Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-α production inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.06.037

A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-α production. We focused on the 1,1-substituted moiety (R¹ and R ²) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-α production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.

Full text of DOI:10.1016/j.bmcl.2010.06.037

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4479–4482
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological evaluation of 1,1,3-substituted urea
derivatives as potent TNF-a production inhibitors
*
Hiroshi Enomoto , Ayako Sawa, Hiroshi Suhara, Noriyoshi Yamamoto, Hiroyuki Inoue, Chikako Setoguchi,
Fumio Tsuji, Masahiro Okamoto, Yoshimasa Sasabuchi, Masato Horiuchi, Masakazu Ban
Nara Research & Development Center, Santen Pharmaceutical Co., Ltd, 8916-16 Takayama, Ikoma, Nara 630-0101, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against
lipopolysaccharide (LPS)-induced TNF-
a
production. We focused on the 1,1-substituted moiety (R1 and
Received 26 April 2010
Revised 2 June 2010
Accepted 7 June 2010
Available online 10 June 2010
R2) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-
a
production
by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-
pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this
moiety showed more potent inhibitory activities than SA13353.
Keywords:
Proinflammatory cytokine
Ó 2010 Elsevier Ltd. All rights reserved.
TNF-a
Anti-TNF-
SA13353
Urea
a agent
2,4-Dinitrobenzenesulfonamide
Manual synthesizer
Cytokines are intercellular messengers responsible for host de-
fense mechanisms such as inflammatory, immune and hematogenic
responses. Although many of them are transient, they are produced
by various cells and act as urgent response mediators in cases of
invasive interventions. Disruption of this biological defense mecha-
nism and continuous and excessive cytokine production contributes
to pathogenesis of inflammatory diseases. One of the representa-
afferent neurons.2a SA13353 is now being evaluated in early phase
II clinical trials in Japan.
SA13353 is a 1,1,3-substituted urea. In the present study, we fo-
cused on the 1,1-substituted moiety of urea (R1 and R2, Fig. 1) and
investigated the substituent effects of this moiety to find more
effective compounds than SA13353 on LPS-induced TNF-a produc-
tion by oral administration in rats.
tives of proinflammatory cytokines is tumor necrosis factor-
(TNF- ). The clinical success of TNF- blocking biologics indicates
that TNF- plays an important role in the pathogenesis of inflamma-
a
The synthesis of SA13353 is shown Scheme 1.3 The tri-substituted
urea, SA13353, was constructed from the primary amine 4 and the
secondary amine 3. 2-(1-Adamantyl)ethyl methanesulfonate (2)
was reacted with an excess of pentylamine to give the secondary
amine, 2-(1-adamantyl)-N-pentylethylamine (3). The primary amine,
3-(4-pyridyl)propylamine (4), was treated with 1,10-carbonyldiimid-
azole to give the intermediate, 1-[3-(4-pyridyl)propylaminocarbon-
a
a
a
tory disease including rheumatoid arthritis, psoriasis and inflamma-
tory bowel disease.1 We have developed an orally active and
low-molecular weight anti-TNF-
SA13353 (1a, Fig. 1).2,3 This compound significantly inhibited lipo-
polysaccharide (LPS)-induced TNF- production by oral administra-
a agent, the novel urea derivative
a
tion in rats, and strongly reduced the hind-paw swelling and joint
destruction associated with collagen-induced arthritis in rats.2a
The mechanism of action of SA13353 was initially not completely
understood because this compound was originally discovered from
R1
H
in vivo screening using LPS-induced TNF-
a production in rats.
N
N
N
R2
R3
However, we recently discovered that SA13353 is an orally active
transient receptor potential vanilloid 1 (TRPV1) agonist and inhibits
H
N
N
O
TNF-a production through the activation of capsaicin-sensitive
O
1
SA13353 (1a)
* Corresponding author. Tel.: +81 743 79 4527; fax: +81 743 79 4608.
E-mail address: hiroshi.enomoto@santen.co.jp (H. Enomoto).
Figure 1. Chemical structures of 1 and SA13353 (1a).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.037

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H. Enomoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4479–4482
a
b
O
O
S
NH
O
N
2
3
c
H
N
N
O
N
N
H
N
H2N
N
N
SA13353 (1a)
4
O
5
Scheme 1. Reagents and conditions: (a) pentylamine, K2CO3, NaI, EtOH, reflux, 17 h; (b) 1,10-carbonyldiimidazole, THF, rt, 20 min; (c) reflux, 1 h.
yl]imidazole (5). The intermediate 5 without isolation was refluxed
with the secondary amine 3 to provide SA13353. However, this syn-
thetic route was not suitable for the efficient conversion of the 1,1-
substituted moietyofurea becausetransformation of primary amines
to the corresponding secondary amines requires excessive primary
amines to avoid the formation of the undesired tertiary amines and
quaternary ammonium salts.
A simple and effective method for the preparation of secondary
amines was reported by Fukuyama et al.4a,b In this method, 2,4-dini-
trobenzenesulfonamides4b which is prepared from primary amines
and 2,4-dinitrobenzenesulfonyl chloride can be converted into N,N-
di-substituted sulfonamides by alkylation using the Mitsunobu
reaction5 or conventional methods in excellent yield. The deprotec-
tion of N,N-di-substituted sulfonamides is accomplished by treat-
ment with a variety of amines and thiol nucleophiles to give the
desired secondary amines. Furthermore, treatment of 2,4-dinitro-
benzenesulfonamides with either thioacids, hydroxamic acids,
dithioacids or dithiocarbamic acids gives directly the corresponding
amides, ureas, thioamides and thioureas, respectively.4c,4d As de-
scribed above, synthesis of secondary amines by using 2,4-dinitro-
benzenesulfonamides is applicable to preparation of not only
diverse combinatorial libraries for exploring new bioactive mole-
cules but also focused libraries in order to reveal structure–activity
relationships.
H
O
O
O
O
Cl
N
S
S
R1
NO2
NO2
a
NO2
NO2
6
7
O
O
R1 =
N
N
O
7a
7b
O
7d
7c
7e
7f
Scheme 2. Reagents and condition: (a) R1NH2, pyridine, CH2Cl2, ꢀ10 °C, 30 min.
We applied the synthesis of secondary amines using 2,4-dini-
trobenzenesulfonamides to the replacement at the 1,1-substituted
moiety (R1 and R2, Fig. 1) of the three substituted urea in order
the by-product, 2,4-dinitrophenylthioacetic acid, is easily removed
by washing with aqueous basic solution. At the third step, the cor-
responding secondary amines are refluxed with 1-[3-(4-pyri-
dyl)propylaminocarbonyl]imidazole (5)7 to give ureas although
the in situ synthesis of ureas has been reported.4d Therefore, the
degrees of swelling of the polymer-supported triphenylphosphine
were investigated in a few solvents in order to use the same sol-
vent for three-step reaction.
The degrees of swelling of the resin against organic solvents
were: tetrahydrofuran, chloroform > ethyl acetate P toluene. The
resin was so swelled in the tetrahydrofuran and chloroform that
the capacity of the reaction vessels of the Quest 210™ (10 mL)
was not enough and the mixing effect seemed to be decreased.
From the results of preliminary experiments, ethyl acetate was
used for the reaction solvent, and the reactions were conducted
under the conditions shown in Scheme 3.8 In each vessel at Side
A on a Quest 210™, a solution of sulfonamide, di-t-butyl azodicar-
boxylate (D-t-BAD), polymer-supported triphenylphosphine (PS-
PPh3) and alcohol (R2-OH which was listed in Table 1) in ethyl ace-
tate were mixed. After the reaction was complete as judged by TLC,
the reaction mixture was treated with triethylamine and mercap-
toacetic acid to give the secondary amine. Finally, reaction with
to elucidate the inhibitory activities on LPS-induced TNF-a produc-
tion by oral administration in rats. Since SA13353 consisted of
hydrophobic substituents at R1 and R2, we chose amines containing
hydrophilic substituents such as morpholine and alkoxy groups
and a hydrophobic substituent such as the cyclohexyl group.
Therefore, the corresponding 2,4-dinitrobenzenesulfonamides
(7a–7f) were prepared by the reaction of six amines and 2,4-dini-
trobenzenesulfonyl chloride (Scheme 2).6
As a one-pot synthesis is an effective method to rapidly synthe-
size derivatives, we designed the reaction conditions of the one-
pot, three step synthesis of urea derivatives containing alkylation
of 2,4-dinitrobenzenesulfonamides, deprotection and urea forma-
tion by using the manual synthesizer Quest 210™ (Argonaut Tech-
nologies). Our strategy for the one-pot synthesis is described
below. At the first step of the synthetic route, alkylation of the
2,4-dinitrobenzenesulfonamides is conducted under the Mitsun-
obu condition by using the polymer-supported triphenylphosphine
because the separation of target materials and the by-product, tri-
phenylphosphine oxide, seems to be complicated. At the second
step, mercaptoacetic acid is used for the deprotection of 2,4-dini-
trobenzenesulfonamides to give the secondary amines because

H. Enomoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4479–4482
4481
R1
N
or weak inhibitory activities in spite of containing hydrophobic
groups at R2. When the R1 moiety was a 2-cyclohexylethyl group,
the introduction of hydrophilic groups at the R2 moiety showed
weak or moderate inhibitory activities (1s–1u). However, introduc-
tion of a 2-cyclohexylethyl group made compound 1r a more potent
inhibitor relative to SA13353. Moreover, the compounds containing
the 3-cyclohexylpropyl group at R1 and hydrophobic groups at R2
displayed an improvement in inhibitory activities (1v–1x). Among
H
O
O
O
O
N
S
R1
S
R2
NO2
NO2
b
a
NO2
NO2
8
7a-f
them, compound 1w exhibited the most potent TNF-
a inhibitory
activity. As described above, hydrophobic substituents, such as a
cycloalkylalkyl group at R1 and R2 made a significant contribution
to the inhibition of TNF-a production. Structure–activity relation-
R1
R1
N
N
c
HN
R2
H
N
R2
ship (SAR) was examined further with 225 calculated molecular
properties with MOE 2009.10 (Chemical Computing Group Inc.,
Montreal, H3A 2R7 Canada) to give significant correlations between
logit-converted activity and PEOE_VSA_FPNEG (r = 0.78), RPC-
(r = 0.71), Q_VSA_HYD (r = 0.73), SlogP_VSA8 (r = 0.71) or SlogP_V-
SA5 (r = 0.71). The results suggested that more the inhibitory
activity was obtained with increasing hydrophobic or with decreas-
ing polar nature of the molecules.
O
9
1b-x
Scheme 3. Reagents and conditions: (a) D-t-BAD, PS-PPh3, R2OH, AcOEt, rt,
overnight; (b) HSCH2COOH, Et3N, rt, 3 h; (c) compound 5, 70 °C overnight.
compound 5 provided the tri-substituted urea (1b–1x, Table 1) in
13–51% yield after standard work-up and column chromatography.
Table 19 outlines the tri-substituted ureas synthesized by
employing the procedure as described above in Scheme 3. In the case
of the morpholinoethyl group at the R1 moiety, compounds 1b–1e
with hydrophobic groups introduced at the R2 moiety demonstrated
moderate inhibitory potency, while compounds 1f–1g containing
hydrophilic groups at the R2 moiety reduced the potency. Com-
pounds 1h–1k having a morpholinopropyl group at the R1 moiety
and hydrophobic groups at the R2 moiety showed also moderate
inhibitory potency and the activities were similar to compounds
1b–1e. In the case of the 3-methoxypropyl or 2-ethoxyethyl group
at the R1 moiety, compounds 1l–1q showed no inhibitory activities
As shown in Figure 2, compound 1w resulted in dose-depen-
dent inhibition of LPS-induced TNF-a production, and the doses
of 0.3, 1 and 3 mg/kg of 1w were statistically significant compared
to the vehicle. The ED50 value for 1w was 0.34 mg/kg. The positive
control, SA13353 (3 mg/kg, po), significantly inhibited LPS-induced
TNF-a production. Compound 1w exhibited more potent inhibitory
activity against TNF-
a production than that of SA13353 because
the ED50 value for SA13353 was 1.7 mg/kg.2a
In conclusion, further optimization of the 1,1-substituted moiety
of SA13353 was carried out. We demonstrated that the synthesis of
a series of urea derivatives possessing a 3-(4-pyridyl)propyl group at
the 3-substituted position (R3, Fig. 1) of urea could be accomplished
by a one-pot method using a manual synthesizer. Several com-
Table 1
Inhibitory effects of compounds 1a–1x on LPS-induced TNF-
a
production in rats
R1
H
N
N
N
R2
O
Compounds
Sulfonamides
R1
R2
Yield (%)
Inhibition %a
SA13353 (1a)
—
2-(1-Adamantyl)ethyl
2-Morpholinoethyl
2-Morpholinoethyl
2-Morpholinoethyl
2-Morpholinoethyl
2-Morpholinoethyl
2-Morpholinoethyl
3-Morpholinopropyl
3-Morpholinopropyl
3-Morpholinopropyl
3-Morpholinopropyl
3-Methoxypropyl
3-Methoxypropyl
3-Methoxypropyl
2-Ethoxyethyl
Pentyl
2-(1-Adamantyl)ethyl
2-Cyclohexylethyl
2-(Bicyclo[2.2.1]hept-2-yl)ethyl
Butyl
2-(7-Aza-bicyclo[2.2.1]hept-7-yl)ethyl
2-(4-Tetrahydropyranyl)ethyl
2-(1-Adamantyl)ethyl
2-Cyclohexylethyl
2-(Bicyclo[2.2.1]hept-2-yl)ethyl
Butyl
2-Cyclohexylethyl
2-(Bicyclo[2.2.1]hept-2-yl)ethyl
Butyl
2-Cyclohexylethyl
2-(Bicyclo[2.2.1]hept-2-yl)ethyl
Butyl
2-Cyclohexylethyl
2-(2-Methoxyethoxy)ethyl
2-(7-Aza-bicyclo[2.2.1]hept-7-yl)ethyl
2-(4-Tetrahydropyranyl)ethyl
Cyclohexylmethyl
—
72 8b
45
50
55
21
8
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
7a
7a
7a
7a
7a
7a
7b
7b
7b
7b
7c
7c
7c
7d
7d
7d
7e
7e
7e
7e
7f
45
33
36
36
13
14
27
25
13
17
13
39
22
41
38
34
18
27
51
28
40
46
18
NI
45
43
44
32
NI
10
21
4
2-Ethoxyethyl
2-Ethoxyethyl
NI
NI
2-Cyclohexylethyl
2-Cyclohexylethyl
2-Cyclohexylethyl
2-Cyclohexylethyl
3-Cyclohexylpropyl
3-Cyclohexylpropyl
3-Cyclohexylpropyl
91
14
48
38
71
92
87
1s
1t
1u
1v
1w
1x
7f
7f
2-Cyclohexylethyl
2-Cyclopentylethyl
a
Values are expressed as the concentration of TNF-
a in the serum of the compounds administration group divided by it of the non-administration
group. The compounds were administrated orally at 2 mg/kg. Values are means of five rats. (NI = not inhibit).
b
The value is expressed as mean SD of 13 tests.

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H. Enomoto et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4479–4482
6. The corresponding experimental procedure to Scheme
2
is shown below.
70000
60000
50000
40000
30000
20000
10000
0
Synthesis of compound 7a; a solution of 4-(2-aminoethyl)morpholine (5.0 ml,
38 mmol) in dichloromethane (20 ml) was slowly added to a solution of 2,4-
dinitrobenzenesulfonyl chloride (10 g, 38 mmol) and pyridine (4.0 ml, 49 mmol)
in dichloromethane (180 ml) dropwise at ꢀ10 °C over 15 min, and the mixture
was stirred at ꢀ10 °C for 30 min. After the reaction mixture was concentrated
under reduced pressure, water (300 ml) was added to the residue and the whole
solution was extracted with ethyl acetate (700 ml). The organic layer was dried
over anhydrous magnesium sulfate. After the solvent was evaporated under
reduced pressure, the resultant solid was washed with diethylether to give the
title compound (11 g) as a brown solid (yield 80 %). 1H NMR (CDCl3, 400 MHz) d
2.37 (t, J = 4.6 Hz, 4H), 2.51–2.54 (m, 2H), 3.19–3.22 (m, 2H), 3.66 (t, J = 4.6 Hz,
4H), 8.38 (d, J = 8.5 Hz, 1H), 8.56 (dd, J = 8.5, 2.2 Hz, 1H), 8.68 (d, J = 2.2 Hz, 1H);
IR (KBr, cmꢀ1) 3299, 2820, 1543, 1356, 1172; MS (ESI, Pos.) 361 [M+H]+, (ESI,
Neg.) 359 [MꢀH]ꢀ.
**
**
7. The experimental procedure of synthesis of compound 5 is shown below. 3-(4-
Pyridyl)propylamine (4, Scheme 1) (5.0 g, 37 mmol) was slowly added to a
solution of 1,10-carbonyldiimidazole (8.9 g, 55 mmol) in anhydrous
tetrahydrofuran (40 ml) dropwise at 0 °C over 30 min, and the mixture was
stirred at room temperature for 30 min. After the reaction mixture was
concentrated under reduced pressure, saturated ammonium chloride solution
(40 ml) was added to the residue and the mixture was extracted with
chloroform (120 ml). The organic layer was washed with brine and dried over
anhydrous sodium sulfate. After the solvent was evaporated under reduced
pressure, the resultant solid was washed with diethylether to give the title
compound (7.4 g) as a colorless solid (yield 88 %). 1H NMR (CDCl3, 400 MHz) d
1.96–2.03 (m, 2H), 2.71 (t, J = 7.4 Hz, 2H), 3.45 (td, J = 7.4, 6.0 Hz, 2H), 7.05 (dd,
J = 1.5, 1.0 Hz, 1H), 7.11–7.15 (m, 3H), 7.40 (dd, J = 1.5, 1.0 Hz, 1H), 8.15 (t,
J = 1.0 Hz, 1H), 8.47 (dd, J = 4.5, 1.5 Hz, 2H); IR (KBr, cmꢀ1) 3443, 3203, 3114,
1719, 1552, 1290; MS (ESI, Pos.) 231 [M+H]+, (ESI, Neg.) 229 [MꢀH]ꢀ.
###
**
Ve hicle
0.1
0.3
1
3
SA13353
3mg/kg
1w (mg/kg)
Figure 2. Effects of compound 1w on LPS-induced TNF-
Compound 1w or SA13353 was administered orally 30 min prior to LPS injection.
Serum was collected 1.5 h after LPS injection. Results are expressed as the mean SEM
of five rats per group. ### p <0.001, Student’s t-test versus vehicle group. p <0.01,
a
production in rats.
**
Dunnett’s t-test versus vehicle group.
8. The corresponding experimental procedure to Scheme
3 is shown below.
Synthesis of compound 1s; In a reaction vessel at Side A on a Quest 210™, the
solution mixture of N-(2-cyclohexylethyl)-2,4-dinitrobenzenesulfonamide (7e)
pounds, especially containing hydrophobic substituents such as 2-
cyclohexyethyl or 3-cyclohexylpropyl groups at R1 and R2, showed
(0.18 g,
triphenylphosphine polystyrene (1 g/1 mmol, 100–200 mesh) (1.0 g,
1.0 mmol), diethylene glycol methyl ether (89 l, 0.75 mmol) in ethyl acetate
0.5 mmol),
di-t-butyl
azodicarboxylate
(0.23 g,
1.0 mmol),
more potent inhibitory activities against LPS-induced TNF-
a pro-
l
duction than SA13353. Among them, compound 1w exhibited the
most potent inhibitory activity. Based on the results described
above, these novel three substituted urea derivatives may prove to
(4 ml) was mixed at room temperature for overnight. Then triethylamine
(0.21 ml, 1.5 mmol) and mercaptoacetic acid (0.10 ml, 1.5 mmol) were added to
the reaction mixture. The solution was mixed at room temperature for 3 h. 1-[3-
(4-Pyridyl)propylaminocarbonyl]imidazole (5) (0.12 g, 0.5 mmol) was added to
the mixture and mixed at 70 °C overnight. The reaction mixture was filtrated to
remove the triphenylphosphine polystyrene. At the same time, the filtrate was
transferred from the vessel at Side A to a vessel at Side B by using a Teflon tube,
and was extracted with 2 M hydrochloric acid (3 ml). After the organic layer was
removed, 4 M sodium hydroxide solution (5 ml) was added to the aqueous layer.
The solution was extracted with chloroform (3 ml ꢁ 2). The solution was dried
over anhydrous magnesium sulfate. After the solvent was evaporated under
reduced pressure, the residue was purified by silica gel column chromatography
(NH silica gel, 50% AcOEt in hexane) to give the title compound (53 mg) as a
slightly brown oil (yield 27 %). 1H NMR (CDCl3, 400 MHz) d 0.89–0.96 (m, 2H),
1.12–1.27 (m, 4H), 1.41–1.45 (m, 2H), 1.68–1.71 (m, 5H), 1.80–1.87 (m, 2H),
2.65 (t, J = 7.9 Hz, 2H), 3.19–3.28 (m, 4H), 3.31 (s, 3H), 3.34 (t, J = 4.8 Hz, 2H),
3.49–3.51 (m, 2H), 3.58–3.62 (m, 4H), 5.57 (s, 1H), 7.13 (dd, J = 4.5, 1.7 Hz, 2H),
8.48 (dd, J = 4.5, 1.7 Hz, 2H); IR (KBr, cmꢀ1) 3355, 2852, 1603, 1533, 1103; MS
(ESI, Pos.) 392 [M+H]+, (ESI, Neg.) 390 [MꢀH]ꢀ.
be potential inhibitors for treatment of diseases mediated by TNF-a.
References and notes
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Patent 7,098,226, 2006.
9. LPS-induced TNF-
were fasted overnight and intravenously injected with lipopolysaccharide (LPS,
Escherichia coli serotype 055:B5) dissolved in saline (100 g/kg). The compounds
were administered orally 30 min prior to LPS injection. Serum was collected 1.5 h
after LPS injection since TNF- levels were maximal at this time. The amounts of
TNF- in serum were measured by enzyme-linked immunosorbent assay (ELISA).
a production in rats; Male normal rats (Lewis, seven weeks old)
4. (a) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36, 6373; (b)
Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan, T. Tetrahedron Lett. 1997, 38,
5831; (c) Messeri, T.; Sternbach, D. D.; Tomkinson, N. C. O. Tetrahedron Lett.
1998, 39, 1669; (d) Messeri, T.; Sternbach, D. D.; Tomkinson, N. C. O. Tetrahedron
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l
a
a
5. Mitsunobu, O. Synthesis 1981, 1.