Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-5-acetylenyl-3- thiophenecarboxamides

Article abstract of DOI:10.1016/j.bmcl.2005.03.090

A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3- thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of th

Full text of DOI:10.1016/j.bmcl.2005.03.090

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2870–2875
Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-
5-acetylenyl-3-thiophenecarboxamides
Dominique Bonafoux,a,* Sheri Bonar,b Lori Christine,b Michael Clare,a Ann Donnelly,b
Julia Guzova,b Nandini Kishore,b Patrick Lennon,a Adam Libby,a Sumathy Mathialagan,b
William McGhee,a Sharon Rouw,b Cindy Sommers,b Michael Tollefson,a
Catherine Tripp,b,ꢀ Richard Weier,a Serge Wolfsona and Yao Minb
aDepartment of Medicinal Chemistry, Pfizer Inc., 700 Chesterfield Parkway West, St Louis, MO 63017, USA
bDepartment of Biological Sciences, Pfizer Inc., 700 Chesterfield Parkway West, St Louis, MO 63017, USA
Received 4 January 2005; revised 21 March 2005; accepted 23 March 2005
Available online 19 April 2005
Abstract—A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for
the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of
the production of inflammatory cytokine IL-8 in IL-1b stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating
their potential usefulness as NF-jB regulators.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Nuclear factor kappa B (NF-jB) is a transcriptional fac-
tor involved in inducing autoimmune and inflammatory
responses1 as well as in regulating apoptosis.2 The IjB
inhibitory proteins sequester NF-jB in the cytoplasm
by masking its Rel homology domain. Cell activation
by proinflammatory cytokines, such as interleukin-1
(IL-1) and tumor necrosis factor alpha (TNF-a), triggers
the activation of IKK-2 that phosphorylates the IjB
proteins, resulting in their degradation by ubiquitin-
mediated proteolysis and thus in the release of NF-jB
in the cytoplasm.3 As a result, control of NF-jB release,
through IKK-2 inhibition, could provide an effective
treatment of inflammatory diseases.
attractive selectivity profile with little or no crossover
to other kinases.16,17 Since the modification of SC-514,
involving the replacement of the 3-aminothiophene-2-
carboxamide with a 2-[(aminocarbonyl)amino]-thio-
phene-3-carboxamide has been shown by others to
dramatically improve the potency on IKK-2,17 we incor-
porated this modification in our design along with the
introduction of an acetylene linker between the two
thiophene rings affording 2-[(aminocarbonyl)amino]-
3-thiophenecarboxamide 1a, a novel lead with a
0.420 lM IC50 on IKK-2 (Fig. 1).
The SAR around new template 1a was investigated by
varying the alkyne at position 5 of the 2-[(aminocar-
bonyl)amino]-thiophene-3-carboxamide. The resulting
2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarb-
oxamides18 were evaluated in vitro on recombinant
human IKK-219,20 and showed moderate inhibition
Even though a variety of structurally distinct molecules
inhibit IKK-2,4–8 small thiophenecarboxamide based
inhibitors have attracted considerable interest in the past
few years.9–15 One of our early hits, the 4-amino-2,30-
bithiophene-5-carboxamide (SC-514), is a poor inhibitor
of IKK-2 with an IC50 of 11.2 lM, but displays a very
Keywords: 2-[(Aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarb-
oxamides; IKK-2 inhibition; NF-jB regulation.
*
Corresponding author. Tel.: +1 617 679 2239; fax: +1 617 579
3635; e-mail: dominique.bonafoux@biogenidec.com
ꢀ Present address: Abbott Bioresearch center, 100 Research drive,
Worcester, MA 01605, USA.
Figure 1.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.090

D. Bonafoux et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2870–2875
2871
(IC50 (IKK-2) P 0.129 lM). Selected inhibitors with an
IC50 (IKK-2) < 0.5 lM were evaluated in a cellular
based assay using IL-1b stimulated rheumatoid arthri-
tis-derived synovial fibroblasts (RASF),16 and showed
significant inhibition of the production of inflammatory
cytokine IL-8, demonstrating the utility of this template
for NF-jB regulation.
obtained in poor yield (6%) from the Sonogashira cou-
pling of 2-[(aminocarbonyl)amino]-5-bromo-3-thiophene-
carboxamide (2) with 3-ethynylthiophene, the
coupling of 2 with the 5-tert-butyldimethylsilyloxy-
methyl-3-ethynylthiophenes 5, 8, and 9 and with 4-ethy-
nylthiophene-2-carbonitrile 12, afforded 10a–c, and 1e in
good to high yields (10a: R = H, 76%, 10b: R = Et, 49%,
10c: R = i-Pr, 71%, 1e, 37%). Subsequent deprotection
of 10 with TBAF afforded the 5-hydroxymethyl-, 5-(3-
hydroxypropyl)-, and 5-(3-hydroxy-2-methylpropyl)-
analogs 1b,c, and 1d.
The 2-[(aminocarbonyl)amino]-5-bromo-3-thiophenecarb-
oxamide (2)15 proved to be a poor substrate for
Sonogashira couplings when using PdCl2(PhCN)2 or
PdCl2 in combination with 2-(di-t-butylphosphino)-
biphenyl or PPh3 as the catalytic system, leading
almost exclusively to the 2-[(aminocarbonyl)amino]-3-
thiophenecarboxamide and bisacetylenes. After screen-
ing a variety of catalysts, we found that, in most
instances, these side reactions could be minimized when
using the air stable premade or in situ generated
dichloropalladium bis(di-isopropylphosphino)ferrocene
(PdCl2[(i-Pr2PC5H4)2Fe]) as the catalyst, in the presence
of CuI and i-Pr2NEt in a 1/1 mixture of EtOH and
dimethylacetamide (DMA) at 65–75 ꢁC. Using these stan-
dard conditions (STD), the 2-[(aminocarbonyl)amino]-
5-acetylenyl-3-thiophenecarboxamides were obtained
with yields up to 80%.
The 2-[(aminocarbonyl)amino]-5-phenylethynyl-3-thio-
phenecarboxamides 1f–h were synthesized at the begin-
ning of our study, prior to the optimization of the
catalytic system, by coupling 2 with commercially
available acetylenes. Thus, as illustrated in Table 1,
the couplings were very low yielding. Even when
PdCl2[(i-Pr2PC5H4)2Fe] was used as the catalyst, the
couplings were inefficient unless a 1/1 mixture of EtOH
and DMA was used as the solvent (Table 1, 1i). The
2-[(aminocarbonyl)amino]-5-[(3-hydroxyphenyl)ethynyl]-
thiophene-3-carboxamide (1j), was obtained in 25%
overall yield from the 3-hydroxyphenylacetylene as
shown in Scheme 2.
As illustrated in Scheme 1, functionalized analogs of 1a
were synthesized using the 4-bromo-2-thiophencarbox-
aldehyde (3) as the key intermediate. In contrast to 1a,
The 2-[(aminocarbonyl)amino]-5-(cyclopropylethynyl)-
thiophene-3-carboxamide (1k) was synthesized in
26% yield from 2 and cyclopropylacetylene under the
Scheme 1. Reagents and conditions: (a) NaBH4; (b) TBDMSCl, imidazole, DMF; (c) TMSCCH, Pd(PPh3)4, CuI, Et3N, THF, 70%; (d) NaOH (1 M,
H2O), MeOH, rt, 2 h; (e) 2, PdCl2 [(i-Pr2PC5H4)2Fe], CuI, i-Pr2NEt, EtOH/DMA, 65–75 ꢁC, 37–76%; (f) TMSCCH, PdCl2(PhCN)2, PPh3, CuI, i-
Pr2NH, benzene, 97%; (g) RMgBr, THF; (h) TBAF, THF, rt, 32–58%; (i) (1) NH2OHÆHCl, Et3N, (2) phthalic anhydride, 99%.

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D. Bonafoux et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2870–2875
Table 1. Yields of 2-[(aminocarbonyl)amino]-5-phenylenyl-3-thiophenecarboxamides (1f–i)
R
Conditions
Yield (%)
1f
1g
1h
1i
3-F–C6H4
2-F–C6H4
2-Cl–C6H4
2-Me–C6H4
PdCl2(PhCN)2, CuI, t-Bu2P(biphenyl), i-Pr2NH, EtOH
PdCl2[(i-Pr2PC5H4)2Fe], CuI, i-Pr2NEt, dioxane
PdCl2[(i-Pr2PC5H4)2Fe], CuI, i-Pr2NEt, EtOH
STD
16
8.6
14.8
34
Scheme 2. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt, 86%; (b) 2, PdCl2[(i-Pr2PC5H4)2Fe], CuI, i-Pr2NEt, EtOH/DMA, 75 ꢁC,
70%; (c) TBAF, THF, rt, 42%.
standard conditions using EtOH as the solvent. The
2-[(aminocarbonyl)amino]-5-[(3-aminophenyl)ethynyl]-
thiophene-3-carboxamide (1l) was obtained in 17% yield
from the coupling of 2 with 3-ethynylaniline and was
subsequently reacted with a variety of acids to afford
the corresponding amides 1m–t (Scheme 3). In addition,
the 2-[(aminocarbonyl)amino]-5-({3-[(methylsulfonyl)ami-
no]-phenyl}ethynyl)thiophene-3-carboxamide (1u) was
synthesized with an 85% yield by reacting 1l with meth-
anesulfonyl chloride in the presence of pyridine at 0 ꢁC
in THF.
ence of the inhibitor, to determine the efficiency of the
inhibition of cytokine IL-8 production, which was mea-
sured by ELISA (IC50 (RASF)).16,21 In a second experi-
ment, after removing the media used for the ELISA,
they were treated with the Alamar Blue Reagent, a dye
commonly used as indicator of cell death. The viable
cells cause a change in the oxidation state of the dye
from an oxidized form (blue) to a reduced fluorescent
form (red). The fluorescence was measured with a Victor
multilable counter to evaluate the LC50 (AB) and thus
the potential cell toxicity of our inhibitors.22
Our inhibitors were evaluated in vitro using a high
throughput screen on human recombinant IKK-2
(IC50 (IKK-2)).19,20 Selected inhibitors with IC50
(IKK-2) < 0.5 lM were then tested in rheumatoid
arthritis-derived synovial fibroblasts (RASF) that were
submitted to two sequential experiments. In the first
experiment, they were stimulated with IL-1b, in the pres-
Most of the inhibitors synthesized in this study (16/21)
showed modest activity with IC50 values ranging from
1 to 0.195 lM. As shown in Table 2, structural modifi-
cation of the alkyne substituent did not, in most
instances, significantly impact the potency of the
2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarb-
oxamides and only 1b,e,f,j, and 1k showed improved
Scheme 3. Reagents and conditions: (a) 2, PdCl2(PhCN)2, (i-Pr2)2Fc, CuI, i-Pr2NEt, EtOH/DMA, 75 ꢁC, 17%; (b) RCO2H, Me2EtN, HBTU, DMF,
rt, 62–93%.

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2873
Table 2. Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides 1
R
IC50 (IKK-2) (lM)a
R
IC50 (IKK-2) (lM)a
1a
1b
0.420
1d
1e
0.563
0.333
0.195
0.465
1c
1f
1g
1h
1i
3-F–C6H4
2-F–C6H4
2-Cl–C6H4
2-Me–C6H4
0.331
0.62
1.01
1j
3-HO–C6H4
Cyclopropyl
3-H2N–C6H4
0.303
0.273
0.709
1k
1l
0.716
1m
>20
1r
3-CF3CONHC6H4
0.980
1n
0.557
1s
1.88
1o
1.85
1t
4.4
1p
1q
3-PhCONHC6H4
3-MeCONHC6H4
0.704
0.964
1u
3-MeSO2NH–C6H4
0.454
a Averaged IC50 (IKK-2) from n = 3.
activity compared to early lead 1a. The most noticeable
effect resulted from the introduction of a hydroxymethyl
group at position 5 of the thiophen-3-yl substituent (1b),
which led to a 2-fold improvement in potency compared
to 1a. In contrast, the introduction of bulkier 5-(3-
hydroxypropyl)- or 5-(3-hydroxy-2-methylpropyl)-group
(1c,d) led to a gradual decrease in potency. It is notewor-
thy that a cyano group was well tolerated at position 5
of the thiophen-3-yl (1e) with an IC50 of 0.333 lM.
served with carboxamides at position 3 of the phenyl
ring regardless of their size (1n–t). It is noteworthy that,
while carboxamides 1n–t exhibited IC50 values ranging
from 0.55 to 4.4 lM, the 2-pyridinyl analog (1m)
showed no activity at all. This lack of activity could be
due to the formation of an intramolecular hydrogen
bond involving the nitrogen of the 2-pyridinyl group
and the NH of the carboxamide forcing 1m to adopt
an unfavorable conformation.
2-[(Aminocarbonyl)amino]-5-phenylenyl-3-thiophenecarb-
oxamides 1f and 1j, respectively, bearing a 3-fluoro- and
3-hydroxy-substituent on the phenyl ring led to a slight
improvement in potency compared to 1a. In contrast
substitution at position 2 of the phenyl ring led to
modest inhibitors (1g–1i). While fluoro- and hydroxyl-
substituents are well tolerated, the introduction of an
amino- group at position 3 of the phenyl ring (1l) led
to a loss of potency. Decreased potency was also ob-
Preliminary results indicate that the replacement of the
arylethynyl- and thienylethynyl-substituents with an
alkylethynyl-substituent is tolerated as illustrated with
the 2-[(aminocarbonyl)amino]-5-cyclopropylethynyl-3-
thiophenecarboxamide (1k) with an IC50 of 0.273 lM.
In spite of their often modest potency on IKK-2, seven
selected 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thio-
phenecarboxamides were tested in RASF in order to

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D. Bonafoux et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2870–2875
Table 3. Inhibition of IL-8 production and cellular toxicity of 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides 1
R
IC50 (IKK-2) (lM)a
IC50 (RASF) (lM)b
IC50 (RASF)/IC50 (IKK-2)
LC50 (AB) (lM)
1a
1b
0.420
1.18
2.8 ·
>30
0.195
0.333
1.13
5.8 ·
2.5 ·
>30
>30
1e
0.832
1f
1j
3-F–C6H4
3-HO–C6H4
0.331
0.303
0.273
0.454
1.65
0.576
3.06
5 ·
>30
2.72
>30
>30
1.9 ·
11 ·
28 ·
1k
1u
Cyclopropyl
3-MeSO2NH–C6H4
12.9
a Averaged IC50 (IKK-2) from n = 3.
b Averaged IC50 (RASF) from n = 2.
3. Senftleben, U.; Karin, M. Crit. Care Med. 2002, 30, S18–
S26.
evaluate whether they would possess cellular activity. As
illustrated in Table 3, all the compounds that were tested
were found not to be lethal to cells with LC50s >30 lM
with the exception of the 3-hydroxyphenylethynyl ana-
log 1j. In contrast to the 3-fluorophenylethynyl-analog
1f that showed efficacy in the RASF assay (IC50
(RASF) = 1.65 lM) with no detectable cell damage
(LC50 (AB) > 30lM), 1j caused severe cell toxicity
(LC50 (AB) = 2.72). All the compounds tested showed
significant inhibition of IL-8 production except for the
cyclopropylethynyl-analog 1k and the 3-methylsulfon-
amide 1u that both exhibited over a 10-fold difference
between their IC50 on IKK-2 and their cellular activity.
Out of the four analogs, 1a,b,e, and 1f, with significant
inhibition of IL-8 production and no cell toxicity, the
cyanothiophenylethynyl-analog 1e stood out with the
strongest inhibition of IL-8 production and thus demon-
strated the potential usefulness of 2-[(aminocarb-
onyl)amino]-5-thiophenethynyl-3-thiophenecarboxamides
for the regulation of NF-jB release through IKK-2
inhibition.
4. Murata, T.; Shimada, M.; Sakakibara, S.; Yoshino, T.;
Masuda, T.; Shintani, T.; Sato, H.; Koriyama, Y.;
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In spite of their often modest activity on IKK-2, we were
able to identify several novel 2-[(aminocarbonyl)amino]-
5-acetylenyl-3-thiophenecarboxamides 1a,b,e, and 1f
that demonstrated reasonable inhibition of IKK-2 and
significant reduction of IL-8 production in IL-1b stimu-
lated RASF without causing noticeable cell toxicity. The
comparable levels of inhibition of IKK-2 and IL-8 pro-
duction by these compounds, especially 1e,23 shows the
potential usefulness of 2-[(aminocarbonyl)amino]-5-
acetylenyl-3-thiophenecarboxamides as regulators of
NF-jB.
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References and notes
16. Kishore, N.; Sommers, C.; Marthialagan, S.; Guzova, J.;
Yao, M.; Hauser, S.; Huynh, K.; Mielke, C.; Albee, L.;
Weier, R.; Graneto, M.; Hanau, C.; Perry, T.; Tripp, C. S.
J. Biol. Chem. 2003, 278, 32861–32871.
1. (a) Roshak, A. K.; Callahan, J. F.; Blake, S. M. Curr.
Opin. Pharmacol. 2002, 2, 316–321; (b) Tak, P. P.;
Firestein, G. S. J. Clin. Invest. 2001, 107, 7–11.
2. (a) Beg, A. A.; Baltimore, D. Science 1996, 274, 782–784; (b)
Foo, S. Y.; Nolan, G. P. Trends Genet. 1999, 15, 229–235.
17. Recently a team at AstraZeneca reported independent
data on SC-514 (IC50 = 1.6 lM on IKK-2), see Ref. 11.

D. Bonafoux et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2870–2875
2875
18. While this manuscript was in preparation, a patent was
issued to Glaxosmithkline, describing various 2-[(amino-
carbonyl)amino]-3-thiophenecarboxamides in which they
report the synthesis of 2-[(aminocarbonyl)amino]-5-acetyl-
enyl-3-thiophenecarboxamides (1) where R = Ph, 4-F–
C6H4, 4-Et–C6H4, 4-MeO–C6H4, 4-Cl–C6H4, 4-CF3–
C6H4, and 3-CF3–C6H4, see Ref. 12.
21. Cellular assay: Adherent RASF cells were isolated via
enzymatic digestions from primary synovial tissue isolated
after knee synovectomy and were cultured in DMEM
High glucose, containing 15% defined bovine serum
(Hyclone) and 50 lg/mL gentamicin. For cytokine release
determination, 1.5 · 104 cells/well were plated in a 96-well
plate and allowed to attach overnight. The growth media
were replaced with fresh DMEM containing 1% serum,
and the cells were pre-treated with increasing doses (seven-
point IC50 curve) of inhibitors in 0.2% Me2SO media for
1 h prior to an overnight stimulation with 1 ng/mL IL-1b.
The supernatant were collected and the amount of
cytokine (IL-8) secreted into the culture media was
measured by ELISA.
22. Alamar Blue Assay: After removing the supernatant
from the cellular assay, the Alamar Blue Reagent (10 lL/
well) was added to all the wells. The mixtures were
incubated for 2.5 h at 37 ꢁC in the presence of 5% CO2
and the fluorescence was read with a Victor multilable
counter.
23. Compound 1e was synthesized by adding PdCl2 [((i-
Pr)2P)2Fc] (0.043 g) to a degassed mixture of 2 (0.54 g,
2 mmol), i-Pr2NEt (0.8 g, 1.09 mL, 6.2 mmol), 4-ethynyl-
thiophene-2-carbonitrile (12) (0.5 g, 3.7 mmol), and CuI
(0.109 g, 0.57 mmol) in 30 mL DMA/EtOH (1/1). The
reaction mixture was stirred overnight at 65 ꢁC. After
cooling to room temperature, the crude reaction mixture
was filtered through Celiteꢂ and the filtrate concentrated
before being partitioned between brine and EtOAc. The
organic phase was dried over MgSO4. Purification by
reverse phase preparative HPLC afforded the title com-
pound as a pale yellow solid, yield 37%. 1H NMR
(400 MHz, DMSO-d6): d 7.07 (br s, 2H), 7.36 (br s, 1H),
7.62 (s, 1H), 7.69 (br s, 1H), 8.08 (s, 1H), 8.25 (s, 1H),
11.13 (s, 1H). 13C NMR (100 MHz, DMSO-d6): d 68.3,
69.4, 92.6, 93.6, 96.6, 98.0, 106.3, 113.8, 121.2, 124.8,
134.8, 138.8, 150.4. HRMS calcd for C13H9N4O2S2
317.0161. Found 317.0196.
19. Kishore, N.; Huynh, Q. K.; Mathialagan, S.; Hall, T.;
Rouw, R.; Creely, D.; Lange, G.; Caroll, J.; Reitz, B.;
Donnelly, A.; Boddupalli, H.; Combs, R. G.; Kretzmer,
K.; Tripp, C. S. J. Biol. Chem. 2002, 277, 13840–13847.
20. Enzyme assay: Kinase activity was measured using a
biotinylated IjBa peptide (Gly-Leu-Lys-Lys-Glu-Arg-Leu-
Leu-Asp-Asp-Arg-His-Asp-Ser32-Gly-Leu-Asp-Ser36-Met-
Lys-Asp-Glu-Glu). The standard reaction mixture for
rhIKK2 assay contained 5 lM biotinylated IjBa peptide,
1 lM (c-33P)ATP (about 1 · 105 CPM), 1 mM DTT,
2 mM MgCl2, 2 mM MnCl2, 10 mM NaF, 25 mM HEPES
buffer, pH 7.6, and increasing doses of inhibitors (seven-
point IC50 curve) and an enzyme solution of 0.2 lg in a
final volume of 50 lL reaction. After incubation at 25 ꢁC
for 30 min, the reaction was stopped by the addition of
150 lL of AG1XB resin in 900 mM sodium formate buffer,
pH 3 (the resin is in slurry of one volume of resin and 3 vol
of sodium formate buffer). The resin was allowed to settle
and 150 lL of supernatant was transferred to a top count
plate followed by the addition of 150 lL of Microscint 40,
mixed well, and incorporation of (c-33P)ATP was mea-
sured using a Top count NXT (Packard Instrument Co).
IC50 validation was done using 96-well streptavidin
Promega plate, and a vacuum system as described previ-
ously (Ref. 19). The reaction mixture (25 lL) described
above was added to a 96-well Promega plate. Each well was
then washed successively with 800 lL of 2 M NaCl, 1.2 mL
of NaCl containing 1% H3PO4, 400 lL of H2O, and 200 lL
of 95% ethanol. The plate was allowed to dry in the hood
for about an hour and then 25 lL of Microscint 20 was
added and the plate was counted using Top count.