Synthesis of symmetrical bis-alkynyl or alkyl pyridine and thiophene derivatives and their antiangiogenic activities

Article abstract of DOI:10.1016/j.bmcl.2004.05.065

Fourteen symmetrical bis-alkynyl pyridine and thiophene derivatives were synthesized and their antiangiogenic activity was evaluated with the proliferation and tube formation inhibitory activity on the human umbilical vein endothelial cells (HUVEC). Compo

Full text of DOI:10.1016/j.bmcl.2004.05.065

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3893–3896
Synthesis of symmetrical bis-alkynyl or alkyl pyridine and
thiophene derivatives and their antiangiogenic activities
d
a
e
Chan Mug Ahn,a,c, Woon-Seob Shin, Ho Bum Woo, Seokjoon Lee and
*
Hyean-Woo Leeb
aDepartment of Basic Sciences, Wonju College of Medicine, Yonsei University, Wonju 220-701, South Korea
bDepartment of Biochemistry, Wonju College of Medicine, Yonsei University, Wonju 220-701, South Korea
cInstitute of Basic Medical Science, Wonju College of Medicine, Yonsei University, Wonju 220-701, South Korea
dDepartment of Microbiology, Kwandong University College of Medicine, Gangneung 210-701, South Korea
eDepartment of Basic Science, Kwandong University College of Medicine, Gangneung 210-701, South Korea
Received 29 March 2004; revised 24 May 2004; accepted 25 May 2004
Available online 17 June 2004
Abstract—Fourteen symmetrical bis-alkynyl pyridine and thiophene derivatives were synthesized and their antiangiogenic activity
was evaluated with the proliferation and tube formation inhibitory activity on the human umbilical vein endothelial cells (HUVEC).
Compounds 6, 8, and 10, rigid mimetic structure of curcumin, showed the potent growth inhibitory activity and the potent tube
formation inhibitory activity.
Ó 2004 Elsevier Ltd. All rights reserved.
Angiogenesis occurs during embryonic development,
wound healing, and the menstruation cycle in physio-
logic conditions. Angiogenesis is an elaborately regu-
lated phenomenon. Unregulated angiogenesis causes
pathological conditions, such as diabetic retinopathy,
psoriasis, arthritis, and cancer.1 During the tumor
growth, angiogenesis need for nourishment and removal
of metabolic wastes from tumor sites. Tumor requires
coordination of angiogenesis with continuous cancer cell
proliferation. Tumor growth and metastasis strictly,
which depend on angiogenesis reminds the idea that
blocking tumor nourishment can be one of the ways to
avoid its spread and growth.2 Therefore, antiangiogenic
treatment has received a lot of attention in cancer
research.3 A lot of antiangiogenic compounds were
reported4 and several antiangiogenic drugs are currently
in clinical trials.5
tor NF-jB, phospholipase A2, arachidonic acid
metabolism, EGF receptor autophosphorylation, and
Ca2þ-ATPase.6 Thus curcumin has a potent antiangio-
genic effect.
Arbiser et al. reported that curcumin (1) and its deriv-
atives including demethoxycurcumin and tetrahydro-
curcumin were discovered as potent antiangiogenic
agents.7 Kwon and co-workers reported that hydrazi-
nocurcumin, which curcumin’s diketone part is modified
to imidazole ring has a potent antiangiogenic activity.8
Recently, Bowen and co-workers reported that aromatic
enone and dienone analogues of curcumin (1) have a
potent antiangiogenic activity.9 Therefore, although
structure–antiangiogenic activity relationship of curcu-
min (1) is not completely understood, curcumin is a
promising lead compound for structural modification.
These studies suggest that phenolic group and aromatic
enone or dienone group is essential to the antiangiogenic
activity of curcumin (Fig. 1).
Curcumin (1) is a natural product isolated from the
spice turmeric. It inhibits several signal transduction
pathways including protein kinase-C, transcription fac-
In addition, based on the previous results,9 we hypoth-
esized that the rigidity of symmetrical aromatic moieties
plays an important role to enhance angiogenic activity.
To certify our assumption, we synthesized symmetrical
bis-aromatic alkynyl pyridine and thiophene derivatives
(2) and bis-aromatic alkyl pyridine and thiophene
Keywords: Bis-alkynyl pyridine; Curcumin; Antiangiogenic activity.
* Corresponding author. Tel.: +82-33-741-0371; fax: +82-33-745-2170;
e-mail: ahn0707@wonju.yonsei.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.065

3894
C. M. Ahn et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3893–3896
and bis-alkynyl thiophene (10) compounds were ob-
O
O
tained by the reaction of 5 with two dibromo pyridines
or diiodothiophene at room temperature for 15–17 h in
the presence of dichloro bis-(triphenylphosphine) pal-
ladium, copper (I) iodide, and diethylamine, as shown in
H3CO
HO
OCH3
OH
1
1
Scheme 1. H, 13C NMR spectrum, and GC/MS spec-
OCH3
OCH3
OH
trum of bis-alkynyl compounds (6, 8, and 10) were
investigated for structural identification.11
HO
To study the relationship between structural rigidity and
activity, we obtained the bis-alkyl compounds (7, 9,
and 11), which are the hydrogenated compounds (6, 8,
and 10) with Pd/C at room temperature in the H2 atmo-
sphere, respectively (Scheme 1). Also, to obtain water-
soluble promising molecules, we reacted bis-alkynyl and
bis-alkyl pyridines (6, 7, 8, and 9) with trimethyloxo-
nium tetrafluoroborate ((Me)3OBF4) in dichloroethane
to transform into their salts (12a, 13a, 14a, and 15a),
and the treatment of methyl trifluoromethanesulfonate
(MeOTf) on 6, 7, 8, and 9 in diethyl ether afforded these
salts (12b, 13b, 14b, and 15b), as shown in Scheme 2.
: heteroaromatic linker
2
Figure 1.
derivatives. The bis-alkynyl compounds (2) are the rigid
structure of curcumin.
The antiangiogenic activities of those synthetic com-
pounds were evaluated with the proliferation and tube
formation inhibitory activities on the human umbilical
vein endothelial cells (HUVEC).
Cell growth inhibitory effect of synthesized compounds
was measured by MTT colorimetric method,12 as shown
in Table 1.
Commercially available aldehyde (3) was reacted with
carbon tetrabromide in the presence of triphenyl phos-
phine in CH2Cl2 at 0 °C for 2 h. The crude product was
twice chromatographed (CHCl3/MeOH ¼ 95/5) on silica
gel to afford dibromoalkene (4). This alkene (4) was
dissolved in dry THF under n-BuLi and stirred at
)78 °C for 5 h to give aromatic alkyne (5). Utilizing
Sonogashira reaction,10 bis-alkynyl pyridines (6 and 8),
As we expected, the bis-alkynyl compounds (6, 8, and
10), rigid mimetic structure of curcumin (1), showed
more potent growth inhibitory activity on HUVEC than
curcumin (1). Especially, bis-alkynyl compounds (6 and
8) have a highly strong inhibition activity upon HU-
VEC. The bis-alkyl compounds (7, 9, and 11) having
more flexible alkyl chain, hydrogenated compounds of
H
H3CO
C
CBr2
H3CO
HO
H3CO
HO
CHO
PPh3, CBr4
CH2Cl2, 0oC
n-BuLi
THF, -78oC
HO
3
4
5
OCH3
OCH3
OH
OCH3
OCH3
Br
N
Br
HO
HO
OH
H2, Pd/C
N
N
7
6
8
OCH3
OCH3
OH
OCH3
OCH3
OH
Br
Br
HO
HO
HO
HO
Pd(PPh3)2Cl2
CuI, Et2NH
H2, Pd/C
N
N
9
N
S
OCH3
OCH3
OH
OCH3
OCH3
OH
I
S
I
H2, Pd/C
S
11
10
Scheme 1. Synthesis of symmetrical bis-alkynyl pyridine and thiophene derivatives, and their hydrogenated compounds.

C. M. Ahn et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3893–3896
3895
OCH3
OCH3
OH
OCH3
OCH3
OH
HO
HO
HO
X
i)
CH3
N
i)
6
7
X
CH3
N
13a, 13b
12a, 12b
OCH3
OCH3
OH
OCH3
OCH3
OH
HO
i)
i)
8
9
N
CH3
N
CH3
X
X
14a, 14b
15a, 15b
Scheme 2. Synthesis of tetrafluoroborate and triflate salts of symmetrical bis-alkynyl pyridine and bis-alkyl pyridine derivatives. Reagents and
conditions: (i) (a) (Me)3OBF4 (1.3 equiv), ClCH2CH2Cl, X ¼ BF4ꢀ, (b) MeOTf (1.6 equiv), diethyl ether, X ¼ OTfꢀ.
Table 1. Inhibitory activity of synthetic compoundsa on HUVEC
growth
Table 2. The rate of tube-formation inhibition by synthesized com-
poundsa
Compounds
Growth inhibition IC50, lg/mL
Compounds
Inhibition percentage (%)
6
2.2
12.6
2.0
At 10 lg/mL
At 5 lg/mL
At 2.5 lg/mL
7
6
97.4
98.2
89.3
41.9
79.4
79.2
71.7
––
31.7
59.9
37.8
––
8
8
9
10
9.3
7.2
10
14a
11
10.8
>50
>50
>50
>50
9.2
a Values expressed in percentage of HUVEC total tube length/field as
compared to untreated control. Total tube length was measured using
Image-pro plus version 3.0(Media Cybernetics, MD, USA).
12a
12b
13a
13b
14a
14b
15a
15b
Curcumin
compounds failed to effectively inhibit the tube forma-
tion.
1.5
22.8
9.9
In conclusion, for the first time, we have shown that
several symmetrical bis-alkynyl pyridine and thiophene
derivatives (6, 8, and 10) readily synthesized by Sono-
gashira reaction, rigid mimetic structure of curcumin,
have a strong antiangiogenic activity and rigidity of
synthetic structures have improved the biological activ-
ity.
10.7
a IC50 was calculated from nonlinear regression by Graphpad Prism
software.
6, 8, and 10, showed similar inhibition activity with
curcumin.
When considered growth inhibition activity upon HU-
VEC, we concluded that the increase of the extent of
unsaturation or rigidity of structure enhanced biological
activity.
References and notes
1. Bikfalvi, A.; Bicknell, R. Trends Pharmacol. Sci. 2002, 23,
576.
2. Folkman, J. New Engl. J. Med. 1971, 285, 1182.
3. Tonini, T.; Rossi, F.; Claudio, P. P. Oncogene 2003, 22,
6549.
4. (a) Oh, S.; Jeong, I. H.; Shin, W.-S.; Lee, S. Bioorg. Med.
Chem. Lett. 2003, 13, 3665; (b) Oh, S.; Jeong, I. H.; Shin,
W.-S.; Lee, S. Bioorg. Med. Chem. Lett. 2003, 13, 2009.
5. (a) Liekens, S.; Clercq, E. D.; Neyts, J. Biochem.
Pharmacol. 2001, 61, 253; (b) Matter, A. Drug Discov.
Today 2001, 6, 1005; (c) Deplanque, G.; Harris, A. L. Eur.
J. Cancer 2000, 36, 1713.
6. (a) Oh, H.; Takagi, H.; Suzuma, K.; Otani, A.; Matsum-
ura, M.; Honda, Y. J. Biol. Chem. 1999, 274, 15732; (b)
Gururaj, A. E.; Belakavadi, M.; Venkatesh, D. A.;
Marme, D.; Salimath, B. P. Biochem. Biophys. Res.
Finally, we examined the antiproliferative effect of salts
(12a,b, 13a,b, 14a,b, and 15a,b) with water solubility.
Disappointingly, except of triflate salt (14b) of 3,5-bis-
alkynyl pyridine, all salt-type molecules have a mild or
weak inhibitory effect.
In the tube-formation assay using HUVEC on the
Matrigel,13 as shown in Table 2, the bis-alkynyl com-
pounds (6, 8, and 10), inhibited the tube formation on
Matrigel as nearly 100% at the concentration of 10 lg/
mL. Even at the concentration of 2.5 lg/mL, compound
8 inhibited about 60%. In expected water-soluble salts,
only 14a have a mild inhibitory activity of tube forma-
tion. The hydrogenated molecules (7, 9, and 11) and salt

3896
C. M. Ahn et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3893–3896
Commun. 2002, 297, 934; (c) Grandjean-Laquerriere, A.;
(2,6)H) ppm; 13C NMR (CDCl3): d 55.8, 86.8, 90.0, 111.3,
114.1, 118.6, 124.4, 125.8, 136.5, 143.8, 146.2, 149.0ppm;
GC/MSD (m=z) 371. For 10: TLC (chloroform/metha-
nol ¼ 95/5) Rf ¼ 0:63; 1H NMR (CDCl3): d 3.91 (6H, s,
OCH3), 5.79 (2H, s, OH), 6.87–6.89 (2H, d, J ¼ 4:9 Hz,
Ar–H), 6.98–6.99 (2H, d, J ¼ 1:8 Hz, Ar–H), 7.05–7.07
(2H, t, J ¼ 8:2 Hz, Ar–H), 7.14 (2H, s, furan-H); 13C
NMR (CDCl3): d 56.0, 79.6, 95.1, 113.6, 114.0, 114.6,
125.6, 132.7, 137.0, 146.2, 146.7 ppm; GC/MSD (m=z) 376.
12. Mosmann, T. J. Immunol. Methods 1983, 65, 55, Assay
method: HUVEC in EBM-2 medium containing the
growth factor (Cambrex, Walkersville, MD) was plated
in a 48-well plate (10,000 cells/well) for 4 h to allow cells to
adhere on the plate. The test compounds, diluted in the
medium, were added to the appropriate wells, and
incubated for 72 h at 37 °C in a 5% CO2 humidified
atmosphere. After incubation, the viability of HUVEC
Gangloff, S. C.; Naour, R. L.; Trentesaux, C.; Hornebeck,
W.; Guenounou, M. Cytokine 2002, 18, 168; (d) Rao, C.
V.; Simi, B.; Reddy, B. S. Carcinogenesis 1993, 14, 2219;
(e) Logan-Smith, M. J.; Locker, P. J.; East, J. M.; Lee, A.
G. J. Biol. Chem. 2001, 276, 46905.
7. Arbiser, J. L.; Klauber, N.; Rohan, R.; van Leeuwen, R.;
Huang, M. T.; Fisher, C.; Flynn, E.; Byers, H. R. Mol.
Med. 1998, 4, 376.
8. Shim, J. S.; Kim, D. H.; Jung, H. J.; Kim, J. H.; Lim, D.;
Lee, S. K.; Kim, K.-W.; Ahn, J. W.; Yoo, J.-S.; Rho, J.-
R.; Shin, J.; Kwon, H. J. Bioorg. Med. Chem. 2002, 10,
2987.
9. Robinson, T. P.; Ehlers, T.; Hubbard, R. B., IV; Bai, X.;
Arbiser, J. L.; Goldsmith, D. J.; Bowen, J. P. Bioorg. Med.
Chem. Lett. 2003, 13, 115.
10. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 16, 4467.
was assayed using
a 3-(4,5-dimethylthiazol-2-yl)-2,5-
11. Analytical data for 6: mp >400 °C; TLC (chloroform/
methanol ¼ 95/5) Rf ¼ 0:31; 1H NMR (CDCl3): d 3.91
(6H, s, OCH3), 5.82 (2H, s, OH), 6.89–6.91 (2H, d,
J ¼ 8:2 Hz, Ar–H), 7.14–7.18 (4H, m, Ar–H), 7.42–7.44
(2H, d, pyridine–(3,5)H), 7.66 (1H, t, J ¼ 7:8 Hz, pyri-
dine–(4)H) ppm; 13C NMR (DMSO-d6): d 56.5, 87.5, 90.9,
112.3, 116.1, 116.7, 126.4, 126.7, 138.3, 144.0, 148.5,
149.4 ppm; GC/MSD (m=z) 371. For 8: mp 175–177 °C;
TLC (chloroform/methanol ¼ 95/5) Rf ¼ 0:33; 1H NMR
(CDCl3): d 3.94 (6H, s, OCH3), 5.83 (2H, s, OH), 6.91–
6.93 (2H, d, J ¼ 8:2 Hz, Ar–H), 7.04 (2H, d, J ¼ 1:8 Hz,
Ar–H), 7.10–7.13 (2H, t, J ¼ 8:2 Hz, Ar–H), 7.94–7.95
(1H, t, J ¼ 1:8 Hz, pyridine–(4)H), 8.64 (2H, s, pyridine–
diphenyltetrazolium bromide (MTT) colorimetric prolif-
eration assay.
13. Aoki, K.; Watanabe, K.; Sato, M.; Ikekita, M.; Haka-
matsuka, T.; Oikawa, T. Eur. J. Pharmacol. 2003, 459,
131, Assay method: Tube formation of HUVEC on
Matrigel (BD Biosciences, Bedford, MA) was assayed in
a 96-well plate. HUVEC was seeded on 50 lL solidified
Matrigel, and incubated for 16 h at 37 °C in a 5% CO2
humidified atomosphere. After incubation, the cells were
stained with toluidin blue in 4% paraformaldehyde, and
photographed. A blinded observer quantified the number
of tube branches. Each concentration of the control or test
compound was assayed in triplicate.