New N-(pyridin-4-yl)-(indol-3-yl)acetamides and propanamides as antiallergic agents

Article abstract of DOI:10.1021/jm981079+

A series of new N-(pyridin-4-yl)-(indol-3-yl)alkylamides 44-84 has been prepared in the search of novel antiallergic compounds. Synthesis of the desired ethyl (2-methyindol-3-yl)acetates 1-4 was achieved by indolization under Fischer conditions; Japp-Klingemann method followed by 2- decarboxylation afforded the ethyl (indol-3-yl)alkanoates 17-25. Amidification was successfully carried out by condensation of the corresponding acids or their N-aryl(methyl) derivatives with 4-aminopyridine promoted by 2-chloro-1-methylpyridinium iodide. Efforts to improve the antiallergic potency of the title series by variation of the indole substituents (R¹, R², R) and the length of the alkanoic chain (n = 1, 2, 3) led to the selection of N-(pyridin-4-yl)-[1-(4-fluorobenzyl)indol-3- yl]acetamide 45, out of 41 compounds. This amide was 406-fold more potent than astemizole in the ovalbumin-induced histamine release assay, using guinea pig peritoneal mast cells, with an IC₅₀ = 0.016 μM. Its inhibitory activity in IL-4 production test from Th-2 cells was identical to that of the reference histamine antagonist (IC₅₀ = 8.0 μM) and twice higher in IL-5 assay: IC₅₀ = 1.5 and 3.3 μM, respectively. In vivo antiallergic activity evaluation confirmed efficiency of 45 in sensitized guinea pig late phase eosinophilia inhibition, after parenteral and oral administration at 5 and 30 mg/kg, respectively. Its efficiency in inhibition of microvascular permeability was assessed in two rhinitis models; ovalbumin and capsaicin- induced rhinorrhea could be prevented after topical application of submicromolar concentrations of 45 (IC₅₀ = 0.25 and 0.30 μM); and it also exerted significant inhibitory effect in the first test after iv and oral administration, with ID₅₀ = 0.005 and 0.46 mg/kg.

Full text of DOI:10.1021/jm981079+

638
J . Med. Chem. 1999, 42, 638-648
New N-(P yr id in -4-yl)-(in d ol-3-yl)a ceta m id es a n d P r op a n a m id es a s An tia ller gic
Agen ts
Cecilia Menciu,† Muriel Duflos,† Fabienne Fouchard,† Guillaume Le Baut,*† Peter Emig,‡ Ute Achterrath,‡
Istvan Szelenyi,‡ Bernd Nickel,‡ J u¨rgen Schmidt,‡ Bernhard Kutscher,‡ and Eckhardt Gu¨nther‡
Department of Organic Chemistry and Medicinal Chemistry, Faculty of Pharmacy, 44035 Nantes Cedex 01, France,
ASTA Medica AG, Weismu¨llerstrasse 45, D-60314 Frankfurt am Main, Germany
Received September 14, 1998
A series of new N-(pyridin-4-yl)-(indol-3-yl)alkylamides 44-84 has been prepared in the search
of novel antiallergic compounds. Synthesis of the desired ethyl (2-methyindol-3-yl)acetates 1-4
was achieved by indolization under Fischer conditions; J app-Klingemann method followed by
2-decarboxylation afforded the ethyl (indol-3-yl)alkanoates 17-25. Amidification was success-
fully carried out by condensation of the corresponding acids or their N-aryl(methyl) derivatives
with 4-aminopyridine promoted by 2-chloro-1-methylpyridinium iodide. Efforts to improve the
antiallergic potency of the title series by variation of the indole substituents (R1, R2, R) and
the length of the alkanoic chain (n ) 1, 2, 3) led to the selection of N-(pyridin-4-yl)-[1-(4-
fluorobenzyl)indol-3-yl]acetamide 45, out of 41 compounds.This amide was 406-fold more potent
than astemizole in the ovalbumin-induced histamine release assay, using guinea pig peritoneal
mast cells, with an IC50 ) 0.016 µM. Its inhibitory activity in IL-4 production test from Th-2
cells was identical to that of the reference histamine antagonist (IC50 ) 8.0 µM) and twice
higher in IL-5 assay: IC50 ) 1.5 and 3.3 µM, repectively. In vivo antiallergic activity evaluation
confirmed efficiency of 45 in sensitized guinea pig late phase eosinophilia inhibition, after
parenteral and oral administration at 5 and 30 mg/kg, respectively. Its efficiency in inhibition
of microvascular permeability was assessed in two rhinitis models; ovalbumin and capsaicin-
induced rhinorrhea could be prevented after topical application of submicromolar concentrations
of 45 (IC50 ) 0.25 and 0.30 µM); and it also exerted significant inhibitory effect in the first test
after iv and oral administration, with ID50 ) 0.005 and 0.46 mg/kg.
In tr od u ction
colony-stimulating factor (GM-CSF), with IL-5 as the
most likely to be a specific stimulator of human eosi-
nophil maturation, proliferation, and function.4-9
Eosinophils produce major basic protein (MBP) and
eosinophil cationic protein (ECP) which are cytotoxic
and may contribute to the denudation of epithelium seen
in asthma.4 Moreover eosinophil granules have the
capacity to generate considerable quantities of leuko-
trienes (LTD4),8 notably after IgE-dependent stimuli10
and of platelet activating factor (PAF), which in turn
induce potent eosinophil chemotaxis.11
Approaches in the discovery of leukotriene modulators
(LT) have resulted in several new classes of com-
pounds: redox inhibitors of 5-LO,12 indole, quinolyl-
methoxy or quindole-containing inhibitors of five lipoxy-
genase activating protein (FLAP),13 styrylquinoline-
containing antagonists of peptidyl LTD4 receptor, and
LTB4 receptor antagonists.14 But evidence to date
indicates that LT modulators can provide symptomatic
relief of inflammatory conditions rather than decrease
process attenuation or reversal.14 Dual cyclooxygenase
and lipoxygenase inhibitors such as BW755C efficiently
inhibit long-lasting ovalbumin-induced eosinophil re-
cruitment in rats,15 but none are in clinical development
and targeted for the treatment of asthma. Among PAF
antagonists, WEB2086, a potent and specific compound,
was experimented in treatment of asthma; but unfor-
tunately it suffered from a short duration of action.12
Allergen-induced eosinophilia could also be inhibited by
phosphodiesterase (PDE) inhibitors; CDP840, a potent
Two of the cytokines, secreted by T-cells in response
to antigen stimulation, are considered to have a par-
ticular important role in atopic allergic disease: inter-
leukin 4 (IL-4) and interleukin 5 (IL-5).
IL-4 and, to a lesser extent, IL-13 play a crucial role
in the regulation of immunoglobulin E (IgE) synthesis
by B-lymphocytes, in normal conditions and in allergic
diseases. The activation effect of IL-4, which requires
physical contact between (CD4+)T-cells and B-cells, is
promoted by IL-5 and IL-6 but inhibited notably by
interferons (IFN)-R and -γ and transforming growth
factor â.1,2 Binding of antigens to specific IgE on mast
cells or basophiles leads to the cross-linking of IgE
receptors, resulting in cell activation and the release of
numerous inflammatory mediators, such as cytokines
(IL-4, IL-5, TNFR), leukotrienes, and histamine.3 These
mediators contribute to bronchoconstriction and re-
cruitement of inflammatory leucocytes and lymphocytes
in atopic disorders.
There is now convincing evidence that activation of
allergen-specific helper (CD4+) T-lymphocytes of the
Th-2 subset induces release of cytokines, including
interleukins 3 and 5 and granulocyte macrophage
* Address correspondence to this author at Department of Organic
Chemistry and Medicinal Chemistry, Faculty of Pharmacy, 1 rue G.
Veil, 44035 Nantes Cedex 01, France. Phone: (33) 2-40-41-28-71. Fax:
(33) 2-40-41-28-76.
† Faculty of Pharmacy, Nantes.
‡ ASTA Medica, Germany.
10.1021/jm981079+ CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/29/1999

New N-(pyridin-4-yl)-(indol-3-yl)acetamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 639
Sch em e 1a
a
Reagents and conditions: (a) (1) ethyl levulinate, CH3COONa, 1 M CH3COOH; (2) H2SO4, EtOH, reflux, 3 h; (b) (1) 12 M HCl, NaNO2,
0 °C, 0.5 h; (2) diethyl 2-acetylglutarate (n ) 1), ethyl 2-oxocyclopentane carboxylate (n ) 2), EtOH, 6 M KOH, 0 °C, then room temperature
for 2 h; (3) H2SO4, EtOH, reflux, 12 h; (c) 6 M NaOH, EtOH, reflux, 1 h; (d) 1 M HCl-EtOH, reflux, 1.5 h; (e) Cu2Cr2O5, quinoline, reflux,
2 h; (f) NaH, DMSO, R1CH2Cl, room temperature for 12 h; (g) 4-iodofluorobenzene, Cu bronze, K2CO3, PhBr, reflux, 24 h; (h) 2-chloro-
1-methylpyridinium iodide, TEA, 4-aminopyridine, CH2Cl2, reflux, 2 h; (i) with 83, BBr3, CH2Cl2, -80 °C, 1 h, then room temperature for
12 h.
novel stereoselective inhibitor of PDE IV,16 which is
predominant in eosinophils, prevents allergen-induced
bronchospasm; but the clinical use of these compounds
could be limited by gastrointestinal side effects, such
as increased acid secretion, nausea, and vomiting.17
Glucocorticoids are highly effective against eosinophil
pulmonary infiltration,18,19 but they often produce sig-
nificant side effects, especially when administered by
oral route in severe asthma. The selective regulation of
IL-4 and IL-5 may therefore offer an alternative ap-
proach to the control of eosinophilic bronchial inflam-
mation in asthma.
In previous works, we reported on the synthesis and
biological evaluation of N-(4,6-dimethylpyridinyl-2-yl)-
heteroaryl carboxamides and acetamides as a novel type
of nonacidic antiinflammatory agents.20 Although the
corresponding indolylalkylamides exhibited only moder-
ate anti-edematous effect, to our surprise, results of
pharmacological screening tests revealed that some of
these compounds significantly inhibited IL-4, IL-5 bio-
synthesis and/or histamine release. These data and the
fact that indole constitutes the central core of numerous
potent LTD4 antagonists,21-23 PAF antagonists,24 or
inhibitors of FLAP,12 prompted us to carry out an
extensive pharmacomodulation in this series. We report
here the results of in vitro and in vivo investigations in
the N-(pyridin-4-yl)-(indol-3-yl)alkylamides 44-84 (Table
3).
Ch em istr y
Preparation of the target (indol-3-yl)alkanoates under
Fischer conditions is depicted in Scheme 1. The inter-
mediate phenylhydrazones were prepared by condensa-
tion of the corresponding substituted phenylhydrazines
with ethyl levulinate (method A) or under J app-Klinge-
mann reaction conditions25 (method B) from substituted
anilines and diethyl 2-acetylglutarate (n ) 1) or ethyl
2-oxocyclopentane carboxylate (n ) 2) as keto com-
pounds.26 The phenylhydrazones were cyclized without
purification to yield ethyl (2-methylindol-3-yl)acetates
1-4 and the indole diesters 5-13, respectively. In-
dolization of 3-methoxyphenylhydrazone afforded a
mixture of 4-and 6-methoxyindole derivatives (1:8)
which were separated by silica gel chromatography. The
hydrolysis of the diesters 5-13, followed by monoes-
terification and C-2 decarboxylation by the couple Cu2-

640 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Menciu et al.
Ta ble 1. Physical and Synthetic Data for Ethyl
(Indol-3-yl)alkanoates 17-25
Ta ble 2. Physical and Synthetic Data for N-Substituted Ethyl
(Indol-3-yl)alkanoates 26-43
no.
R
n
yield (%)a
formula
mp (°C) solvent
method
mp (°C)
solvent
no.
n
R
R1
R2 yield (%)
formula
17 5-OCH3
18 5-OCH3
19 5-Cl
20 5-Cl
21 5-F
1
2
1
2
1
2
1
2
1
16
28
15
22
18
25
14
17
12
C13H15NO3
C14H17NO3
C12H12ClNO2
C13H14ClNO2
C12H12FNO2
C13H14FNO2
C15H19NO2
C16H21NO2
C13H15NO3
90b
74b
90b
75b
76b
54b
46b
41b
55c
26
1
H
H
H
H
4-FPh
H
C
79
D
60
D
75
D
66
C
60
D
70
D
72
D
69
C
85
D
69
D
70
D
72
D
80
D
52
D
65
D
C
C
C
C
18H16FNO2
96a
62a
56a
46a
98a
61a
63a
65a
110a
65a
61a
62c
69c
85c
66c
58c
81c
102a
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
1
2
3
1
2
2
1
1
1
2
1
2
1
2
2
1
1
4-FBn
4-FBN
4-FBn
H
19H18FNO2
20H20FNO2
21H22FNO2
22 5-F
H
23 5-i-Pr
24 5-i-Pr
25 6-OCH3
H
5-OCH3 4-FPh
5-OCH3 Bn
H
C19H18FNO3
C21H23NO3
C21H22FNO3
a
Overall yield after the four-step sequence (steps b-e, Scheme
b
1). Petroleum ether. c Diisopropyl ether.
H
Cr2O5/quinoline, afforded the desired ethyl (indol-3-yl)-
alkanoates 17-25 (Table 1). Ethyl (indol-3-yl)alkanoates
14, 15, 16 (n ) 1, 2, 3) were obtained by refluxing the
corresponding commercially available acids in HCl
ethanol (Scheme 1). N-substitution of indole esters 1-4
and 14-25 was carried out either with 4-iodofluoroben-
zene under Ullmann conditions, using Cu/K2CO3 in
refluxing bromobenzene (method C), or with benzyl
halogenides after metalation with NaH in DMSO
(method D), affording the esters 26-43 in fair yields
(Table 2). These esters failed to undergo aminolysis with
4-aminopyridine, even in the presence of a catalytic
amount of 2-hydroxypyridine.27 On the contrary, activa-
tion of the corresponding acids, resulting from alkaline
hydrolysis of the esters, by reaction with 2-chloro-1-
methylpyridinium iodide (method E) afforded the target
N-(pyridin-4-yl)-(indol-3-yl)alkylamides 44-46 and 48-
83 in moderate to excellent yields; the N-unsubstituted
4-(indol-3-yl) butanamide 47 was obtained by the same
method, starting from the corresponding commercially
available acid (Table 3). Last, demethylation of the
methoxy compound 83 was carried out by BBr3 (method
F), in a 71% yield, leading to the 6-hydroxy derivative
84.
5-OCH3 4-FBn
H
5-Cl
5-F
4-FBn
4-FPh
Bn
H
C
C
C
C
C
C
19H17ClFNO2
18H15F2NO2
19H18FNO2
20H20FNO2
19H17F2NO2
20H19F2NO2
H
5-F
H
5-F
Bn
H
5-F
4-FBn
Bn
H
5-F
H
5-F
Bn
CH3
H
C20H20FNO2
5-i-Pr
5-i-Pr
5-i-Pr
Bn
C
C
23H27NO2
4-FBn
H
23H26FNO2
68
D
42
D
4-FBn CH3
C23H26FNO2
C20H20FNO3
6-OCH3 4-FBn
H
42
Diisopropyl ether. c Petroleum ether.
a
Among the three types of substituents introduced at
indolic nitrogen (4-fluorophenyl, benzyl, and 4-fluo-
robenzyl), generally speaking, the last one exerted the
most favorable influence; nevertheless the 4-fluorophe-
nyl derivative 67, contrary to 70, was active and
equipotent to 45 (Table 3). Tentative increase or emer-
gence of activity by methylation at C-2 remained unsuc-
cessful in 9 compounds out of 10 experimented; only 74
was more active than its nonmethylated counterpart 70.
Introduction of different substituents at C-5 (OCH3, Cl,
F, i-Pr) exerted a detrimental effect (44 vs 51 and 45
vs 61, 70, 78); on the contrary, activity was maintained
by a methoxy group fixed at C-6 in 83 (IC50 ) 9.8 µM).
Increasing of the alkanoate chain of indolyl acetamide
45 (leading to 46, n ) 2) reduced IL-4 inhibition, and
no marked increase of this activity was observed in 48
(n ) 3); so further pharmacomodulation at this level was
limited to n ) 1 and 2. Replacement of the acetamide
chain by a propanamide one did not increase IL-4
inhibition, except in the 5-F and 5-Cl subseries: com-
pounds 58 and 69. Nevertheless, even in the acetamide
subseries, inhibition remained moderate and only 4
compounds (45, 67, 74, 83) exhibited IC50 in the range
of 8-10 µM (Table 4). Although these amides showed 3
orders of magnitude weaker activity against IL-4 ex-
Str u ctu r e-Activity Rela tion sh ip s
Antiallergic activity was first evaluated by three in
vitro assays: inhibition of IL-4 and IL-5 production in
Th-2 cells and inhibition of ovalbumin-induced hista-
mine release in guinea pig peritoneal mast cells. Results
expressed in percent inhibition for IL-4 and IL-5 and
by IC50 for histamine antagonism are shown in Table
3. IC50 toward IL-4 and IL-5 inhibition are displayed in
Table 4 for the most potent compounds. Among these,
actetamide 45 was selected for further evaluation in
three complementary assays: inhibition of ionomycin-
induced histamine release, inhibition of acetylcholine
hyperreactivity of rat trachea induced by tetraethylam-
monium chloride, and inhibition of carbachol-induced
contraction of isolated guinea pig trachea.
IL-4 In h ibition . It was previously established that
amides issued from 2- and 3-aminopyridines were
regularly less active than their 4-pyridinyl congeners:
none of them exhibited an inhibition percentage greater
than 35% at 10 µM; this statement prompted us to limit
pharmacomodulation to the last subseries.

New N-(pyridin-4-yl)-(indol-3-yl)acetamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 641
Ta ble 3. N-(Pyridin-4-yl)-ω-(indol-3-yl)alkylamides 44-84
IL-4
IL-5
histamine
b
% inhibitiona
% inhibitiona
IC50
no.
n
R
R1
R2
at 10 µM
at 10 µM
(µM)
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
1
1
2
3
3
1
2
1
1
2
2
1
1
1
2
1
2
1
2
1
1
1
2
1
1
2
1
2
1
1
1
1
H
H
H
H
H
4-FPh
4-FBn
4-FBn
H
4-FBn
H
H
4-FPh
Bn
Bn
4-FBn
H
4-FBn
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
CH3
CH3
CH3
H
H
H
31
63
2.9
20
31
NA
NA
24
48
100
22
NA
42
NA
26
0.302
0.016
0.220
2.9
0.51
>10
NT
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-F
5-F
5-F
5-F
5-F
22
38
NT
33
2.80
3.900
NT
NA
NA
NA
NA
NA
26
NA
NA
NA
22
NA
25
49
NA
54
41
NA
26
NA
25
71
48
42
43
37
NA
41
61
29
28
NT
5.00
4.500
1.460
NT
0.74
0.230
1.30
>10
NT
>10
2.210
NT
0.060
0.420
NT
0.380
10.13
0.480
0.40
4.92
3.120
6.000
NT
5.340
2.800
7.54
6.900
0.330
0.940
b
Bn
Bn
39
NA
NA
NA
NA
28
NA
NA
51
4-FBn
4-FBn
H
4-FBn
H
H
4-FPh
Bn
NA
27
Bn
5-F
5-F
5F
5-F
4-FBn
4-FBn
H
Bn
4-FBn
H
H
Bn
4-FBn
4-FBn
H
NA
NA
NA
NA
49
5F
5-i-Pr
5-i-Pr
5-i-Pr
5-i-Pr
5-i-Pr
5-i-Pr
5-i-Pr
5-i-Pr
6-OCH3
6-OH
33
2
2
1
2
1
1
1
1
NA
NA
NA
NA
NA
NA
NA
51
25
23
35
NA
34
NA
76
37
H
H
CH3
CH3
CH3
H
Bn
4-FBn
4-FBn
4-FBn
1
H
29
66
astemizole
74
a
b
Mean of three individual experiments each performed in duplicate ((SEM); NA, no activity; NT, not tested. IC50 (µM) of reference
drugs. Astemizole: 6.5 ( 1.3; Terfenadine: 4.7 ( 0.1. Cyclosporin A: 1.1 ( 0.4.
Ta ble 4. IL-4 and IL-5 Production Inhibition of Compounds
45, 61, 67, 74, and 83
inhibited more regularly and more efficiently IL-5
production: 13 acetamides (instead of 5) possessed
percent inhibition greater than 40%, at 10 µM (Table
3). The presence of a methyl group at C-2 induced the
same deleterious effect, except in 74. Introduction of
methoxy and isopropyl groups at C-5 resulted in a
marked decrease of activity; on the other hand, the 5-Cl,
5-F, and 6-OCH3 derivatives 59, 61, 74, and 83, al-
though less active than the non-homocycle-substituted
congener 45, exerted significant activity at 10 µM, and
the 5-F derivative 67 was nearly twice as active as its
5-H counterpart 44. Although 2 orders of magnitude less
active than dexamethasone and cyclosporin A (Table 4),
[1-(4-fluorobenzyl)indol-3-yl]acetamides 45 and 83 showed
higher potency than astemizole: IC50 ) 1.50, 2.28, and
3.30 µM, respectively.
IL-4
IC50 (µM)
IL-5
IC50 (µM)
no.
45
61
67
74
8.00
>10
9.70
10.20
9.80
1.50
8.80
4.56
7.87
2.28
3.30
3.96
0.0035
0.242
83
astemizole
terfenadine
dexamethasone
cyclosporin A
8.00
4.95
0.0047
0.073
pression than dexamethasone (IC50 ) 4.7 nM) and
cyclosporin A (IC50 ) 73 nM), they were as active as
astemizole (IC50 ) 8.0 µM).
IL-5 In h ibition . The stucture-activity profile of
these N-pyridinyl alkylcarboxamides does not markedly
vary between IL-4 and IL-5 assays. Nevertheless, they
H ist a m in e R elea se In h ib it ion . Among the 32
amides tested in the ovalbumin-induced histamine

642 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Menciu et al.
Ta ble 5. Inhibition of Late Phase Eosinophilia in
Bronchoalveolar Lavage (BAL) of Guinea Pigs after Parenteral
or Oral Administration of 45 and Zardaverine
Ta ble 6. In Vivo Activity of 45 in Rhinitis Model: Inhibition of
Ovalbumin-Induced Rhinitis in Anesthetized Rats after Topical,
Intravenous, or Oral Administration of 45
% inhibition
administration route
compd
IC50 (µM) ID50 (µg/kg)
administration
route
dose
2 h before 4 h after
topical application
(nasal perfusion)
45
0.25
1.26
7.41
5a
compd
(mg/kg) challenge challenge
antazoline
mepyramine
45
IP
IP
IP
PO
PO
45
45
10
5
5
30
10
59
NI
41
45
28
60
IV
PO
NIa
NI
35
460b
zardaverine
45
zardaverine
a
b
Administered 30 min before challenge. Administered 60 min
before challenge.
67
a
NI: no inhibition.
cant inhibition (∼30%) 2 h before and 4 h after chal-
lenge, at a dose of 30 mg/kg.
release assay, 12 compounds possessed IC50 in the
submicromolar range (Table 3). In line with precedent
observations about optimal chain length, propanamide
46 and butyramide 48 were 14-fold and 32-fold less
efficacious than acetamide 45; the same phenomenon
was observed, in the 5-Cl and 5-F subseries, by com-
parison of propanamides 62 and 69 with their respective
acetamides 61 and 68. As observed for IL-4 and IL-5
inhibition, removal of the ether functionality of 83,
leading to phenol 84, exerted a deleterious effect: IC50
) 0.330 and 0.940 µM. Comparison with astemizole
points to the very high potency of acetamides 45 and
68 which were 406-fold and 108-fold more efficient than
the reference histamine antagonist: IC50 ) 0.016 and
0.060 and 6.5 µM, respectively.
The interest of 45, as histamine release inhibitor, was
confirmed by its high activity in the ionomycin-induced
histamine release inhibition: IC50 ) 0.011 µM. In the
same conditions, IC50 of disodium cromoglycate (DSCG),
used as a reference drug, was >1 µM.
In h ibition of Acetylch olin e Hyp er r ea ctivity. In-
hibition by 45 of tetraethylammonium chloride (TEA)-
induced acetylcholine hyperreactivity was carried out
in rat tracheal segments. It exerted a potent inhibitory
In h ibition of Aller gica lly In d u ced or Ca p sa icin -
In d u ced Rh in itis. On the first rhinitis model, 45
possessed a very potent inhibitory activity after topical
administration: IC50 ) 0.25 µM, (Table 6). With regard
to this mainly histamine-mediated phenomenon, it was
markedly more efficient than were classical antihista-
mines such as antazoline and mepyramine, whose IC50
were 5-fold and 30-fold higher than that of 45. Its
inhibitory activity was confirmed after iv administration
at a very low dose: ID50 ) 5 µg/kg. Although markedly
less active per os, due likely to poor oral bioavailability,
its ID50 remained nevertheless inferior to 0.5 mg/kg.
This effect, in line with in vitro data, indicates that 45
could also be able to inhibit histamine release in vivo.
Surprisingly, 45 was roughly 10-times more potent
than morphine in the inhibition of the capsaicin-induced
increase in nasal vascular permeability of rats, after
topical application: IC50 ) 0.3 and 4.1 µM, respectively.
Capsaicin stimulates the sensory c fibers, resulting in
the release of substance P (SP) which is responsible for
the increased vascular leakage. Morphine inhibits the
capsaicin-induced SP release by stimulation of its pre-
synaptic receptor located on c fiber terminals. Such a
mechanism for 45 might be excluded because there is
large evidence that capsaicin does not influence the
histamine content in the guinea pig nasal mucosa.28
effect at a very low micromolar concentration: IC50
)
0.021 µM, i.e., at a 60-fold lower concentration than
verapamil (IC50 ) 1.3 µM).
In h ibition of Ca r ba ch ol-In d u ced Con tr a ction .
Relaxation of carbachol-precontracted guinea pig tra-
chea, induced by 45, was very moderate at the high
concentration of 100 µM (36%); higher concentrations
(up to 500 µM) did not cause further increase in the
relaxatory activity of 45. This weak inhibitory effect on
the carbachol-induced contraction can be considered as
unspecific.
Con clu sion
We prepared a series of N-(4-pyridinyl)-(indol-3-yl)
acetamides and propanamides and conducted the struc-
ture-activity relationship studies on their inhibitory
activities toward IL-4 and IL-5 production in Th-2 cells
and histamine release from mast cells. The results may
be summarized as follows: (1) inhibitory activity was
increased by placement of 4-fluorophenyl or (4-fluoro)-
benzyl groupings at indolic nitrogen; (2) generally
speaking, the presence of a methyl group at the C-2
position of indole was detrimental; (3) the incorporation
of an acetamide chain at C-3 provided the optimal
potency, and (4) introduction of halogen, isopropyl,
methoxy, or hydroxy groups at C-5 or C-6 failed to
improve potency.
Biologica l P r op er ties of Com p ou n d 45
In vivo efficacy of 45 in allergic disorders was con-
firmed by carrying out two assays: inhibition of late
phase eosinophilia in actively sensitized and challenged
guinea pigs and inhibition of rhinitis in sensitized and
challenged rats or induced by capsaicin in naive rats.
Inhibition of allergically induced late phase eosino-
philia. The results are summarized in Table 5. Com-
pound 45 significantly decreased the eosinophil number
in bronchoalveolar lavage of ovalbumin-treated guinea
pigs, after parenteral and oral administration. After ip
administration of 5 mg/kg, percent inhibition, 4 h after
challenge, was comparable to that of zardaverine: 41
and 45%, respectively. Although less potent than zar-
daverine after oral administration, 45 induced signifi-
Indolylacetamide 45 is an extremely strong inhibitor
of the allergically induced histamine release from peri-
toneal mast cells; morever, it was able to inhibit the IL-4
and IL-5 biosynthesis with IC50 values in low micromo-
lar ranges. In keeping with its in vitro profile, 45
potently inhibited the mainly histamine-mediated in-
crease of allergically induced vascular permeability in
the nasal cavity of actively sensitized rats and the
allergen-induced late phase eosinophilia in actively

New N-(pyridin-4-yl)-(indol-3-yl)acetamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 643
cooled to 0 °C. NaNO2 (5.1 g, 73.9 mmol) in H2O (30 mL) was
added dropwise so that the temperature remained below 3 °C.
Separately was prepared a mixture of ethyl 2-oxocyclopentane
carboxylate (10.5 g, 67.5 mmol) in ethanol (75 mL), ice (125
g), and 4 M KOH (60 mL). The solution of diazonium salt was
added to this mixture with stirring. After 2 h, the mixture was
extracted with diethyl ether (3 × 100 mL). The combined
organic layers were washed with brine, dried (Na2SO4), and
concentrated to leave a dark oil which was added to a solution
of sulfuric acid (10 mL) in absolute ethanol (100 mL). After
refluxing for 12 h, the mixture was concentrated and dichlo-
romethane (200 mL) was added, washed with 10% Na2CO3 and
brine. The organic layer was dried (Na2SO4), concentrated, and
purified by chromatography on silica gel, eluting with dichlo-
romethane to give 10 (6.2 g, 28%) as white crystals: mp 73
°C; 1H NMR (DMSO-d6) δ 1.14 (t, 3H, J ) 7.1 Hz), 1.28 (d,
6H, J ) 6.9 Hz), 1.38 (t, 3H, J ) 7.1 Hz), 2.61 (t, 2H, J ) 7.8
Hz), 2.99 (m, 1H, J ) 6.9 Hz), 3.32 (t, 2H, J ) 7.8 Hz), 4.04 (q,
2H, J ) 7.1 Hz), 4.37 (q, 2H, J ) 7.1 Hz), 7.21 (d, 1H, J ) 8.5
Hz), 7.37 (d, 1H, J ) 8.5 Hz), 7.49 (s, 1H), 11.48 (s, 1H). Anal.
(C19H25NO4) C, H, N.
sensitized guinea pigs. Although no direct study of the
mechanism of action of compound 45 was carried out
up to now, it could be assumed that this amide is liable
to down regulate Ca2+ concentration in lymphocytes,
insofar as enhancing intracellular Ca2+ concentration
with the ionophor (A23187) induces an incrase of IL-4
production.29 In the same way, histamine release inhibi-
tion could be linked to a decrease in intracellular Ca2+
concentration via interaction with the chloride channel,
like DSCG.30 Finally, literature data dealing with TEA-
induced acetylcholine hyperreactivity,31 as well as cap-
saicin-induced rhinitis32 could also suggest the involve-
ment of acetamide 45 in the regulation of Ca2+-activated
ion channels.
The present results suggest that 45 may prove to be
a valuable candidate as lead structure for further
synthetic work aimed at reducing IL-4 and IL-5 release
and thereby IgE biosynthesis production. This conclu-
sion is in agreement with recent literature data on the
high IgE biosynthesis suppressive activity of N(R-
carboxyphenyl)phenylacetamides.33
Step 2: 3-(2-Ca r boxy-5-isop r op ylin d ol-3-yl)p r op a n oic
a cid . Diester 10 (6.2 g, 18.8 mmol) was dissolved in ethanol
(150 mL). 6 M NaOH (20 mL) was added, and the mixture
was refluxed for 1 h. After evaporation to a volume of 50 mL,
1 M HCl was added, up to pH 6 and the mixture was extracted
with ethyl acetate (3 × 50 mL). The organic layers were dried
(Na2SO4) and concentrated to provide the 3-(2-carboxy-5-
isopropylindol-3-yl)propanoic acid (4.9 g, 95%) as white crys-
Exp er im en ta l Section
A. Ch em ica l Syn th esis. Melting points were determined
in open glass capillaries and are uncorrected. 1H NMR spectra
were recorded on a Bruker AC 250 spectrometer (250 MHz)
(Bruker, Wissenbourg, France), using DMSO-d6 as solvent;
chemical shifts are reported in parts per million (ppm) relative
to tetramethylsilane as an internal standard. IR spectra were
run with KBr pellets with a Beckman IR 4230 grating infrared
spectrophotometer (Beckman, Gagny, France). Microanalyses
were performed on a Perkin-Elmer CHN 240 apparatus.
Elemental analyses were within 0.4% of the calculated value.
Silica gel 60 (E. Merck, 230-400 mesh) was used for prepara-
tive column chromatography. Diethyl ether was freshly dis-
tilled from sodium benzophenone. Reagents were obtained
commercially (Aldrich Chemical Co.) and used without further
purification. Chemical yields reported are unoptimized specific
examples of one preparation. The following ethyl (5-substituted-
2-methylindol-3-yl)acetates have been previously described:
5-F,34 5-Cl,35 and 5-OCH3.36 The ethyl (indol-3-yl)alkanoates
(n ) 1, 2, 3) 14, 15, and 16 were obtained by HCl-ethanol es-
terification of the corresponding acids, purchased from Aldrich
Chemical Co. The acids of the following ethyl esters have been
previously synthesized starting from â-ketoesters or ethyl
γ-oxobutyrate: 17,37 18,38 20,38 22,39 2537 and 19,40 21.41
Eth yl (5-Isopr opyl-2-m eth ylin d ol-3-yl)a ceta te (4). 4-Iso-
propylphenylhydrazine hydrochloride (7.3 g, 39.1 mmol) was
dissolved on heating in a solution of sodium acetate (5.9 g, 43
mmol) in 1 M CH3COOH (80 mL). Ethyl levulinate (6.7 g, 46.9
mmol) was added, and after cooling the precipitated hydrazone
was filtered, washed with water, and dried in vacuo to provide
10.2 g of crude hydrazone. The hydrazone was then added to
a solution of sulfuric acid (10 mL) in absolute ethanol (100
mL) and heated at reflux for 3 h. The mixture was concen-
trated to a volume of 50 mL, the pH was adjusted to pH 6
with 1 M NaOH, and the mixture was extracted with dichlo-
romethane (2 × 100 mL). The organic layer was dried (Na2-
SO4) and concentrated. The residue was purified by chroma-
tography on silica gel, eluting with dichloromethane to give 4
(7.6 g, 75%) as beige crystals: mp 38 °C; 1H NMR (DMSO-d6)
δ 1.22 (t, 3H, J ) 7.0 Hz), 1.27 (d, 6H, J ) 7.0 Hz), 2.37 (s,
3H), 2.98 (m, 1H, J ) 7.0 Hz), 3.68 (s, 2H), 4.04 (q, 2H, J )
7.0 Hz), 6.96 (dd, 1H, J ) 8.7 Hz, 2.1 Hz), 7.22 (d, 1H, J ) 8.7
1
tals: mp 181 °C; H NMR (DMSO-d6) δ 1.28 (d, 6H, J ) 6.9
Hz), 2.53 (t, 2H, J ) 7.3 Hz), 2.99 (m, 1H, J ) 6.9 Hz), 3.28 (t,
2H, J ) 7.3 Hz), 7.18 (dd, 1H, J ) 8.6 Hz, 1.4 Hz), 7.35 (d, 1H,
J ) 8.6 Hz), 7.51 (d, 1H, J ) 1.4 Hz), 11.35 (s, 1H), 12.51 (m,
2H). Anal. (C15H17NO4) C, H, N.
Step 3: Eth yl 3-(2-Ca r boxy-5-isop r op ylin d ol-3-yl)p r o-
p a n oa te. A solution of 3-(2-carboxy-5-isopropylindol-3-yl)-
propanoic acid (4.9 g, 17.8 mmol) in 1 M HCl in ethanol (50
mL) was refluxed for 1.5 h. After evaporation to a volume of
20 mL, the mixture was cooled and filtered. The crystals were
washed with diisopropyl ether to give ethyl 3-(2-carboxy-5-
isopropylindol-3-yl)propanoate (4.3 g, 79%) as white crystals:
mp 186 °C; 1H NMR (DMSO-d6) δ 1.21 (t, 3H, J ) 7.1 Hz),
1.27 (d, 6H, J ) 6.8 Hz), 2.56 (t, 2H, J ) 7.5 Hz), 2.98 (m, 1H,
J ) 6.8 Hz), 3.29 (t, 2H, J ) 7.5 Hz), 4.02 (q, 2H, J ) 7.1 Hz),
7.18 (d, 1H, J ) 8.6 Hz,), 7.35 (d, 1H, J ) 8.6 Hz), 7.48 (s,
1H), 11.53 (s, 1H), 13.01 (s, 1H). Anal. (C17H21NO4) C, H, N.
Step 4: Eth yl 3-(5-Isop r op ylin d ol-3-yl)p r op a n oa te (24).
To a solution of ethyl 3-(2-carboxy-5-isopropylindol-3-yl)-
propanoate (4.3 g, 14.1 mmol) in quinoline (80 mL), copper
chromite (0.8 g, 2.6 mmol) was added and the mixture was
refluxed under N2 for 2 h. After cooling, diethyl ether (200 mL)
was added and the mixture was filtered. The filtrate was
washed with 2 M HCl (3 × 100 mL) and brine, then dried (Na2-
SO4), filtered, concentrated, and purified by chromatography
on silica gel, eluting with dichloromethane to provide 24 (3.0
g, 81%) as beige crystals: mp 41 °C; 1H NMR (DMSO-d6) δ
1.22 (t, 3H, J ) 7.1 Hz), 1.27 (d, 6H, J ) 6.9 Hz), 2.63 (t, 2H,
J ) 7.8 Hz), 2.95 (t, 2H, J ) 7.8 Hz), 4.11 (q, 2H, J ) 7.1 Hz),
7.02 (dd, 1H, J ) 8.7 Hz, 1.7 Hz), 7.23 (d, 1H, J ) 1.7 Hz),
7.30 (d, 1H, J ) 8.4 Hz), 7.36 (s, 1H), 10.84 (s, 1H). Anal.
(C16H21NO2) C, H, N.
Compounds 17-23 and 25 were prepared in a similar
manner; see Table 1.
Gen er a l P r oced u r e for P r ep a r a tion of Eth yl N-(4-
F lu or op h en yl)-(in d ol-3-yl)a ceta tes (Meth od C). Eth yl
[1-(4-F lu or op h en yl)-5-flu or oin d ol-3-yl]a ceta te (34). To a
solution of ethyl (5-fluoroindol-3-yl)acetate 21 (3.9 g, 17.6
mmol) in bromobenzene (70 mL) was added 4-iodofluoroben-
zene (4.3 g, 19.3 mmol), anhydrous K2CO3 (4.9 g, 35.2 mmol),
and Cu bronze (2 g). The mixture was refluxed under N2 for
24 h. After cooling and filtration, the filtrate was concentrated
and purified by chromatography on silica gel, eluting with
dichloromethane to provide 34 (4.7 g, 85%) as beige crystals:
mp 110 °C; 1H NMR (DMSO-d6) δ 1.25 (t, 3H, J ) 7.1 Hz),
Hz), 7.28 (d, 1H, J ) 2.1 Hz), 10.71 (m, 1H). Anal. (C16H21
NO2) C, H, N.
-
Gen er a l P r oced u r es for P r ep a r a tion of Eth yl ω-(in d ol-
3-yl)alkan oates. Eth yl 3-(5-Isopr opylin dol-3-yl)pr opan oate
(24) (Meth od B). Step 1: Eth yl 3-(2-Eth oxyca r bon yl-5-
isop r op ylin d ol-3-yl)p r op a n oa t e (10). 4-Isopropylaniline
(9.1 g, 67.5 mmol) was dissolved in 12 M HCl (35 mL) and

644 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Menciu et al.
3.86 (s, 2H), 4.15 (q, 2H, J ) 7.1 Hz), 7.09 (dd, 1H, J ) 9.2
Hz, 2.6 Hz), 7.41-7.67 (m, 6H), 7.70 (s, 1H). Anal. (C18H15F2-
NO2) C, H, N.
Compounds 26 and 30 were prepared in a similar manner;
see Table 2.
) 8.0 Hz), 7.57-7.60 (m, 3H), 8.43 (d, 2H, J ) 6.3 Hz), 10.30
(s, 1H). Anal. (C24H22FN3O) C, H, N.
N-(P yr id in -4-yl)-(5-m eth oxyin d ol-3-yl)a ceta m id e (49).
Meth od E: 50% yield; mp 166 °C; 1H NMR (DMSO-d6) δ 3.77
(s, 3H), 3. 81 (s, 2H), 6.76 (dd, 1H, J ) 8.7 Hz, 2.5 Hz), 7.13
(d, 1H, J ) 2.5 Hz), 7.26 (s, 1H), 7.28 (d, 1H, J ) 8.7 Hz), 7.63
(dd, 2H, J ) 5.0, 1.5 Hz), 8.44 (d, 2H, J ) 5.0, 1.5 Hz), 10.54
(s, 1H), 10.83 (m, 1H). Anal. (C16H15N3O2) C, H, N.
Gen er a l P r oced u r e for P r ep a r a tion of Eth yl N-Ben -
zyla ted ω-(In d ol-3-yl)a lk a n oa tes (Meth od D). Eth yl [1-(4-
F lu or oben zyl)-6-m eth oxyin d ol-3-yl]a ceta te (43). To a sus-
pension of NaH (0.3 g, 12.5 mmol) in dry DMSO (40 mL), under
N2, at room temperature, was carefully added a solution of
ethyl (6-methoxyindol-3-yl)acetate 25 (2.3 g, 9.7 mmol) in
DMSO (40 mL), and the mixture was stirred for 0.5 h.
4-Fluorobenzyl chloride (1.4 g, 9.7 mmol) was added, and
reaction was completed overnight. H2O (300 mL) was added,
and the mixture was extracted with diethyl ether (3 × 80 mL).
The organic layers were washed with brine, dried (Na2SO4),
concentrated, and purified by chromatography on silica gel,
eluting with dichloromethane to provide 43 (1.4 g, 42%) as
beige crystals: mp 102 °C; 1H NMR (DMSO-d6) δ 1.20 (t, 3H,
J ) 7.1 Hz), 3.72 (s, 2H), 3.78 (s, 3H), 4.10 (q, 2H, J ) 7.1 Hz),
5.35 (s, 2H), 6.72 (dd, 1H, J ) 8.6 Hz, 2.2 Hz), 7.04 (d, 1H, J
) 2.2 Hz), 7.17 (dd, 2H, J ) 8.8 Hz), 7.26 (s, 1H), 7.30 (dd,
N -(P y r id in -4-y l)-3-(5-m e t h o x y in d o l-3-y l)p r o p a n a -
1
m id e (50). Meth od E: 52% yield; mp 140-141 °C; H NMR
(DMSO-d6) δ 2.76 (t, 2H, J ) 7.9 Hz), 3.03 (t, 2H, J ) 7.9 Hz),
3.78 (s, 3H), 6.74 (dd, 1H, J ) 8.7, 2.4 Hz), 7.07 (d, 1H, J )
2.4 Hz), 7.12 (d, 1H, J ) 2.2 Hz), 7.25 (d, 1H, J ) 8.7 Hz),
7.63 (d, 2H, J ) 5.0 Hz), 8.45 (d, 2H, J ) 5.0 Hz), 10.42 (s,
1H), 10.65 (m, 1H). Anal. (C17H17N3O2) C, H, N.
N-(P yr id in -4-yl)-[1-(4-flu or op h en yl)-5-m eth oxyin d ol-3-
yl]a ceta m id e (51). Meth od E: 79% yield; mp 218 °C; 1H
NMR (DMSO-d6) δ 3.80 (s, 3H), 3.87 (s, 2H), 6.87 (dd, 1H, J )
8.9, 2.5 Hz), 7.23 (d, 1H, J ) 2.5 Hz), 7.39-7.61 (m, 6H), 7.61
(d, 2H, J ) 6.0 Hz), 8.45 (d, 2H, J ) 6.0 Hz), 10.60 (s, 1H).
Anal. (C22H18FN3O2) C, H, N.
N-(P yr id in -4-yl)-(1-ben zyl-5-m eth oxyin d ol-3-yl)a ceta -
m id e (52). Meth od E: 30% yield; mp 145 °C; 1H NMR
(DMSO-d6) δ 3.77 (s, 3H), 3. 81 (s, 2H), 5.38 (s, 2H), 6.78 (dd,
1H, J ) 8.8, 2.5 Hz), 7.15 (d, 1H, J ) 2.5 Hz), 7.20-7.36 (m,
6H), 7.41 (s, 1H), 7.62 (d, 2H, J ) 6.2 Hz), 8.44 (d, 2H, J ) 6.2
Hz), 10.56 (s, 1H). Anal. (C23H21N3O2) C, H, N.
2H, J ) 8.8 Hz, 5.7 Hz), 7.42 (d, 1H, J ) 8.6 Hz). Anal. (C20H20
FNO3) C, H, N.
-
Compounds 27, 28, 29, 31, 32, 33, and 35-42 were prepared
in a similar manner; see Table 2.
Gen er a l P r oced u r e for P r ep a r a tion of N-(P yr id in -4-
yl)-ω-(in d ol-3-yl)a lk a n a m id es (Meth od E). N-(P yr id in -4-
yl)-[1-(4-flu or oben zyl)-6-m eth oxyin dol-3-yl]acetam ide (83).
To a solution of [1-(4-fluorobenzyl)-6-methoxyindol-3-yl]acetic
acid (1.2 g, 3.8 mmol) in dichloromethane (100 mL) were added
triethylamine (1.0 g, 9.5 mmol), 2-chloro-1-methylpyridinium
iodide (1.16 g, 4.5 mmol), and 4-aminopyridine (0.36 g, 3.8
mmol). The mixture was refluxed for 2 h and, after cooling,
washed with H2O, dried (Na2SO4), concentrated and purified
by chromatography on silica gel, eluting with dichloromethane/
ethanol (97:3) to provide 83 (1.1 g, 71%) as yellow crystals:
N-(P yr id in -4-yl)-3-(1-ben zyl-5-m eth oxyin d ol-3-yl)p r o-
1
p a n a m id e (53). Meth od E: 60% yield; mp 135 °C; H NMR
(DMSO-d6) δ 2.75 (t, 2H, J ) 7.0 Hz), 3.04 (t, 2H, J ) 7.0 Hz),
3.78 (s, 3H), 5.32 (s, 2H), 6.76 (dd, 1H, J ) 9.0, 2.5 Hz), 7.10-
7.32 (m, 8H), 7.61 (d, 2H, J ) 4.5 Hz), 8.44 (d, 2H, J ) 4.5
Hz), 10.32 (s, 1H). Anal. (C24H23N3O2) C, H, N.
N-(P yr id in -4-yl)-3-[1-(4-flu or oben zyl)-5-m eth oxyin d ol-
3-yl]p r op a n a m id e (54). Meth od E: 81% yield; mp 144 °C;
1H NMR (DMSO-d6) δ 2.75 (t, 2H, J ) 7.0 Hz), 3.05 (t, 2H, J
) 7.0 Hz), 3.78 (s, 3H), 5.32 (s, 2H), 6.76 (dd, 1H, J ) 8.9, 2.1
Hz), 7.02-7.22 (m, 5H), 7.27 (s, 1H), 7.32 (d, 1H, J ) 8.9 Hz),
7.60 (d, 2H, J ) 4.5 Hz), 8.45 (d, 2H, J ) 4.5 Hz), 10.32 (s,
1H). Anal. (C24H22FN3O2) C, H, N.
N-(P yr id in -4-yl)-(5-m eth oxy-2-m eth ylin d ol-3-yl)a ceta -
m id e (55). Meth od E: 50% yield; mp 203 °C; 1H NMR (DMSO-
d6) δ 2.39 (s, 3H), 3.72 (s, 2H), 3.74 (s, 3H), 6.66 (dd, 1H, J )
8.2, 2.0 Hz), 7.07 (d, 1H, J ) 2.0 Hz), 7.16 (d, 1H, J ) 8.5 Hz),
7.61 (d, 2H, J ) 4.5 Hz), 8.43 (d, 2H, J ) 4.5 Hz), 10.48 (s,
1H), 10.72 (s, 1H). Anal. (C17H17N3O2) C, H, N.
1
mp 152 °C; H NMR (DMSO-d6) δ 3.77 (s, 2H), 3.75 (s, 3H),
5.36 (s, 2H), 6.72 (dd, 1H, J ) 8.6, 1.9 Hz), 7.04 (d, 1H, J )
1.9 Hz), 7.17 (dd, 2H, J ) 8.8 Hz), 7.27 (s, 1H), 7.30 (dd, 2H,
J ) 8.8, 5.9 Hz), 7.49 (d, 1H, J ) 8.6 Hz), 7.60 (d, 2H, J ) 6.2
Hz), 8.43 (d, 2H, J ) 6.2 Hz), 10.52 (s, 1H). Anal. (C23H20
FN3O2) C, H, N.
-
N-(P yr id in -4-yl)-[1-(4-flu or op h en yl)in d ol-3-yl]a cet a -
m id e (44). Meth od E: 67% yield; mp 152 °C; 1H NMR
(DMSO-d6) δ 3.92 (s, 2H), 7.15-7.74 (m, 9H), 7.62 (d, 2H, J )
6.0 Hz), 8.45 (d, 2H, J ) 6.0 Hz), 10.62 (s, 1H). Anal. (C21H16
FN3O) C, H, N.
-
N-(P yr id in -4-yl)-[1-(4-flu or oben zyl)-5-m eth oxy-2-m eth -
ylin d ol-3-yl]a ceta m id e (56). Meth od E: 72% yield; mp 162
1
N-(P yr id in -4-yl)-[1-(4-flu or ob en zyl)in d ol-3-yl]a cet a -
°C; H NMR (DMSO-d6) δ 2.37 (s, 3H), 3.76 (s, 3H), 3.79 (s,
1
m id e (45). Meth od E: 71% yield; mp 140-142 °C; H NMR
2H), 5.38 (s, 2H), 6.71 (dd, 1H, J ) 8.7 2.5 Hz), 7.09-7.19 (m,
5H), 7.30 (d, 1H, 8.7 Hz), 7.61 (dd, 2H, J ) 4.7 Hz, 1.5 Hz),
(DMSO-d6) δ 3.81 (s, 2H), 5.38 (s, 2H), 7.02 (dd, 1H, J ) 7.2
Hz), 7.08-7.17 (m, 3H), 7.27 (dd, 2H, J ) 8.8, 5.6 Hz), 7.43 (s,
1H), 7.45 (d, 1H, J ) 8.5 Hz), 7.59 (d, 2H, J ) 5.4 Hz), 7.61 (d,
1H, J ) 8.1 Hz), 8.41 (d, 2H, J ) 5.4 Hz), 10.53 (s, 1H). Anal.
(C22H18FN3O) C, H, N.
8.44 (dd, 2H, J ) 4.7, 1.5 Hz), 10.54 (s, 1H). Anal. (C24H22
FN3O2) C, H, N.
-
N-(P yr id in -4-yl)-(5-ch lor oin d ol-3-yl)a cet a m id e (57).
1
Meth od E: 40% yield; mp 200 °C dec; H NMR (DMSO-d6) δ
3.80 (s, 2H), 7.10 (d, 1H, J ) 9.0 Hz), 7.39 (s, 1H), 7.41 (d, 1H,
J ) 9.0 Hz), 7.60 (d, 2H, J ) 4.5 Hz), 7.67 (s, 1H), 8.44 (d, 2H,
J ) 4.5 Hz), 10.53 (s, 1H), 11.20 (s, 1H) Anal. (C15H12ClN3O)
C, H, N.
N-(P yr id in -4-yl)-3-[1-(4-flu or ob en zyl)in d ol-3-yl]p r o-
1
p a n a m id e (46). Meth od E: 53% yield; mp 124 °C; H NMR
(DMSO-d6) δ 2.77 (t, 2H, J ) 7.2 Hz), 3.08 (t, 2H, J ) 7.2 Hz),
5.36 (s, 2H), 7.03-7.16 (m, 4H), 7.22 (dd, 2H, J ) 8.6 Hz, 5.4
Hz), 7.32 (s, 1H), 7.45 (d, 1H, J ) 8.0 Hz), 7.60 (d, 2H, J ) 6.0
Hz), 7.63 (d, 1H, J ) 8.0 Hz), 8.45 (d, 2H, J ) 6.0 Hz), 10.33
(s, 1H). Anal. (C23H20FN3O) C, H, N.
N-(P yr idin -4-yl)-3-(5-ch lor oin dol-3-yl)pr opan am ide (58).
Meth od E: 65% yield; mp 188 °C; 1H NMR (DMSO-d6) δ 2.75
(t, 2H, J ) 7.0 Hz), 3.05 (t, 2H, J ) 7.0 Hz), 7.10 (dd, 1H, J )
8.5 Hz, 1.5 Hz), 7.26 (s, 1H), 7.39 (d, 1H, J ) 8.5 Hz), 7.60 (d,
2H, J ) 4.5 Hz), 7.66 (d, 1H, J ) 1.5 Hz), 8.45 (d, 2H, J ) 4.5
Hz), 10.34 (s, 1H), 11.05 (s, 1H). Anal. (C16H14ClN3O) C, H, N.
N-(P yr id in -4-yl)-(1-b en zyl-5-ch lor oin d ol-3-yl)a cet a -
m id e (59). Meth od E: 62% yield; mp 138 °C; 1H NMR
(DMSO-d6) δ 3.82 (s, 2H), 5.43 (s, 2H), 7.12-7.53 (m, 8H), 7.60
(d, 2H, J ) 5.0 Hz), 7.70 (s, 1H), 8.44 (d, 2H, J ) 5.0 Hz),
10.56 (s, 1H). Anal. (C22H18ClN3O) C, H, N.
N-(P yr id in -4-yl)-4-(in d ol-3-yl)bu tyr a m id e (47). Meth od
1
E: 71% yield; mp 91 °C; H NMR (DMSO-d6) δ 2.01 (m, 2H),
2.45 (t, 2H, J ) 7.5 Hz), 2.77 (t, 2H, J ) 7.5 Hz), 6.69 (dd, 1H,
J ) 7.9 Hz), 7.09 (dd, 1H, J ) 7.9 Hz), 7.17 (d, 1H, J ) 2.1
Hz), 7.37 (d, 1H, J ) 7.9 Hz), 7.56 (d, 1H, J ) 7.9 Hz), 7.60 (d,
2H, J ) 6.3 Hz), 8.43 (d, 2H, J ) 6.3 Hz), 10.30 (s, 1H), 10.82
(m, 1H). Anal. (C17H17N3O) C, H, N.
N-(P yr idin -4-yl)-4-[1-(4-flu or oben zyl)-in dol-3-yl]bu tyr a-
m id e (48). Meth od E: 73% yield; mp 56 °C; 1H NMR (DMSO-
d6) δ 2.01 (m, 2H), 2.46 (t, 2H, J ) 7.4 Hz), 2.78 (t, 2H, J )
7.4 Hz), 5.37 (s, 2H), 7.03 (dd, 1H, J ) 8.0 Hz), 7.09-7.19 (m,
3H), 7.28 (dd, 2H, J ) 8.5, 5.6 Hz), 7.33 (s, 1H), 7.44 (d, 1H, J
N-(P yr idin -4-yl)-3-(1-ben zyl-5-ch lor oin d ol-3-yl)p r op a n -
a m id e (60). Meth od E: 70% yield; mp 160 °C; 1H NMR
(DMSO-d6) δ 2.74 (t, 2H, J ) 7.2 Hz), 3.06 (t, 2H, J ) 7.2 Hz),
5.38 (s, 2H), 7.09-7.25 (m, 6H), 7.40 (s, 1H), 7.46 (d, 1H, J )

New N-(pyridin-4-yl)-(indol-3-yl)acetamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 645
8.5 Hz), 7.58 (d, 2H, J ) 6.0 Hz), 7.69 (d, 1H, J ) 1.8 Hz),
8.44 (d, 2H, J ) 6.0 Hz), 10.31 (s, 1H). Anal. (C23H20ClN3O) C,
H, N.
(d, 2H, J ) 6.2 Hz), 10.49 (s, 1H), 11.01 (m, 1H). Anal. (C16H14-
FN3O) C, H, N.
N-(P yr id in -4-yl)-(1-b en zyl-5-flu or o-2-m et h ylin d ol-3-
yl)a ceta m id e (73). Meth od E: 61% yield; mp 181 °C; 1H
NMR (DMSO-d6) δ 2.40 (s, 3H), 3.82 (s, 2H), 5.45 (s, 2H), 6.90
(ddd, 1H, J ) 9.1, 2.4 Hz), 7.01-7.43 (m, 7H), 7.61 (d, 2H, J
N-(Pyridin-4-yl)-[5-chloro-1-(4-fluorobenzyl)indol-3-yl]aceta-
mide (61). Method E: 51% yield; mp 150 °C; 1H NMR (DMSO-
d6) δ 3.82 (s, 2H), 5.42 (s, 2H), 7.13-7.21 (m, 3H), 7.30 (dd,
2H, J ) 8.7 Hz, 5.6 Hz), 7.52 (d, 1H, J ) 8.5 Hz), 7.54 (s, 1H),
7.61 (d, 2H, J ) 4.5 Hz), 7.70 (d, 1H, J ) 2.0 Hz), 8.44 (d, 2H,
J ) 4.5 Hz), 10.57 (s, 1H). Anal. (C22H17ClFN3O) C, H, N.
N-(P yr id in -4-yl)-3-[5-ch lor o-1-(4-flu or oben zyl)in d ol-3-
) 4.9 Hz), 8.44 (d, 2H, J ) 4.9 Hz), 10.54 (s, 1H). Anal. (C23H20
FN3O) C, H, N.
-
N-(P yr id in -4-yl)-[5-flu or o-1-(4-flu or ob en zyl)-2-m et h -
ylin d ol-3-yl]a ceta m id e (74). Meth od E: 65% yield; mp 142
1
1
yl]p r op a n a m id e (62). Meth od E: 56% yield; mp 58 °C; H
°C; H NMR (DMSO-d6) δ 2.40 (s, 3H), 3.81 (s, 2H), 5.44 (s,
NMR (DMSO-d6) δ 2.73 (t, 2H, J ) 7.0 Hz), 3.05 (t, 2H, J )
7.0 Hz), 5.37 (s, 2H), 7.06 (dd, 2H, J ) 8.6 Hz), 7. 12 (dd, 1H,
J ) 8.7 Hz, 2.0 Hz), 7.21 (dd, 2H, J ) 8.6 Hz, 5.6 Hz), 7.40 (s,
1H), 7.48 (d, 1H, J ) 8.7 Hz), 7.58 (d, 2H, J ) 5.7 Hz), 7.68 (d,
1H, J ) 2.0 Hz), 8.44 (d, 2H, J ) 5.7 Hz), 10.30 (s, 1H). Anal.
(C23H19ClFN3O) C, H, N.
2H), 6.91 (ddd, 1H, J ) 8.9 Hz, 2.5 Hz), 7.04-7.44 (m, 6H),
7.61 (d, 2H, J ) 6.0 Hz), 8.45 (d, 2H, J ) 6.0 Hz), 10.54 (s,
1H) Anal. (C23H19F2N3O) C, H, N.
N-(P yr id in -4-yl)-(5-isop r op ylin d ol-3-yl)a ceta m id e (75).
Meth od E: 61% yield; mp 175 °C dec; 1H NMR (DMSO-d6) δ
1.26 (d, 6H, J ) 7.0 Hz), 2.96 (m, 1H, J ) 7.0 Hz), 3.78 (s,
2H), 7.02 (dd, 1H, J ) 8.7, 1.5 Hz), 7.25 (d, 1H, J ) 1.5 Hz),
7.30 (d, 1H, J ) 8.7 Hz), 7.45 (s, 1H), 7.61 (dd, 2H, J ) 5.0,
1.5 Hz), 8.44 (d, 2H, J ) 5.0, 1.5 Hz), 10.52 (s, 1H), 10.84 (m,
1H). Anal. (C18H19N3O) C, H, N.
N -(P yr id in -4-yl)-3-(5-isop r op ylin d ol-3-yl)p r op a n a -
m id e (76). Meth od E: 64% yield; mp 134 °C dec; 1H NMR
(DMSO-d6) δ 1.29 (d, 6H, J ) 6.9 Hz), 2.78 (t, 2H, J ) 7.1 Hz),
2.99 (m, 1H, J ) 6.9 Hz), 3.19 (t, 2H, J ) 7.1 Hz), 6.84 (dd,
1H, J ) 8.9, 2.1 Hz), 7.10 (d, 1H, J ) 2.1 Hz), 7.12 (d, 1H, J
) 2.1 Hz), 7.28 (d, 1H, J ) 8.9 Hz), 7.60 (d, 2H, J ) 5.8 Hz),
8.44 (d, 2H, J ) 5.8 Hz), 10.40 (s, 1H), 10.71 (m, 1H). Anal.
(C19H21N3O) C, H, N.
N-(P yr id in -4-yl)-(5-ch lor o-2-m et h ylin d ol-3-yl)a cet a -
m id e (63). Meth od E: 63% yield; mp >250 °C; 1H NMR
(DMSO-d6) δ 2.39 (s, 3H), 3.75 (s, 2H), 7.01 (dd, 1H, J ) 8.5,
2.0 Hz), 7.29 (d, 1H, J ) 8.5 Hz), 7.59-7.62 (m, 3H), 8.44 (d,
2H, J ) 4.5 Hz), 10.50 (s, 1H), 11.12 (s, 1H). Anal. (C16H14
ClN3O) C, H, N.
-
N-(P yr id in -4-yl)-[5-ch lor o-1-(4-flu or ob en zyl)-2-m et h -
ylin d ol-3-yl]a ceta m id e (64). Meth od E: 81% yield; mp 145
1
°C; H NMR (DMSO-d6) δ 2.39 (s, 3H), 3.83 (s, 2H), 5.44 (s,
2H), 7.06-7.19 (m, 5H), 7.46 (d, 1H, J ) 8.5 Hz), 7.61 (d, 2H,
J ) 5.2 Hz), 7.68 (s, 1H), 8.44 (d, 2H, J ) 5.2 Hz), 10.55 (s,
1H). Anal. (C23H19ClFN3O) C, H, N.
N-(P yr id in -4-yl)-(5-flu or oin d ol-3-yl)a cet a m id e (65).
Meth od E: 50% yield; mp 200 °C dec; 1H NMR (DMSO-d6) δ
3.78 (s, 2H), 6.94 (ddd, 1H, J ) 8.7 Hz, 2.5 Hz), 7.34-7.41 (m,
3H), 7.60 (d, 2H, J ) 6.2 Hz), 8.43 (d, 2H, J ) 6.2 Hz), 10.53
(s, 1H), 11.09 (m, 1H). Anal. (C15H12FN3O) C, H, N.
N-(P yr idin -4-yl)-3-(5-flu or oin dol-3-yl)pr opan am ide (66).
Meth od E: 51% yield; mp >250 °C; 1H NMR (DMSO-d6) δ
2.74 (t, 2H, J ) 7.0 Hz), 3.03 (t, 2H, J ) 7.0 Hz), 6.94 (ddd,
1H, J ) 8.5, 1.5 Hz), 7.25 (s, 1H), 7.33-7.39 (m, 2H), 7.60 (d,
2H, J ) 4.5 Hz), 8.44 (d, 2H, J ) 4.5 Hz), 10.33 (s, 1H), 10.92
(s, 1H). Anal. (C16H14FN3O) C, H, N.
N-(P yr id in -4-yl)-3-(1-ben zyl-5-isop r op ylin d ol-3-yl)p r o-
p a n a m id e (77). Meth od E: 50% yield; mp 116 °C; H NMR
1
(DMSO-d6) δ 1.26 (d, 6H, J ) 6.9 Hz), 2.75 (t, 2H, J ) 7.0 Hz),
2.96 (m, 1H, J ) 6.9 Hz), 3.06 (t, 2H, J ) 7.0 Hz), 5.33 (s, 2H),
7.01 (d, 1H, J ) 8.5 Hz), 7.19-7.27 (m, 6H), 7.33 (d, 1H, J )
8.5 Hz), 7.42 (s, 1H), 7.61 (d, 2H, J ) 5.4 Hz), 8.44 (d, 2H, J
) 5.4 Hz), 10.33 (s, 1H). Anal. (C26H27N3O) C, H, N.
N-(P yr id in -4-yl)-[1-(4-flu or oben zyl)-5-isop r op ylin d ol-
3-yl]a ceta m id e (78). Meth od E: 75% yield; mp 88 °C; 1H
NMR (DMSO-d6) δ 1.25 (d, 6H, J ) 7.0 Hz), 2.96 (m, 1H, J )
7.0 Hz), 3.80 (s, 2H), 5.37 (s, 2H), 7.04 (d, 1H, J ) 8.5 Hz),
7.16 (dd, 2H, J ) 8.7 Hz), 7.27-7.47 (m, 5H), 7.61 (d, 2H, J )
N-(P yr id in -4-yl)-[5-flu or o-1-(4-flu or op h en yl)in d ol-3-yl]-
1
6.0 Hz), 8.44 (d, 2H, J ) 6.0 Hz), 10.56 (s, 1H). Anal. (C25H24
FN3O) C, H, N.
-
a ceta m id e (67). Meth od E: 68% yield; mp 186 °C; H NMR
(DMSO-d6) δ 3.90 (s, 2H), 7.09 (ddd, J ) 9.2 Hz, 2.5 Hz), 7.42-
7.67 (m, 6H), 7.62 (d, 2H, J ) 6.1 Hz), 7.71 (s, 1H), 8.45 (d,
2H, J ) 6.1 Hz), 10.61 (s, 1H). Anal. (C21H15F2N3O) C, H, N.
N -(P yr id in -4-yl)-(1-b e n zyl-5-flu or oin d ol-3-yl)a ce t a -
m id e (68). Meth od E: 70% yield; mp 137 °C; 1H NMR (DMSO-
d6) δ 3.81 (s, 2H), 5.43 (s, 2H), 6.99 (dd, 1H, J ) 8.8 Hz), 7.23-
7.50 (m, 7H), 7.54 (s, 1H), 7.61 (d, 2H, J ) 5.2 Hz), 8.45 (d,
2H, J ) 5.2 Hz), 10.57 (s, 1H). Anal. (C22H18FN3O) C, H, N.
N-(P yr id in -4-yl)-3-(1-b en zyl-5-flu or oin d ol-3-yl)p r op i-
on a m id e (69). Meth od E: 80% yield; mp 184 °C; 1H NMR
(DMSO-d6) δ 2.75 (t, 2H, J ) 6.9 Hz), 3.05 (t, 2H, J ) 6.9 Hz),
5.39 (s, 2H), 6.97 (dd, 1H, J ) 8.8 Hz), 7.14-7.44 (m, 8H),
7.60 (d, 2H, J ) 5.5 Hz), 8.45 (d, 2H, J ) 5.5 Hz), 10.32 (s,
1H). Anal. (C23H20FN3O) C, H, N.
N-(P yr idin -4-yl)-3-[1-(4-flu or oben zyl)-5-isopr opylin dol-
3-yl]p r op a n a m id e (79). Meth od E: 60% yield; mp 107 °C;
1H NMR (DMSO-d6) δ 1.29 (d, 6H, J ) 6.9 Hz), 2.78 (t, 2H, J
) 7.1 Hz), 3.02 (m, 1H, J ) 6.9 Hz), 3.20 (t, 2H, J ) 7.1 Hz),
5.17 (s, 2H), 6.91 (d, 1H, J ) 8.9 Hz), 7.12-7.42 (m, 5H), 7.44
(s, 1H), 7.61 (d, 2H, J ) 5.5 Hz), 8.44 (d, 2H, J ) 5.5 Hz),
10.42 (s, 1H). Anal. (C26H26FN3O) C, H, N.
N-(P yr id in -4-yl)-(2-m eth yl-5-isop r op ylin d ol-3-yl)a ceta -
m id e (80). Meth od E: 75% yield; mp 208 °C; 1H NMR (DMSO-
d6) δ 1.24 (d, 6H, J ) 7.0 Hz), 2.39 (s, 3H), 2.96 (m, 1H, J )
7.0 Hz), 3.73 (s, 2H), 6.92 (dd, 1H, J ) 8.2, 2.0 Hz), 7.18 (d,
1H, J ) 8.2 Hz), 7.37 (d, 1H, J ) 2.0 Hz), 7.61 (d, 2H, J ) 4.5
Hz), 8.44 (d, 2H, J ) 4.5 Hz), 10.49 (s, 1H), 10.74 (s, 1H). Anal.
(C19H21N3O) C, H, N.
N-(P yr id in -4-yl)-[5-flu or o-1-(4-flu or oben zyl)in d ol-3-yl]-
1
a ceta m id e (70). Meth od E: 60% yield; mp 156 °C; H NMR
N-(P yr id in -4-yl)-(1-ben zyl-2-m eth yl-5-isop r op ylin d ol-
1
(DMSO-d6) δ 3.80 (s, 2H), 5.42 (s, 2H), 7.00 (ddd, 1H, J ) 9.0,
2.5 Hz), 7.17 (dd, 2H, J ) 8.9 Hz), 7.31 (dd, 2H, J ) 8.9 Hz,
5.7 Hz), 7.40 (dd, 1H, J ) 9.0, 2.5 Hz), 7.49 (dd, 1H, J ) 9.0,
4.4 Hz), 7.54 (s, 1H), 7.61 (d, 2H, J ) 4.5 Hz), 8.44 (d, 2H, J
) 4.5 Hz), 10.56 (s, 1H). Anal. (C22H17F2N3O) C, H, N.
N-(P yr id in -4-yl)-3-[5-flu or o-1-(4-flu or oben zyl)in d ol-3-
yl]p r op a n a m id e (71). Meth od E: 58% yield; mp 131 °C; 1H
NMR (DMSO-d6) δ 2.73 (t, 2H, J ) 7.1 Hz), 3.03 (t, 2H, J )
7.1 Hz), 5.36 (s, 2H), 6.97 (dd, J ) 8.7 Hz), 7.06 (dd, 2H, J )
8.9 Hz), 7.22 (dd, 2H, J ) 8.9, 5.6 Hz), 7.38-7.57 (m, 3H), 7.59
(d, J ) 6.2 Hz), 8.44 (d, 2H, J ) 6.2 Hz), 10.31 (s, 1H). Anal.
(C23H19F2N3O) C, H, N.
3-yl)a ceta m id e (81). Meth od E: 70% yield; mp 164 °C; H
NMR (DMSO-d6) δ 1.25 (d, 6H, J ) 7.0 Hz), 2.38 (s, 3H), 2.95
(m, 1H, J ) 7.0 Hz), 3.82 (s, 2H), 5.40 (s, 2H), 6.87 (dd, 1H, J
) 8.5, 1.5 Hz), 7.05 (d, 1H, J ) 8.5 Hz), 7.21-7.35 (m, 5H),
7.46 (d, 1H, J ) 1.5 Hz), 7.62 (d, 2H, J ) 4.7 Hz), 8.44 (d, 2H,
J ) 4.7 Hz), 10.55 (s, 1H). Anal. (C26H27N3O) C, H, N.
N-(P yr id in -4-yl)-[1-(4-flu or oben zyl)-2-m eth yl-5-isop r o-
p ylin d ol-3-yl]a ceta m id e (82). Meth od E: 79% yield; mp 172
°C; 1H NMR (DMSO-d6) δ 1.26 (d, 6H, J ) 7.0 Hz), 2.39 (s,
3H), 2.98 (m, 1H, J ) 7.0 Hz), 3.81 (s, 2H), 5.41 (s, 2H), 6.86
(d, 1H, J ) 8.4 Hz), 7.10-7.41 (m, 6H), 7.60 (d, 2H, J ) 5.4
Hz), 8.44 (d, 2H, J ) 5.4 Hz), 10.40 (s, 1H). Anal. (C25H24FN3O)
C, H, N.
N-(P yr id in -4-yl)-(5-flu or o-2-m et h ylin d ol-3-yl)a cet a -
1
m id e (72). Meth od E: 64% yield; mp 200 °C dec; H NMR
Meth od F . N-(P yr id in -4-yl)-[1-(4-flu or oben zyl)-6-h y-
d r oxyin d ol-3-yl]a ceta m id e (84). A solution of acetamide 83
(1.9 g, 4.9 mmol) in dichloromethane (45 mL) was cooled to
(DMSO-d6) δ 2.42 (s, 3H), 3.73 (s, 2H), 6.85 (ddd, 1H, J ) 8.5
Hz, 2.4 Hz), 7.23-7.33 (m, 2H), 7.61 (d, 2H, J ) 6.2 Hz), 8.44

646 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Menciu et al.
-80 °C under N2. 1 M Boron tribromide in dichloromethane
(12.25 mL) was added dropwise. After removal of the cooling
bath, the mixture was stirred overnight. A saturated solution
of NaHCO3 was added slowly up to pH 7, cooling with an ice
bath. The mixture was washed with H2O, dried (Na2SO4),
concentrated, and purified by chromatography on silica gel,
eluting with dichloromethane/ethanol (95:5) to provide 6-hy-
droxy derivative 84 (0.77 g, 42%) as beige crystals: mp 213
The effects of the test compounds on anti-CD3 mAb induced
IL-4 and IL-5 production were tested at 10 µM and are
depicted in Table 3. Inhibition of IL-4 and IL-5 production by
the most potent compounds was determined over a range of
concentrations and the corresponding estimated IC50 values
are listed in Table 4.
In h ibition of Hista m in e Relea se. In vitro study of
inhibition of allergically induced histamine release was carried
out with rat peritoneal mast cells by test compounds. Spra-
gue-Dawley rats were sensitized by sc injection of 30 mg of
whole egg white (WEW) and killed Bordetella pertussis organ-
isms (5 × 1010); 4 weeks later, peritoneal cells were harvested.
The cells were centrifuged and suspended in Tris-Gel CM
buffer of the following composition (mM): Tris, 25; NaCl, 120;
KCl, 5; CaCl2, 0.6; MgCl2, 1; and 0.01% gelatin; pH 7.6. The
reaction mixture containing 2 × 106 peritoneal cells (2-6%
mast cells) and phosphatidylserine (10 µg/mL, known to
enhance allergic histamine release) was preincubated in the
presence of the drug in polypropylene tubes at 37 °C for 15
min. After antigen challenge (WEW, 50 µg/mL), the cell
suspensions were incubated for an additional 30 min at 37 °C
and were then centrifuged at 2000 rpm for 5 min at 4 °C.
Histamine in the supernatant is measured by a fluometric
method.42 Histamine release, corrected for the spontaneous
release, is expressed as a percentage of the total cell content.
Total histamine from a separate, duplicate cell suspension is
released by boiling for 10 min. Histamine release, induced by
whole egg white, is assayed in the presence and absence of
drug. Test compounds were first screened at the fixed concen-
tration of 10 µM (data not shown) and then at 4-6 increasing
concentrations for the calculation of the half-maximal inhibi-
tion (IC50).
1
°C; H NMR (DMSO-d6) δ 3.74 (s, 2H), 5.27 (s, 2H), 6.59 (dd,
1H, J ) 8.3 Hz, 1.9 Hz), 6.70 (d, 1H, J ) 1.9 Hz), 7.17 (dd, 2H,
J ) 8.8 Hz), 7.21 (s, 1H), 7.22 (dd, 2H, J ) 8.8, 5.9 Hz), 7.39
(d, 1H, J ) 8.3 Hz), 7.59 (d, 2H, J ) 6.1 Hz), 8.43 (d, 2H, J )
6.1 Hz), 9.01 (s, 1H), 10.51 (s, 1H). Anal. (C22H18FN3O2) C, H,
N.
B. Biologica l Tests. IL-4 a n d IL-5 Relea se In h ibition .
Complete medium was RPMI 1640 containing HEPES (Boe-
hringer Mannheim, Germany) supplemented with 2 mM
glutamine (Boehringer Mannheim), 50 µM mercaptoethanol
(Sigma Chemie GmbH, Germany), 100 IU/mL penicillin/100
µg/mL streptomycin (Biochrom, Germany), and 10% fetal calf
serum (Boehringer Mannheim). Rat T-STIM was purchased
from Collaborative Biomedical Products (Beldford, MA), Cy-
closporin A (Sandimmun ad Infus.) was obtained from Sandoz
AG, Nu¨rnberg, Germany. Dimethylsufoxyde (DMSO), dexam-
ethasone and mitomycin C were obtained from Sigma Chemie
GmbH.
The hamster anti-mouse-CD3 mAb (145-2C11) was obtained
from Cedar Lane Laboratories Hornby, Ontario, Canada.
P r ep a r a t ion of Syn gen eic Sp len ocyt es. Spleens from
male AKR/J (H-2k) mice (Charles River, Wiga Laboratories,
Germany) were aseptically removed and minced with scissors
and gently pressed through a No. 60 sieve. Cells were collected
by centrifugation. The cell pellet was resuspended in 5 mL of
lysing buffer (8.29 g/L NH4Cl, 1.0 g/L KHCO3, 0.037 g/L EDTA)
and incubated for 2-5 min to lyse the red cells. Medium was
added (45 mL) and spun down. The cell pellet was resuspended
in phosphate buffered saline (PBS) containing 50 µg/mL
mitomycin C with a cell concentration of 2 × 107 cells/mL and
incubated for 30 min at 37 °C. Immediately the cells were
diluted with PBS and washed exhaustively.
Cell Cu ltu r e. The Th-2 clone D10.G4.1 (D10) is specific for
conalbumin in the context of H-2k and was obtained from
American Type Culture Collection (ATCC), Rockville, MD. It
was stimulated every 10 days with the antigen conalbumin
(100 µg/mL) and mitomycin C treated syngeneic splenocytes
as antigen presenting cells (APC). The ratio of D10 to APC
was 1:4. As a source of lymphokines, 5% Rat T-STIM was
added to the culture medium. After several cycles of restimu-
lation and 3 days after the last stimulation with antigen and
APC, several freezes were made, and all experiments reported
here were performed with this batch of cells. After thawing,
cells were cultured for 3 days in complete medium with Rat
T-STIM.
Exp osition of Cells a n d Stim u la tion by An ti-CD3 m Ab.
Cells were cultivated in 96-well microtiter plates (4 × 104 cells/
well/100 µL) for 3.5 h in complete medium without Rat
T-STIM. Stock solutions of the various drugs were made in
DMSO (1000 times concentrated vs final concentration) and
subsequent dilutions were made in complete medium. Fifty
microliters of drug solutions and solvent, respectively (4 times
concentrated), were added to the cultures and incubated for
30 min. Cells were stimulated by application of 50 µL of anti-
CD3 mAb (final dilution 1:100) and further incubated for 16
h. Supernatants were centrifuged and stored at -70 °C until
estimation of their cytokine contents.
In the ionomycin-induced histamine release assay, perito-
neal mast cells were obtained from naive animals. The final
concentration of ionomycin was 0.300 µM.
In h ibition of TEA-In d u ced Hyp er r ea ctivity to Acetyl-
ch olin e in Ra t Tr a ch ea l Segm en ts. Male Wistar rats (200-
250 g) obtained from Charles River Wiga, Sulzfeld, (Germany)
were sacrificed by CO2 inhalation, and the tracheas were taken
out. Tracheal segments (rings, 2-3 cartilages) were prepared
and set up in organ baths under isotonic conditions with 1 g
of preload in Krebs-Henseleit solution at 37 °C gassed with
carbogen. After an equilibration period of 30 min, the cumula-
tive concentration curve (CRC) for acetylcholine (0.01 µM to 1
M) was determined. After the sample was washed and rested,
another CRC for acetylcholine in the presence of TEA (10 mM,
30 min) was established. Under these conditions TEA-induced
hyperreactivity was about 30-50% at 100 µM acetylcholine.
After the sample was washed and rested, another CRC for
acetylcholine was estimated in the presence of the test drug
(30 min equilibration before TEA) and TEA (30 min equilibra-
tion before acetylcholine). Millimeter contraction (mean and
standard deviation) for 100 µM acetylcholine from 3 to 5
preparations was calculated and percent inhibition of hyper-
reactivity calculated by the following formula
(A - C) - (B - C)/(A - C) × 100 ) percent inhibition
where A ) millimeters of contraction for 100 µM acetylcholine
in the presence of TEA alone, B ) millimeters of contraction
for 100 µM acetylcholine in the presence of TEA and test drug,
and C ) millimeters of contraction for 100 µM acetylcholine
alone.
Usually the screening concentration of the drug is 30 µM.
If more than 50% inhibition is achieved, the IC50 value is
determined from 3 to 4 concentrations by regression analysis.43
R ela xa t ion of Ca r ba ch ol-P r econ t r a ct ed Gu in ea P ig
Tr a ch ea . Guinea pigs (250-400 g) obtained from Charles-
River Wiga (Sulzfeld, Germany) are stunned by a blow on the
neck and exsanguinated. Tracheas were taken out, freed from
surrounding tissue, cut into spirals, and set up in organ baths
in Tyrode solution, under isotonic conditions with preload of
2 g. The temperature was 37 °C, and the bath was gassed with
carbogen. After washing and equilibration period of 30 min,
carbachol (0.164 µM) is injected to give contraction. After a
Deter m in a tion of Cytok in e Relea se. The supernatants
were assayed for their cytokine contents by using the com-
mercially available ELISA kits for murine IL-4 and IL-5,
respectively (Endogen Inc., Boston, MA).
Resu lts. Sixteen hours following stimulation with anti-CD3
mAb D, 10 cells produced 461 ( 54 pg IL-5/mL and 5210 (
1319 pg IL-4/mL. Without stimulation no cytokine levels were
detectable. DMSO, in concentrations up to 0.1%, affects neither
IL-4 nor IL-5 production (data not shown).

New N-(pyridin-4-yl)-(indol-3-yl)acetamides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 647
stable contraction is achieved (3-4 equal spasms ( 10% from
the mean), isoprenaline is injected cumulatively every 5 min
until full relaxation is achieved. After the sample was washed
and rested, carbachol is given, and then the cumulative
concentration response curve for the test compound is esti-
mated. Percentage relaxation for each concentration is calcu-
lated. IC50 values for the reference drug salbutamol (0.050 µM)
and the test drug were calculated from 3 to 5 preparations in
parallel by regression analysis.43
In h ibition of Aller gica lly In d u ced La te P h a se Eosi-
n op h ilia . Male Dunkin-Hartley guinea pigs (200-250 g)
obtained from Charles-River Wiga (Sulzfeld, Germany) were
actively sensitized with two ip injections of a suspension of 20
µg of ovalbumin (OVA) (Sigma, Germany) + 20 mg of Al(OH)3
in 0.5 mL of saline, on two consecutive days. Fourteen days
after the second injection, animals were preteated with me-
pyramine (10 mg/kg, ip) to protect against lethal anaphylaxis.
Thirty minutes later, guinea pigs were placed in a chamber
and exposed to an OVA aerosol for 30 s. The OVA aerosol was
In h ibition of Ca p sa icin -In d u ced Rh in itis in An esth e-
tized Ra ts. Male Sprague Dawley rats (280-320 g) obtained
from Charles-River Wiga (Sulzfeld, Germany) were used. The
experimental protocol was identical to that described previ-
ously. The only difference was that the increase in the nasal
vascular permeability was induced by a PBS solution contain-
ing 0.5 mg/mL capsaicin (Sigma, Germany). Morphine was
used as the reference compound.
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