Synthesis and 5-lipoxygenase inhibitory activity of new cinnamoyl and caffeoylclusters

Article abstract of DOI:10.1016/j.bmcl.2008.12.108

Novel cinnamoyl and caffeoyl clusters were synthesized by multiple Cu(I)-catalyzed [1,3]-dipolar cycloadditions and their anti-5-lipoxygenase inhibitory activity was tested. Caffeoyl cluster showed an improved 5-lipoxygenase inhibitory activity compared t

Full text of DOI:10.1016/j.bmcl.2008.12.108

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1118–1121
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and 5-lipoxygenase inhibitory activity of new cinnamoyl
and caffeoylclusters
*
Jérémie Doiron, Luc H. Boudreau, Nadia Picot, Benoît Villebonet, Marc E. Surette, Mohamed Touaibia
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada E1A 3E9
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel cinnamoyl and caffeoyl clusters were synthesized by multiple Cu(I)-catalyzed [1,3]-dipolar cyc-
loadditions and their anti-5-lipoxygenase inhibitory activity was tested. Caffeoyl cluster showed an
improved 5-lipoxygenase inhibitory activity compared to caffeic acid, with caffeoyl trimer 16 and tetra-
mer 19 showing the best 5-lipoxygenase inhibitory activity.
Received 4 November 2008
Revised 23 December 2008
Accepted 30 December 2008
Available online 3 January 2009
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Caffeic acid
Cinnamic acid
5-Lipoxygenase inhibitors
Clusters
Triazole
5-Lipoxygenase (5-LO) is the key enzyme in the metabolism of
arachidonic acid (AA) to leukotriene A4 (LTA4).1 Further metabo-
lism of LTA4 produces LTB4, a potent chemotactic agent for leuko-
cytes that is thought to be a key component in a variety of
diseases,2 including inflammatory bowel disease and atherosclero-
sis. LTA4 can also be converted to the peptidoleukotrienes LTC4,
LTD4, and LTE4,3 which are implicated in allergic hyper reactivity
disorders such as asthma.2b Elevated levels of these LTs, associated
with several inflammatory and allergic disorders, have been found
in various pathologic tissues.1
fects on prostaglandin H synthase and have potential for the inhi-
bition of 5-LO.13 However, despite the large number of therapeutic
indications and the strong need for efficient and safe drugs that
target the 5-LO pathway, zileuton is the only 5-LO inhibitor mar-
keted for the therapy of human subjects.1
Multivalent interactions have several advantages over mono-
meric ones and are often used by nature to control a wide variety
of cellular processes.14 Consequently, multivalent arrangements of
ligands generally favor the formation of physiologically relevant
associations.14
In search of pharmacological strategies that intervene with LTs,
a large number of different types of low molecular weight inhibi-
tors that potently suppress LTs synthesis have been developed in
the past 20 years.4 Among the known inhibitors of the 5-LO are a
variety of polyhydroxylated natural products such as caffeic acid,5
and flavonoids.6 The rational design of efficient inhibitors requires
perfect knowledge about the target itself. In the case of 5-LO, much
of our knowledge is based upon studies on soybean lipoxygenase.7
Caffeic and cinnamic acid derivatives are widely distributed in
plants and exhibit a broad spectrum of biological activities includ-
ing anti-oxidative,8 anti-inflammatory,9 antiviral,10 and anti-can-
cer effects.11
Thus, the development of novel multivalent macromolecules or
dendrimers bearing caffeoyl or cinnamoyl moieties that inhibit 5-
LO is an important challenge. To the best of our knowledge, clus-
ters having peripheral covalently-bound caffeoyl or cinnamoyl
units have not been described. Such inhibitors may provide thera-
peutic benefit for the pharmacological treatment of inflammatory
and allergic disorders, cardiovascular diseases, and cancer.
Here we present the design and synthesis of novel caffeoyl clus-
ters that proved to be potent inhibitors of 5-LO. Since the hydroxyl
groups within the caffeic acid catechol moiety may also play an
important role in the inhibitory activity, we also examined the ef-
fect of the presence of these groups by the synthesis of the cinnam-
Several modified caffeic acid amides were recently demon-
strated for anti-lipoxidation and exhibited more stable characteris-
tics.12 Furthermore, some caffeic acid amide analogs, such as
N-caffeoyl-b-phenethylamine were reported to have inhibitory ef-
oyl cluster analogs. A simple approach to the synthesis of
multivalent caffeoyl- or cinnamoyl-bearing clusters is the attach-
ment of acid moieties to structurally simple hyper-branched mol-
ecules. Taking advantage of the click ligation technique, we were
able to use terminal alkyne- or azide-functionalized cores to intro-
duce caffeoyl or cinnamoyl units into dendrimers in a ‘‘clicked”
divergent fashion.
* Corresponding author. Tel.: +1 506 858 4493; fax: +1 506 858 4541.
E-mail address: mohamed.touaibia@umoncton.ca (M. Touaibia).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.108

J. Doiron et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1118–1121
1119
Click chemistry has attracted much attention recently because
of its high specificity, quantitative yield, and tolerance to various
groups. Several reports have described the synthesis of clusters
or dendrimers through the click reaction in either a convergent
or a divergent manner.15 The synthetic route to the mono and biva-
lent derivatives is outlined in Scheme 1. The novel propargyl 1, 3
and azido 2, 4 esters and were prepared from propargyl alcohol
or azidoethanol16 with cinnamic or acetylated caffeic acid in pyri-
dine in quantitative yields. Using the combination of caffeic/cin-
namic propargyl esters 1, 3 and the azido analogs 2, 4 as starting
materials, we carried out the 1,3-dipolar cycloaddition15 under
standards conditions (CuSO4, ascorbic acid, THF: H2O, rt) to give,
after hydrolysis, the corresponding triazole linked derivatives 5,
7, 9, and 11 in 69%, 74%, 78%, and 82% yields, respectively (Scheme
1).
The pentaerythrityl core acts as a versatile building block that
has been employed in dendrimer syntheses. To the best of our
knowledge, no pentaerythritol-based cinnamoyl or caffeoyl clus-
ters have been reported. Using pentaerythritol-based azide plat-
forms functionalized with three 12 and four 13 azide groups,17
click chemistry provided, as expected, tris and tetrameric clusters
14, 15, 17, and 1818 according to Scheme 2. Finally, complete
hydrolysis of the acetyl ester protecting groups in 15 and 18 to af-
ford 16 and 1918 was accomplished under catalytic trans-esterifi-
cation conditions using K2CO3 in CH2Cl2: MeOH (16: 69%, 19: 72%).
A retrosynthetic analysis reveals two possibilities for the syn-
thesis of new clusters bearing 1,2,3-triazole linkages, the azide or
the alkyne functions can be located on either the pentaerythritol
core or on the cinnamoyl or the caffeoyl moieties. According to
Scheme 3, nitrogen linked caffeic and cinnamic moieties were ob-
tained this time with the tetra alkyne 2019 and the corresponding
azide 2 or 4 under the same click reaction conditions. Thus, acetyl
hydrolysis (K2CO3, CH2Cl2: MeOH) provided the extended cluster
analogs.
For the next higher member of the family, the same synthetic
strategy was used. Under the same conditions described above,
cinnamoyl and caffeoyl hexamers could be obtained starting from
the hexa-azide 2415,20 and the corresponding alkynes 1 and 3.
(Scheme 4).
In all cases, analysis of the 1H, 13C NMR spectra of our clusters
revealed calculated integrations for the triazole protons respective
to the vinylic protons and complete disappearance of the acety-
lenic signals, thus confirming, together with MS and IR data, com-
pletion of the multivalent Cu(I)-catalyzed azide–alkyne
cycloadditions.
The unprecedented clusters prepared in this study displayed
some structural differences that are governed by the pentaerythri-
tol and bis-pentaerythritol scaffolds as well as by the number of
cinnamoyl/caffeoyl units and their relative orientation. These dis-
parities offer a unique opportunity to estimate their 5-LO inhibi-
tory activities. The inhibitory activity of our compounds, with
valencies ranging from 1 to 6 U, was measured in cell lysates of
HEK 293 cells stably transfected with a pcDNA3.1 vector express-
O
O
R2
R2
R1
R1
O
O
O
O
N
3
+
O
R2
R2
N3
+
O
1 R1 = H
3 R1 = OAc
O
O
2 R2 = H
O
2 or 4
i
4 R2 = OAc
i
20
O
R2
R2
O
O
R2
R2
R2
R2
R1
R1
O
N
N
O
O
N
N
N
N
N
N
N
O
N
5 (1 + 2) R1 = R2 = H
6 (1 + 4) R1 = H; R2 = OAc
7 R1 = H; R2 = OH
O
O
ii
ii
ii
O
O
R2
R2
O
N
8 (3 + 2) R1 = OAc; R2 = H
9 R1 = OH; R 2 = H
O
N
R2
R2
N
N
N
O
10 (3 + 4) R1 = R2 = OAc
11 R1 = R2 = OH
21 R2 = H
O
22 R2 = OAc
23 R2 = OH
ii
Scheme 1. Reagents and conditions: (i) CuSO4, ascorbic acid, THF: H2O, rt, 12 h, 5
(69%), 6 (80%), 8 (76%), 10 (78%); (ii) K2CO3 in CH2Cl2/MeOH (1:1, v/v), rt, 4 h, 7
(74%), 9 (78%), 11 (82%).
Scheme 3. Reagents and conditions: (i) CuSO4, ascorbic acid, THF: H2O, rt, 12 h, 21
(82%), 22 (75%); (ii) K2CO3 in CH2Cl2/MeOH (1:1, v/v), rt, 4 h, 23 (69%).
R1
O
O
R1
R1
R1
HO
N3
O
O
N3
N3
N3
O
N
R1
R1
N
+
R1
N
14 R1 = H
1 or 3
R1
R1
N
N
N
O
15 R1 = OAc
16 R1 = OH
R1
N
N
N
N3
N3
ii
24
O
HO
O
N3
O
12
O
O
i
R1
R1
N3
i
R1
O
O
N3
O
O
R1
R1
O
N
N
N
R1
R1
N
R1
R1
N
N
N
O
O
N
N
N
O
N
O
R1
R1
R1
N
N
O
i
O
N
17 R1 = H
18 R1 = OAc
19 R1 = OH
1 or 3
N3
N
N
N
N
N3
N
N
N
N
N
O
N
N
N
N
ii
N3
13
O
O
N3
O
O
N
N
R1
R1
R1
R1
R1
O
O
25 R1 = H
O
N
O
R1
R1
26 R1 = OAc
27 R1 = OH
O
R1
ii
Scheme 2. Reagents and conditions: (i) CuSO4, ascorbic acid, THF: H2O, rt, 12 h, 14
(80%), 15 (78%), 17 (82%), 18 (79%); (ii) K2CO3 in CH2Cl2/MeOH (1:1, v/v), rt, 4 h, 16
(69%), 19 (72%).
Scheme 4. Reagents and conditions: (i) 1 or 3, CuSO4, ascorbic acid, THF: H2O, rt,
12 h, 25 (82%), 26 (78%); (ii) K2CO3 in CH2Cl2/MeOH (1:1, v/v), rt, 4 h, 27 (69%).

1120
J. Doiron et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1118–1121
Table 1
ing human 5-LO. Measurement of 5-LO activity in cell lysates was
performed as previously described with modifications.21
Determination of IC50 values of selected inhibitors
M) per Caf. Ac.a
R IC50
b
From the structure-activity point of view, all of the clusters
bearing the cinnamic acid moiety 5, 14, 17, 21, and 25, showed less
inhibitory activity than the corresponding clusters bearing the caf-
feic acid moiety 7, 9, 11, 16, 19, 23, and 27 (Fig. 1), reinforcing the
pharmacophoric contribution of the catechol entity to the mecha-
nism of action against 5-LO activity.
Compound
IC50
(
l
M)
IC50
(
l
7
11
16
19
0.68
0.74
0.79
0.66
0.68
1.48
2.37
2.46
36.7
16.8
10.5
10.1
1
Caf. Ac.
25
25
a
IC50 on a per caffeoyl residue basis.
Relative IC50 values based on caffeic acid as standard.
As shown in Figure 1, compounds exhibiting one (7,9), two (11),
three (16) or four (19) caffeic acid entities were equivalent and po-
tent inhibitors of 5-LO activity compared to caffeic acid itself. The
trimer 16 and tetramer 19 showed the most potent inhibition of
the 5-LO activity (91% and 92%, respectively, Fig. 1). The inhibitory
activity of tetramer 19 is more pronounced than that of tetramer
23 suggesting that the position of the triazole may be important
with regard to modulating the inhibitory activity since these two
compounds differ by the relative positioning of the triazole ring.
The noticeable activity enhancement observed for 16 and 19
must be ascribed to the special geometry arrangement and thus
can accommodate the clustering of a few trimeric or tetrameric
targets. The incorporation of additional caffeoyl units in hexamer
27 resulted in a less potent inhibition of 5-LO. This result would
be consistent with a loss of activity due to enthalpy-entropy com-
pensation for higher valency compounds.
Although the 5-LO activity shown in Figure 1 represents the
sum of all 5-LO products detected (5-hydroxyeicosatetraenoic acid,
LTB4 and its trans isomers), similar results were observed for each
of these 5-LO products separately (data not shown).
To better determine the potency of the compounds that demon-
strated significant inhibitory activities against 5-LO at 1 lM, four
promising inhibitors were selected (7, 11, 16, and 19) for further
investigation in concentration-response studies, and the results
are summarized in Table 1.
b
are good lead compounds in the design and synthesis of more po-
tent 5-LO inhibitors.
Acknowledgment
This research was financially supported by the New Brunswick
Innovation Foundation, The Medical Research Fund of New Bruns-
wick and the Canada Research Chairs Program.
References and notes
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Thorsteinsdottir, U.; Samani, N. J.; Gudmundsson, G.; Grant, S. F.; Thorgeirsson,
G.; Sveinbjornsdottir, S.; Valdimarsson, E. M.; Matthiasson, S. E.; Johannsson,
H.; Gudmundsdottir, O.; Gurney, M. E.; Sainz, J.; Thorhallsdottir, M.;
Andresdottir, M.; Frigge, M. L.; Topol, E. J.; Kong, A.; Gudnason, V.;
Hakonarson, H.; Gulcher, J. R.; Stefansson, K. Nat. Genet. 2004, 36, 233; (c)
Romano, M.; Claria, J. FASEB J. 2003, 17, 1986.
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Biochim. Biophys. Acta 1984, 792, 92.
The inhibitory activities of selected compounds were compared
based on the number of caffeic acid moieties they contained (IC50
per Caf. Ac. in Table 1). All these compounds concentration-depen-
dently inhibited 5-LO products synthesis and showed prominent
inhibitory activities with IC50 values ranging from 0.66 to
6. Baumann, J.; Von Bruchhausen, F.; Wurm, G. Prostaglandins 1980, 20, 627.
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Masferrer, J. L.; Carothers, A. M.; Subbaramaiah, K.; Zweifel, B. S.; Koboldt, C.;
Mestre, J. R.; Grunberger, D.; Sacks, P. G.; Tanabe, T.; Dannenberg, A. J. Cancer
Res. 1999, 59, 23472.
10. (a) Burke, T. R., Jr.; Fesen, M. R.; Mazumder, A.; Wang, J.; Carothers, A. M.;
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(b) King, P. J.; Ma, G.; Miao, W.; Jia, Q.; McDougall, B. R.; Reinecke, M. G.;
Cornell, C.; Kuan, J.; Kim, T. R.; Robinson, W. E., Jr. J. Med. Chem. 1999, 42, 497.
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Ferraro, T.; Conney, A. H.; Grunberger, D. Cancer Res. 1993, 53, 1255; (b) Lee, Y.
J.; Liao, P. H.; Chen, W. K.; Yang, C. Y. Cancer Lett. 2000, 153, 51.
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Bioorg. Med. Chem. Lett. 2001, 11, 215.
13. Tseng, C. F.; Iwakama, S.; Mikajiri, A.; Shibuya, M.; Hanaoka, F.; Ebizuka, Y.;
Padmawinata, K.; Sankawa, U. Chem. Pharm. Bull. 1992, 40, 396.
14. Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem.. Int. Ed. 1998, 37,
2754.
0.79
ton (IC50 = 0.5–1
l
M, which were comparable to the inhibitory activity of zileu-
l
M22). On the basis of corrected values on a per
caffeoyl residue, dimer 11, trimer 16, and tetramer 19, readily sur-
passed the activity of caffeic acid by more than 10-fold.
In conclusion, an alkyne-azide cycloaddition based route to var-
ious cinnamoyl and caffeoyl clusters featuring 1,2,3-triazole rings
has been achieved. The results demonstrate that caffeoyl clusters
100
Cinnamic acid series
Control
Caffeic acid series
80
60
40
20
0
15. For recent reviews on the Huisgen 1,3-dipolar cycloaddition reaction see
Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952.
16. Smith, R. H.; Mehl, A. F.; Shantz, D. L.; Chmurny, G. N.; Michejda, C. J. J. Org.
Chem. 1988, 53, 1467.
17. (a) Wilson, E. R.; Frankel, M. B. J. Org. Chem. 1985, 50, 3211; (b) Hayes, W.;
Osborn, H. M. I.; Osborne, S. D.; Rastall, R. A.; Romagnoli, B. Tetrahedron 2003,
59, 7983.
18. Compound 18. To
a magnetically stirred solution of azide 13 (40 mg,
0.17 mmol) and propargyl derivative 1 (246 mg, 0.81 mmol), dissolved in a
1:1 mixture of water and tetrahydrofuran (3 mL), were sequentially added
CuSO4 (8.5 mg, 0.034 mmol, 5% per azide), and ascorbic acid (6 mg,
0.034 mmol, 5% per azide). The mixture was stirred for about 12 h until
disappearance of the starting material (TLC, 5% MeOH/CH2Cl2). After addition
of water (15 mL), the crude reaction was repeatedly extracted with ethyl
acetate (4 Â 10 mL). The combined organic extracts were treated with brine
Figure 1. 5-LO Activity in cell lysates preincubated with the different test
compounds (1 lM).

J. Doiron et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1118–1121
1121
(20 mL), and then dried over anhydrous magnesium sulfate. After
concentration under reduced pressure, the residue was purified by silica gel
CDCl3): d 44.5, 50.2, 59.3, 127.5, 142.9, 115.3, 117.6, 118.2, 121.3, 129.4, 143.2,
145.6, 142.5, 166.3; HRMS Calcd for C53H48O16N12 + (H+): 1109.33895. Found:
1109.33678.
circular chromatography (chromatotronÒ
, model 7924, Harrison Research)
eluting with dichloromethane and gradually increasing the polarity to MeOH/
CH2Cl2 (3:97) to afford 193 mg (79% yield) of the required tetra cluster 18.
Colorless oil; Rf = 0.33 (5% MeOH/CH2Cl2). 1H NMR (200 MHz, CDCl3): d 2.25 (s,
24H, 8Â OAc), 4.44 (s, 8H, CH2N), 5.38 (s, 8H, CH2O), 6.41 (d, 4H, J = 17.6 Hz,
@CHCO), 7.20 (d, 4H, J = 8.2 Hz, Har), 7.35 (br s, 8H, Har), 7.63 (d, 4H,
J = 17.6 Hz, @CHCar), 8.30 (s, 4H, @CHN); 13C NMR (50 MHz, CDCl3): d 20.6,
20.6, 46.6, 49.2, 57.5, 86.1, 118.4, 122.8, 123.9, 126.5, 127.8, 132.9, 132.4,
19. Korostova, S. E.; Mikhaleva, A. I.; Shevchenko, S. G.; Sobenina, L. N.; Fel’dman,
V. D.; Shishov, N. I. Zh. Prikl. Khim. 1990, 63, 234.
20. Touaibia, M.; Wellens, A.; Shiao, T. C.; Wang, Q.; Sirois, S.; Bouckaert, J.; Roy, R.
ChemMedChem 2007, 2, 1190.
21. (a) Cells were lysed in phosphate-buffered saline containing 1 g/L glucose.
Following the addition of 2 mM EDTA and 5 mM CaCl2 lysates were then pre-
incubated with each of our compounds at the indicated concentration for
5 min at 37 °C. The 5-LO reaction was initiated with the addition of 1 mM ATP
142.43, 143.6, 166.1, 167.9, 168.0; HRMS Calcd for C69H64O24N12
+
(H+):
1445.42347. Found: 1445.42314. Compound 19. To a solution of 18 (62 mg,
0.043 mmol) in acetone (5 mL) was added 3N HCl (2 mL). The reaction mixture
was refluxed for 3 h. The solution was then diluted with EtOAc (25 mL),
washed with brine, dried (MgSO4), concentrated, and purified silica gel circular
chromatography (chromatotronÒ, model 7924, Harrison Research) eluting with
(CH3CN/H2O; 95:5) to give 34 mg (72% yield) of the free hydroxyl tetra cluster
19. 1H NMR (200 MHz, DMSO-d6): d 4.72 (s, 8H, CH2N), 5.30 (s, 8H, CH2O), 6.30
(d, 4H, J = 18.6 Hz, @CHCO), 6.85 (m, 8H, Har), 7.05 (m, 4H, Har), 7.12 (br s, 8H,
OH), 7.60 (d, 4H, J = 17.6 Hz, @CHCar), 8.35 (s, 4H, @CHN); 13C NMR (50 MHz,
and 40 l
M arachidonic acid followed by incubation at 37 °C for 20 min.21b
Reactions were stopped with addition of 0.5 vol methanol and samples were
processed for separation and quantification of 5-LO products (5-HETE, LTB4 and
its trans isomers) by reverse-phase HPLC with UV detection as previously
described;21c.; (b) Werz, O.; Szellas, D.; Steinhilber, D. Eur. J. Biochem. 2000,
267, 1263; (c) Borgeat, P.; Picard, S.; Vallerand, P.; Bourgoin, S.; Odeimat, A.;
Sirois, P.; Poubelle, P. E. Methods Enzymol. 1990, 187, 98.
22. Carter, G. W.; Young, P. R.; Albert, D. H.; Bouska, J.; Dyer, R.; Bell, R. L.;
Summers, J. B.; Brooks, D. W. J. Pharmacol. Exp. Ther. 1991, 256, 929.