Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 1

Article abstract of DOI:10.1016/j.bmcl.2009.06.094

We investigated proinflammatory cytokine TNFα production inhibitors in order to develop novel anti-inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetrahydropyridine group at the β-position, showed potent inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS) induced TNFα production in human whole blood, IC₅₀ = 1.86 μM) and in vivo (inhibition of LPS induced TNFα production in mice, ID₅₀ = 5.98 mg/kg). Crown Copyright

Full text of DOI:10.1016/j.bmcl.2009.06.094

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4607–4610
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetrahydropyridine derivatives with inhibitory activity on the production
of proinflammatory cytokines: Part 1
b
c
d
d
Akira Nakao a, , Nobuyuki Ohkawa , Takayoshi Nagasaki , Takashi Kagari , Hiromi Doi ,
*
Takaichi Shimozato d, Shigeru Ushiyama e, Kazumasa Aoki a
a Medicinal Chemistry Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Research Department II, Daiichi Sankyo RD Associe Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Biological Research Laboratories III, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
e R&D Planning Department, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We investigated proinflammatory cytokine TNF
inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetra-
hydropyridine group at the b-position, showed potent inhibitory activity in vitro (inhibition of lipopoly-
a production inhibitors in order to develop novel anti-
Received 27 April 2009
Revised 20 June 2009
Accepted 24 June 2009
Available online 30 June 2009
saccharide (LPS) induced TNFa production in human whole blood, IC50 = 1.86 lM) and in vivo (inhibition
of LPS induced TNF production in mice, ID50 = 5.98 mg/kg).
a
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Keywords:
Anti-inflammatory agent
p38 MAP kinase
Proinflammatory cytokine
TNFa
Proinflammatory cytokines such as TNFa
and IL-1b,1 are associ-
N
ated with the onset of inflammatory diseases as well as several
autoimmune diseases,2 including rheumatoid arthritis (RA),3 toxic
shock syndrome, osteoarthritis, and inflammatory bowel dis-
ease.4,5 The recent success of anti-cytokine biological agents has
demonstrated clinical benefits in the treatment of inflammatory
diseases.6 However, due to the well known disadvantages common
to these protein-based therapies, such as high cost and subcutane-
ous or intravenous administration, orally active small molecules
that can effectively act as anti-cytokine agents would clearly be
of additional benefit to patients.5
O
N
S
N
H
SB203580
F
N
N
O
N
O
N
N
S
N
H
The p38 mitogen-activated protein kinase (MAPK) pathway has
been proven to play a central role in the regulation of the proin-
L-167307
N
SB210313
F
F
flammatory cytokines, TNF
a
and IL-1b.7 The discovery of a series
of triaryl-imidazoles as p38 inhibitors, as exemplified by
SB2035807,8 (Fig. 1), was seminal. The clinical proof of concept in
rheumatoid arthritis was achieved with VX-7459 and BIRB-795,10
which validated the MAP kinase pathway as a useful mechanism
for intervention in inflammatory disease.
Merck reported that L-167307 with an imidazole ring of
SB203580 modified to a pyrrole ring, showed more potent inhibi-
tory activity in vivo in a rat adjuvant-induced arthritis model (rat
R
R : basic polar groups
N
H
A
F
Figure 1.
AA model).11 These results suggested that the pyrrole ring could
be explored as a possible alternative to the imidazole scaffold.
* Corresponding author.
E-mail address: nakao.akira.g5@daiichisankyo.co.jp (A. Nakao).
0960-894X/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.094

4608
A. Nakao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4607–4610
which were expected to increase potency in vitro and in vivo.
N
Herein, we report our initial results, which have led to the identi-
fication of a unique and potent pyrrole-based inhibitor of the pro-
inflammatory cytokine TNFa, 17.
The preparation of 4, 6 and 11, each of which has a distal amino
alkyl group at the b-position of the pyridylpyrrole, and their deriv-
atives are summarized in Schemes 1–3. The key intermediate 3
Tol
CO2Et
2
b, c
a
SO2
NC
N
H
F
1
F
N
N
was provided from a-(p-toluene-sulfonyl)-4-fluorobenzylisonitrile
1,13 which was condensed with ethyl (2E)-3-pyridin-4-ylacrylate
to give pyrrole ester 2.14 Reduction of the ester by diisobutylalumi-
num hydride (DIBAL) gave a corresponding alcohol and subsequent
oxidation by MnO2 provided the aldehyde 3.
CHO
3
NH2
d, e
2HCl
4
N
N
H
H
F
F
The 3-methylaminopyrrole 4 was synthesized by treatment of 3
with O-methyl hydroxylamine hydro-chloride to give oxime, and
subsequent catalytic hydrogenation was carried out to afford 4
(Scheme 1). We next prepared the 3-(3-amino)propylpyrrole 6 by
a Horner–Emmons reaction of 3, and subsequent hydrogenation
of olefin and a nitrile reduction with lithium aluminum hydride
(LiAlH4). Several amide derivatives, 7a and 7b from 6, were pre-
pared by using corresponding anhydrides. Reduction of the N-
tert-butoxy carbonyl derivative 7b with LiAlH4 gave N-methyl
derivative 7c (Scheme 2).
The 3-(4-amino)butylpyrrole 11 was obtained as follows
(Scheme 3). In the same manner as described above, a Horner–Em-
mons reaction of 3 and subsequent reductions gave the alcohol 9.
The corresponding tosylate 10 was converted to a nitrile, which
was then reduced to obtain 11. As well, the tosylate 10 was con-
verted by displacement with a desired secondary amine to dial-
kylaminopropyl derivatives 12a–f. Catalytic hydrogenation of 12f
gave the piperazine derivative 12g.
Scheme 1. Reagents and conditions: (a) n-BuLi, LiBr, THF, À45 °C then ethyl (2E)-3-
pyridin-4-ylacrylate, 89%; (b) DIBAL, THF, 0 °C then rt, 99%; (c) MnO2, DMSO, 50 °C,
69%; (d) MeONH2ÁHCl, Et3N, MeOH, reflux, 77%; (e) H2, 10% Pd/C, MeOH/10% HCl
(1:1), 68%.
N
N
CN
b, c
NH2
a
3
N
H
N
H
5
6
F
F
N
NHR
d
N
7a R=COMe (99%)
7b R=CO2t-Bu (68%)
7c R=Me
7
c 31 %
H
F
We now describe the syntheses of pyridyl pyrrole derivatives
possessing a cyclicaminoalkyl group at the b-position of the pyr-
role, which are shown in Scheme 4. Removal of the ethoxycarbonyl
group of 2 by an acidic condition and the subsequent protection of
the pyrrole nitrogen by a triisopropylsilyl (TIPS) group15 followed
by selective b-bromination using N-bromo succinimide (NBS) gave
13. Introduction of a cyclicamino group to the b-position of the
pyrrole ring was carried out by bromine-lithium exchange fol-
lowed by 1,2-addition with 1-benzyl-4-piperidin-1-one. Debenzy-
lation and subsequent deprotection of the TIPS group on the
obtained tert-alcohol derivative 14 was performed to give 16.
Dehydroxylation of the tert-alcohol in 15 was carried out in excel-
lent yield by exposure to trifluoroacetic acid (TFA) concurrently
with deprotection of the TIPS group to give the tetrahydropyridine
derivative 17. Compound 17 was further transformed to the piper-
idine 18 and the pyridyl derivative 19.
Scheme 2. Reagents and conditions: (a) NaH, THF, 0 °C then diethylphosphono-
acetonitrile, rt, 68%; (b) H2, 10% Pd/C, MeOH, 81%; (c) LiAlH4, THF, 60 °C, 94%; (d)
acyl anhydride, THF, rt.
N
CO2Et
a, b
N
c
3
91 %
N
H
8
F
N
NH2
OR
f, c (43 %)
F
N
H
N
H
9 R=H
11
d
F
F
10 R=Ts
The designed and synthesized 2-(4-fluorophenyl)-3-(4-pyri-
dyl)pyrrole derivatives were evaluated in terms of their inhibitory
e
activities in LPS induced TNFa
production in human whole blood.16
12a R=thiomorpholine (83%)
12b R=morpholine (45%)
12c R=NMe2 (66 %)
N
SB203580 was used as a reference for a comparison of the in vitro
potencies of the new analogues (Table 1).
R
12d R=piperidine (63%)
12e R=4-Me-piperazine( 64%)
12f R=4-Bn-piperazine (57%)
12g R=piperazine
Compound 6, possessing a 3-aminopropyl group (n = 3) at the b-
position of the pyridylpyrrole, was proven to be more effective
than 4 (n = 1), having an aminomethyl group, 11 (n = 4) having a
4-aminobutyl group and SB-203580. Therefore, for inhibitory
activity the most suitable linker connecting the amino group and
pyrrole ring was the propyl group.17
b 98 %
N
H
12
Scheme 3. Reagents and conditions: (a) NaH, THF, 0 °C then triethyl phosphono-
acetate, rt, 60%, 73%; (b) H2, 10% Pd/C, EtOH, rt, 73%; (c) LiAlH4, THF, 60 °C; (d) Ts2O,
Et3N, CH2Cl2, rt, 53%; (e) secondary amine, K2CO3, acetonitrile, reflux; (f) KCN, DMF,
100 °C, 42%.
Then, we carried out a replacement of the amino moiety of 6
(Table 2).
The corresponding amido analogues (7a) resulted in a loss of
activity. Furthermore, in aminoalkyl analogues such as 7c and
12c, which possess methyl and dimethyl groups, respectively, the
increasing bulkiness of the substituent led to decreased potency.
We next examined various cyclicaminopropyl derivatives, such
as 12a, b and d–g, which showed no potencies. These results indi-
cate that the nitrogen atom of the aminopropyl substituent should
On the other hand, SmithKline reported that SB210313, an imidaz-
ole derivative, which substituted the basic polar group at the N-1-
position, showed an anti-inflammatory effect.12
On the basis of these reports, we investigated simple diaryl-pyr-
role derivatives possessing basic polar groups at b-position (A)

A. Nakao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4607–4610
4609
N
N
NBn
N
NH
N
Br
CO2Et
OH
OH
a, b, c
d
e
N
TIPS
13
N
TIPS
14
N
TIPS
15
N
H
2
98 %
F
F
F
F
f
79 %
N
NH
N
NH
g
15
N
OH
H
F
17
N
H
F
h
e 88 %
16
N
NH
N
N
N
N
H
19
H
F
F
18
Scheme 4. Reagents and conditions: (a) AcOH, H2SO4, H2O, 100 °C, 99%; (b) n-BuLi, THF, À78 °C then TIPSOTf, rt, quant.; (c) NBS, THF, À78 °C then rt, 43%; (d) n-BuLi, THF,
À78 °C then 1-benzylpiperidin-4-one, À78 °C then rt, 60%; (e) H2, 10% Pd/C, EtOH; (f) TBAF, THF, rt, 79%; (g) TFA, CH2Cl2, rt then TBAF, THF, rt, 93%; (h) 10% Pd/C, K2CO3,
xylene/MeOH (25: 2), 140 °C, 53%.
Table 1
Table 2
Activities of aminoalkyl derivatives
Activities of aminopropyl derivatives
R1
N
N
N
R2
(CH2)nNH2
N
H
N
H
F
F
Compd
N
IC50
(
lM)
Compd
R1
R2
IC50 (lM)
a
a
SB203580
4
6
11
6.40
>30
4.76
13.8
6
7a
7c
12a
12b
12c
12d
12e
12f
12g
H
COMe
Me
Thiomorphorine
Morphorine
Me
H
H
H
4.76
>30
14.7
>30
>30
22.4
28.4
>30
>30
>30
1
3
4
a
Inhibition of LPS induced TNFa production in human whole blood. N = 3–4.
Me
Piperidine
4-Methylpiperazine
4-Benzylpiperazine
Piperazine
be located at a length of 4 atoms from the b-position of the pyrrole,
should have basicity and should be sterically unhindered, in order
to show inhibitory activity.
a
Inhibition of LPS induced TNFa production in human whole blood. N = 3–4.
Based on the information above, we designed further pyrrole
derivatives possessing a cyclicaminoalkyl group at the b-position
(Table 3) to fix the lone-pair on the nitrogen atom.
The 4-hydroxypiperidine analogue 16 unexpectedly showed no
potency, while the tetrahydropyridine analogue 17 and its satu-
rated analogue, piperidine derivative 18, showed good inhibitory
From these results, the tetrahydropyridine group which has ba-
sic nitrogen and an olefin bond at the b-position of the pyrrole ring
showed good inhibitory activities in vitro and in vivo. The results of
the in vivo assay were reflected in those of the in vitro assay and
we found that the tetrahydropyridine group was the effective
group in this template.
activity (IC50 = 1.86 and 2.98 lM, respectively). The derivative 19,
In order to develop new anti-inflammatory agents, we synthe-
sized and evaluated derivatives possessing basic polar functional
groups at the b-position of the pyrrole ring. The analogue 17, which
has a tetrahydropyridine group, showed good inhibitory activity in
possessing pyridine, which is a more planar and more oxidized
substituent compared to tetrahydropyridine, showed however no
potency.
We evaluated the in vivo efficacy of these derivatives (16–19) in
LPS induced TNFa
production in mice.18 The analogue 17 showed
LPS induced TNFa production in vitro (human whole blood) and
in vivo (mice) compared to that of SB203580. Based on these re-
sults, we plan to further investigate pyridylpyrrole possessing
tetrahydropyridine groups.
the most effective activity (ID50 = 5.98 mg/kg), which was more
than four times as potent as that of SB203580. However, the ana-
logue 18 showed several times lower activity than 17.

4610
A. Nakao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4607–4610
8. (a) Adams, J. L.; Bohem, J. C.; Kassis, S.; Corycki, P. D.; Ewdd, E. F.; Hall, R.;
Table 3
Sorenson, M.; Ayrton, A.; Griswold, D. E.; Gallagfer, T. F. Bioorg. Med. Chem. Lett.
1998, 8, 3111; (b) Godl, K.; Wissing, J.; Kurtenbach, A.; Habenberger, P.; Blenke,
S.; Gutbrod, H.; Salassidis, K.; Stein-Gerlach, M.; Missio, A.; Cotten, M.; Daub, H.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15434.
In vitro and in vivo activities of piperidine, tetrahydropyridine, and pyridine
derivatives
N
9. Ferracioli, G. F. Curr. Opin. Anti-inflamm. Immunomod. Invest. Drugs 2000, 2, 74.
10. Dominguez, C.; Powers, D. A.; Tamayo, N. Curr. Opin. Drug Discovery Dev. 2005,
8, 421.
R
11. Stephen, E. D. L.; Denise, V.; Lily, A.; Linda, C.; Jayne, C.; Gist, C.; Amy, F.; Daniel,
F.; Betsy, F.; Candice, H.; William, H.; Coral, H.; Matthew, K.; Bing, L.; Sylvie, L.;
Malcolm, M.; Nathan, M.; Edward, A. O.; Chad, O.; Margaret, P.; Janey, P.; Anna,
R.; Yousif, S.; Kelley, S.; Rock, W.; Stephen, J. O. Bioorg. Med. Chem. Lett. 1998, 8,
2689.
12. Jeffrey, C. B.; Juanita, M. S.; Margaret, E. S.; Ravi, S. G.; Timothy, F. G.; Peter, L. S.;
Jeremy, B.; Alison, M. B.; Jeffrey, T. L.; John, C. L.; Leonard, M. H.; Dnald, E. G.;
John, J. B.; Marie, C. C.; Jerry, L. A. J. Med. Chem. 1996, 39, 3929.
13. Adams, J. L.; Garigipati, R. S.; Boehm, J. C. U.S. Patent 5,670,527, 1997.
14. Sisko, J.; Mellinger, M.; Sheldrake, P. W.; Baine, N. H. Tetrahedron Lett. 1996, 37,
8113.
15. Bray, B. L.; Mathies, P. H.; Naef, R.; Solas, D. R.; Tidwell, T. T. J. Org. Chem. 1990,
55, 6317.
16. Fresh blood was collected aseptically in the presence of heparin by
venipuncture from healthy adult volunteers. The subjects did not have any
apparent inflammatory conditions and had taken no drug for at least 7 days
prior to blood collection. Written informed consent was obtained from all
N
H
F
a
b
Compds
R
IC50
(l
M)
ID50 (mg/kg)
SB203580
6.40
4.76
27.9
NH2
NH
c
6
—
16
17
18
>30
1.86
2.98
>30
À2.7%d
5.98
OH
volunteers before the experiments. Blood aliquots of 988
either 2 L of a test compound solution or 2 L of DMSO, and with 10
1.0 mg/mL LPS (dissolved in PBS, final concentration: 10 g/mL) in Eppendorf
l
L were mixed with
NH
NH
N
l
l
l
L of
l
tubes. The compounds were dissolved and diluted to the appropriate
concentrations with DMSO. The compound solutions were prepared
immediately before use. The mixture was incubated at 37 °C for 6 h. Control
17.9
assays were carried out in additional tubes by mixing blood with 2
lL of DMSO
and 10 L of PBS instead of the test compounds and LPS (negative control),
l
respectively. After the incubation was finished, the mixture was immediately
chilled at 4 °C and centrifuged at 15,300g for 5 min, and the plasma was stored
at À20 °C until the assay. The levels of cytokines in the plasma were
determined by using commercially available immunoassay kits. The percent
inhibition of cytokine production by the compounds was calculated by the
following equation: Percent inhibition of cytokine production =
{1 À (concentration of cytokine in the reaction mixture of test
compound À concentration of cytokine in the reaction mixture of negative
control)/(concentration of cytokine in the reaction mixture of
control À concentration of cytokine in the reaction mixture of the negative
control)} Â 100.
19
24.5%e
a
Inhibition of LPS induced TNF
Inhibition of LPS induced TNF
Not tested.
a
a
production in human whole blood. N = 3–4.
production in mice. N = 5.
b
c
d
e
% Inhibition at 20 mg/kg.
% Inhibition at 50 mg/kg.
17. We tried to synthesize an analogue possessing an aminoethyl group (n = 2), but
it was too unstable a compound to isolate.
Supplementary data
18. TNF
a production was induced in mice by the procedure of Griswold et al. (J.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.094.
Immunol. Methods 1996, 195, 1–5.) with a slight modification. In brief, mice
were deprived of food overnight, but not water. The next day, these mice were
orally administered with test compounds suspended in 0.5% CMC aqueous
solution at 10 mL/kg except normal and control mice that were administered
with 0.5% CMC aqueous solution. Thirty minutes later, LPS solution dissolved in
physiological saline was intravenously injected at 0.45-mg/10 mL/kg except for
the normal mice, which were injected with physiological saline at 10 mL/kg.
One hour later, blood samples were taken from the mice from the vena cava
inferior into a syringe containing heparin sodium. After centrifugation of the
blood samples at 13,230g for 3 min at 4 °C, plasma samples were taken
immediately and frozen at À20 °C until measurement of the concentration of
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McLaughlin, M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J. R.; Adams, J.
L.; Young, P. R. Nature 1994, 372, 739.
TNF
were measured with commercially available ELISA kits. The percent inhibition
of TNF production was obtained by the following equation: Percent
inhibition = {1 À (concentration of TNF in plasma sample of mice
administrated test compounds À mean concentration of TNF in the plasma
samples of normal mice)/(concentration of TNF in the plasma samples in
in the plasma samples of normal
a in the plasma sample. The concentrations of TNFa in the plasma samples
a
a
a
a
control mice À mean concentration of TNF
a
mice)} Â 100.