1H-imidazo[4,5-c]quinoline derivatives as novel potent TNF-α suppressors: Synthesis and structure-activity relationship of 1-, 2-and 4-substituted 1H-imidazo[4,5-c]quinolines or 1H-imidazo[4,5-c]pyridines

Article abstract of DOI:10.1016/S0968-0896(03)00178-0

Structural modification of imiquimod (1), which is known as an interferon-α (IFN-α) inducer, for the aim of finding a novel and small-molecule tumor necrosis factor-α (TNF-α) suppressor and structure-activity relationship (SAR) are described. Structural m

Full text of DOI:10.1016/S0968-0896(03)00178-0

Bioorganic & Medicinal Chemistry 11 (2003) 2541–2550
1H-Imidazo[4,5-c]quinoline Derivatives as Novel Potent TNF-ꢀ
Suppressors: Synthesis and Structure–Activity Relationship of
1-, 2-and 4-Substituted 1H-imidazo[4,5-c]quinolines
or 1H-imidazo[4,5-c]pyridines
Tomoyuki Izumi,* Jun Sakaguchi, Makoto Takeshita, Harumi Tawara, Ken-ichi Kato,
Hitomi Dose, Tomomi Tsujino, Yoshinari Watanabe and Hideo Kato
Research Division, R&D Headquarters, Hokuriku Seiyaku Co., Ltd., 37-1-1, Inokuchi, Katsuyama, Fukui, 911-8555, Japan
Received 16 January 2003; accepted 10 March 2003
Abstract—Structural modification of imiquimod (1), which is known as an interferon-a (IFN-a) inducer, for the aim of finding a
novel and small-molecule tumor necrosis factor-a (TNF-a) suppressor and structure–activity relationship (SAR) are described.
Structural modification of a imiquimod analogue, 4-amino-1-[2-(1-benzyl-4-piperidyl)ethyl-1H-imidazo[4,5-c]quinoline (2), which
had moderate TNF-a suppressing activity without IFN-a inducing activity, led to a finding of 4-chloro-2-phenyl-1-[2-(4-piper-
idyl)ethyl]-1H-imidazo[4,5-c]quinoline (10) with potent TNF-a suppressing activity. The relation between conformational direction
of 2-(4-piperidyl)ethyl group at position 1 and TNF-a suppressing activity is also demonstrated by NMR.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
such as rheumatoid arthritis (RA), Crohn’s disease
Immunostimulants have a rationale for use in primary
or acquired immunodeficiency. In particular, interferon
(IFN) inducers are examples of this approach. Imiqui-
mod (1) has been known as a low molecular weight
IFN-a inducer, and was approved by the US Food and
Drug Administration (FDA) in 1997 for the topical
treatment of external genital warts (Fig. 1).1 The drug 1
is recognized by antigen presenting cells including
monocytes, macrophages, B-cells and dendritic cells and
induces these cells to produce cytokines including IFN-
a and tumor necrosis factor-a (TNF-a) and others.1 For
the cytokine inducer like imiquimod, however, the
selectivity against a kind of proinflammatory cytokine
such as TNF-a is sometimes critical issue. On the other
hand, TNF-a was defined as an endotoxin-induced
serum factor, which produced necrosis of tumors both
in vitro and in vivo, and identified as a proin-
flammatory cytokine produced in response to infection
and other cellular stresses.2 Overproduction of TNF-a
can lead to the aggravation of the immune diseases
(CD) and endotoxic shock.2 At present, infliximab
(RemicadeTM), a chimeric monoclonal anti-TNF-a
antibody, and etanercept (EnbrelTM), a recombinant
human soluble TNF-a p75 receptor fusion protein, have
been used for treatment of RA and it has been proved
that anti-TNF-a therapy was effective against RA.3
Therefore, low molecular weight TNF-a suppressors
seemed to be promising drugs for treatment of RA. By
the way, the structural resemblance between agonist and
antagonist was well known, for instance, the theory of
Ariens,4 antimetabolites,5 conversion of muscarinic or
g-aminobutylic acid (GABA) receptor agonists into
antagonists.6 From this point of view, we coun-
terscreened analogues without IFN-a inducing activity
of 1 for lipopolysaccharide (LPS)-stimulated TNF-a
production in human peripheral blood mononuclear
cells (PBMCs) and consequently found that 4-amino-1-
[2-(1-benzyl-4-piperidyl)ethyl]-1H-imidazo[4,5-c]quino-
line (2) possessed weak suppressing effect on the TNF-a
production (IC50=4810 nM). This paper describes fur-
ther structural modification of 2 for lead-finding of a
novel and low molecular weight TNF-a suppressor and
their structure–activity relationship (SAR) on TNF-a
suppressing activity.
*Corresponding author. Tel.: +81-779-88-8012; fax: +81-779-88-
8024; e-mail: tomoyuki.izumi@abbott.com
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00178-0

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T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
Compounds bearing an unsubstituted piperidine ring (9,
10, 12, 14 and 15) were prepared as shown in Scheme 1.
Dichlorination of 2,4-dihydroxy-3-nitroquinoline (3)7
with phosphorus oxychloride gave 2,4-dichloro-3-nitro-
quinoline (4)8 with a large amount of overchlorinated
byproduct, 2,3,4-trichloroquinoline. In contrast, the
reaction of
3
with phenylphosphonic dichloride
(PhPOCl2) gave 4 in 90% yield without any significant
byproducts. The dichloride 4 was condensed with tert-
butyl 4-(2-aminoethyl)-1-piperidinecarboxylate9 to afford
5, followed by the reduction with nickel(II) chloride
.
hexahydrate (NiCl2 6H2O) and sodium borohydride
(NaBH4) to give 6. Cyclization of 6 with triethyl ortho-
formate [HC(OEt)3] or triethyl orthobenzoate
[PhC(OEt)3] in the presence of catalytic amount of
Figure 1.
Chemistry
.
p-toluenesulfonic acid monohydrate (p-TsOH H2O)
The synthetic methods of 1H-imidazo[4,5-c]quinolines
and 1H-imidazo[4,5-c]pyridines are illustrated in
Schemes 1–3.
gave compounds 7 or 8, respectively. The reaction of 7
with phenol (PhOH) and potassium hydroxide (KOH)
yielded 4-phenoxylated compound (11). Conversion of
.
Scheme 1. Reagents and conditions: (a) PhPOCl2; (b )tert-butyl 4-(2-aminoethyl)-1-piperidinecarboxylate, Et3N, DMF; (c) Ni(II)Cl2 6H2O, NaBH4,
.
MeOH, THF; (d) HC(OEt)3, p-TsOH H2O; (e) PhC(OEt)3, p-TsOH H2O; (f) TFA, 1,2-dichloroethane; (g) PhOH, KOH; (h) NH4OAc; (i) H2, Pd/C,
.
MeOH.
Scheme 2. Reagents and conditions: (a) n-BuBr, K2CO3, DMF; (b) BnBr, K2CO3, DMF; (c) Ac2O, pyridine; (d) KOH, PhOH; (e) NH4OAc.

T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
2543
.
Scheme 3. Reagents and conditions: (a) 1-triphenylmethyl-2-(4-piperidyl)ethylamine, Et3N, DMF; (b) Ni(II)Cl2 6H2O, NaBH4, MeOH, THF; (c)
.
HC(OEt)3, p-TsOH H2O; (d) TFA, 1,2-dichloroethane; (e) n-BuBr, K2CO3, DMF; (f) BnBr, K2CO3, DMF.
11 into 4-amino compound (13) was achieved with
ammonium acetate (NH4OAc). Final deprotection of
tert-butoxycarbonyl (Boc) group of the compounds (7,
8, 11 or 13) with trifluoroacetic acid (TFA) afforded the
desired compounds (9, 10, 12 or 14). On the other hand,
4-dechlorinated compound (15) was prepared from 9 by
catalytic hydrogenolysis.
Table 1. In vitro inhibitory activity of TNF-a production for 1H-
imidazoquinolines
Compounds with substituted piperidine rings (2 and 16–
22) were prepared as shown in Scheme 2. Alkylation of
secondary amines (9 or 10) with n-butyl bromide or
benzyl bromide, and acetylation with acetic anhydride
and pyridine afforded compounds 16–19. The reaction of
17 or 18 with PhOH and KOH yielded 4-phenoxylated
compounds (20 or 21), which were easily converted into
4-amino compounds (2 or 22) as described above.
Compd
R1
R3
Inhibition of TNF-a production
IC50 (nM)
2
9
Benzyl
H
H
H
H
n-Butyl
Benzyl
Acetyl
Acetyl
NH2
Cl
OPh
NH2
H
Cl
Cl
Cl
NH2
4810
205
3660
1380
3680
8020
2670
12
14
15
16
17
18
22
1
>10,000
>10,000
>10,000
Imidazo[4,5-c]pyridine derivatives were prepared as
shown in Scheme 3. The condensation of 2,4-dichloro-3-
nitropyridine (23)10 with 1-triphenylmethyl-2-(4-piper-
idyl)ethylamine gave the nitro compound (24). The
.
reduction with NiCl2 6H2O and NaBH4, the cyclization
with triethyl orthoformate and the following acidic
deprotection afforded the secondary amine (27), which
was treated with n-butyl or benzyl bromide to give 28 or
29, respectively.
showed similar activities, that for chlorine atom (9)
enhanced approximately 23-fold TNF-a suppressing
activity as compared to 2. As for the substitution at the
piperidyl nitrogen atom, n-butyl (16), benzyl (17) or
acetyl compound (18) showed 13 times or more
decreased activity as compared to 9. These results sug-
gested that the combination of the unsubstituted piper-
idine and C-4 chlorine atom would be optimal for
TNF-a suppressing activity.
Results and Discussion
A series of 1H-imidazo[4,5-c]quinolines or 1H-imi-
dazo[4,5-c]pyridines were evaluated for the inhibitory
activity of LPS-stimulated TNF-a production in human
PBMCs and their potencies were expressed by IC50
(inhibitory concentration 50%) values.
Next, we compared the 1H-imidazo[4,5-c]quinoline com-
pound with the corresponding 1H-imidazo[4,5-c]pyridine
one (Table 2). However, none of 1H-imidazo[4,5-c]pyri-
dine compounds (27–29) showed significant TNF-a sup-
pressing activity. The fused benzene ring with
imidazo[4,5-c]pyridine nucleus would be essential for
TNF-a suppressing activity.
First of all, we assessed some C-2 unsubstituted com-
pounds (9, 12, 14–18 and 22) for SARs regarding the
substituents at piperidyl nitrogen atom and C-4 position
of 2 (Table 1). Debenzylation at piperidyl nitrogen atom
(14) provided 3.5 times improved TNF-a suppressing
activity. Although substitution of amino group for
phenoxy group (12) or hydrogen atom (15) at position 4
As concerned with 1H-imidazo[4,5-c]pyridine com-
pounds without TNF-a suppressing activity, we inferred
that the fused benzene ring contributed to stabilizing the

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T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
Table 2. In vitro inhibitory activity of TNF-a production for 1H-
imidazopyridines
Table 3. In vitro inhibitory activity of TNF-a production for 2-
phenyl-1H-imidazoquinolines
Compd
R1
Inhibition of TNF-a production
IC50 (nM)
Compd
R1
Inhibition of TNF-a production
IC50 (nM)
27
28
29
H
n-Butyl
Benzyl
>10,000
>10,000
>10,000
10
19
H
Acetyl
52
611
spatial and conformational direction of 2-(4-piper-
idyl)ethyl group at position 1. In order to elucidate the
relation between the conformational direction of N-1
substituent and TNF-a suppressing activity, we moved
on to the introduction of phenyl ring at C-2 position,
which made 2-(4-piperidyl)ethyl group at position 1 to
be conformational restricted (Fig. 2 and Table 3). As we
had expected, 2-phenyl-1H-imidazoquinolines (10 and
19) showed improved TNF-a suppressing activity,
compared to C-2 unsubstituted compounds (9 and 18).
In particular, 10 showed the highest TNF-a suppressing
activity (IC50=52 nM) with no significant IFN-a indu-
cing activity at the concentration of 10ꢀ6 M in human
PBMCs (data not shown). Compound 10 also showed a
moderate interleukin-1b (IL-1b) inhibitory activity
(data not shown). These results indicated that a kind of
conformational restriction of 2-(4-piperidyl)ethyl group
at position 1 would be considerably related with TNF-a
suppressing activity. We would suggest that both C-2
phenyl ring and C-9 proton on the fused benzene ring
can stabilize the conformation of the 2-(4-piper-
idyl)ethyl group, and prevent bending to the unfavor-
able direction of that.
protons (d 1.92 and d 3.28) of the piperidine ring would
not be spatially located near C-9 proton but C-2 proton.
The 2-(4-piperidyl)ethyl group of 9 was considered to
fold to the imidazole ring side as shown in Figure 3. On
the other hand, apparent conformational differences of
N-1 aminoalkyl moiety between 10 and 27 were
observed. In contrast with compound 9, compound 27
showed a long-range NOE between C-20 methylene
proton resonance (d 1.80) and C-7 proton signal (d
7.74), but no long-range NOE between the C-20 protons
and C-2 proton (d 8.49). Besides, the medium-range
NOEs between C-6 proton signal (d 8.16) and piperidine
NH and two equatorial protons adjacent to nitrogen
atom (d 8.45 and d 3.15–3.30, respectively) were also
observed. From the above results, it was suggested that
the conformation of N-1 side chain of 27 would be near
to the left side of pyridine ring. Regarding 10, there
were three long-range NOEs. One NOE is observed
between C-20 methylene protons (d 1.75) and C-9 proton
signal (d 8.36–8.42). The second one is observed
between ortho and meta C-2 phenyl proton signals (d
7.60–7.68 and d 7.75–7.85) and two equatorial proton
signals of the piperidine ring adjacent to nitrogen atom
(d 3.03). The other is between ortho C-2 phenyl proton
signal (d 7.75–7.85) and two axial proton signals of the
piperidine ring adjacent to nitrogen atom (d 2.58). In
summary, the piperidine ring of 10 was in the relatively
near to the C-2 phenyl ring, similarly to compound 9.
The TNF-a suppressing activity of 9, 10 and 27 seemed
to be significantly affected by the spatial position of
piperidyl nitrogen atom. In other word, we proved our
hypothesis that the fused benzene ring of 9 and 10
would hinder the 2-(4-piperidyl)ethyl group at position
1 from approaching to the tricyclic core.
In order to demonstrate the conformational restriction
of 2-(4-piperidyl)ethyl group at position 1, we measured
the nuclear Overhauser effect (NOE) of compounds 9,
10 and 27 in the DMSO solution with the 2 dimensional
NMR (NOESY) experiments (Fig. 3). Judging from the
line width or link number of the NOESY spectra for the
C-20 methylene proton resonance (d 1.89) of 9, the NOE
with C-9 proton signal (d 8.35) was observed more
strongly than that of C-2 proton (d 8.52). Furthermore,
the long-range NOEs of 9 indicated that four equatorial
Figure 2.

T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
2545
Figure 3. NOESY Experiments for compounds 9, 10 and 27. aM represents the medium-range NOE judged from links whose sequence separation is
three or four. bL represents the long-range NOE judged from links whose sequence separation is one or two.
Conclusion
recorded with JEOL JMS-DX 300 or Waters 2690 mass
We developed modification of substituents at piperidyl
nitrogen atom, position 2 and 4 of compound 2, which
was a imiquimod analogue without IFN-a inducing
activity, and found a promising lead compound of
TNF-a suppressor, 4-chloro-2-phenyl-1-[2-(4-piper-
idyl)ethyl]-1H-imidazo[4,5-c]quinoline (10), which had
about 100-fold TNF-a suppressing activity in compar-
ison with 2. In addition, compound 10 showed a mod-
erate IL-1b suppressing activity and no significant
IFN-a inducing activity at the concentration of 10ꢀ6 M
in human PBMCs. On the other hand, NMR experi-
ments indicated that C-2 phenyl ring and C-9 proton on
the fused benzene ring could contribute the conforma-
tional restriction of 2-(4-piperidyl)ethyl group at posi-
tion 1 related with TNF-a suppressing activity. Further
detailed optimization with 10 is under investigation.
spectrometer. Elemental analyses were performed using
Yanagimoto MT-5 or MT-6 elemental analysis appara-
tus, and analytical results were within ꢁ0.4% of theo-
retical values. Column chromatography was carried out
with Kieselgel 60 (Merck) or Aluminum oxide 90
(Merck). TLC was conducted on a 0.25 mm pre-coated
silica gel plate (60F254, Merck), and spots were detected
by inspection under short (254 nm) wavelength UV
light, or by the colors developed with iodine. Organic
extracts were dried over anhydrous Na2SO4. The sol-
vent was concentrated under reduced pressure.
The following known compounds were prepared essen-
tially according to the literature: 2,4-dihydroxy-3-nitro-
quinoline
(3),7
tert-butyl 4-(2-aminoethyl)-1-
2,4-dichloro-3-nitropyridine
piperidinecarboxylate,9
(24).10
2,4-Dichloro-3-nitroquinoline (4).8
A mixture of 3
Experimental
Chemistry
(0.50 g, 2.43 mmol) and PhPOCl2 (1.40 mL, 10.1 mmol)
was stirred at 140 ꢂC for 3 h. The reaction mixture was
poured into water. The resulting precipitate was col-
lected by filtration and washed with water to give 0.53 g
(90%) as a brown crystal, which was used for the next
All melting points were measured on a Yanagimoto melt-
ing point apparatus and are uncorrected. 1H NMR spec-
tra were measured with JEOL Lambda-300 (300MHz) or
JEOL Alpha-500 (500MHz) spectrometer; chemical shifts
are expressed as d (ppm) values with tetramethylsilane
(TMS) as an internal standard (in NMR description,
s=singlet, d=doublet, t=triplet, q=quartet, m=multi-
plet, and br=broad peak). IR spectra were measured with
Hitachi 270-30 spectrometer. Mass spectra (MS) were
1
step without further purification. H NMR (CDCl3) d
7.81 (1H, t, J=8 Hz), 7.95 (1H, t, J=8 Hz), 8.12 (1H, d,
J=8 Hz), 8.28 (1H, d, J=8 Hz); IR n (KBr) cmꢀ1: 1546,
1296.
tert-Butyl 4-[2-[(2-chloro-3-nitroquinoline-4-yl)amino]e-
thyl]-1-piperidinecarboxylate (5). To a mixture of 4

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T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
(4.52 g, 18.6 mmol) and triethylamine (Et3N) (2.80 mL,
20.1 mmol) in N,N-dimethylformamide (DMF) (11 mL),
from 24, colorless crystals [ethanol (EtOH)]: mp 206.5–
208.5 ꢂC; H NMR (DMSO-d6) d 1.15–1.40 (3H, m),
1
a
solution of tert-butyl 4-(2-aminoethyl)-1-piper-
1.49 (2H, q, J=11 Hz), 1.54 (2H, q, J=7 Hz), 1.68 (2H,
d, J=11 Hz), 2.85–3.00 (2H, m), 3.10 (2H, q, J=7 Hz),
4.62 (2H, s), 5.51 (1H, t, J=7 Hz), 7.15 (3H, t,
J=7.5 Hz), 7.28 (6H, t, J=7.5 Hz), 7.25–7.35 (1H, m),
7.35–7.45 (6H, m), 7.38 (1H, d, J=5.5 Hz); IR n (KBr)
idinecarboxylate (4.30 g, 18.8 mmol) in DMF (3 mL)
was added under ice cooling, followed by stirring at
room temperature for 1 h. The reaction mixture was
poured into ice-water and extracted with ethyl acetate
(EtOAc). The extract was washed with water, dried and
concentrated. The residue was washed with a mixture of
EtOAc and diisopropyl ether (iso-Pr2O) to give 6.85 g
(85%) of 5 as a yellow crystal, which was recrystallized
from a mixture of EtOAc and iso-Pr2O to afford yellow
cmꢀ1: 3460, 3360. Anal. calcd for C31H33ClN4O2 1/
.
2H2O: C, 73.57; H, 6.77; N, 11.07. Found: C, 73.61; H,
6.55; N, 10.92.
tert-Butyl 4-[2-(4-chloro-2-phenyl-1H-imidazo[4,5-c]qui-
nolin-1-yl)ethyl]-1-piperidinecarboxylate (8). A mixture
ꢂ
1
crystals: mp 133–134 C; H NMR (CDCl3) d 1.10–1.20
(2H, m), 1.46 (9H, s), 1.50–1.60 (1H, m), 1.60–1.75 (4H,
m), 2.60–2.80 (2H, m), 3.45–3.55 (2H, m), 4.00–4.20
(2H, m), 5.80–5.90 (1H, m), 7.50–7.60 (1H, m), 7.70–
of
6
(21.1 g, 52.1 mmol), PhC(OEt)3 (23.6 mL,
.
104 mmol) and p-TsOH H2O (0.99 g, 5.20 mol) in tolu-
ene (100 mL) was refluxed for 5 h. The reaction mixture
was concentrated and the residue was washed with Et2O
to afford 21.1 g (83%) of 8 as a colorless crystal, which
was recrystallized from a mixture of EtOAc and n-hep-
tane to afford colorless crystals: mp 159–161 ꢂC; 1H
NMR (DMSO-d6) d 0.87 (2H, qd, J=12.5, 3 Hz), 1.23–
1.34 (3H, m), 1.36 (9H, s), 1.74 (2H, q, J=7.5 Hz), 2.48–
2.57 (2H, m), 3.76 (2H, d, J=12.5 Hz), 4.71 (2H, t,
J=7.5 Hz), 7.62–7.67 (3H, m), 7.75–7.83 (4H, m), 8.10–
7.80 (1H, m), 7.85–7.95 (2H, m); IR n (KBr) cmꢀ1
:
1660. Anal. calcd for C21H27ClN4O4: C, 57.99; H, 6.26;
N, 12.88. Found: C, 57.99; H, 6.34; N, 12.85.
2-Chloro-3-nitro-4-[2-(1-triphenylmethyl-4-piperidyl)ethyl]
amino]pyridine (24). By using the similar procedure as
for 5, compound 24 was prepared in 97% yield from 23,
yellow crystals (iso-Pr2O): mp 128–130 ꢂC; 1H NMR
(CDCl3) d 1.15–1.45 (3H, m), 1.45–1.75 (6H, m), 3.00–
3.20 (2H, m), 3.25 (2H, q, J=7 Hz), 6.50–6.60 (1H, m),
6.59 (1H, d, J=6 Hz), 7.10–7.20 (3H, m), 7.20–7.30 (6H,
m), 7.35–7.60 (6H, m), 8.01 (1H, d, J=6 Hz); IR n
(KBr) cmꢀ1: 1602, 1518. Anal. calcd for C31H31ClN4O2:
C, 70.64; H, 5.93; N, 10.63. Found: C, 70.70; H, 6.03; N,
10.52.
8.15 (1H, m), 8.37–8.43 (1H, m); IR n (KBr) cmꢀ1
:
1696; MS m/z (3:1, M)+ 490, 492. Anal. calcd for
C28H31ClN4O2: C, 68.49; H, 6.36; N, 11.41. Found: C,
68.36; H, 6.27; N, 11.37.
tert-Butyl 4-[2-(4-chloro-1H-imidazo[4,5-c]quinolin-1-
yl)ethyl]-1-piperidinecarboxylate (7). By using the simi-
lar procedure as for 8, compound 7 was prepared in
96% yield from 6, colorless crystals (EtOAc): mp 188–
tert-Butyl 4-[2-[(3-amino-2-chloroquinolin-4-yl)amino]-
ethyl]-1-piperidinecarboxylate (6). To a mixture of
.
NiCl2 6H2O (1.85 g, 7.78 mmol) and NaBH4 (0.30 g,
189 ꢂC; H NMR (CDCl3) d 1.20–1.35 (2H, m), 1.46
1
(9H, s), 1.50–1.60 (1H, m), 1.70–1.80 (2H, m), 1.98 (2H,
q, J=7.5 Hz), 2.65–2.80 (2H, m), 4.00–4.25 (2H, m),
4.63 (2H, t, J=7.5 Hz), 7.67 (1H, td, J=8.5, 1.5 Hz),
7.72 (1H, td, J=8.5, 1.5 Hz), 7.97 (1H, s), 8.11 (1H, dd,
J=8.5, 1.5 Hz), 8.21 (1H, dd, J=8.5, 1.5 Hz); IR n
(KBr) cmꢀ1: 1682; MS m/z (3:1, M)+ 414, 416. Anal.
calcd for C22H27ClN4O2: C, 63.68; H, 6.56; N, 13.50.
Found: C, 63.45; H, 6.60; N, 13.40.
7.93 mmol) in methanol (MeOH) (140 mL), a solution
of 5 (6.75 g, 15.5 mmol) in tetrahydrofuran (THF)
(20 mL) was added under ice-cooling. To the mixture,
NaBH4 (1.76 g, 46.5 mmol) was added portionwise, and
then the reaction mixture was stirred at room tempera-
ture for 0.5 h. The precipitate was filtered off, and the
filtrate was concentrated. To the resulting residue,
EtOAc and 10% aqueous ammonium chloride (NH4Cl)
was added and passed through a pad of Celite. The
organic layer of the filtrate was separated, washed suc-
cessively with 10% aqueous NH4Cl and saturated aqu-
eous sodium chloride (NaCl), dried and concentrated.
The resulting residue was washed with diethyl ether
(Et2O) to give 5.04 g (80%) of 6 as a pale brown crystal,
which was recrystallized from a mixture of EtOAc and
iso-Pr2O to afford colorless crystals: mp 115.5–116 ꢂC;
1H NMR (CDCl3) d 1.10–1.20 (2H, m), 1.45 (9H, s),
1.50–1.70 (5H, m), 2.60–2.75 (2H, m), 3.25–3.35 (2H,
m), 3.75–3.80 (1H, m), 4.00–4.20 (4H, m), 7.46 (1H, td,
J=8.5, 2 Hz), 7.50 (1H, td, J=8.5, 2 Hz), 7.75 (1H, dd,
J=8.5, 2 Hz), 7.90 (1H, dd, J=8.5, 2 Hz); IR ꢀ (KBr)
cmꢀ1: 3436, 3376, 3312, 1682. MS m/z (3:1, M)+ 404,
406. Anal. calcd for C21H29ClN4O2: C, 62.29; H, 7.22;
N, 13.84. Found: C, 61.99; H, 7.28; N, 13.73.
4-Chloro-1-[2-(1-triphenylmethyl-4-piperidyl)ethyl]-1H-
imidazo[4,5-c]pyridine (26). By using the similar proce-
dure as for 8, compound 26 was prepared in 80% yield
from 25, pale brown crystals [iso-propyl alcohol (iso-
PrOH)]: mp 202–203 ꢂC; H NMR (DMSO-d6) d 1.00–
1
1.10 (1H, m), 1.20–1.30 (2H, m), 1.51 (2H, q, J=11 Hz),
1.67 (2H, d, J=11 Hz), 1.78 (2H, q, J=7 Hz), 2.85–3.00
(2H, m), 4.29 (2H, t, J=7 Hz), 7.15 (3H, t, J=7.5 Hz),
7.29 (6H, t, J=7.5 Hz), 7.30–7.45 (6H, m), 7.69 (1H, d,
J=5.5 Hz), 8.12 (1H, d, J=7.5 Hz), 8.44 (1H, s). Anal.
calcd for C32H31ClN4: C, 75.80; H, 6.16; N, 11.05.
Found: C, 75.66; H, 6.23; N, 10.99.
4 - Chloro - 2 - phenyl - 1 - [2 - (4 - piperidyl)ethyl] - 1H - imi-
dazo[4,5-c]quinoline (10).
A mixture of 8 (15.4 g,
31.4 mmol), TFA (46 mL) and 1,2-dichloroethane
(77 mL) was stirred at room temperature for 1 h. The
reaction mixture was concentrated and the residue was
washed with a mixture of iso-PrOH and EtOAc to give
the trifluoroacetate as a colorless crystal, which was
3-Amino-2-chloro-4-[2-(1-triphenylmethyl-4-piperidyl)-
ethyl]amino]pyridine (25). By using the similar proce-
dure as for 7, compound 25 was prepared in 72% yield

T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
2547
added to a mixture of MeOH/1,2-dichloroethane (1:10)
and water, and adjusted to pH 10 with 5% aqueous
sodium hydroxide (NaOH). The organic layer was
separated, washed with saturated aqueous NaCl, dried
and concentrated to afford pale brown oil. The residue
was converted to the fumarate in the usual manner to
afford 13.9 g (87%) of 10 as a colorless crystal, which
was recrystallized from MeOH to afford colorless crys-
compound 27 was prepared quantitatively as the tri-
fluoroacetateꢂfrom 26, pale brown crystals (iso-PrOH):
1
mp 149–150 C; H NMR (DMSO-d6) d 1.33 (2H, q,
J=12 Hz), 1.43–1.55 (1H, m), 1.80 (2H, q, J=7.5 Hz),
1.86 (2H, d, J=12 Hz), 2.83 (2H, t, J=12 Hz), 3.15–3.30
(2H, m), 4.34 (2H, t, J=7.5 Hz), 7.74 (1H, d,
J=5.5 Hz), 8.16 (1H, d, J=5.5 Hz), 8.19 (1H, br-s), 8.45
(1H, br-s), 8.49 (1H, s); IR n (KBr) cmꢀ1: 3460, 1682;
MS m/z (3:1, M+1)+ 265, 267. Anal. calcd for
tals: mp 185.5–186.5 ꢂC (dec.); H NMR (DMSO-d6) d
1
.
1.15 (2H, q, J=12.5 Hz), 1.32–1.46 (3H, m), 1.75 (2H,
q, J=7.5 Hz), 2.58 (2H, t, J=12.5 Hz), 3.03 (2H, d,
J=12.5 Hz), 4.71 (2H, t, J=7.5 Hz), 6.44 (2H, s), 7.60–
7.68 (3H, m), 7.75–7.85 (4H, m), 8.10–8.16 (1H, m),
8.36–8.42 (1H, m); IR n (KBr) cmꢀ1: 3464, 1712; MS m/
C13H17ClN4 3/2C2HF3O: C, 44.10; H, 4.28; N, 12.86.
Found: C, 44.15; H, 4.43; N, 12.88.
tert-Butyl 4-[2-(4-phenoxy-1H-imidazo[4,5-c]quinolin-1-
yl)ethyl]-1-piperidinecarboxylate (11). A mixture of 7
(4.46 g, 10.7 mmol), PhOH (10.1 g, 107 mmol) and KOH
(1.80 g, 32.1 mmol) was heated at 120 ꢂC for 7 h. The
reaction mixture was basified with 10% aqueous
NaOH, and extracted with EtOAc. The extract was
washed successively with 10% aqueous NaOH and
saturated aqueous NaCl, dried and concentrated to give
a brown oil. The residue was purified by column chro-
matography (SiO2, EtOAc) to afford 3.59 g (71%) of 11
as a colorless solid, which was recrystallized from a
mixture of EtOAc and n-hexane to afford colorless
z
(3:1, M+1)+391, 393. Anal. calcd for
.
.
C23H23ClN4 C4H4O4 H2O: C, 61.77; H, 5.57; N, 10.67.
Found: C, 62.04; H, 5.40; N, 10.70.
4-Chloro-1-[2-(4-piperidyl)ethyl]-1H-imidazo[4,5-c]quino-
line (9). By using the similar procedure as for 10, com-
pound
9
trifluoroacetate from 7, colorless crystals (EtOH/iso-
was prepared in 98% yield as the
ꢂ
1
Pr2O): mp 182.5–185.5 C; H NMR (DMSO-d6) d 1.40
(2H, qd, J=13.0, 3.5 Hz), 1.61–1.72 (1H, m), 1.89 (2H,
q, J=7.5 Hz), 1.92 (2H, d, J=13 Hz), 2.87 (2H, t,
J=13 Hz), 3.28 (2H, d, J=13 Hz), 4.76 (2H, t,
J=7.5 Hz), 7.76 (2H, td, J=7, 1.5 Hz), 8.10 (1H, dd,
J=7, 1.5 Hz), 8.35 (1H, dd, J=7.0, 1.5 Hz), 8.52 (1H, s);
IR n (KBr) cmꢀ1: 3448, 1682. Anal. calcd for
crystals: mp 130.5–132.5 ꢂC; H NMR (CDCl3) d 1.22–
1
1.33 (2H, m), 1.47 (9H, s), 1.52–1.62 (2H, m), 1.68–1.82
(1H, m), 2.00 (2H, q, J=7.5 Hz), 2.66–2.78 (2H, m),
4.13 (2H, br-s), 4.64 (2H, t, J=7.5 Hz), 7.26 (1H, t,
J=7.5 Hz), 7.40 (2H, d, J=7.5 Hz), 7.45 (2H, t,
J=7.5 Hz), 7.51 (1H, td, J=8, 1 Hz), 7.57 (1H, td, J=8,
1 Hz), 7.91 (1H, dd, J=8, 1 Hz), 7.95 (1H, s), 8.06 (1H,
dd, J=8, 1 Hz); IR n (KBr) cmꢀ1: 1694; MS m/z (M)+
472. Anal. calcd for C28H32N4O3: C, 71.16; H, 6.83; N,
11.86. Found: C, 71.10; H, 7.10; N, 11.69.
.
.
C17H19ClN4 C2HF3O 1/4H2O: C, 52.66; H, 4.77; N,
12.93. Found: C, 52.43; H, 4.77; N, 12.76.
4 - Phenoxy - 1 - [2 - (4 - piperidyl)ethyl] - 1H - imidazo[4,5 -
c]quinoline (12). By using the similar procedure as for
10, compound 12 was prepared in 100% yield as the tri-
fluoroacetate from 11, colorless crystals (EtOH/
CH2Cl2): mp 211–216 ꢂC (dec.); 1H NMR (DMSO-d6) d
1.62–1.73 (1H, m), 1.39 (2H, qd, J=12, 4 Hz), 1.93 (2H,
q, J=7 Hz), 2.88 (2H, t, J=12 Hz), 3.28 (2H, d,
J=12 Hz), 4.75 (2H, t, J=7 Hz), 7.30 (1H, d, J=7.5 Hz),
7.47 (2H, t, J=7.5 Hz), 7.54–7.62 (2H, m), 7.74 (1H, d,
J=7.5 Hz), 8.26 (1H, d, J=7.5 Hz), 8.42 (1H, s); IR n
(KBr) cmꢀ1: 1676; MS m/z (M)+ 372. Anal. calcd for
1-[2-(1-Benzyl-4-piperidyl)ethyl]-4-phenoxy-1H-imi-
dazo[4,5-c]quinoline (20). By using the similar procedure
as for 11, compound 20 was prepared in 87% yield from
ꢂ
1
17, colorless crystals (MeOH): mp 152.5–153.5 C; H
NMR (CDCl3) d 1.40–1.50 (3H, m), 1.70–1.80 (2H, m),
1.95–2.05 (4H, m), 2.92 (2H, d, J=11 Hz), 3.51 (2H, s),
4.61 (2H, t, J=7.5 Hz), 7.20–7.35 (6H, m), 7.40 (2H, d,
J=7.5 Hz), 7.44 (2H, t, J=7.5 Hz), 7.49 (1H, td, J=8.5,
1 Hz), 7.56 (1H, td, J=8.5, 1 Hz), 7.90 (1H, dd, J=8.5,
1 Hz), 7.93 (1H, s), 8.06 (1H, dd, J=8.5, 1 Hz); MS m/z
(M)+ 462. Anal. calcd for C30H30N4O: C, 77.89; H,
6.54; N, 12.11. Found: C, 78.00; H, 6.29; N, 12.05.
.
.
C23H24ClN4O C2HF3O 1/4H2O: C, 61.16; H, 5.23; N,
11.41. Found: C, 61.26; H, 5.05; N, 11.47.
4-Amino-1-[2-(4-piperidyl)ethyl]-1H-imidazo[4,5-c]quino-
line (14). By using the similar procedure as for 10, com-
pound 14 was prepared in 86% yield as the
hydrochloride from 13, colorless crystals (EtOH): mp
243–244 ꢂC (dec.); 1H NMR (DMSO-d6) d 1.40–1.50
(2H, m), 1.60–1.70 (1H, m), 1.85 (2H, q, J=7.5 Hz),
1.89 (2H, d, J=13 Hz), 2.83 (2H, td, J=13, 2.5 Hz),
3.23 (2H, d, J=13 Hz), 4.65 (2H, t, J=7.5 Hz), 6.99
(2H, br-s), 7.34 (1H, t, J=7 Hz), 7.51 (1H, t, J=7 Hz),
7.69 (1H, d, J=7 Hz), 8.06 (1H, d, J=7 Hz), 8.30 (1H,
s), 8.87 (2H, br-s); IR n (KBr) cmꢀ1: 3376, 3320, 3204;
4-[2-(4-Phenoxy-1H-imidazo[4,5-c]quinolin-1-yl)ethyl]-1-
piperidineacetamide (21). By using the similar procedure
as for 11, compound 21 was prepared in 82% yield from
18, colorless crystals (EtOAc/iso-Pr2O): mp 187–
189.5 ꢂC; 1H NMR (CDCl3) d 1.28 (2H, qd, J=13,
4.5 Hz), 1.60–1.72 (1H, m), 1.79 (1H, td, J=13 Hz), 1.85
(1H, d, J=13 Hz), 2.01 (2H, q, J=7.5 Hz), 2.10 (3H, s),
2.56 (1H, td, J=13, 2.5 Hz), 3.05 (1H, td, J=13,
2.5 Hz), 3.84 (1H, d, J=13 Hz), 4.58–4.72 (3H, m), 7.26
(1H, t, J=7 Hz), 7.40 (2H, d, J=7 Hz), 7.45 (2H, t,
J=7 Hz), 7.50 (1H, td, J=8, 1 Hz), 7.57 (1H, td, J=8,
1 Hz), 7.91 (1H, dd, J=8, 1 Hz), 7.94 (1H, s), 8.04 (1H,
dd, J=8, 1 Hz); IR n (KBr) cmꢀ1: 1640; MS m/z (M)+
414. Anal. calcd for C25H26N4O2: C, 72.44; H, 6.32; N,
13.52. Found: C, 72.35; H, 6.26; N, 13.42.
MS m/z (M)+ 295. Anal. calcd for C17H21N5 HCl 3/
4H2O: C, 59.12; H, 6.86; N, 20.28. Found: C, 59.10; H,
6.83; N, 20.30.
.
.
4-Chloro-1-[2-(4-piperidyl)ethyl]-1H-imidazo[4,5-c]pyri-
dine (27). By using the similar procedure as for 10,

2548
T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
tert-Butyl
4-[2-(4-amino-1H-imidazo[4,5-c]quinolin-1-
afford 0.47 g (52%) of 15 as a colorless crystal, which
was recrystallized from a mixture of MeOH and
1,2-dichloroethane (1:10) to afford colorless crystals: mp
yl)ethyl]-1-piperidinecarboxylate (13). A mixture of 11
(4.40 g, 9.31 mmol) and NH4OAc (34.5 g, 448 mmol)
was heated at 140 ꢂC for 3 h. The reaction mixture was
adjusted to pH 9 with 10% aqueous NaOH and extrac-
ted with CH2Cl2. The extract was washed with saturated
aqueous NaCl, dried and concentrated. The residue was
purified by column chromatography [Al2O3, CH2Cl2/
MeOH (100:1–20:1)] and washed with iso-Pr2O to
afford 1.88 g (51%) of 13 as a colorless crystal, which
was recrystallized from EtOAc to afford colorless crys-
230–233 ꢂC (dec.); H NMR (DMSO-d6) d 1.48 (2H, q,
1
J=12 Hz), 1.65–1.77 (1H, m), 1.88–1.97 (4H, m), 2.84
(2H, q, J=12 Hz), 3.25 (2H, d, J=12 Hz), 4.86 (2H, t,
J=7.5 Hz), 7.97 (1H, t, J=8 Hz), 8.02 (1H, t, J=8 Hz),
8.49 (1H, d, J=8 Hz), 8.57 (1H, d, J=8 Hz), 8.83 (2H,
br-s), 8.99 (1H, br-s), 9.70 (1H, s); IR n (KBr) cmꢀ1
:
3416; MS m/z (M)+ 280. Anal. calcd for
.
.
C17H20N4 2HCl 1/2H2O: C, 56.36; H, 6.40; N, 15.46.
Found: C, 56.36; H, 6.18; N, 15.35.
tals: mp 193–193.5 ꢂC; H NMR (CDCl3) d 1.20–1.30
1
(2H, m), 1.46 (9H, s), 1.50–1.60 (1H, m), 1.60–1.80 (2H,
m), 1.96 (2H, q, J=7.5 Hz), 2.60–2.80 (2H, m), 4.00–
4.25 (2H, m), 4.56 (2H, t, J=7.5 Hz), 5.42 (2H, br-s),
7.35 (1H, t, J=8 Hz), 7.55 (1H, t, J=8 Hz), 7.79 (2H, s),
7.84 (1H, d, J=8 Hz), 7.93 (1H, d, J=8 Hz); IR n (KBr)
cmꢀ1: 3476, 1694; MS m/z (M)+ 395. Anal. calcd for
C22H29N5O2: C, 66.81; H, 7.39; N, 17.71. Found: C,
66.93; H, 7.48; N, 17.66.
1-[2-(1-n- Butyl-4-piperidyl)ethyl] - 4-chloro-1H-imi-
dazo[4,5-c]quinoline (16). To a suspension of tri-
fluoroacetate of 9 (1.20 g, 2.80 mmol) and potassium
carbonate (K2CO3) (0.77 g, 5.57 mmol) in DMF (6 mL),
n-butyl bromide (0.30 mL, 2.80 mmol) was added and
stirred at room temperature for 5 h. The reaction mix-
ture was adjusted to pH 10 with 10% aqueous NaOH
and extracted with EtOAc. The extract was washed
successively with water and saturated aqueous NaCl,
dried and concentrated to give pale brown oil. The
residue was dissolved in THF and passed through a pad
of SiO2. The filtrate was concentrated to give 0.87 g
(84%) of 16 as a colorless solid, which was converted to
the hydrochloride in the usual manner and recrystallized
from a mixture of MeOH and EtOAc to afford colorless
1-[2-(1-Benzyl-4-piperidyl)ethyl]-1H-imidazo[4,5-c]quino-
line-4-amine (2). By using the similar procedure as for
13, compound 2 was prepared in 89% yield from 20,
colorless crystals (EtOH): mp 191.5–192 ꢂC; H NMR
1
(DMSO-d6)
d 1.20–1.40 (3H, m), 1.70 (2H, d,
J=11.5 Hz), 1.81 (2H, q, J=7 Hz), 1.92 (2H, t,
J=11.5 Hz), 2.78 (2H, d, J=11.5 Hz), 3.20 (2H, t,
J=11.5 Hz), 3.44 (2H, s), 4.61 (2H, t, J=7 Hz), 6.41
(2H, s), 7.20–7.35 (6H, m), 7.43 (1H, td, J=8, 1 Hz),
7.62 (1H, dd, J=8, 1 Hz), 8.01 (1H, dd, J=8, 1 Hz),
8.18 (1H, s); IR n (KBr) cmꢀ1: 3360, 3304; MS m/z
(M)+ 385. Anal. calcd for C24H27N5: C, 74.77; H, 7.06;
N, 18.17. Found: C, 74.87; H, 7.18; N, 18.06.
ꢂ
1
crystals: mp 144–146 C; H NMR (DMSO-d6) d 0.91
(3H, t, J=7.5 Hz), 1.32 (2H, sextet, J=7.5 Hz), 1.55–
1.72 (5H, m), 1.88 (2H, q, J=7.5 Hz), 1.94 (2H, d,
J=11 Hz), 2.83 (2H, q, J=11 Hz), 2.91–2.98 (2H, m),
3.43 (2H, d, J=11 Hz), 4.77 (2H, t, J=7.5 Hz), 7.75
(1H, td, J=7.5, 2 Hz), 7.78 (1H, td, J=7.5, 2 Hz), 8.10
(1H, dd, J=7.5, 2 Hz), 8.36 (1H, dd, J=7.5, 2 Hz), 8.54
(1H, s), 10.19 (1H, br-s); MS m/z (M)+ 370, 372. Anal.
4-[2-(4-Amino-1H-imidazo[4,5-c]quinolin-1-yl)ethyl]-
1-piperidineacetamide (22). By using the similar proce-
dure as for 13, compound 22 was prepared in 48% yield
from 21, colorless crystals (MeOH): mp 231.5–232.5 ꢂC;
1H NMR (DMSO-d6) d 1.00–1.25 (2H, m), 1.55–1.65
(1H, m), 1.70–1.80 (2H, m), 1.82 (2H, q, J=7.5 Hz),
1.97 (3H, s), 2.52 (1H, d, J=12.5 Hz), 2.99 (1H, t,
J=12.5 Hz), 3.77 (1H, d, J=12.5 Hz), 4.34 (1H, d,
J=12.5 Hz), 4.63 (2H, t, J=7.5 Hz), 6.42 (2H, br-s),
7.27 (1H, td, J=8, 1 Hz), 7.44 (1H, td, J=8, 1 Hz), 7.63
(1H, dd, J=8, 1 Hz), 8.02 (1H, dd, J=8, 1 Hz), 8.20
(1H, s); IR n (KBr) cmꢀ1: 3480, 3284, 1638; MS m/z
(M)+ 337. Anal. calcd for C19H23N5O: C, 67.63; H,
6.87; N, 20.76. Found: C, 67.46; H, 6.79; N, 20.63.
.
.
calcd for C21H27ClN4 2HCl 1/2H2O: C, 55.70; H, 6.68;
N, 12.37. Found: C, 55.80; H, 6.65; N, 12.44.
1-[2-(1-n- Butyl-4-piperidyl)ethyl] - 4-chloro-1H-imi-
dazo[4,5-c]pyridine (28). By using the similar procedure
as for 16, compound 28 was prepared in 69% yield from
27, pale brown prisms (iso-Pr2O): mp 107.5–109 ꢂC; H
1
NMR (DMSO-d6) d 0.86 (3H, t, J=7.5 Hz), 1.10–1.25
(3H, m), 1.26 (2H, sextet, J=7.5 Hz), 1.37 (2H, quintet,
J=7.5 Hz), 1.60–1.70 (2H, m), 1.70–1.80 (4H, m), 2.20
(2H, t, J=7.5 Hz), 2.78 (2H, d, J=11 Hz), 4.32 (2H, t,
J=7.5 Hz), 7.71 (1H, d, J=5.5 Hz), 8.14 (1H, d,
J=5.5 Hz), 8.47 (1H, s); MS m/z (M)+ 320, 322. Anal.
calcd for C17H25ClN4: C, 63.64; H, 7.85; N, 17.46.
Found: C, 63.56; H, 7.71; N, 17.46.
1-[2-(4-Piperidyl)ethyl]-1H-imidazo[4,5-c]quinoline (15).
A mixture of trifluoroacetate 9 (1.10 g, 2.57 mmol) and
5% palladium on carbon (0.55 g) in MeOH (100 mL) was
stirred at room temperature and an atmospheric pressure
of H2 for 4 h. The catalyst was removed by filtration and
the filtrate was concentrated. The resulting residue was
dissolved in a small amount of water and washed with
EtOAc. Then the aqueous layer was adjusted to pH 10
with 10% aqueous NaOH and extracted with a mixture
of MeOH and 1,2-dichloroethane (1:10). The extract was
washed with saturated aqueous NaCl, dried and con-
centrated to afford pale brown oil. The residue was con-
verted to the hydrochloride in the usual manner to
1 - [2 - (1 - Benzyl - 4 - piperidyl)ethyl] - 4 - chloro - 1H - imi-
dazo[4,5-c]quinoline (17). To a suspension of tri-
fluoroacetate of 9 (0.50 g, 1.17 mmol) and K2CO3
(0.32 g, 2.32 mmol) in DMF (3 mL), benzyl bromide
(0.14 mL, 1.18 mmol) was added and stirred at room
temperature for 3 h. The reaction mixture was basified
with 10% aqueous NaOH and extracted with EtOAc.
The extract was washed successively with water and
saturated aqueous NaCl, dried and concentrated to give
a pale brown oil. The residue was purified by column
chromatography [SiO2, n-heptane/THF (4:1)] to afford

T. Izumi et al. / Bioorg. Med. Chem. 11 (2003) 2541–2550
2549
0.41 g (87%) of 17 as a colorless crystal, which was
recrystallized from a mixture of EtOAc and iso-Pr2O to
afford colorless crystals: mp 116.5–118 ꢂC; 1H NMR
(CDCl3) d 1.38–1.48 (3H, m), 1.74 (2H, d, J=11 Hz),
1.92–2.03 (4H, m), 2.93 (2H, d, J=11 Hz), 3.51 (2H, s),
4.60 (2H, t, J=8 Hz), 7.23–7.28 (1H, m), 7.30–7.35 (4H,
m), 7.64 (1H, td, J=7, 1.5 Hz), 7.71 (1H, td, J=7,
1.5 Hz), 7.96 (1H, s), 8.11 (1H, dd, J=7, 1.5 Hz), 8.20
(1H, dd, J=7, 1.5 Hz); MS m/z (3:1, M)+ 404, 406.
Anal. calcd for C24H25ClN4: C, 71.19; H, 6.22; N, 13.84.
Found: C, 70.97; H, 6.44; N, 13.69.
by venipuncture, and poured into plastic tubes which
contains 170 mL of Novo-heparin 1000 (Novo-Nordisk
A/S). Then, PBMCs were separated using Lympho-
prepTM (NYCOMED PHARMAAS) lymphocyte
separation tube, and cultured with RPMI-1640 medium
(Nissui Pharmaceutical Co.) containing 2 mM l-gluta-
mine (Life Technologies), 2.5 U/mL penicillin-2.5 mg/
mL streptomycin solution (Life Technologies) supple-
mented with 10% fetal calf serum (Intergen Company)
at 1.11ꢃ106 cells/mL (final 1ꢃ106 ceus/mL).
Preparation of test compounds. Test compounds were
dissolved in distilled ultra-pure water, DMSO or 0.1 N
hydrochloric acid at 20 mM and were diluted with saline
sequentially. Test compounds were evaluated at con-
centrations from 10ꢀ10 M to 10ꢀ5 M.
1 - [2 - (1 - Benzyl - 4 - piperidyl)ethyl] - 4 - chloro - 1H - imi-
dazo[4,5-c]pyridine (29). By using the similar procedure
as for 17, compound 29 was prepared in 48ꢂ% yield from
27, colorless prisms (toluene): mp 150–151 C; 1H NMR
(DMSO-d6) d 1.15–1.30 (3H, m), 1.60–1.70 (2H, m),
1.76 (2H, q, J=7 Hz), 1.87 (2H, t, J=11 Hz), 2.76 (2H,
d, J=11 Hz), 3.42 (2H, s), 4.31 (2H, t, J=7 Hz), 7.15–
7.35 (5H, m), 7.71 (1H, d, J=6 Hz), 8.14 (1H, d,
J=6 Hz), 8.47 (1H, s); MS m/z (3:1, M)+ 354, 356.
Anal. calcd for C20H23ClN4: C, 67.69; H, 6.53; N, 15.79.
Found: C, 67.65; H, 6.50; N, 15.73.
Treatment of cells with test compounds. 180 mL of pre-
pared human PBMCs was added to each well of 96-well
plate (MicroTest IIITM tissue culture plate; FALCON)
containing 10 mL of adequate concentrations solution of
test compounds or solvent. Then after 30 min, 10 mL of
20 mg/mL LPS (final 1 mg/mL) was added to each well,
and cultured for 16 h at 37 ꢂC in humidified atmosphere
of 5% CO2 and 95% air.
4-[2-(4-Chloro-1H-imidazo[4,5-c]quinolin-1-yl)ethyl]-
1-piperidineacetamide (18). A mixture of trifluoroacetate
of 9 (0.60 g, 1.40 mmol), acetic anhydride (2.00 mL,
21.2 mmol) and pyridine (4.00 mL, 49.5 mmol) was stir-
red at room temperature for 1 h. The reaction mixture
was concentrated, and the resulting residue was washed
with a mixture of iso-PrOH and iso-Pr2O to give 0.45 g
(90%) of 18 as a colorless crystal, which was recrys-
tallized from a mixture of CH2Cl2 and iso-Pr2O to
afford colorless crystals: mp 183–186.5 ꢂC; 1H NMR
(CDCl3) d 1.28 (2H, qd, J=12.5, 4 Hz), 1.61–1.71 (1H,
m), 1.79 (1H, d, J=12.5 Hz), 1.85 (1H, d, J=12.5 Hz),
1.99 (2H, q, J=7 Hz), 2.10 (3H, s), 2.55 (1H, td,
J=12.5, 2.5 Hz), 3.06 (1H, td, J=12.5, 2.5 Hz), 3.84
(1H, d, J=12.5 Hz), 4.58–4.72 (3H, m), 7.67 (1H, td,
J=8, 1 Hz), 7.73 (1H, td, J=8, 1 Hz), 7.98 (1H, s), 8.09
(1H, dd, J=8, 1 Hz), 8.21 (1H, dd, J=8, 1 Hz); IR ꢀ
(KBr) cmꢀ1: 1642; MS m/z (3:1, M)+ 356, 358. Anal.
calcd for C19H21ClN4O: C, 63.95; H, 5.93; N, 15.70.
Found: C, 63.81; H, 5.87; N, 15.61.
Measurement of human TNF-ꢀ. The level of human
TNF-a in culture supernatant was measured by con-
structed sandwich enzyme-linked immunosorbent assay
method. 96-well microtiter plates were coated with anti-
cytokine antibody (first-antibody) by introducing ade-
quately diluted capture antibody. After washing the
wells, the culture supernatant was diluted appropriately,
added to each well and incubated. Then second-anti-
body against cytokine and third-antibody against sec-
ond-antibody were introduced to wells sequentially,
with interposed washing periods. After the final washing
period, reaction of colorimetric quantification was star-
ted by addition of tetramethylbenzidine solution
(DAKO) to each well. Optical absorbance at 450 nm of
each well was measured by M-VmaxTM kinetic micro-
plate reader (Molecular Devices) after the quenching by
addition of 1 N sulfuric acid. Concentrations of cyto-
kines were determined by SoftmaxTM (Molecular Devi-
ces) quantification software in comparison with
calibration curve derived from corresponding recombi-
nant cytokines as standard. For measurement of human
TNF-a, monoclonal anti-human TNF-a (ENDOGEN),
polyclonal rabbit anti-human TNF-a (Pharma Bio-
technologie Hannover), peroxidase conjugated donkey
anti-rabbit IgG (Jackson ImmunoRes. Labs.), and
recombinant human TNF-a (INTERGEN Company)
were used for first-, second-, third-antibody and stan-
dard for calibration curve, respectively. In measurement
of effect on TNF-a, the activities of test compounds are
shown as percentages of cytokine production with being
treated by LPS and test compounds against by LPS only.
4-[2-(4-Chloro-2-phenyl-1H-imidazo[4,5-c]quinolin-1-yl)-
ethyl]-1-piperidineacetamide (19). By using the similar
procedure as for 18, compound 19 was prepared in 48%
yield from 10, colorless crystals (CH2Cl2/iso-Pr2O): mp
154.5–156 ꢂC; H NMR (DMSO-d6) d 0.76–0.88 (1H,
1
m), 0.90–1.02 (1H, m), 1.25–1.43 (3H, m), 1.71–1.82
(2H, m), 1.91 (3H, s), 2.33 (1H, t, J=12 Hz), 2.83 (1H, t,
J=12 Hz), 3.63 (1H, d, J=12 Hz), 4.18 (1H, d,
J=12 Hz), 4.72 (2H, t, J=7.5 Hz), 7.63–7.67 (3H, m),
7.77–7.82 (4H, m), 8.11–8.15 (1H, m), 8.37–8.42 (1H,
m); IR n (KBr) cmꢀ1: 1642; MS m/z (3:1, M)+ 432, 434.
.
Anal. calcd for C25H25ClN4O 1/4H2O: C, 68.64; H,
5.88; N, 12.81. Found: C, 68.78; H, 5.78; N, 12.71.
References and Notes
Biology
Preparation of PBMCs for culture. About 50 mL of
whole blood was collected from adult healthy volunteers
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