Vascular barrier protective effects of 3-N- or 3-O-cinnamoyl carbazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.07.079

In this Letter, we investigated the barrier protective effects of 3-N-(MeO)_n-cinnamoyl carbazoles (BS 1; n = 1, BS 2; n = 2, BS 3; n = 3) and 3-O-(MeO)₃-cinnamoyl carbazole (BS 4) against high-mobility group box 1 (HMGB1)-mediated vascular disruptive responses in human umbilical vein endothelial cells (HUVECs) and in mice for the first time. Data showed that BS 2, BS 3, and BS 4, but not BS 1, inhibited HMGB1-mediated vascular disruptive responses and transendothelial migration of human neutrophils to HUVECs. BS 2, BS3, and BS 4 also suppressed HMGB1-induced hyperpermeability and leukocyte migration in mice. Interestingly, the barrier protective effects of BS 3 and BS 4 were better than those of BS 2. These results suggest that the number of methoxy groups substituted on the cinnamamide or cinnamate moiety of the 9H-3-carbazole derivative is an important pharmacophore for the barrier protective effects of these compounds.

Full text of DOI:10.1016/j.bmcl.2015.07.079

Accepted Manuscript
Vascular barrier protective effects of 3-N- or 3-O-cinnamoyl carbazole deriva-
tives
Sae-Kwang Ku, Jee Hyun Lee, O. Yuseok, Wonhwa Lee, Gyu-Yong Song,
Jong-Sup Bae
PII:
S0960-894X(15)00783-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.079
BMCL 22968
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 April 2015
4 July 2015
23 July 2015
Please cite this article as: Ku, S-K., Lee, J.H., Yuseok, O., Lee, W., Song, G-Y., Bae, J-S., Vascular barrier protective
effects of 3-N- or 3-O-cinnamoyl carbazole derivatives, Bioorganic & Medicinal Chemistry Letters (2015), doi:
http://dx.doi.org/10.1016/j.bmcl.2015.07.079
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Vascular barrier protective effects of 3-N- or 3-O-cinnamoyl carbazole derivatives
Sae-Kwang Kua†, Jee Hyun Leeb†, Yuseok Ob, Wonhwa Leec,d, Gyu-Yong Songb*, and Jong-Sup
Baec*
a
From Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany
University, Gyeongsan 712-715, Republic of Korea; bCollege of Pharmacy, Chungnam National
c
University, Daejon 305-764, Republic of Korea; College of Pharmacy, CMRI, Research
Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701,
d
Republic of Korea; Department of Biochemistry and Cell Biology, BK21 Plus KNU
Biomedical Convergence Program, School of Medicine, Kyungpook National University,
Daegu 702-422, Republic of Korea
†First two authors contributed equally to this work.
* Corresponding Authors:
Gyu-Yong Song, Ph.D.
College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejon 305-
764, Republic of Korea
1

Phone, 82-42-821-5926; Fax, 82-42-823-6566; E-mail, gysong@cnu.ac.kr
and
Jong-Sup Bae, Ph.D.
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National
University
80 Daehak-ro, Buk-gu, Daegu 702-701, Republic of Korea
Phone, 82-53-950-8570; Fax, 82-53-950-8557; E-mail, baejs@knu.ac.kr
2

Abstract
In this letter, we investigated the barrier protective effects of 3-N-(MeO)n-cinnamoyl carbazoles
(BS 1; n = 1, BS 2; n = 2, BS 3; n = 3) and 3-O-(MeO)3-cinnamoyl carbazole (BS 4) against
high-mobility group box 1 (HMGB1)-mediated vascular disruptive responses in human
umbilical vein endothelial cells (HUVECs) and in mice for the first time. Data showed that BS 2,
BS 3, and BS 4, but not BS 1, inhibited HMGB1-mediated vascular disruptive responses and
transendothelial migration of human neutrophils to HUVECs. BS 2, BS3, and BS 4 also
suppressed HMGB1-induced hyperpermeability and leukocyte migration in mice. Interestingly,
the barrier protective effects of BS 3 and BS 4 were better than those of BS 2. These results
suggest that the number of methoxy groups substituted on the cinnamamide or cinnamate
moiety of the 9H-3-carbazole derivative is an important pharmacophore for the barrier
protective effects of these compounds.
Key words: 3-N-cinnamoyl carbazole, 3-O-cinnamoyl carbazole, vascular barrier integrity,
HMGB1
3

The vascular endothelium has diverse functions, including prevention of coagulation,
regulation of vascular tonus and permeability, coordination of blood cell migration, and
production of chemo-attractants 1-2. Endothelial cells (ECs) provide a semi-selective barrier
between the blood and underlying tissue interstitium, and the disruption of barrier integrity
caused by cellular injury or stress induces vascular hyperpermeability, which is typically found
3
in inflammation, tumor angiogenesis, and atherosclerosis . Thus, maintaining endothelial
integrity against vascular stress has emerged as an effective therapeutic strategy for the
alleviation of inflammatory-associated diseases 3-4. Among the numerous approaches used to
enhance vascular integrity, inhibition of high-mobility group box 1 (HMGB1) has been
recognized as a sensible protocol. HMGB1 is released into the extracellular space from necrotic
cells and/or immune cells upon inflammatory stimuli. The released HMGB1 induces the
expression of cell adhesion molecules and facilitates the production of an array of pro-
inflammatory cytokines that mediate leukocyte adherence, increased vascular permeability,
coagulation activation, and microvascular thrombosis 5-6. Therefore, anti-HMGB1 agents
capable of inhibiting HMGB1 activity may be developed into a therapeutic intervention for the
treatment of vascular inflammatory disorders given the significant role of HMGB1 in rendering
endothelial hyperpermeability 7-8. In our continuing efforts to discover anti-HMGB1 agents for
the intervention of pathogenic conditions associated with vascular dysfunction 9-11, we herein
4

elaborate the synthesis and structural elucidation of 3-N- and 3-O-(MeO)n-cinnamoyl carbazoles
(BS 1–4). We also address whether these compounds could be developed for the treatment of
vascular dysfunction by employing relevant cellular and animal models of vascular disruptive
complications.
Carbazole derivatives are a significant class of heterocyclic compounds and have been
reported to have various biological activities, such as anticancer, antimicrobial, antiviral,
antioxidant, anti-platelet aggregation, and anti-inflammatory. In particular, we found that many
compounds with a carbazole moiety exhibit anti-inflammatory activities through a variety of
14
15
mechanisms, such as carprofen 12-13, 3-pyrazolyl-9-methyl carbazole , AF3442 , LCY-2CHO
16-17, and 3’-N-substituted carbazoles 18 (Fig. 1).
OCH3
OH
OCH3
N
Cl
O
N
N
H
N
N
H
OH
O
Cl
3-pyrazolyl-9-methyl carbazole
LCY-2-CHO
Carprofen
O
N
H
N
N
(CH2)n
H
H
R
F3C
N
N
H
n= 0 - 4
R= H, NO2, NH2
3-N-substituted carbazoles
AF3442
Figure 1. Anti-inflammatory active carbazole compounds
In a previous study, we identified that 3’-N-substituted carbazole derivatives with a urea
linkage introduced could be new candidate drugs for severe vascular inflammatory diseases,
5

such as sepsis. Meanwhile, it has been found that some compounds with the cinnamoyl moiety
possess anti-inflammatory effects, such as tranilast 19, avenanthramide 20, piperlongumine 21, and
lupeol cinnamate 22 (Fig. 2).
O
O
O
OH
OCH3
OCH3
OCH3
OCH3
H
N
N
CH2
H
O
H3C
OCH3
Tranilast
Piperlongumine
H
OH
CH3
CH3 CH3
H
O
HO
N
H
H
n
R
O
O
R= H, OH, OCH3
H3C CH3
O
OH
Avenanthramide
Lupeol cinnamate
Figure 2. Anti-inflammatory active compounds with a cinnamoyl moiety
Therefore, the combination of a carbazole moiety and cinnamoyl moiety are expected
have a synergistic effect on anti-inflammatory activity (Fig. 3).
O
X
(OCH3)n
N
X= N or O
H
3-N or -O-cinnamoyl carbazoles
( BS series )
Figure 3. Combination of a carbazole moiety and cinnamoyl moiety
In this study, we designed and synthesized novel 3-N- or -O-cinnamoyl carbazole
derivatives (BS 1–4), and introduced a cinnamamide and cinnamate linkage in carbazole’s
framework, instead of a urea linkage. Moreover, the effects of BS 1–4 on vascular barrier
6

integrity in both cellular systems and animal models have not yet been elucidated. The objective
of the present study was to determine the vascular barrier protective effect of BS 1–4 on ECs
and mice.
To determine the effects of synthesized BS 1–4 (supplementary data) on the HMGB1-mediated
hyperpermeability primary HUVECs, the flux of albumin in a dual-chamber system was
23
23-26
monitored as described previously . HUVECs
were treated with each compound for 6 h
after the addition of HMGB1 (1 g/mL). The results show that BS 2–4 at 10 M induced the
down-regulation of HMGB1-mediated membrane disruption and the vascular protective effects
of BS 3 and BS 4 were better than that of BS 2 (Table 1).
Table 1. Effects of different BS compounds on HMGB1-mediated hyperpermeability in
HUVECs
Compound
DMSO
HMGB1
BS 1
Dose
ELISA OD650
0.116 ± 0.021
0.531 ± 0.039
0.512 ± 0.019
0.313 ± 0.023*
0.168 ± 0.021*
0.158 ± 0.031*
Inhibition (%)
1 g/mL
10 M
10 M
10 M
10 M
3.6
42.9
68.4
70.2
BS 2
BS 3
BS 4
*
Each entry represents the mean ± SEM of three different experiments. p < 0.05 significantly
7

different from HMGB1.
DMSO: dimethyl sulfoxide; HMGB1: high-mobility group box 1; HUVECs: human umbilical
vein endothelial cells; SEM: standard error of the mean.
27
The in vivo effects of BS 2–4 on vascular permeability were assessed in order to
corroborate the in vitro results. Thus, as can be seen in Table 2, BS 2–4 at 10 M induced
marked inhibition of the peritoneal leakage of dye induced by HMGB1. Because the average
circulating blood volume for mice is 72 mL/kg 28, the average weight of the mice used was 27 g,
and the average blood volume was 2 mL. The amount of BS 1 (342.4 g/kg), BS 2 (372.4
g/kg), BS 3 (402.4 g/kg), and BS 4 (403.4 g/kg) injected yielded a maximum concentration
of 10 M in the peripheral blood.
Table 2. Effects of different BS compounds on HMGB1-mediated hyperpermeability in mice
Compound
DMSO
HMGB1
BS 1
Dose
Inhibition (%)
g/mouse (n=5)
0.35 ± 0.05
5.72 ± 0.29
5.64 ± 0.35
4.22 ± 0.42*
2.43 ± 0.21*
2.35 ± 0.22*
100.0 g/kg
342.4 g/kg
372.4 g/kg
402.4g/kg
403.4 g/kg
1.4
26.2
57.5
58.9
BS 2
BS 3
BS 4
8

The amount of BS 1 (342.4 g/kg), BS 2 (372.4 g/kg), BS 3 (402.4 g/kg), and BS 4 (403.4
g/kg) injected yielded a concentration of 10 M in the peripheral blood.
*
Each entry represents the mean ± SEM of three different experiments. p < 0.05 significantly
different from HMGB1.
DMSO: dimethyl sulfoxide; HMGB1: high-mobility group box 1; SEM: standard error of the
mean.
To exclude the possibility that the inhibition of permeability was due to cytotoxicity
caused by each compound, cellular viability assays 27 were performed in HUVECs treated with
each compound for 24 h. At the concentrations used (up to 20 M), BS 1–4 did not affect cell
viability (data not shown).
The migration of leukocytes to ECs is an important step in the HMGB1-mediated
29
inflammatory response . We conducted studies to determine whether BS 2–4 could block the
migration of human neutrophils 30-31 to HMGB1-stimulated HUVECs as described previously 23.
We demonstrated that BS 2–4 at 10 M effectively inhibited the migration of human
neutrophils to HMGB1-stimulated ECs (Table 3).
Table 3. Effects of different BS compounds on HMGB1-mediated neutrophil migration in
HUVECs
Compound
DMSO
Dose
Migration Index
35.3 ± 2.7
Inhibition (%)
9

HMGB1
BS 1
188.2 ± 13.7
179.2 ± 14.5
106.4 ± 9.4*
71.6 ± 6.1*
65.2 ± 5.1*
1 g/mL
10 M
10 M
10 M
10 M
4.8
43.5
62.0
65.4
BS 2
BS 3
BS 4
*
Each entry represents the mean ± SEM of three different experiments. p < 0.05 significantly
different from HMGB1.
DMSO: dimethyl sulfoxide; HMGB1: high-mobility group box 1; HUVECs: human umbilical
vein endothelial cells; SEM: standard error of the mean.
To confirm this effect in vivo, HMGB1-induced leukocyte migration in mice was
examined. HMGB1 significantly stimulated leukocyte migration into the peritoneal cavity of
mice and BS 2–4 at 10 M significantly decreased leukocyte counts (Table 4).
Table 4 Effects of different BS compounds on HMGB1-mediated leukocyte migration in mice
Compound
DMSO
HMGB1
BS 1
Dose
x 106
Inhibition (%)
1.45 ± 0.13
7.35 ± 0.72
7.29 ± 0.65
4.48 ± 0.35*
2.92 ± 0.21*
100.0 g/kg
342.4 g/kg
372.4 g/kg
402.4g/kg
0.8
39.0
60.3
BS 2
BS 3
10

BS 4
2.85 ± 0.25*
61.2
403.4 g/kg
The amount of BS 1 (342.4 g/kg), BS 2 (372.4 g/kg), BS 3 (402.4 g/kg), and BS 4 (403.4
g/kg) injected yielded a concentration of 10 M in the peripheral blood.
*
Each entry represents the mean ± SEM of three different experiments. p < 0.05 significantly
different from HMGB1.
DMSO: dimethyl sulfoxide; HMGB1: high-mobility group box 1; HUVECs: human umbilical
vein endothelial cells; SEM: standard error of the mean.
In addition, the anti-inflammatory effects of BS 3 and BS 4 were better than that of BS
2 (Tables 2–4). The structure–activity relationship (SAR) of 3-cinnamoyl carbazole derivatives
(BS 1–4) revealed that the more methoxy groups present on the cinnamoyl moiety, the more
potent was the anti-inflammatory activity, regardless of whether cinnamamide or cinnamate
linkage was used.
The present work aimed to study the vascular barrier integrity protection offered by
the three new structurally similar compounds, BS 2–4. Maintenance of vascular barrier integrity
is emerging as an important therapeutic strategy. Along with involvement in the progression of
sepsis, HMGB1 has also been implicated as a proinflammatory cytokine and a proangiogenic
factor in the sustained proinflammatory responses observed in severe vascular inflammatory
32
diseases, such as sepsis or septic shock . The results of this study suggest that the anti-septic
11

effects of the three new structurally similar compounds, BS 2–4, occur through inhibition of
HMGB1-mediated inflammatory responses.
Acknowledgements
This study was supported by the National Research Foundation of Korea (NRF) funded by the
Korea government [MSIP] (Grant Nos. 2012R1A5A2A42671316) and by the Priority Research
Centers Program through the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2009-0093815).
12

References and notes
1.
2.
3.
4.
5.
Luescher, T. F.; Barton, M. Clin. Cardiol. 1997, 20 (Suppl. II), II.
Wu, M., KK; Thiagarajan, M., P. Annu. Rev. Med. 1996, 47, 315.
Komarova, Y. A.; Mehta, D.; Malik, A. B. Sci. Signal. 2007, 2007, re8.
Dudek, S. M.; Garcia, J. G. J. Appl. Physiol. 2001, 91, 1487.
Fiuza, C.; Bustin, M.; Talwar, S.; Tropea, M.; Gerstenberger, E.; Shelhamer, J. H.;
Suffredini, A. F. Blood 2003, 101, 2652.
6.
Treutiger, C. J.; Mullins, G. E.; Johansson, A. S. M.; Rouhiainen, A.; Rauvala, H.
M. E.; Erlandsson-Harris, H.; Andersson, U.; Yang, H.; Tracey, K. J.; Andersson,
J.; Palmblad, J. E. W. J. Intern. Med. 2003, 254, 375.
7.
8.
9.
Lee, W.; Kim, T. H.; Ku, S.-K.; Min, K.-j.; Lee, H.-S.; Kwon, T. K.; Bae, J.-S.
Toxicol. Appl. Pharml. 2012, 262, 91.
Yang, E.-J.; Ku, S.-K.; Lee, W.; Lee, S.; Lee, T.; Song, K.-S.; Bae, J.-S. J. Cell.
Physio. 2013, 228, 975.
Kim, D.-C.; Lee, W.; Bae, J.-S. Inflamm. Res. 2011, 60, 1161.
10. Zhou, W.; Oh, J.; Wonhwa, L.; Kwak, S.; Li, W.; Chittiboyina, A. G.; Ferreira, D.;
Hamann, M. T.; Lee, S. H.; Bae, J.-S. Biochim. Biophys. Acta, Gen. Subj. 2014,
1840, 2042.
13

11. Lee, W.; Ku, S.-K.; Bae, J. W.; Bae, J.-S. Food. Cheml. Toxicol. 2012, 50, 1826.
12. Fox, S. M.; Johnston, S. A. J. Am. Vet. Med. Assoc. 1977, 210, 1493.
13. Zall, A.; Kieser, D.; Hotteche, N.; Naumann, E. C.; Thomaszewski, B.; Schneider,
K.; Steinbacher, D. T.; Schubenel, R.; Nasur, S.; Baumann, K; Schmidt, B. Bioorg.
Med. Chem. 2011, 19, 4093.
14. Bandgar, B. P.; Adsul, L. K.; Chavan, H. V.; Jalde, S. S.; Shringare, S. N.; Shaikh,
R.; Meshram, R. J.; Gacche, R. N.; Masand, V. Bioorg. Med. Chem. Lett. 2012,
22, 5839.
15. Bruno, A.; Francesco, L. D.; Coletta, I.; Mangano, G.; Alisi, M. A.; Polenzani, L.;
Milanese, C.; Anzellotti, P.; Ricciotti, E.; Dovizio, M.; Francesco, A. D; Tacconelli,
S.; Capone, M. L.; Patrignani, P. Biochem. Pharmacol. 2010, 79, 974.
16. Lee, C. Y.; Huang, L. J.; Wang, J. P.; Kuo, S. C. Chin. Pharm. J. 2002, 54, 35
17. Ho, F. M.; Kang, H. C.; Lee, S. T.; Chao, Y.; Chen, Y. C.; Huang, L. J.; Lin, W. W.
Biochem. Pharmacol. 2007, 74, 298
18. Lee, W. H.; Kwak, S.; Yun,.E.; Lee, J. H.; Na, M. K, Song, G. Y.; Bae, J. S.
Inflammation. 2015, in press.
19. Pae, H. O.; Jeong, S. O.; Koo, B. S.; Ha, H. Y.; K Lee, K. M.; Chung, H. T.
Biochem. Biophy. Res. Communi. 2008, 371, 361.
14

20. Sur, R.; Nigam, A.; Grote, D.; Liebel, F.; Southall, M. D. Arch. Dermatol. Res.
2008, 300, 569.
21. Seo, Y. H.; Kim, J. K.; Jun, J. G. Bioorg. Med. Chem. Lett. 2014, 24, 5727.
22. Akihisa, T.; Kojima, N.; Kikuchi, T.; Yasukawa, K.; Tokuda, H.; Masters, E. T.;
Manosroi, A.; Manosroi, J. J. Oleo. Sci. 2010, 59, 273.
23. Bae, J. S.; Lee, W.; Nam, J. O.; Kim, J. E.; Kim, S. W.; Kim, I. S. Am J Respir Crit
Care Med 2014, 189, 779.
24. Ku, S. K.; Han, M. S.; Lee, M. Y.; Lee, Y. M.; Bae, J. S. BMB Rep 2014, 47, 336.
25. Ku, S. K.; Bae, J. S. Arch Pharm Res 2014, 37, 1454.
26. Ku, S. K.; Bae, J. S. BMB Rep 2014, 47, 376.
27. Kwak, S.; Ku, S. K.; Han, M. S.; Bae, J. S. Toxicol Appl Pharmacol 2014, 281, 30.
28. Diehl, K. H.; Hull, R.; Morton, D.; Pfister, R.; Rabemampianina, Y.; Smith, D.; Vidal,
J. M.; van de Vorstenbosch, C. J Appl Toxicol 2001, 21, 15.
29. Luo, Y.; Li, S. J.; Yang, J.; Qiu, Y. Z.; Chen, F. P. Biochem Biophys Res Commun
2013, 438, 732.
30. Hofbauer, R.; Moser, D.; Salfinger, H.; Frass, M.; Kapiotis, S. Anesth Analg 1998,
87, 1181.
31. Bae, J. S.; Rezaie, A. R. BMB Rep 2013, 46, 544.
15

32. Bae, J. S. Arch Pharm Res 2012, 35, 1511.
16

NO2
NH2
a
b
N
H
N
H
N
H
1
2
3
c
H
R
N
O
BS 1. R= 4-OCH3
BS 2. R= (3,4-OCH3)2
BS 3. R= (3,4,5-OCH3)3
N
H
Scheme 1. Reagents and conditions: (a) HNO3, water, RT  80°C, 3 h, (b) Pd/C 10%, ethanol,
3 h, (c) EDC, 4-DMAP, substituted cinnamic acid, THF anhydrous, RT, 24 h
OMe
Br
b
a
N
H
N
N
H
H
6
1
5
R
O
OH
c
d
O
N
N
H
H
7
BS 4. R= (3,4,5-OCH3)3
Scheme 2. Reagents and conditions: (a) NBS, THF, RT, 5 h, (b) CuI, DMF, MeONa/MeOH,
120°C, 20 h, (c) BBr3 1 M solution, dry MC, RT, 3 h, (d) EDC, 4-DMAP, substituted cinnamic
acid, THF anhydrous, 24 h.
17

Graphical abstract
18