Synthesis and mode of action of 125I- and 3H-labeled thieno[2,3-c]pyridine antagonists of cell adhesion molecule expression

Article abstract of DOI:10.1021/jo016171j

A series of thieno[2,3-c]pyridine antagonists of cell adhesion molecule (CAM) expression, such as A-205804 (1) and A-249377 (2), selectively suppressed the induced expression of E-selectin and ICAM-1 over VCAM-1. In an effort to explore the biological mechanism of action of these inhibitors, we synthesized ¹²⁵I- and ³H-labeled thieno[2,3-c]pyridines 5 and 6. An isolated diazonium tetrafluoroborate salt efficiently trapped Na¹²⁵I on very small scale (7.5 μg of Na¹²⁵I), providing the corresponding ¹²⁵I-labeled thieno[2,3-c]pyridine in modest yield. Preliminary mechanistic investigations using these radiolabeled compounds revealed that, upon incubation with human umbilical vein endothelial cells (HUVECs), these inhibitors of CAM expression translocated to the cell nucleus and were noncovalently associated with macromolecules of molecular weight greater than 650 kDa.

Full text of DOI:10.1021/jo016171j

J . Org. Chem. 2002, 67, 943-948
943
3
Syn th esis a n d Mod e of Action of 125I- a n d H-La beled
Th ien o[2,3-c]p yr id in e An ta gon ists of Cell Ad h esion Molecu le
Exp r ession
Gui-Dong Zhu,* Verlyn Schaefer, Steven A. Boyd, and Gregory F. Okasinski
Abbott Laboratories, Metabolic Diseases Research, Pharmaceutical Products Division, Department 04MJ ,
Building AP10, 100 Abbott Park Road, Abbott Park, Illinois 60064-3500
gui-dong.zhu@abbott.com
Received October 2, 2001
A series of thieno[2,3-c]pyridine antagonists of cell adhesion molecule (CAM) expression, such as
A-205804 (1) and A-249377 (2), selectively suppressed the induced expression of E-selectin and
ICAM-1 over VCAM-1. In an effort to explore the biological mechanism of action of these inhibitors,
we synthesized 125I- and 3H-labeled thieno[2,3-c]pyridines 5 and 6. An isolated diazonium
tetrafluoroborate salt efficiently trapped Na125I on very small scale (7.5 µg of Na125I), providing
the corresponding 125I-labeled thieno[2,3-c]pyridine in modest yield. Preliminary mechanistic
investigations using these radiolabeled compounds revealed that, upon incubation with human
umbilical vein endothelial cells (HUVECs), these inhibitors of CAM expression translocated to the
cell nucleus and were noncovalently associated with macromolecules of molecular weight greater
than 650 kDa.
In tr od u ction
rat rheumatoid arthritis model and in a mouse asthma
model.7
Cell adhesion molecules (CAMs) are a group of cell-
surface proteins that are involved in mediating adhesion
of cells to each other and to extracellular matrix proteins.
Binding of CAMs on vascular endothelial cells (e.g.,
E-selectin, ICAM-1, and VCAM-1) to their counter-
receptors on circulating leukocytes (e.g., Lewis-X anti-
gens, â1 and â2 integrins) lead to transmigration of
leukocytes to the site of injury.1 Continuous recruitment2
of leukocytes from blood vessels may result in disease
states, such as perpetuation of tissue injury in chronic
inflammation.3 An early and critical step in stimulated
leukocyte recruitment is the induced expression of cell
adhesion molecules on the lumenal surface of vascular
endothelial cells.4,5 We have discovered a series of thieno-
[2,3-c]pyridine antagonists such as A-205804 (1) and
A-249377 (2) that selectively suppressed the tumor
necrosis factor-R (TNFR)-induced expression of E-selectin
and ICAM-1 over VCAM-1 (1: IC50 ∼ 25 nM for E-selectin
and ICAM-1, IC50 > 1 µM for VCAM-1; 2: IC50 ∼ 4 nM
for E-selectin and ICAM-1, IC50 ) 48 µM for VCAM-1).6,7
Significant clinical benefit was demonstrated by 2 in a
Preliminary biological characterization using a series
of structurally diverse inhibitors indicated that the
compounds were effective regardless of method of activa-
tion of the endothelial cells [e.g., interleukin-1 â (IL-1â)].
These compounds were not general inhibitors of protein
synthesis or gene transcription and had little effect on
T-cell function. However, the mode of action of our lead
compounds on the molecular level has not been eluci-
dated. To explore the biomolecular target of our thieno-
pyridine inhibitors, a tritium (3H), or preferably the
higher energy emitting 125I-labeled thienopyridine inhibi-
tor, would be required. Since compound 3 and 4 were very
similar to our lead 2 and 1 in all aspects of biological
characterizations (3: IC50 ) 25 nM for ICAM-1, 4: IC50
) 4 nM for ICAM-1) and represented two series of thieno-
[2,3-c]pyridine antagonists of cell adhesion molecule
expression, herein we report the synthesis of their 125I-
and 3H-labeled compound 5 and 6 and preliminary
investigation toward elucidation of their mechanism of
action.
* Address for correspondence: D47S, AP-10, Abbott Laboratories,
100 Abbott Park Rd., Abbott Park, IL 60064-6101. Tel: 847-935-1305.
FAX: 847-935-5165.
(1) (a) Carter, W.; Robinson, J . In Phagocyte Function; Robinson,
J .; Babcock, G., Eds.; Wiley-Liss: New York, 1998; pp 253-276. (b)
Forlow, S. B.; Ley, K. Am. J . Physiol. Heart Circ. Physiol. 2001, 280,
H634-H641.
(2) Leukocyte Recruitment, Endothelial Cell Adhesion Molecules, and
Transcriptional Control: Insight for Drug Discovery,; Collins, T., Ed.;
Kluwer: Boston, MA, 2001; pp 1-327.
(3) Winn, R.; Vedder, N.; Ramamoorthy, C.; Sharar, S.; Harlan, J .
Blood Coagulation Fibrinolysis 1998, 9(2), S17-S22
(4) Kubes, P.; Ward, P. A. Brain Pathol. 2000, 10, 127-135.
(5) (a) Newman, W.; Mirabelli, C. Expert Opin. Invest. Drugs 1998,
7, 19-25. (b) Panes, J .; Granger, D. Gastroenterology 1998, 114, 1066-
1090.
(6) Stewart, A.; Bhatia, P.; McCarty, C.; Patel, M.; Staeger, M.;
Arendsen, D.; Gunawardana, I.; Melcher, L.; Zhu, G.-D.; Boyd, S.; Fry,
D.; Cool, B.; Kifle, L.; Lartey, K.; Marsh, K.; Kempf-Grote, A.;
Kilgannon, P.; Wisdom, W.; Meyer, J .; Gallatin, M.; Okasinski, G. J .
Med. Chem. 2001, 44, 988-1002.
(7) Zhu, G.-D.; Arendsen, D.; Gunawardana, I.; Boyd, S.; Stewart,
A.; Fry, D.; Cool, B.; Kifle, L.; Schaefer, V.; Meuth, J .; Marsh, K.;
Kempf-Grote, A.; Kilgannon, P.; Gallatin, M.; Okasinski, G. J . Med.
Chem. 2001, 44, 3469-3487.
10.1021/jo016171j CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/11/2002

944 J . Org. Chem., Vol. 67, No. 3, 2002
Zhu et al.
Sch em e 1
one-pot synthesis of the 4-phenoxythieno[2,3-c]pyridine
carboxylate (e.g., 18), we first used parent phenol to
explore the reaction and avoid the complicating func-
tional groups. Indeed, displacement of the chloride in 7
with a phenol was much slower and required assistance
of a base (Scheme 1, X ) O). Under conditions similar to
the synthesis of 15 (see Experimental Section, method
A of 16), the reaction of 7 with phenol afforded only 20%
of desired compound 16. Using potassium phenoxide as
the nucleophile gave a cleaner reaction, and generated
16 in 40% yield (method B). Interestingly, when 7 was
heated with two equiv of potassium phenoxide at 70 °C
for 3 h, both chlorides were displaced. Substitution of one
of the phenoxides by methyl thioglycolate proceeded
smoothly at room temperature, followed by cyclization,
to give 16 in a respectable 75% yield (method C).
With an efficient protocol to construct the 4-phen-
oxythieno[2,3-c]pyridine-2-carboxylate core in hand, we
next explored the reaction of 7 with 4-aminophenol 11
and 4-nitrophenol 10. The former phenol would provide
the requisite amino compound 18 for the 125I-labeling, and
the nitro-product 17 could be easily converted to 18 via
hydrogenation (Scheme 1). Unfortunately, no desired
product (17 and 18) was detected for both reactions under
all three conditions described for the unsubstituted
phenol.
To circumvent this problem, we explored a direct
nitration of 16. There are several electron-rich sites in
16; therefore, a traditional H2SO4/HNO3 protocol would
not be desired because of the challenges in preventing
over-nitration. We envisioned that addition of 1 equiv of
solid potassium nitrate to concentrated sulfuric acid
would generate 1 equiv of fuming nitric acid that could
be directly used for nitration. Indeed, nitration of 16 in
sulfuric acid with 1 equiv of solid KNO3 gave a spot-to-
spot conversion to 17, specifically at the para-position of
the phenoxide. The major advantage of this nitration
strategy included easy operation for smaller scale reac-
tions. Hydrogenation of the nitrophenyl ether 17 in the
presence of 10% Pd/C afforded 18.
When working on the nitration strategy to access
aniline 18, we also screened the potential protecting
groups for the troublesome amino functionality in 11.
Under the best conditions for synthesis of 16 (method C
of Experimental Section), the reaction of 4-tert-butoxy-
carbonylaminophenol (12) with 7 did not provide any
detectable desired product 19. By employing the Cs2CO3/
DMF protocol (method A), only 10% desired 19 was
obtained. When we first isolated intermediate 14 in pure
form by column chromatography (67%) and resubjected
it to methyl thioglycolate substitution/cyclization, the
same reaction furnished 19 in 37% overall yield. Heating
ester 19 in a methanolic ammonia solution afforded
amide 21. The BOC protecting group of 21 was removed
by treating with trifluoroacetic acid (TFA) to provide
aniline 22.
Resu lts a n d Discu ssion
To save the expensive isotopically enriched reagents
and reduce the number of steps involving radioactive
intermediates, an ideal process of radiolabeling intro-
duces the radioactive reagent at the end of synthesis.
In our case, Sandmeyer reaction of aniline 22, followed
by Na125I trapping, and hydrogenation of 20 under
tritium would serve as the ideal labeling steps for the
synthesis of 5 and 6. We envisioned that substitution of
one chloride of 3,5-dichloropyridinecarboxaldehyde (7)
with a functionalized thio- or regular phenol would install
the requisite bromide and amino groups. Displacement
of the second chloride of 7 by a methyl thioglycolate,
followed by a base-catalyzed Aldol type cyclization of the
glycolate with the 4-carbonyl, would construct the thieno-
[2,3-c]pyridine core of 20 and 22. The detailed syntheses
are outlined in Scheme 1. Treatment of dichloropyridine-
carboxaldehyde 7 with 1 equiv of 4-bromothiophenol (8)
afforded a monosubstituted diaryl sulfide 13. The diaryl
sulfide 13 was not purified but rather was reacted with
methyl thioglycolate in the same pot. The Aldol type
cyclization needed assistance of a weak base such as
potassium carbonate, giving the 4-substituted thieno[2,3-
c]pyridine-2-carboxylate 15 in 50% yield over two steps.
Heating of the methyl ester 15 in a methanolic ammonia
solution at 45 °C furnished 20 in 95% yield. Hydrogena-
tion of 20 in ethanol under tritium (10% Pd/C, 0.125 atm
3H2) afforded the desired tritium labeled thieno[2,3-c]-
pyridine 6. This tritium-labeled compound was not
characterized spectroscopically, but was identical by
HPLC and TLC with protio compound 4 that was
prepared by the same protocol.
The results of Sandmeyer reactions of 22 are sum-
marized in Table 1. The Sandmeyer reaction of 22 on a
30 mg scale with 3 equiv of sodium iodide afforded
82% of desired iodide 3. A major side reaction was
reduction of the diazonium salt 24 to give the unsubsti-
tuted phenyl ether 23. There was still about 5% diazo-
nium salt 24 that was not trapped by NaI, as determined
by HPLC.
Since phenols are less nucleophilic than thiophenols,
and because additional challenges were expected for a
Since Na125I was much more expensive and less avail-
able than 22, our goal was to increase the yield of

Thieno[2,3-c]pyridine Antagonists Cell Adhesion Molecule Expression
J . Org. Chem., Vol. 67, No. 3, 2002 945
Ta ble 1. Sa n d m eyer Rea ction sa of 22
22/NaI
yield (3)
yield (3)
yield (23),
22 (mg) mol/mol (from 22), % (from NaI), %
%
30
5
3
1/3
2/1
5/1
82
25
3
27
50
17
5
10
20
6
1
5/1
1
6
0.5
0.5
5/1
35/1
<1
0
2
0
2
0
a
Aniline 22 was treated with 1 equiv of KNO2 in 3 M H2SO4.
Sch em e 2
F igu r e 1. Kinetics of 6 in HUVECs. Essentially all of the
radioactivity which bound to the cell was translocated to the
nucleus. The nucleus was saturated about 5 h after treatment
of the cells with radiolabeled compound. Less than 1% of the
radioactivity counts were detected in the cytoplasmic fraction.
Ta ble 2. Tr a p p in g Na I w ith 25
25/Na125
I
Yield (5)
Yield (5)
Yield (23),
25 (mg)
mol/mol
(from 25), % (from NaI), %
%
2
1
0.5
0.5
0.5
1/1a
4/1a
44
11
23
2.5
1
44
42
42
38
35
35
40
40
42
45
2/1a
18/1
35/1
a
Naturally occurring NaI was used in the reaction. The product
was 3 (instead of 5).
radiolabeled compound 5 based on consumed Na125I.
When less NaI (0.5 equiv) was used in the reaction, a
higher 50% yield was achieved for NaI, even though the
yield based on 22 was much lower (25%) on 5 mg scale.
However, when the reaction scale was reduced from 3
mg to 0.5 mg, the yield for iodine incorporation to 3
dropped dramatically from 17% to 2%. Other than small
amount of 23 (2%), the majority of the derived diazonium
salt 24 remained unreacted in the 0.5 mg reaction. On a
practical scale of 125I labeling in a regular biological
laboratory, we attempted to trap 7.5 µg of Na125I with
the diazonium salt 24 from 0.5 mg of 22. In this reaction,
none of the desired product 5 was detected by HPLC.
F igu r e 2. Binding saturation of 6. With increasing concentra-
tion of 6, the total CPM increased nearly linear to the
concentration (not shown in chart). The radioactivity in the
nucleus increased and saturated at about 500 nM of 6. Less
than 1% of the counts was found in the cytoplasm, and the
percentage was not dependent on the concentration of 6.
bound to any enzymes that involved in the signaling
pathway leading to activation of adhesion molecule
expression. Thus, the more readily available 6 was
incubated with HUVEC lysates at 37 °C for 2 and 18 h
and subjected to a BioRad P30 spin column assay. The
void volume containing molecules with molecular weight
>40 kDa was collected after centrifugation at 600 g for
4 min. Less than 0.1% of the total radioactivity was
detected in the void volume, demonstrating lack of
interaction of 6 with any molecule with a molecular
weight of 40 kDa or greater in the HUVEC cell lysates.
We then incubated 6 with HUVECs in log phase
growth at 37 °C, with samples taken over 20 h. The cells
were fractionated according to a modified protocol by
Goldstein et al.8 Figure 1 shows the distribution of
radiolabeled compounds in HUVECs.
We suspected that the strongly acidic conditions em-
ployed (3 M H2SO4) could be a major factor in the NaI
trapping. The diazonium tetrafluoroborate 25 was then
prepared by reaction of 24 with sodium tetrafluoroborate.
Indeed, when treating 25 with NaI in DMSO, desired
product 5 was isolated consistently in modest yield. The
amount of NaI employed had no significant affect on the
yield (Scheme 2 and Table 2). The reaction of 0.5 mg of
25 with 7.5 µg of Na125I essentially gave the same yield
of 5 (35%) compared to a reaction on a much larger scale
(44% for 1 mg of NaI).
As illustrated in Figure 1, with increasing time of
incubation (∼5 h), the thienopyridine 6 became localized
to the cell nucleus. The cytoplasm contained less than
1% of the total radioactivity. Figure 2 shows that the
translocation of 6 to nucleus increased with increasing
concentration of radiolabeled drug (total CPM was linear
With the two radiolabeled compounds 5 and 6 in hand,
we first performed a direct binding experiment with
HUVEC lysates to evaluate if our thienopyridine leads
(8) Wang, X.; Sato, R.; Brown, M.; Hua, X.; Goldstein, J . Cell 1994,
77, 53-62.

946 J . Org. Chem., Vol. 67, No. 3, 2002
Zhu et al.
F igu r e 3. Gel filtration of the nuclear fraction of 5 treated HUVECs. The major peak associated with 5 had a molecular weight
greater than 650 kDa as deduced from the markers of 670, 158, 44, 17 kDa and 1350 Da. The gel filtration was performed on a
Beckman Gold HPLC chromatography system with a SynChropak GPC-300 column (250 × 4.6 mm). Mobile phase: 20 mM sodium
phosphate (pH 7.4) with 135 mM NaCl. Flow rate: 100 µL/min.
Flash column chromatography was performed on silica gel 60
(Merck, 230-400 mesh) using the indicated solvent. For
routine aqueous workup, the reaction mixture was partitioned
between brine and EtOAc, and the organic layer was washed
with brine and dried over MgSO4.
with the concentration of 6 and is not shown in Figure
2). The radioactivity counts, however, saturated at about
500 nM of 6. Only a very small amount of thienopyridine
(<1%) was found in the cytoplasm, and the percentage
was not dependent on the concentration of 6.
Meth yl 4-[(4-Br om op h en yl)th io]th ien o[2,3-c]p yr id in e-
2-ca r boxyla te (15). To a solution of pyridine aldehyde 76 (400
mg, 2.27 mmol) in DMF (10 mL) were added 4-bromoben-
zenethiol 8 (429 mg, 2.27 mmol) and powered K2CO3 (627 mg,
4.54 mmol) at 0 °C. The reaction mixture was stirred at the
same temperature for 1 h and at rt for 3 h. After the reaction
mixture was cooled to 0 °C again, methyl thioglycolate (241
mg, 2.27 mmol) and K2CO3 (314 mg, 2.27 mmol) were added.
The mixture was stirred at rt for 0.5 h and at 70 °C for 2 h.
After being cooled to rt, the reaction mixture was partitioned
between ethyl acetate and water. The organic layer was
washed with water, dried, and concentrated. The residual
material was purified by flash chromatography (15% EtOAc
These two experiments clearly showed that 6 did not
bind to any macromolecules in the cytoplasm of HUVECs
with a molecular weight >40 kDa. Since tritium has
relative low energy, further exploration by the tritium-
labeled 6 on the biomolecular target was not conclusive.
When the higher energy emitting 125I-labeled 5 was
employed, the tris glycine native gel of the nuclear
fraction revealed that 5 was associated with very large
molecules in the cell nucleus. When treated with SDS
detergent, 5 was released from the nuclear fraction,
indicating a noncovalent binding of 5 with the macro-
molecule. Gel filtration of the nuclear lysates confirmed
that the macromolecule had a molecular weight greater
than 650 kDa (Figure 3).
1
in hexane) to provide 15 (432 mg, 50%). H NMR (300 MHz,
CDCl3) δ 3.97 (s, 3 H), 7.14 (d, J ) 8.5 Hz, 2 H), 7.41 (d, J )
8.5 Hz, 2 H), 8.13 (s, 1 H), 8.50 (s, 1H), 9.13 (s, 1 H). MS (DCI/
NH3) m/z 380, 382 (M + H)+. Anal. Calcd for C15H10BrNO2S2:
C, 47.38; H, 2.65; N, 3.68. Found: C, 47.45; H, 2.60; N, 3.71.
In summary, the reaction of the diazonium salt 25 with
Na125I in DMSO represented a practical and efficient
method for 125I-labeling of our thienopyridine antagonist
of cell adhesion molecule expression. Preliminary inves-
4-[(4-Br om op h en yl)t h io]t h ien o[2,3-c]p yr id in e-2-ca r -
boxa m id e (20). Ester 15 (381 mg, 1 mmol) was dissolved in
2 M methanolic ammonia (10 mL) and warmed to 45 °C in a
sealed tube for 18 h. The precipitate was filtered, washed with
methanol-diethyl ether (1:1), and dried under vacuum to give
amide 20 (250 mg). The mother liquor was concentrated, and
the residual material was purified by flash chromatography
to provide additional 20 (97 mg). Combined yield: 95%. 1H
NMR (300 MHz, DMSO-d6) δ 7.20 (dt, J ) 8.5, 2.0 Hz, 2H),
7.53 (dt, J ) 8.6, 2.0 Hz, 2H), 7.87 (br s, 1H), 8.21 (s, 1H),
8.51 (br s, 1H), 8.54 (s, 1H), 9.36 (s, 1H); MS (DCI/NH3) m/z
365 (M + H)+. Anal. Calcd for C14H9BrN2OS2: C, 46.03; H,
2.48; N, 7.67. Found: C, 46.21; H, 2.57; N, 7.77.
3
tigation using our H- and 125I-labeled compounds showed
that our thienopyridine inhibitors noncovalently bound
to a macromolecule or complex in the cell nucleus with a
molecular weight greater than 650 kDa.
Exp er im en ta l Section
Gen er a l Sp ectr oscop ic a n d Exp er im en ta l Da ta . The
NMR spectra were obtained on Varian UP-300, Varian M-300,
Bruker AMX-400, and Varian U-400 magnetic resonance
spectrometer (300/400 MHz for 1H and 75/100 MHz for 13C)
with deuteriochloroform as solvent and internal standard
unless otherwise indicated. The chemical shifts are given in
delta (δ) values and the coupling constants (J ) in hertz (Hz).
Infrared spectra were recorded on Nicolet 5SX and Nicolet
Magna-IR 750 spectrometer. Mass spectra were acquired on
a J EOL J MS-SX-102 spectrometer. Elemental analysis was
performed by Robertson Microlit Laboratories, Inc., Madison,
NJ . All manipulations were performed under nitrogen atmo-
sphere unless otherwise mentioned. All solvents and reagents
were purified when necessary using standard procedures.
4-(P h en ylth io)th ien o[2,3-c]pyr idin e-2-car boxam ide (4).
To a solution of 20 (20 mg) in a 1:1 mixture of ethanol and
DMF (5 mL) were added triethylamine (20 mg) and 10% Pd/C
(20 mg) under nitrogen. This suspension was purged with
hydrogen and was stirred under hydrogen (1 atm) for 72 h.
The solid material was filtered off and washed with ethanol.
The filtrate was concentrated, and the residue was purified
by flash chromatography (50% EtOAc in hexane) to provide 4
1
(13 mg, 83%). H NMR (300 MHz, DMSO-d6) δ 7.29-7.40 (m,
5H), 7.86 (br s, 1H), 8.25 (s, 1H), 8.46 (s, 1H), 8.52 (br s, 1H),
9.31 (s, 1H); MS (DCI/NH3) m/z 287 (M + H)+. Anal. Calcd for

Thieno[2,3-c]pyridine Antagonists Cell Adhesion Molecule Expression
J . Org. Chem., Vol. 67, No. 3, 2002 947
C14H10N2OS2: C, 58.72; H, 3.52; N, 9.78. Found: C, 58.90; H,
3.50; N, 9.89.
purified by flash chromatography (20% EtOAc in hexane) to
give 17 (36.4 mg, 62%). H NMR (300 MHz, CDCl3) δ 3.97 (s,
1
4-(4-3H-P h en ylth io)th ien o[2,3-c]p yr id in e-2-ca r boxa m -
id e (6). To a solution of 20 (5 mg, 0.014 mmol) in a 1:1 mixture
of ethanol and DMF (2 mL) were added triethylamine (5 mg)
and 10% Pd/C (5 mg) under nitrogen. This suspension was
degassed under vacuum. Tritium (0.125 atm) was then intro-
duced, and the suspension was stirred at room temperature
under tritium for 72 h. The solid material was filtered off and
washed with ethanol. The filtrate was concentrated and the
residue was purified by HPLC (C-18, CH3CN/H2O containing
0.1% TFA) to provide 6 (3.2 mg, 80%). The radiochemical yield
was 70%, and the specific activity of 6 was 29 Ci/mmol. 6 was
identical by HPLC and TLC with protio compound 4 and had
an IC50 of 25 nM against E-selectin and 20 nM against ICAM-1
in a cell ELISA assay.
3 H), 7.10 (d, J ) 9.2 Hz, 2 H), 7.97 (s, 1 H), 8.26 (d, J ) 9.2
Hz, 2 H), 8.36 (s, 1 H), 9.12 (s, 1 H); MS (DCI) m/z 331 (M +
H)+. Anal. Calcd for C15H10N2O5S: C, 54.54; H, 3.05; N, 8.48.
Found: C, 54.65; H, 3.15; N, 8.40.
Met h yl 4-(4-Am in op h en oxy)t h ien o[2, 3-c]p yr id in e-2-
ca r boxyla te (18). To a solution of 17 (40 mg) in ethyl acetate
(5 mL) was added 10% Pd/C (30 mg) under nitrogen. This
suspension was purged with hydrogen and was stirred at rt
under hydrogen (1 atm) for 42 h. The solid material was
filtered off and washed with ethyl acetate. The filtrate was
concentrated, and the residue was purified by flash chroma-
tography (70% EtOAc in hexane) to provide 18 (27 mg, 75%).
1H NMR (300 MHz, CDCl3) δ 3.97 (s, 3 H), 6.71 (d, J ) 8.6
Hz, 2 H), 6.94 (d, J ) 8.7 Hz, 2 H), 8.00 (s, 1 H), 8.21 (s, 1 H),
8.87 (s, 1 H); MS (DCI) m/z 301 (M + H)+. Anal. Calcd for
C15H12N2O3S: C, 59.99; H, 4.03; N, 9.33. Found: C, 60.06; H,
4.10; N, 9.21.
Meth yl 4-P h en oxyth ien o[2, 3-c]p yr id in e-2-ca r boxyla te
(16). Method A: To a solution of phenol (213 mg, 2.27 mmol)
and pyridinealdehyde 7 (400 mg, 2.27 mmol) in DMF (8 mL)
was added cesium carbonate (740 mg, 2.27 mmol) at rt. The
reaction mixture was stirred at 70 °C for 1 h and was cooled
to 0 °C. Methyl thioglycolate (241 mg, 2.27 mmol) was added.
After the reaction mixture was stirred at rt for 1 h, more
cesium carbonate (740 mg, 2.27 mmol) was added. The reaction
mixture was stirred at rt for 1 h and at 70 °C for 0.5 h. After
being cooled to rt, the reaction mixture was partitioned
between ethyl acetate and water. The organic layer was
washed with water, dried, and concentrated. The residual
material was purified by flash chromatography (20% EtOAc
[4-(5-Ch lor o-4-for m ylp yr id in -3-yloxy)p h en yl]ca r ba m -
ic Acid ter t-Bu tyl Ester (14). To a solution of pyridinecar-
boxaldehyde 7 (2.0 g, 11.4 mmol) and phenol 12 (2.38 g, 11.4
mmol), which was prepared according to literature method,9
in DMF (30 mL) was added cesium carbonate (3.70 g, 11.4
mmol) at rt. The reaction mixture was stirred at rt for 1 h
and at 70 °C for 0.5 h. After being cooled to rt, the reaction
mixture was partitioned between ethyl acetate and brine. The
organic layer was washed with water, dried, and concentrated.
The residual material was purified by flash chromatography
1
1
in hexane) to provide 16 (129 mg, 20%). H NMR (400 MHz,
(15% EtOAc in hexane) to provide 14 (2.66 g, 67%). H NMR
CDCl3) δ 3.95 (s, 3 H), 7.07 (d, J ) 7.7 Hz, 2 H), 7.17 (t, J )
7.3 Hz, 1 H), 7.37 (t, J ) 8.0 Hz, 2 H), 8.10 (s, 1 H), 8.12 (s, 1
H), 8.92 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 52.8, 118.5,
124.2, 125.8, 129.9, 132.3, 136.5, 138.0, 139.2, 139.9, 149.0,
156.2, 162.2; IR (KBr) ν 1717 (s) cm-1; MS (DCI) m/z 286 (M
+ H)+. Anal. Calcd for C15H11NO3S: C, 63.14; H, 3.89; N, 4.91.
Found: C, 63.25; H, 3.87; N, 4.97.
(300 MHz, CDCl3) δ 1.53 (s, 9 H), 6.52 (s, 1 H), 7.03 (d, J )
9.2 Hz, 2 H), 7.43 (d, J ) 9.2 Hz, 2 H), 8.17 (s, 1 H), 8.43 (s, 1
H), 10.54 (s, 1 H); MS (DCI) m/z 349 (M + H)+. Anal. Calcd
for C17H17ClN2O4: C, 58.54; H, 4.91; N, 8.03. Found: C, 58.55;
H, 4.97; N, 8.10.
Meth yl 4-(4-ter t-Bu toxycar bon ylam in oph en oxy)th ien o-
[2,3-c]p yr id in e-2-ca r boxyla te (19). To a solution of 14 (2.64
g, 7.58 mmol) in THF (30 mL) was added methyl thioglycolate
(804 mg, 7.58 mmol) at 0 °C. The reaction mixture was stirred
at rt for 1 h after which cesium carbonate (2.47 g, 7.58 mmol)
was added. The mixture was stirred at rt for 1 h and at 70 °C
for 0.5 h. After cooled to rt, the reaction mixture was
partitioned between ethyl acetate and brine. The organic layer
was washed with water, dried, and concentrated. The residual
material was purified by flash chromatography (20% EtOAc
in hexane) to provide 19 (1.66 g, 55%). 1H NMR (300 MHz,
DMSO-d6) δ 1.48 (s, 9 H), 3.91 (s, 3 H), 7.09 (d, J ) 8.8 Hz, 2
H), 7.51 (d, J ) 9.2 Hz, 2 H), 7.98 (s, 1 H), 8.06 (s, 1 H), 9.14
(s, 1 H), 9.40 (s, 1 H); 13C NMR (400 MHz, DMSO-d6) δ 28.06,
53.1, 79.0, 119.1, 119.7, 124.9, 131.6, 135.4, 136.2, 137.9, 138.6,
140.4, 148.9, 150.4, 152.7, 161.7; IR (KBr) ν 3371 (m), 1731
(s), 1701 (s) cm-1; MS (DCI) m/z 401 (M + H)+. Anal. Calcd
for C20H20N2O5S: C, 59.99; H, 5.03; N, 7.00. Found: C, 60.08;
H, 5.15; N, 7.10.
4-(4-Am in op h en oxy)th ien o[2, 3-c]p yr id in e-2-ca r boxa -
m id e (22). A solution of ester 19 (800 mg, 2 mmol) in 2 M
methanolic ammonia (100 mL) was heated at 55 °C in a sealed
tube for 24 h. Concentration of the reaction mixture gave
essentially pure 4-[(4-tert-Butyloxycarbonylamino)phenoxy]-
thieno[2,3-c]pyridine-2-carboxamide (21). This material was
not purified further and was dissolved in trifluoroacetic acid
(20 mL). The formed yellow solution was kept at room
temperature for 1 h before TFA was distilled off under reduced
pressure. The residual oil was triturated with a mixture of
ethyl acetate and aqueous NaHCO3 solution. The resultant
solid was collected by filtration, washed successively with ethyl
acetate, aqueous NaHCO3 solution, water, methanol, and ethyl
acetate, and dried to provide 22 (492 mg, 86% over 2 steps) as
a yellow solid. mp > 250 °C; 1H NMR (300 MHz, DMSO-d6) δ
5.62 (br s, 2H), 6.65 (d, J ) 8.8 Hz, 2H), 6.93 (d, J ) 8.8 Hz,
2H), 7.86 (s, 1H), 8.30 (s, 1H), 8.44 (s, 1H), 9.00 (br s, 1H); IR
(KBr) ν 3347 (m), 3136 (m), 1676 (s) cm-1; MS (DCI/NH3) m/e
Method B: To a solution of phenol (213 mg, 2.27 mmol) in
THF (8 mL) was added potassium tert-butoxide (1 M solution
in THF, 2.27 mL, 2.27 mmol) at 0 °C. The solution was kept
at rt for 1 h, and was then cooled to 0 °C. Pyridinecarboxal-
dehyde 7 (400 mg, 2.27 mmol) was added as solid. The reaction
mixture was stirred at rt for 0.5 h, and at 70 °C for 1 h. After
the reaction mixture was cooled to 0 °C again, methyl thiogly-
colate (241 mg, 2.27 mmol) was added and stirred for 1 h.
Cesium carbonate (740 mg, 2.27 mmol) was added. The
reaction mixture was stirred at rt for 1 h and at 70 °C for 0.5
h. After being cooled to rt, the reaction mixture was partitioned
between ethyl acetate and water. The organic layer was
washed with water, dried, and concentrated. The residual
material was purified as above to give 16 (258 mg, 40%).
Method C: A solution of phenol (0.94 g, 10 mmol) in THF
(10 mL) was treated with a solution of potassium tert-butoxide
(1.0 M solution in THF, 10 mL, 10 mmol) at 0 °C. This solution
was stirred at 25 °C for 1 h and was cooled to 0 °C. A solution
of 3,5-dichloropyridine-4-carboxaldehyde (7) (0.88 g, 5 mmol)
in THF (5 mL) was added, and the reaction mixture was
heated at 70 °C for 3 h. After cooled to 0 °C, methylthiogly-
colate (0.53 g, 5 mmol) and Cs2CO3 (1.63 g, 5 mmol) were
added. The mixture was heated at 70 °C for 0.5 h and was
cooled to room temperature and filtered. The filtrate was
diluted with ethyl acetate, washed sequentially with water and
brine, dried (MgSO4), filtered, and concentrated. Purification
of the residue by flash chromatography afforded 16 (1.06 g,
75%).
Met h yl 4-(4-Nit r op h en oxy)t h ien o[2, 3-c]p yr id in e-2-
ca r boxyla te (17). Compound 16 (50 mg, 0.175 mmol) was
dissolved in concentrated H2SO4 (3 mL) at rt, and the solution
was cooled to 0 °C. Potassium nitrate (18 mg, 0.175 mmol)
was then added as solid in several portions. The solution was
kept at 0 °C for 5 min and was then poured into ice. The
mixture was basified with sodium bicarbonate to pH 8 and
was extracted with ethyl acetate. The combined ethyl acetate
solution was washed with water, dried over MgSO4, and
filtered. The filtrate was concentrated, and the residue was
(9) Vigroux, A.; Bergon, M.; Zedde, C.; J . Med. Chem. 1995, 38, 3983.

948 J . Org. Chem., Vol. 67, No. 3, 2002
Zhu et al.
286 (M + H)+. Anal. Calcd for C14H11N3O2S‚0.5 CH3OH: C,
57.79; H, 3.85; N, 13.94. Found: C, 57.69; H, 3.95; N, 13.77.
expression (IC50 ) 5 nM for ICAM-1 versus 4 nM for 3) in a
cell ELISA assay.
Dir ect Bin d in g St u d ies of 6 w it h HUVE C Nu clei.
Primary HUVECs (total 9 × 107 cells) were washed with PBS,
scraped, and pelleted. The cell pellet was resuspended in 1
mL of imidazole HCl buffer (pH7.4) that contained 1 mM
EGTA and Boehringer Mannheim complete protease inhibitor
cocktail (imidazole cocktail). After douncing, the cytosol was
separated from nuclear pellet. The nuclear pellet was sonicated
in 0.5 mL of imidazole cocktail and was then clarified by
centrifigation. The nuclear lysate was incubated with 6 at 37
°C for 2 and 18 h separately. BioRad P30 spin columns were
preequilibrated with imidazole cocktail by gravity and then
centrifugated at 300 g for 2 min prior to loading 50 µL of the
nuclear lysates. The radiocounts were evaluated before and
after the BioRad P30 spin column fractionation. Void volume
containing molecules with a molecular weight >40 kDa was
collected after centrifigation at 600 g for 4 min.
Gel F iltr a tion Exp er im en t of th e Nu clea r F r a ction of
5 Tr ea ted HUVECs. Primary HUVECs (total 9 × 107 cells)
were incubated in EBM/2% FBS/human epidermal growth
factor/bovine brain extract/gentamicin (Clonetics/BioWhitak-
er). The following day labeled compound 5 was added, and the
plate was incubated at 37 °C for 18 h. The plate was washed
once with D-PBS (Gibco/BRL), and the cells were scraped and
pelleted. The cell pellet was resuspended in 1 mL of imidazole
HCl buffer (pH7.4) that contained 1 mM EGTA and Boehringer
Mannheim complete protease inhibitor cocktail (imidazole
cocktail). After douncing, the cytosol was separated from the
nuclear pellet. The nuclear pellet was sonicated in 0.5 mL
imidazole cocktail and was then subjected to a gel filtration
that was performed on a Beckman Gold HPLC chromatogra-
phy system with a SynChropak GPC-300 column (250 × 4.6
mm). Mobile phase: 20 mM sodium phosphate (pH 7.4) with
135 mM NaCl. Flow rate: 100 µL/min.
Gen er a l Cell ELISA Assa y for Com p ou n d s 1-6. Pri-
mary HUVECs were plated in 96 well plates at 5 × 104 cells/
mL in EBM/2% FBS/human epidermal growth factor/bovine
brain extract/gentamicin (Clonetics/BioWhitaker). The follow-
ing day test compounds were added and the plates incubated
24 h at 37 °C. TNFR (Gibco/BRL) then was added to a final
concentration of 5 ng/mL, and the plates were incubated an
additional 6 h at 37 °C. The plates were washed once with
D-PBS (Gibco/BRL), and primary antibody (Becton Dickinson)
was added in D-PBS/2% BSA (Sigma)/0.01% NaN3. Antibodies
used were mouse monoclonal anti-ELAM-1, anti-ICAM-1, and
anti-VCAM-1. The plates were stored overnight at 4 °C and
then washed three times with D-PBS. Secondary antibody,
HRP-conjugated donkey anti-mouse IgG (J ackson Labs) in
D-PBS/2%BSA, was added, and the plates were incubated 1-2
h at room temperature and then washed three times with
D-PBS. OPD solution (Abbott) was added to the wells, the
plates were developed for 15-20 min and neutralized with 1
N sulfuric acid, and the absorbance was read at 490 nm.
4-[4-Iod op h en oxy]t h ien o[2,3-c]p yr id in e-2-ca r b oxa m -
id e (3) a n d 4-P h en oxyth ien o[2,3-c]p yr id in e-2-ca r boxa -
m id e (23). Compound 22 (30 mg, 0.105 mmol) was dissolved
o
in 30% H2SO4 (2 mL), and the solution was cooled to 0 C with
an ice bath. A solution of sodium nitrite (8.0 mg, 0.116 mmol)
in H2O (80 µL) was then added, and the reaction mixture was
stirred at 0 °C for 1 h. Sodium iodide (48 mg, 0.315 mmol) as
a solution in H2O (0.3 mL) was added. The reaction mixture
was stirred at 0 °C for 1 h and was allowed to warm to rt for
3 h. Ethyl acetate (30 mL) was added, and the mixture was
washed with saturate aqueous sodium bicarbonate solution,
5% aq sodium bisulfite solution, and water. The EtOAc solution
was dried over MgSO4, filtered, and concentrated. The residual
material was separated by flash chromatography to give 34
1
mg of 3 (82%) and 2.8 mg of 23 (10%). Data for 3: H NMR
(300 MHz, DMSO-d6) δ 6.94 (d, J ) 8.8 Hz, 2H), 7.74 (d, J )
8.8 Hz, 2H), 7.86 (br s, 1H), 8.13 (s, 1H), 8.17 (s, 1H), 8.44 (br
s, 1H), 9.16 (s, 1H); 13C NMR (400 MHz, DMSO-d6) δ 87.5,
119.9, 120.2, 133.1, 137.5, 138.2, 138.8, 141.3, 146.5, 147.0,
156.7, 162.3; IR (KBr) ν 3351 (m), 3199 (m), 1663 (s) cm-1
;
MS (DCI/NH3) m/z 397 (M + H)+. Anal. Calcd for C14H9-
IN2O2S: C, 42.44; H, 2.29; N, 7.07. Found: C, 42.35; H, 2.19;
1
N, 6.99. Data for 23. H NMR (300 MHz, DMSO-d6) δ 7.15 (d,
J ) 8.0 Hz, 2H), 7.20 (t, J ) 7.8 Hz, 1H), 7.45 (t, J ) 8.0 Hz,
2H), 7.85 (br s, 1H), 8.10 (s, 1H), 8.20 (s, 1H), 8.45 (br s, 1H),
9.15 (s, 1H); MS (DCI/NH3) m/z 271 (M + H)+. Anal. Calcd for
C
14H10N2O2S: C, 62.21; H, 3.73; N, 10.36. Found: C, 62.35;
H, 3.79; N, 10.45.
4-(2-Ca r ba m oylth ien o[2,3-c]p yr id in -4-yloxy)ben zen e-
d ia zon iu m Tetr a flu or obor a te (25). To a solution of 22 (30
mg, 0.105 mmol) in 30% H2SO4 (2 mL) was added sodium
nitrite (10 mg, 0.145 mmol) in 100 µL of H2O at 0 °C. The
reaction mixture was stirred at the same temperature for 2 h,
and the solid material was removed by filtration. NaBF4 (32
mg, 2.1 mmol) in 0.25 mL of H2O was added, and the solution
was stirred at 0 °C for 30 min. The formed slightly yellow solid
was collected by filtration and triturated with 100 µL of H2O.
After filtration, the solid was washed with ether (200 µL) and
methanol (200 µL) and dried to give 25 (23 mg, 55%). This
material was not characterized spectroscopically and was
directly used for the following iodination reaction.
4-[4-Iod op h en oxy]t h ien o[2,3-c]p yr id in e-2-ca r b oxa m -
id e (3) fr om 25. To a solution of diazonium salt 25 (500 µg,
1.3 µmol) in DMSO (25 µL) was added a solution of sodium
iodide (100 µg, 0.65 µmol) in H2O (5 µL) at rt. The solution
was stirred at rt for 2 h, and was then separated by HPLC
(C-18, CH3CN/H2O containing 0.1% TFA) to give 3 (113 µg as
determined by analytical HPLC, 42%).
(
125I)-(4-Iod op h en oxy)th ien o[2,3-c]p yr id in e-2-ca r boxa -
m id e (5). A solution of diazonium salt 25 (500 µg, 1.3 µmol)
in DMSO (25 µL) was added to a 1.5 mL polypropylene vial
that contained 2 mCi of Na125I (Amersham Corporation, ∼15
µg) in H2O (20 µL). The solution was stirred at rt for 2 h and
was then separated by HPLC (C-18, CH3CN/H2O containing
0.1% TFA) to give 5 (15 µg as determined by analytical HPLC
using 3 as standard). The radiochemical yield was 35-40% in
several trials. The obtain material had a same retention time
with 3 on HPLC by coinjection and showed similar activity as
compared with 3 on inhibition of cell adhesion molecule
Ack n ow led gm en t. The authors thank the Abbott
Analytical Department for assistance in acquiring NMR,
IR, and mass spectra. We are grateful to Drs. Bruce
Surber and J oseph Meuth for technical assistance in
radiolabeling and gel filtration experiments.
J O016171J