Synthesis and initial evaluation of novel, non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

Article abstract of DOI:10.1016/j.bmcl.2008.11.074

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS com

Full text of DOI:10.1016/j.bmcl.2008.11.074

Bioorganic & Medicinal Chemistry Letters 19 (2009) 352–355
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and initial evaluation of novel, non-peptidic antagonists
of the
av-integrins
avb3 and avb5
b
a
a
a
a
Jeffrey J. Letourneau a, , Jinqi Liu , Michael H. J. Ohlmeyer , Chris Riviello , Yajing Rong , Hong Li ,
*
Kenneth C. Appell b, Shalini Bansal b, Biji Jacob b, Angela Wong b, Maria L. Webb b
a Department of Chemistry, Pharmacopeia, Inc., PO Box 5350, Princeton, NJ 08543, USA
b Department of Biology, Pharmacopeia, Inc., PO Box 5350, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the av-inte-
grins
libraries led to the identification of compound 1 as a dual inhibitor of the
mization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of
compounds 15a and 15b with reduced PSA and much improved potency for both the vb3 and vb5 inte-
avb3 and a
vb5 is described. High-throughput screening of an extensive series of ECLiPSTM compound
Received 4 November 2008
Revised 18 November 2008
Accepted 20 November 2008
Available online 24 November 2008
av-integrins avb3 and avb5. Opti-
a
a
grins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and
compound 15a also exhibited a promising Caco-2 permeability profile.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Integrin
Inhibitors
Integrins belong to a superfamily of heterodimeric cell surface
receptors with critical roles in regulating both physiological and
pathological processes. They are involved in a variety of cell signal-
ing pathways by mediating cell adhesion, migration and prolifera-
tion.1 Many of the receptors in the integrin family recognize the
tripeptidic RGD-sequence present in the respective ligands. Two
obstacles to overcome. One of the reasons for this is the zwiterionic
character of the RGD mimetics. In the past few years a number of
groups have reported
avb3 inhibitors with good oral bioavailabil-
ity.6 The majority of these compounds incorporate basic groups
with relatively low pKa values (7–8) such as aminopyridyl variants.
High-throughput screening of an extensive series of ECLiPSTM li-
braries7 employing a time resolved fluorescence (TRF) assay8,9 re-
sulted in the identification of compound 1 as an antagonist of
integrins that have received considerable attention are
av-inte-
grins vb3 and vb5. Upregulation of these integrins has been asso-
a
a
ciated with various pathological angiogenesis including ocular
a
vb3 (Fig. 1). Subsequent evaluation of the binding activity of 1
measured on the human vb5 receptor using a TRF assay with
vitronectin as the ligand revealed this compound to be a dual
inhibitor of vb3 and vb5, with better potency against vb3.
diseases, tumorigenesis and metastasis.2 Recently a dual inhibitor
a
of avb3 and avb5 integrins (SCH221153) has been reported to inhi-
bit angiogenesis and tumor growth in vivo.3 In addition, in clinical
trials for recurrent glioblastoma multiforme, a highly invasive and
vascular cancer, single-agent Cilengitide (i.e., EMD 121974, a pep-
a
a
a
Consideration of the physicochemical properties of compound 1
revealed it to be drug-like with respect to Lipinski’s ‘rule of 5’.10 As
mentioned earlier, most avb3 inhibitors with good oral bioavailabil-
ity have basic groups with relatively low pKa values (i.e., between 7
and 8). We have estimated the basic aminobenzimidazole moiety in
our molecule to be in this range, having a calculated pKa value of
6.9. However, the polar surface area (PSA) of compound 1 is poten-
tially high as indicated by calculation of ‘fast polar surface area’
tidic dual inhibitor of
a
vb3 and
a
vb5 integrins)4 has demonstrated
antitumor benefits and minimal toxicity.5 As such there is signifi-
cant interest in the identification and development of dual inhibi-
tors of avb3 and avb5 integrins.
Many groups have reported small molecule integrin inhibitors
designed as RGD (Arg-Gly-Asp) mimetics.6 These RGD mimetics
typically consist of: (i) a basic group as an arginine mimic, (ii) a
subunit containing a carboxylic acid as an aspartic acid mimic,
(iii) and a molecular core which can organize these two pharmaco-
phores in the proper spatial orientation with respect to one an-
O
O
HO
N
H
other. The difficulty in obtaining
avb3 inhibitors with good oral
O
N
absorption has been recognized for many years as one of the major
1
N
αvβ3: IC50 = 0.23 μM
αvβ5: IC50 = 2.05 μM
NH2
* Corresponding author. Tel.: +1 609 452 3748; fax: +1 609 655 4187.
E-mail address: jletourn@pcop.com (J.J. Letourneau).
Figure 1. Dual avb3/avb5 antagonist 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.074

J. J. Letourneau et al. / Bioorg. Med. Chem. Lett. 19 (2009) 352–355
353
(FPSA).11 The calculated FPSA of 1 is 120.3 Å2, and we reasoned that
Table 1
this may potentially limit the prospect for good absorption.12
Since the high FPSA value for compound 1 is due in large part to
the presence of four H-bond donors (COOH, amide NH, and NH2 of
aminobenzimidazole), we examined the incorporation of con-
straints into the molecule as shown in Figure 2. Each of these con-
straints reduces polar surface area (PSA) by eliminating an H-bond
donor (i.e., amide NH and NH of amino group) and has the poten-
In vitro biological activity of compounds 2a, 2b, 2c, 10, 15a and 15b
O
R O
HO
N
R2
R1
a
O
N
n
N
HN
R1
R2
a
vb3 IC50
a
vb5 IC50
M)
a
IIbb3 IC50
a
a
Compound
n
R
tial to improve absorption while maintaining potency against av
-
(l
M)
(
l
(lM)
integrins vb3 and vb5.
a
a
2a
2b
2c
10
15a
15b
0
1
2
1
1
1
H
H
H
H
H
H
H
Me
Me
Me
0.18
0.039
0.37
4.8
0.60
4.3
0.31
0.014
0.005
>10
>10
>10
6.3
0.167
0.323
The compounds shown in Table 1 were synthesized via the gen-
eral synthetic routes outlined in Schemes 1–3 and the synthesis of
compounds 2a, 2b, 10 and 15a has been previously described.13
Compound 2a was prepared as shown in Scheme 1. First, treatment
of benzylbromide 4 with tert-butyl-3-aminopropionate in the pres-
ence of base with heating followed by BBr3-mediated demethyla-
tion gave the phenol 5. SNAr2 reaction of phenol 5 with 2,6-
difluoronitrobenzene followed by a second fluoro displacement
with methylamine and subsequent nitro-reduction gave aminoan-
iline 6. Finally, treatment of intermediate 6 with cyanogen bromide
to form the aminobenzimidazole followed by ester hydrolysis gave
desired compound 2a.
The synthesis of compounds 2b, 2c and 10 is shown in Scheme
2. Compounds 2b and 2c were prepared from the commercially
available lactams 7a and 7b, respectively. First the phenol was pro-
tected as the benzyl ether by benzylation with benzyl bromide.
Next the lactams were N-alkylated with ethyl 3-bromopropanoate
to give intermediates 8a and 8b. After removal of the benzyl pro-
tecting group by hydrogenolysis, the conversion of 8a and 8b to
2b and 2c was carried out in a similar manner to that for the con-
version of 5 to 2a. The synthesis of compound 10 was similar but
involved extra steps to prepare the tricyclic benzimidazole moiety
via intermediate 9c. Briefly, after removal of the benzyl protecting
group of intermediate 8a, SNAr2 reaction of the resultant phenol
–(CH2)3– 0.007
3,4-Benzdioxolyl –(CH2)3– 0.002
3-Pyridyl –(CH2)3– 0.001
a
Values are means of at least two experiments with standard deviation less than
20%.
The preparation of compounds 15a and 15b is summarized in
Scheme 3. First phenol 7a was reacted with benzylbromide to pro-
tect the phenol as the benzyl ether. This benzyl ether intermediate
was then converted into intermediates 12a and 12b using chemis-
try developed by Katritzky14 involving lanthanide triflate catalyzed
reaction of benzotriazole intermediates 11a and 11b with the com-
mercially available silylketene acetal 1-(tert-butyldimethylsilyl-
oxy)-1-methoxyethene. Conversion of intermediates 12a and 12b
to compounds 15a and 15b was similar to that described for the
conversion of intermediate 8a to compound 10.
The compounds synthesized in this study were initially evalu-
ated using a TRF assay for their ability to antagonize integrin–li-
gand interactions for
avb3 and avb5. The fibrinogen receptor
(aIIbb3) was also tested using the same assay for selectivity charac-
terization.9 As illustrated in Table 1, replacement of the benzamide
spacer with bicyclic lactam scaffolds in which the ring size of the
lactam ring was varied from 5 to 7 atoms (i.e., constraint A in
Fig. 2) was investigated first. The 6-membered lactam analog 2b
was preferred and represented a 6-fold increase in potency at
with 2,6-difluoronitrobenzene followed by
a second fluoro
displacement with 3-amino-1-propanol gave intermediate 9c.
After nitro-reduction of intermediate 9c, followed by reaction of
the resultant aminoaniline intermediate with cyanogen bromide
to form the aminobenzimidazole, the hydroxyl group was
converted to a chloride by treatment with thionyl chloride. The
chloro intermediate then underwent cyclization upon heating to
form the 6-membered ring constraint. Ester hydrolysis then gave
compound 10.
a
vb3 and a 3-fold increase in potency at
compound 1. The 5-membered lactam analog 2a was about 2-fold
more active at vb3 but 2-fold less active at vb5 compared to the
library hit 1. The 7-membered lactam analog 2c showed decreased
potency versus compound 1 at both vb3 and vb5. Compounds 2a,
2b and 2c were all virtually inactive (IC50 > 10 M) at the related
integrin IIbb3.
To further increase the binding affinity for
duce PSA (by deletion of an H-bond donor), our efforts focused on
avb5 compared to the lead
a
a
a
a
l
a
a
vb3 and avb5 and re-
O
O
HO
N
O
N
O
O
n
N
a,b
n = 0; 2a
n = 1; 2b
n = 2; 2c
O
O
NH2
N
O
OH
O
Br
4
5
constraint A
O
O
O
O
c,d,e
f,g
N
HO
N
H
O
N
O
O
N
H
NH2
N
6
A
N
1
B
O
NH2
constraint B
O
N
O
O
O
HO
N
NH2
HO
N
2a
H
O
N
Scheme 1. Reagents and conditions: (a) tert-butyl-3-aminopropionateÃHCl, Et3N,
MeOH, reflux; (b) BBr3, CH2Cl2, À78 °C to 23 °C; MeOH; (c) 2,6-difluoronitroben-
zene, CsCO3, DMF, 80 °C; (d) MeNH2 (2.0 M in MeOH), DMF, 23 °C; (e) H2 (g) (1 atm),
10% Pd/C, EtOH; (f) BrCN, MeOH; (g) LiOH, MeOH/THF/H2O (3:2:1).
N
3
HN
Figure 2. Two possible constraints of compound 1.

354
J. J. Letourneau et al. / Bioorg. Med. Chem. Lett. 19 (2009) 352–355
also enhanced activity at
a
IIbb3. Still, analog 15a exhibits over 80-
IIbb3 and greater than 10-fold
IIbb3. Analog 15b demonstrates an even
better selectivity profile, with greater than 300-fold selectivity for
vb3 versus IIbb3 and over 60-fold selectivity for vb5 versus IIbb3.
Because of their excellent potency at both vb3 and vb5, and
their good selectivity profile versus integrin IIbb3, analogs 15a
O
O
O
a,b
O
fold selectivity for
avb3 versus a
HN
O
N
selectivity for vb5 versus
a
a
OH
OBn
n
n
a
a
a
a
7a; n=1
7b; n=2
8a; n=1
8b; n=2
a
a
a
O
c,d,e
and 15b were further evaluated in functional cellular assays (see
Table 2). To predict their potential for GI absorption, Caco-2 per-
meability was also determined. Both analogs demonstrated good
potency in inhibiting FGF and VEGF-induced human umbilical vein
endothelial cells (HUVEC) proliferation15,16 and HUVEC migra-
tion.17,18 In fact, both analogs were essentially equipotent in inhib-
iting proliferation and were approximately 10-fold more active in
O
N
R
O
or c,d,f
N
n
H
NO2
9a; n= 1, R= Me
9b; n= 2, R= Me
9c; n= 1, R= (CH2)3OH
O
O
reducing cell migration compared to the known
avb3 and avb5
HO
N
antagonist cycloRGDfV.19 In early in vitro ADME profiling, com-
pound 15a displayed a promising Caco-2 permeability profile
(Papp = 89.6 nm/s), but compound 15b exhibited low permeability.
g,h,i
9a,b
9c
O
N
n
N
NH2
2b; n=1
2c; n=2
In summary, a novel, non-peptidic, dual inhibitor of the
av-inte-
grins vb3 and vb5 was identified in a broad screen of Pharmaco-
a
a
O
O
N
g,h,j,k,i
peia’s ECLiPSTM library collection. Introduction of two novel
constraints, aimed in part at minimizing PSA, and incorporation
of a 3,4-benzdioxolyl or 3-pyridyl substituent at the 3-position of
the propionic acid side-chain resulted in the identification of com-
pounds 15a and 15b which display nanomolar affinity for both
HO
O
N
N
10
HN
Scheme 2. Reagents and conditions: (a) benzylbromide, K2CO3, DMF; (b) NaH, ethyl
3-bromopropanoate, DMF; (c) H2 (g) (1 atm), 10% Pd/C, EtOH; (d) 2,6-difluoroni-
trobenzene, CsCO3, DMF, 80 °C; (e) MeNH2 (2.0 M in THF), DMF, 23 °C; (f) 3-amino-
1-propanol, EtOH, reflux; (g) H2 (g) (1 atm), 10% Pd/C, EtOH; (h) BrCN, MeOH; (i)
LiOH, MeOH/THF/H2O (3:2:1); (j) SOCl2, reflux; (k) K2CO3, TBAI (cat.), DMSO, 120 °C.
avb3 and
avb5 in binding assays. These analogs also display good
selectivity over the related integrin
aIIbb3. In addition, both analogs
show promising efficacy in functional cellular assays and com-
pound 15a displayed promising Caco-2 permeability. Therefore,
compound 15a was chosen as a lead structure for further SAR stud-
the aminobenzimidazole moiety next. Specifically, the exocyclic
primary amine substituent contributes substantially to the overall
high PSA of our molecules since it contains two H-bond donors.
One way to eliminate one of the H-bond donors on the exocyclic
primary amine substituent (and therefore decrease PSA) would
be to simply introduce an alkyl group (e.g., methyl) onto the amino
substituent. However, in earlier SAR studies around the amino-
benzimidazole moiety, we demonstrated that introduction of a
methyl substituent onto the exocyclic amino group (i.e., –NHMe
vs –NH2) resulted in a large decrease in binding affinity at both
ies in pursuit of potent dual inhibitors for
potentially good oral bioavailability.
a
vb3 and avb5 with
R
O
O
a,b
N
N
N
HN
N
or a,c
OBn
OH
O
7a
11a; R = [3,4]benzdioxolyl
11b; R = 3-pyridyl
avb3 and avb5 (not shown). We reasoned that this loss in potency
could be attributable to an unfavorable steric interaction between
the methyl group on the exocyclic amine substituent and the
methyl substituent of the imidazole nitrogen which would
disrupt the correct orientation of the remaining H-bond donor
(i.e., –NHMe) for binding to the integrin receptors.
To test this hypothesis, we prepared constrained analogs in
which the alkyl group on the exocyclic nitrogen and the alkyl
group on the imidazole nitrogen were joined together forming a
6-membered ring (see Fig. 2, constraint B). This constraint led to
R
O
e,f,g
OBn
d
O
N
12a,b
O
R
O
h,i
O
N
N
OH
N
O
H
NO2
13a,b
O
R
O
a substantial (5-fold) increase in binding affinity at
at vb5 was more moderate, resulting in a 2-fold increase in po-
tency (compare analogs 10 and 2b). Interestingly, the activity of
compound 10 at the integrin IIbb3 was enhanced compared to
analog 2b, but compound 10 is still highly selective for both
and vb5 versus IIbb3. Binding affinities at both vb3 and
avb3. The effect
OH
j,k,l
O
a
O
N
N
a
14a,b
NH2
a
a
vb3
vb5
O
R
O
a
a
a
HO
N
could be further enhanced by incorporating certain aryl and
heteroaryl substituents at the 3-position of the propionic acid
O
N
N
side-chain which have been utilized in potent
avb3 antagonists
15a,b
HN
described by Merck.6g For example, introduction of a 3,4-benzdiox-
olyl substituent to give compound 15a resulted in a 3- to 4-fold
Scheme 3. Reagents and conditions: (a) benzylbromide, K2CO3, DMF; (b) piperonal,
benzotriazole, TsOH (cat.), toluene, reflux, ÀH2O (Dean–Stark); (c) nicotinaldehyde,
benzotriazole, TsOH (cat.), toluene, reflux, –H2O (Dean–Stark); d) 1-(tert-butyldi-
methylsilyloxy)-1-methoxyethene, Yb(OTf)2, CH2Cl2; (e) H2 (g) (1 atm), 10% Pd/C,
MeOH; (f) 2,6-difluoronitrobenzene, CsCO3, DMF, 80 °C; (g) 3-amino-1-propanol,
EtOH, reflux; (h) H2 (g) (1 atm), 10% Pd/C, MeOH; (i) BrCN, MeOH; (j) SOCl2, reflux;
(k) DIPEA, NMP, 120 °C; (l) 1 N NaOH (aq), MeOH, 60 °C; H+.
increase in potency at
vb5 compared to the unsubstituted analog 10. Analog 15b
containing a 3-pyridyl substituent was even more active at both
vb3 and vb5 with single digit nanamolar potency at both recep-
tors. Introduction of the 3,4-benzdioxolyl or 3-pyridyl substituents
avb3 and a 22-fold increase in potency at
a
a
a

J. J. Letourneau et al. / Bioorg. Med. Chem. Lett. 19 (2009) 352–355
355
Table 2
9. Time resolved fluorescence (TRF) assay was used for initial high-throughput
compound library screening and subsequent lead optimization. The general
Functional cell-based activity of compounds 15a, 15b and cycloRGDfV and Caco-2
permeability of compounds 15a and 15b
protocol of the TRF assay is described in Ref. 8. The TRF assays for
vb5 and IIbb3 were essentially the same with only minor modifications.
Briefly, 384 solid black plate was coated with vb3 or vb5 or IIbb3 in buffer
avb3,
a
a
Compound
HUVEC
HUVEC
HUVEC
Caco-2
a
a
a
A containing 50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 1 mM
CaCl2, 1 mM MnCl2 overnight at 4 °C followed by blocking the well with 1%
BSA for 1 h at room temperature. The plate was washed with buffer A.
proliferation/
proliferation/
migration
permeability
Papp (nm/s)
a
a
a
FGF IC50
(lM)
VEGF IC50
(l
M) IC50
,
(
lM)
15
l
L of compound solution and 5
vb5; fibrinogen for IIbb3) in buffer A plus 20 lM DTPA, 0.05% BSA
l
L of 20 nM Eu3+-ligand (vitronectin for
15a
15b
2.9 ( 1)
1.1 ( 1)
CycloRGDfV 3.1 ( 3)
1.1( 0.5)
4.3( 2)
4.5( 1.5)
0.040 ( 0.020) 89.6
0.037 ( 0.028)
0.53 ( 0.22)
0.0
ND
a
vb3 and
a
a
were then added to the wells followed by 1-h incubation at room
temperature. The plate was washed with buffer A. The enhancement
solution was added and the fluorescence was measured after 10-min
incubation on the shaker.
a
Values are means of two experiments, standard deviation is given in paren-
theses (ND, not determined).
10. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
1997, 23, 3.
11. Physicochemical properties, including FPSA, were calculated using ADME
Profiler: Program for discreet compound analysis and calculation of
physicochemical properties, version 1.6.0; Pharmacopeia Inc., 2004.
12. (a) Egan, W. J.; Merz, K. M.; Baldwin, J. J. J. Med. Chem. 2000, 43, 3867; (b) Veber,
D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D. J. Med.
Chem. 2002, 45, 2615.
13. Letourneau, J. J.; Paradkar, V.; Ohlmeyer, M. H. J.; Dillard, L. W.; Baldwin, J. J.;
Riviello, C. M.; Wong, A.; Rong, Y. U.S. Patent 7,365,209, April 29, 2008.
14. Kobayashi, S.; Ishitani, H.; Komiyama, S.; Oniciu, D. C.; Katritzky, A. R.
Tetrahedron Lett. 1996, 37, 3731.
References and notes
1. Hynes, R. O. Cell 1992, 69, 11.
2. Trikha, M.; Zhou, Z.; Timar, J.; Raso, E.; Kennel, M.; Emmell, E.; Nakada, M. T.
Cancer Res. 2002, 62, 2824.
3. Kumar, C. C.; Malkowski, M.; Yin, Z.; Tanghetti, E.; Yaremko, B.; Nechuta, T.;
Varner, J.; Liu, M.; Smith, E. M.; Neustadt, B.; Presta, M.; Armstrong, L. Cancer
Res. 2001, 61, 2232.
4. Stupp, R.; Ruegg, C. J. Clin. Oncol. 2007, 25, 1637.
5. Reardon, D. A.; Nabors, B. L.; Stupp, R.; Mikkelsen, T. Expert Opin. Investig. Drugs
2008, 17, 1225.
15. Kohn, E. C.; Alessandro, R.; Spoonster, J.; Wersto, R. P.; Liotta, L. A. Proc. Natl.
Acad. Sci. U.S.A. 1995, 92, 1307.
6. (a) Nagarajan, S. R.; Devadas, B.; Malecha, J. W.; Lu, H.-F.; Ruminski, P. G.; Rico,
J. G.; Rogers, T. E.; Marrufo, L. D.; Collins, J. T.; Kleine, H. P.; Lantz, M. K.; Zhu, J.;
Green, N. F.; Russell, M. A.; Landis, B. H.; Miller, L. M.; Meyer, D. M.; Duffin, T.
D.; Engleman, V. W.; Finn, M. B.; Freeman, S. K.; Griggs, D. W.; Williams, M. L.;
Nickols, M. A.; Pegg, J. A.; Shannon, K. E.; Steininger, C.; Westlin, M. M.; Nickols,
G. A.; Keene, J. L. Bioorg. Med. Chem. 2007, 15, 3783; (b) Manzoni, L.; Bassanini,
M.; Belvisi, L.; Motto, I.; Scolastico, C.; Castorina, M.; Pisano, C. Eur. J. Org. Chem.
2007, 8, 1309; (c) Marugan, J. J.; Manthey, C.; Anaclerio, B.; Lafrance, L.; Lu, T.;
Markotan, T.; Leonard, K. A.; Crysler, C.; Eisennagel, S.; Dasgupta, M.; Tomczuk,
B. J. Med. Chem. 2005, 48, 926; (d) Leonard, K.; Pan, W.; Anaclerio, B.; Gushue, J.
M.; Guo, Z.; DesJarlais, R. L.; Chaikin, M. A.; Lattanze, J.; Crysler, C.; Manthey, C.
L.; Tomczuk, B. E.; Marugan, J. J. Bioorg. Med. Chem. Lett. 2005, 15, 2679; (e)
Zartman, A. E.; Duong, L. T.; Fernandez-Metzler, C.; Hartman, G. D.; Leu, C.-T.;
Prueksaritanont, T.; Rodan, G. A.; Rodan, S. B.; Duggan, M. E.; Meissner, R. S.
Bioorg. Med. Chem. Lett. 2005, 15, 1647; For an excellent review see: (f) Cacciari,
B.; Spalluto, G. Curr. Med. Chem. 2005, 12, 51 (and references therein); (g)
Meissner, R. S.; Perkins, J. J.; Duong, L. T.; Hartman, G. D.; Hoffman, W. F.; Huff,
J. R.; Ihle, N. C.; Leu, C. T.; Nagy, R. M.; Naylor-Olsen, A.; Rodan, G. A.; Whitman,
D. B.; Wesolowsky, G. A.; Duggan, M. E. Bioorg. Med. Chem. Lett. 2002, 12, 25.
7. (a) Baldwin, J. J.; Burbaum, J. J.; Henderson, I.; Ohlmeyer, M. H. J. J. Am. Chem.
Soc. 1995, 117, 5588; (b) Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L. W.;
Reader, J. C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 10922; (c) Nestler, H. P.; Bartlett, P. A.; Still, W. C. J. Org.
Chem. 1994, 59, 4723.
16. Assays for FGF or VEGF-induced HUVEC proliferation were performed as
described in Ref. 15 with modifications. Briefly, HUVEC cells were seeded at
1000 cells per well in 200
cells were cultured at 37 °C with 5% CO2 for 24 h. The media was then decanted
and 200 L of proliferation media containing EBM-2 plus 2% FBS and 3 ng/mL
lL of complete EBM-2 media in 96 well-format. The
l
FGF with the compound was added and the cells were incubated for an
additional 48 h. Cells were then fixed with 2.5% glutaraldehyde for 30 min,
washed and stained with 0.1% crystal violet. Absorbance at 595 nM was
measured after being washed and destained using 10% acetic acid.
17. Yoon, M.-J.; Cho, C.-H.; Lee, C. S.; Jang, I.-H.; Ryu, S. H.; Koh, G. Y. Biochem.
Biophys. Res. Commun. 2003, 308, 101.
18. HUVEC migration assay was performed essentially as described in Ref.17.
Briefly, the polycarbonate filters from a 24-well Boyden chamber were coated
with
2 lg/mL of vitronectin in coating buffer containing 50 mM Hepes,
100 mM NaCl, 1 mM of MgCl2, CaCl2 and MgCl2 for 1 h at 37 °C, followed by
blocking with 2% BSA in the coating buffer for 1 h at 37 °C. HUVEC cells were
then seeded to the upper chamber at 20,000 per well in 100
lL of migration
buffer in the presence or absence of the compound. Migration buffer (500
lL)
was added to the bottom chamber followed by incubation at 37 °C for 4 h. The
non-migrated cells were removed from the upper side of the filters with a
cotton ball. The migrated cells were fixed with 2.5% glutaraldehyde followed
by washing and staining using 0.1% crystal violet. The stained cells were
photographed and analyzed using ScanImage.
19. Chavakis, E.; Riecke, B.; Lin, J.; Linn, T.; Bretzel, R. G.; Preissner, K. T.; Brownlee,
M.; Hammes, H.-P. Diabetologia 2002, 45, 262.
8. Kapyla, J.; Ivaska, J.; Riikonen, R.; Nykvist, P.; Pentikainen, O.; Johnson, M.;
Heino, M. J. Biol. Chem. 2000, 275, 3348.