT-29 cells
Cl
Cl
Compounds
Cytotoxicity IC50a, (
lM)
NCN
NHR
2a-e
N
MCF-7
MDA-MB 231
HT-29
N
(a)
+
H3CS
3a
3b
3c
3d
3e
3f
3g
3h
3.9 0.3
3.5 0.1
1.4 0.2
3.9 1.0
4.0 0.3
2.9 0.4
2.3 0.7
7.1 0.4
8.0 0.3
7.5 0.7
10.5 0.6
4.7 0.4
3.1 0.5
1.7 0.1
4.1 1.1
1.9 0.4
6.9 1.0
2.7 1.0
2.6 0.4
18.2 0.2
12.9 1.2
8.4 1.3
12.1 1.1
9.6 0.3
4.0 0.2
4.4 0.2
2.0 0.2
4.0 0.9
3.7 1.0
3.1 0.5
3.2 0.7
5.7 1.6
6.5 1.1
6.2 2.4
11.2 0.7
7.3 1.0
N
N
H
RHN
NCN
3a-e
1
n-C4H9
2a, 3a
n-C5H11
2b, 3b
n-C8H17
2c, 3c
R =
3i
Loratadine
Desloratadine (1)
5-FU
2e, 3e
2d, 3d
Scheme 1. Synthetic routes of 3a–e. Reagents and conditions: (a) toluene, 6.5 h,
reflux, 37.2–71.1%.
a
The IC50 values represent the concentration which results in a 50% decrease in
cell growth after 72 h of incubation.
cytotoxicity against breast (MCF-7 and MDA-MB 231) and colon
(HT-29) cancer cell lines. The experiments were performed accord-
ing to established procedures.12,27–29
The experimental cytotoxic activity data suggested that substit-
uents at the aromatic ring of the N0-phenylethyl residue might re-
duce the cytotoxic potency. The 3,4-dimethoxy (3h) and the 3,4-
methylenedioxy (3i) derivatives were less effective than the
unsubstituted 3g.
Loratadine caused comparable effects as 5-FU against MDA-MB
231 (loratadine: IC50 = 8.4
(loratadine: IC50 = 6.2 M; 5-FU: IC50 = 7.3
cells, loratadine (IC50 = 7.5 M) was less active than 5-FU
(IC50 = 4.7 M). For desloratadine IC50 values of about 10–12
were calculated at all cell lines.
As outlined in Table 1, most of the target compounds (3a to 3g)
displayed IC50 values (1.4–6.9 M) lower than 5-FU and loratadine.
Especially the most promising compound 3c (IC50 (MCF-
7) = 1.4 M; IC50 (MDA-MB 231) = 4.1 M; IC50 (HT-29) = 2.0 M)
l
M; 5-FU: IC50 = 9.6
lM) and HT-29
l
l
M) cells. At MCF-7
l
Compound 3c was further investigated in a time-dependent test
for antiproliferative activity. The time over activity (T/Ccorr) corre-
lation is shown in Figure 2 and indicates only a marginal recuper-
ation of cells after a prolonged time of exposition. The minimal T/
Ccorr (maximum of growth inhibition) after an incubation time of
48 h remained nearly unchanged or increased only slowly. There-
fore, the development of drug resistance is very unlikely.
Next, as the thiourea derivatives containing a benzo[5,6]cyclo-
hepta[1,2-b]pyridine moiety showed anti-inflammatory activity
in the well-known xylene-induced ear swelling assay in
mice12,27,30 we investigated the cyanoguanidine derivatives in the
same in vivo assay, too. As shown in Table 2, 3a–i exhibited anti-
inflammatory activity at a dose of 4 mg/kg b.w. comparable to
l
lM
l
l
l
l
possessed at least 2-fold higher cytotoxic potential than 5-FU
and loratadine. In addition, the IC50 values of 3a–g were lower than
those of the thiourea derivatives (IC50 = 4.7–10.4 lM) which indi-
cate that the introduction of the substituted cyanoguanidine moi-
ety in loratadine was more effective than the substituted thiourea
moiety.12
Cl
Cl
Cl
N
N
(a)
(b)
N
NCN
SCH3
+
N
N
H3CS
N
H
1
RHN
H3CS
NCN
3f-i
NCN
4
O
O
H3CO
H3CO
R =
3g
3f
3i
3h
Scheme 2. Synthetic routes of 3f–i. Reagents and conditions: (a) absolute ethanol, rt, over 2 h, 81,2%; (b) substituted amine, toluene, 9 h, reflux, 53.2–79.2%.

6078
W. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6076–6080
b.w.). To achieve the same activity as 3e ibuprofen has to be used
at a dose of 30 mg/kg b.w. (39.13% inhibition).
120
100
80
Finally, in order to study if the inhibition of COX-1/2 enzymes
(common targets for both antitumor and anti-inflammation) con-
tributed to the anti-inflammatory and antitumor properties, we
exemplarily examined 3e by ELISA.12,27,28,31 In these experiments,
3e caused only a weak COX inhibition (COX-1 (4.9%) and COX-2
60
(18.9%)) at 10 lM (indomethacin as reference: COX-1 inhibition:
40
56%; COX-2 inhibition: 100%). Therefore, 3e and its derivatives
may not interfere in the arachidonic acid cascade via COX enzymes.
In conclusion, a series of cyanoguanidine derivatives of lorata-
dine was designed and synthesized. All compounds have potential
antitumor activity in the preliminary cytotoxic bioassay. 3c was
the most cytotoxic compound against tumor cells used in this
study. Furthermore, the in vivo anti-inflammatory tests indicated
that these compounds were more active than ibuprofen in the xy-
lene-induced ear swelling assay. Additional investigations on the
biological activity as well as an extended structure–activity rela-
tionship study are in progress and will be part of a forthcoming
paper.
20
0
-20
-40
0
20
40
60
80
100
120
5 µM
140
Incubation time (h)
0.63 µM
1.25 µM
2.5 µM
10 µM
120
100
80
Acknowledgements
The authors are grateful for the financial supports of National
Natural Science Foundation of China (No. 30973638) and Jiangsu
Enterprise-Academics-Research Innovation Found-Prospective
Joint Research Project (No. BY2011158). The China scholarship
council and the Deutsche Forschungsgemeinschaft (FOR 630) are
gratefully acknowledged.
60
40
References and notes
20
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S. Mol. Carcinog. 2006, 45, 461.
-20
0
20
40
60
80
100
120
5 µM
140
Incubation time (h)
0.63 µM
1.25 µM
2.5 µM
10 µM
Figure 2. Time activity curves of 3c on MCF-7 (up) and HT-29 (left) cell lines. Error
bars are hidden behind the symbols in some cases.
8. Robak, T.; Szmigielska-Kaplon, A.; Pluta, A.; Grzybowska-Izydorczyk, O.;
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Table 2
ꢀ
Effect of the compounds on xylene-induced ear swelling in mice (n = 8, x S)
Dose (mg/kg)
Swollen extent; weight (g)
Inhibition (%)
Control
3a
3b
3c
3d
3e
3f
3g
3h
3i
Ibuprofen
Ibuprofen
0.023 0.003
0.019 0.005*
0.020 0.006
0.019 0.004*
0.020 0.001
0.017 0.008*
0.020 0.003
0.020 0.003
0.018 0.006*
0.020 0.002
0.021 0.002*
0.014 0.004*
4
4
4
4
4
4
4
4
4
4
30
17.39
13.04
15.55
17.39
26.09
13.04
13.04
21.73
13.04
8.22
39.13
18. Binderup, E.; Bjorkling, F.; Hjarnaa, P. V.; Latini, S.; Baltzer, B.; Carlsen, M.;
Binderup, L. Bioorg. Med. Chem. Lett. 2005, 15, 2491.
*
P<0.05, data were subjected to one-way ANOVA.
19. Li, S. L.; Huang, C. H.; Lin, C. C.; Huang, Z. N.; Chern, J. H.; Lien, H. Y.; Wu, Y. Y.;
Cheng, C. H.; Chang, C. Y.; Chuu, J. J. Invest. New Drugs 2011, 29, 195.
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James, L.; Kirschmeier, P.; Bishop, W. R. Bioorg. Med. Chem. Lett. 1998, 8, 2521.
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Bishop, W. R.; Weber, P. C.; Girijavallabhan, V. Bioorg. Med. Chem. Lett. 2002, 12,
601.
the thiourea derivatives.12 Especially the high anti-inflammatory
inhibition rate of 3e (26.09%) is worthy to mention. In addition,
all compounds (13.04–26.09% inhibition) were more active than
ibuprofen (8.22% inhibition) used at the same dose (4 mg/kg
22. Florence, X.; Sebille, S.; Tullio, P.; Lebrun, P.; Pirotte, B. Bioorg. Med. Chem. 2009,
17, 7723.

W. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6076–6080
6079
23. Shi, Y.; Zhang, J.; Shi, M.; O’Connor, S. P.; Bisaha, S. N.; Li, C.; Sitkoff, D.;
Pudzianowski, A. T.; Chong, S.; Klei, H. E.; Kish, K.; Yanchunas, J., Jr.; Liu, E. C.;
Hartl, K. S.; Seiler, S. M.; Steinbacher, T. E.; Schumacher, W. A.; Atwal, K. S.;
Stein, P. D. Bioorg. Med. Chem. Lett. 2009, 19, 4034.
24. General procedure for the synthesis of 3a–e. 2.0 g (6.4 mmol) of 1 (desloratadine)
and 3.8 mmol of substituted methyl N’-cyanocarbamimidothioate 2a–e were
combined and refluxed in 25 mL of toluene for 6.5 h. Then the solvent was
removed under reduced pressure. The crude product was purified by flash
column chromatography with a mixture eluent of petroleum ether and ethyl
acetate to give the corresponding product.
8.35 (dd, 1H, J = 4.6 Hz, pyridine-H). Anal. calcd for C28H26ClN5: C, 71.86; H,
5.50; N, 14.96. Found C, 71.77; H, 5.62; N, 14.21.
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-(2-phenylethyl)-1-piperidinecarboximidamide (3g). Yield: 79.2% of a
white solid; mp: 178–180 °C. ESI-MS: 482.1 [M+H]+. IR (cmꢀ1): 3226, 3085,
2925, 2882, 2856, 2163, 1556, 1456, 1436, 995, 829, 699. 1H NMR (CDCl3) d:
2.27–2.34 (m, 2H, @C(CH2)2); 2.39–2.60 (m, 2H, @C(CH2)2); 2.75–2.84 (m, 2H,
N(CH2)2); 2.89 (t, 2H, CH2Ar); 3.15–3.22 (m, 2H, N(CH2)2); 3.27–3.37 (m, 2H,
PhCH2); 3.55–3.65 (m, 2H, CH2); 3.67–3.71 (m, 2H, NCH2); 4.82–4.95 (br, 1H,
NH); 7.06–7.32 (m, 9H, Ar-H, pyridine-H); 7.46 (dd, 1H, J = 6.9 Hz, pyridine-H);
8.38 (dd, 1H, J = 2.2 Hz, pyridine-H). Anal. calcd for C29H28ClN5: C, 72.26; H,
5.86; N, 14.53. Found C, 72.23; H, 5.74; N, 14.61.
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-(n-butyl)-1-piperidinecarboximidamide (3a). Yield: 42.1% of a white
solid; mp: 151–152 °C. ESI-MS: 434.1 [M+H]+, 456.1 [M+Na]+. IR (cmꢀ1): 3443,
3292, 2954, 2926, 2868, 2163, 1574, 1556, 1436, 1324, 1298, 1246, 997, 828.
1H NMR (CDCl3) d: 0.9–0.94 (t, 3H, CH3); 1.25–1.42 (m, 2H, CH2); 1.51–1.60 (m,
2H, CH2); 2.33–2.43 (m, 2H, @C(CH2)2); 2.48–2.67 (m, 2H, @C(CH2)2); 2.76–
2.89 (m, 2H, N(CH2)2); 3.26–3.40 (m, 6H, PhCH2, NCH2, N(CH2)2); 3.67–3.71 (m,
2H, CH2); 4.81 (br, 1H, NH); 7.09–7.17 (m, 4H, Ar-H, pyridine-H); 7.49 (dd, 1H,
J = 7.6 Hz, pyridine-H); 8.40 (dd, 1H, J = 1.4 Hz, pyridine-H). Anal. Calcd for
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-(2-(3,4-dimethoxyphenyl)ethyl)-1-piperidinecarboximidamide
(3h).
Yield: 65.3% of a yellow solid; mp: 112–113 °C. ESI-MS: 542.3 [M+H]+, 564.2
[M+Na]+. IR (cmꢀ1): 3372, 3268, 2931, 2858, 2832, 2165, 1571, 1545, 1510,
1437, 1261, 1236, 1156, 1141, 1028, 993, 829, 811, 746, 557. 1H NMR (CDCl3) d:
2.27–2.36 (m, 2H, @C(CH2)2); 2.45–2.65 (m, 2H, @C(CH2)2); 2.77–2.89 (m, 4H,
N(CH2)2); 3.21–3.28 (m, 2H, ArCH2); 3.30–3.38 (m, 2H, PhCH2); 3.57–3.70 (m,
4H, NCH2, CH2); 3.84 (s, 3H, OCH3); 3.87 (s, 3H, OCH3); 4.78 (br, 1H, NH); 6.71–
6.83 (m, 3H, Ar-H); 7.11–7.24 (m, 4H, Ar-H, pyridine-H); 7.58 (dd, 1H,
J = 7.1 Hz, pyridine-H); 8.42 (dd, 1H, J = 4.8 Hz, pyridine-H). Anal. calcd for
C
25H28ClN5: C, 69.19; H, 6.50; N, 16.14. Found C, 69.14; H, 6.52; N, 16.20.
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-(n-amyl)-1-piperidinecarboximidamide (3b). Yield: 37.2% of a yellow
solid; mp: 155–156 °C. ESI-MS: 448.2 [M+H]+, 470.1 [M+Na]+. IR (cmꢀ1): 3290,
2953, 2928, 2868, 2158, 1573, 1553, 1503, 1435, 1322, 1295, 999, 993, 827,
810. 1H NMR (CDCl3) d: 0.89 (t, 3H, CH3); 1.25–1.33 (m, 4H, 2CH2); 1.52–1.61
(m, 2H, CH2); 2.36–2.39 (m, 2H, @C(CH2)2); 2.41–2.81 (m, 2H, @C(CH2)2); 2.83–
2.84 (m, 2H, N(CH2)2); 3.27–3.39 (m, 6H, PhCH2, NCH2, N(CH2)2); 3.67–3.73 (m,
2H, CH2); 4.82 (br, 1H, NH); 7.09–7.17 (m, 4H, Ar-H, pyridine-H); 7.48 (dd, 1H,
J = 7.5 Hz, pyridine-H); 8.40 (dd, 1H, J = 3.9 Hz, pyridine-H). Anal. calcd for
C
31H32ClN5O2.H2O: C, 66.48; H, 6.12; N, 12.50. Found C, 66.35; H, 6.23; N,
12.39.
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-(2-(3,4-methylenedioxyphenyl)ethyl)-1-piperidinecarboximidamide (3i).
Yield: 65.5% of a white solid; mp: 214–216 °C. ESI-MS: 526.2 [M+H]+, 548.1
[M+Na]+. IR (cmꢀ1): 3428, 3272, 2922, 2889, 2164, 1548, 1500, 1488, 1438,
1261, 1247, 1158, 1042, 992, 814, 779, 549. 1H NMR (CDCl3) d: 2.23–2.36 (m,
2H, @C(CH2)2); 2.42–2.66 (m, 2H, @C(CH2)2); 2.73–2.89 (m, 4H, N(CH2)2);
3.17–3.28 (m, 2H, ArCH2); 3.30–3.39 (m, 2H, PhCH2); 3.57–3.69 (m, 4H, NCH2,
CH2); 4.95 (br, 1H, NH); 5.91 (s, 2H, OCH2O); 6.61–6.74 (m, 3H, Ar-H); 7.06–
7.17 (m, 4H, Ar-H); 7.46 (dd, 1H, J = 6.8 Hz, pyridine-H); 8.38 (dd, 1H, J = 1.5 Hz,
pyridine-H). Anal. calcd for C30H28ClN5O2: C, 66.50; H, 5.37; N, 13.31. Found C,
66.62; H, 5.41; N, 13.27.
C
26H30ClN5: C, 69.70; H, 6.75; N, 15.63. Found C, 69.69; H, 6.81; N, 15.71.
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-(n-octyl)-1-piperidinecarboximidamide (3c). Yield: 41.4% of a white
solid; mp: 136–137 °C. ESI-MS: 490.2 [M+H]+, 512.3 [M+Na]+. IR (cmꢀ1): 3285,
2952, 2920, 2852, 2161, 1571, 1538, 1436, 1329, 1297, 1254, 1174, 1085, 993,
829, 783. 1H NMR (CDCl3) d: 0.86 (t, 3H, CH3); 1.25–1.26 (m, 10H, 5CH2); 1.53–
1.57 (m, 2H, CH2); 2.35–2.40 (m, 2H, @C(CH2)2); 2.50–2.78 (m, 2H, @C(CH2)2);
2.80–2.84 (m, 2H, N(CH2)2); 3.24–3.39 (m, 6H, PhCH2, NCH2, N(CH2)2); 3.65–
3.72 (m, 2H, CH2); 4.88 (br, 1H, NH); 7.08–7.16 (m, 4H, Ar-H, pyridine-H); 7.46
(dd, 1H, J = 7.6 Hz, pyridine-H); 8.38 (dd, 1H, J = 3.5 Hz, pyridine-H). Anal. calcd
for C29H36ClN5: C, 71.07; H, 7.40; N, 14.29. Found C, 71.01; H, 7.38; N, 14.32.
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-cyclohexyl-1-piperidinecarboximidamide (3d). Yield: 71.1% of a white
solid; mp: 219–220 °C. ESI-MS: 460.2 [M+H]+, 482.1 [M+Na]+. IR (cmꢀ1): 3293,
3117, 2924, 2853, 2159, 1576, 1528, 1478, 1436, 1329, 1298, 1246, 1174, 1086,
995, 826. 1H NMR (CDCl3) d: 1.12–2.00 (m, 10H, 5CH2); 2.34–2.43 (m, 2H,
@C(CH2)2); 2.51–2.77 (m, 2H, @C(CH2)2); 2.80–2.92 (m, 2H, N(CH2)2); 3.30–
3.40 (m, 4H, N(CH2)2, PhCH2); 3.67–3.77 (m, 3H, CH, CH2); 4.56 (br, 1H, NH);
7.12–7.20 (m, 4H, Ar-H, pyridine-H); 7.56 (dd, 1H, J = 7.4 Hz, pyridine-H); 8.43
(dd, 1H, J = 4.9 Hz, pyridine-H). Anal. calcd for C27H30ClN5.H2O: C, 67.84; H,
6.75; N, 14.65. Found C, 67.79; H, 6.79; N, 14.52.
27. Liu, W.; Zhou, J.; Bensdorf, K.; Zhang, H.; Liu, H.; Wang, Y.; Qian, H.; Zhang, Y.;
Wellner, A.; Rubner, G.; Huang, W.; Guo, C.; Gust, R. Eur. J. Med. Chem. 2011, 46,
907.
28. Liu, W.; Zhou, J.; Liu, Y.; Liu, H.; Bensdorf, K.; Guo, C.; Gust, R. Arch. Pharm. 2011,
344, 487.
29. Cell culture: The human MCF-7, MDA-MB 231 breast and HT-29 colon cancer
cell lines were obtained from the American Type Culture Collection. All cell
lines were maintained as
a monolayer culture in L-glutamine containing
Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose (PAA
Laboratories, Austria), supplemented with 5% fetal bovine serum (FBS;
Biochrom, Germany) in a humidified atmosphere (5% CO2) at 37 °C.
Cytotoxicity: In 96 well plates, 100 lL of a cell suspension in culture medium at
7500 cells/mL (MCF-7 and MDA-MB 231) or 3000 cells/mL (HT-29) were plated
into each well and incubated for three days under culture conditions. After the
addition of various concentrations of the test compounds, cells were incubated
for up to 144 h. Then the medium was removed, the cells were fixed with
glutardialdehyde solution (1%) and stored under phosphate buffered saline
(PBS) at 4 °C. Cell biomass was determined by a crystal violet staining, followed
by extracting of the bound dye with ethanol and a photometric measurement
at 590 nm. Mean values were calculated and the effects of the compounds
were expressed as % Treated/Controlcorr values according to the following
equations:
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-(1-phenylethyl)-1-piperidinecarboximidamide (3e). Yield: 38.3% of a
white solid; mp: 135–136 °C. ESI-MS: 482.1 [M+H]+, 504.1 [M+Na]+. IR (cmꢀ1):
3425, 3259, 2974, 2920, 2853, 2168, 1572, 1527, 1478, 1436, 1329, 1292, 1174,
1087, 993, 829, 762, 699. 1H NMR (CDCl3) d: 1.56 (d, 3H, CH3, J = 6.5 Hz); 2.30–
2.38 (m, 2H, @C(CH2)2); 2.48–2.76 (m, 2H, @C(CH2)2); 2.78–2.88 (m, 2H,
N(CH2)2); 3.30–3.38 (m, 4H, N(CH2)2, PhCH2); 3.66–3.85 (m, 2H, CH2); 4.98–
5.08 (m, 2H, NHCH); 7.14–7.33 (m, 9H, Ar-H, pyridine-H); 7.55 (dd, 1H,
J = 3.4 Hz, pyridine-H); 8.41 (dd, 1H, J = 4.4 Hz, pyridine-H). Anal. calcd for
T ꢀ C0
T=Ccorr½%ꢁ ¼
ꢂ 100
29H28ClN5.: C, 72.26; H, 5.86; N, 14.53. Found C, 72.31; H, 5.89; N, 14.45.
C ꢀ C0
C
25. 4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-1-piperidinecarboximidothioic acid methyl ester (4). The flask was charged
with 60 mL of absolute ethanol, 6.0 g (19.3 mmol) of 1 (desloratadine) and
2.9 g (20 mmol) of dimethyl cyanocarbonimidodithioate. The resulting
solution was stirred at rt for over 2 h. Then the white suspension was
filtered under suction and the solid was washed with a small amount of cold
ethanol to give the white product 4 (6.4 g). Yield: 81.2% of a white solid; mp:
162–164 °C. ESI-MS: 409.0 [M+H]+. IR (cmꢀ1): 3080, 2929, 2894, 2852, 2180,
1548, 1540, 1456, 1434, 991, 834. 1H NMR (CDCl3) d: 2.36–2.44 (m, 2H,
@C(CH2)2); 2.47–2.72 (m, 2H, @C(CH2)2); 2.76 (s, 3H, SCH3); 2.78–2.90 (m, 2H,
N(CH2)2); 3.28–3.39 (m, 2H, N(CH2)2); 3.50–3.60 (m, 2H, PhCH2); 4.03–4.15 (m,
2H, CH2); 7.09–7.18 (m, 3H, Ar-H); 7.26 (m, 1H, pyridine-H); 7.47 (dd, 1H,
J = 7.3 Hz, pyridine-H); 8.40 (dd, 1H, J = 4.7 Hz, pyridine-H).
26. General procedure for the synthesis of 3f–i. 1.0 g (2.4 mmol) of 4 and 3.3 mol of
the respectively substituted amine were combined and refluxed in 25 mL of
toluene for 9 h. Then the solvent was removed under reduced pressure. The
crude product was purified by flash column chromatography with a mixture
eluent of petroleum ether and ethyl acetate to give the corresponding product.
4-(8-Chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-N-
cyano-N’-benzyl-1-piperidinecarboximidamide (3f). Yield: 53.2% of a white solid;
mp: 177–178 °C. ESI-MS: 468.1 [M+H]+. IR (cmꢀ1): 3238, 3116, 3055, 3028,
2924, 2856, 2156, 1556, 1536, 1495, 1435, 1356, 1301, 990, 828, 695. 1H NMR
(CDCl3) d: 2.33–2.39 (m, 2H, @C(CH2)2); 2.46–2.65 (m, 2H, @C(CH2)2); 2.75–
2.87 (m, 2H, N(CH2)2); 3.27–3.37 (m, 4H, N(CH2)2, PhCH2); 3.69–3.73 (m, 2H,
CH2); 4.52–4.54 (m, 2H, NCH2); 5.41–5.50 (br, 1H, NH); 7.06–7.16 (m, 4H, Ar-
H); 7.26–7.33 (m, 5H, Ar-H, pyridine-H); 7.44 (dd, 1H, J = 7.6 Hz, pyridine-H);
(C0 control cells at the time of compound addition; C control cells at the time of
test end; T probes/samples at the time of test end).
The IC50 value was determined as the concentration causing 50% inhibition of
cell proliferation and calculated as mean of at least three independent experi-
ments (OriginPro 8).
30. Xylene-induced ear edema. Animals: The experiments applied with animals
were approved by Research Ethic Committee of Jiang-Shu province, China.
Kunming male mice of approximately 20 g were obtained from experimental
animal centre of China Pharmaceutical University, and fed with food and water
ad libitum. All animals were fasted for 12 h before the experiments. The
temperature (25 °C) and humidity (60%) in the animal room were well
controlled.
Method: Mice were allotted to groups of 8 animals each. One group of mice,
which served as control was given vehicle (0.5% CMC in water in a volume of
20 mL/kg) only. Test compounds (4 mg/kg b.w.) and ibuprofen (4 mg/kg b.w.
and 30 mg/kg b.w.) suspended in vehicle (20 mL/kg) were administered ip to
respective groups for 5 days. Thirty minutes after the last administration,
0.1 mL of xylene was applied to the anterior and posterior surfaces of the right
ear. The left ear was considered as control. One hour after xylene application,
mice were killed and both ears were removed. Circular sections were taken,
using a cork borer with a diameter of 9 mm, weighed and measured. The degree
of ear swelling was calculated based on the weight of left ear without xylene.
Statistical analysis: The measurement data were expressed as the mean SD.
Data were subjected to one-way analysis of variance (ANOVA), followed by
multiple comparison with least significant differences (LSD) test or Dunnett’s

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W. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6076–6080
test as appropriate. Statistical significance was considered with P <0.05. The
analysis of data was performed by software SPSS 13.0.
31. The inhibition of isolated ovine COX-1 and human recombinant COX-2 was
screening assay’, Cayman Chemicals). Experiments were performed according
to the manufacturer’s instructions. Absorption was measured at 415 nm
(Victor2, Perkin Elmer). Results were calculated as the means of duplicate
determinations.
determined with 10 lM of the respective compound by ELISA (‘COX inhibitor