Carbamazepine derivatives with P2X4 receptor-blocking activity

Article abstract of DOI:10.1016/j.bmc.2013.12.035

Antagonists for the P2 receptor subtype P2X4, an ATP-activated cation channel receptor, have potential as novel drugs for the treatment of neuropathic pain and other inflammatory diseases In the present study, a series of 47 carbamazepine derivatives including 32 novel compounds were designed, synthesized, and evaluated as P2X4 receptor antagonists Their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor was determined Additionally, species selectivity (human, rat, mouse) and receptor subtype selectivity (P2X4 vs P2X1, 2, 3, 7) were investigated for selected derivatives The most potent compound of the present series, which exhibited an allosteric mechanism of P2X4 inhibition, was N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide (34, IC₅₀ of 3.44 μM) The present study extends the so far very limited knowledge on structure-activity relationships of P2X4 receptor antagonists

Full text of DOI:10.1016/j.bmc.2013.12.035

Bioorganic & Medicinal Chemistry 22 (2014) 1077–1088
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbamazepine derivatives with P2X4 receptor-blocking activity
Maoqun Tian, Aliaa Abdelrahman, Stephanie Weinhausen, Sonja Hinz, Stefanie Weyer, Stefan Dosa,
⇑
Ali El-Tayeb , Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Antagonists for the P2 receptor subtype P2X4, an ATP-activated cation channel receptor, have potential as
novel drugs for the treatment of neuropathic pain and other inflammatory diseases. In the present study,
a series of 47 carbamazepine derivatives including 32 novel compounds were designed, synthesized, and
evaluated as P2X4 receptor antagonists. Their potency to inhibit ATP-induced calcium influx in 1321N1
astrocytoma cells stably transfected with the human P2X4 receptor was determined. Additionally, spe-
cies selectivity (human, rat, mouse) and receptor subtype selectivity (P2X4 vs P2X1, 2, 3, 7) were inves-
tigated for selected derivatives. The most potent compound of the present series, which exhibited an
allosteric mechanism of P2X4 inhibition, was N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide
Received 15 November 2013
Revised 15 December 2013
Accepted 16 December 2013
Available online 25 December 2013
Keywords:
Heterocycles
Inflammation
Ligand-gated ion channel
Negative allosteric modulator
Neuropathic pain
P2X4 receptor
(34, IC50 of 3.44
lM). The present study extends the so far very limited knowledge on structure–activity
relationships of P2X4 receptor antagonists.
Ó 2013 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Synthesis
Tricyclics
1. Introduction
channel pore, which is caused by the flexing of the lower body do-
main and expanding the region of the receptor proximal to the ion
channel pore.6
Purinergic receptors, which were first defined in 1976 by Burn-
stock, are cell-surface proteins activated by ATP and its metabo-
lites.1,2 There are two major families of purinergic receptors, P1
and P2, activated by extracellular adenosine and ATP/ADP, respec-
tively.3,4 The P2 receptors can be further subdivided into two sub-
groups, ATP-gated cation channels (P2X) and G protein-coupled
receptors (P2Y). Seven P2X receptor subunits (P2X1–P2X7) have
been identified in mammalian cells, which form homo- or het-
ero-trimeric channels.3 The recently published X-ray structures
of the zebrafish P2X4 receptor represent the very first crystal struc-
tures of a P2X receptor.5,6 A chalice-shaped trimeric structure, pre-
viously suggested by Nicke et al.7 was confirmed. Each subunit is
composed of a large rigid ectodomain, two continuous transmem-
P2X receptors are widely distributed throughout many tissues,
and play key physiological roles in nerve transmission, pain sensa-
tion and inflammation.3 The P2X4 subtype is expressed in the cen-
tral nervous system as well as peripheral tissues, for example in
microglia, in testis, colon and endothelial cells.3 After peripheral
nerve injury, microglia in the dorsal horn of the spinal cord be-
comes activated and upregulates the expression of the P2X4 recep-
tors, which has been identified as an important determinant of
neuropathic pain.8–11
P2X4 receptor knockout mice showed decreased inflammatory
and neuropathic pain and reduced allodynia.12 Therefore, P2X4
receptor antagonists are of great interest as potential new drugs
for the treatment of neuropathic pain, a severe chronic condition,
which is difficult to treat with current analgesic drugs. Further-
more, recent evidence has accumulated that P2X4 antagonists
may also be useful for the treatment of other inflammatory dis-
eases including diabetic nephropathy,13 joint inflammation and
arthritis,14 spinal cord injury,15 and the prevention of excitotoxic
damages after epileptic seizures.16
brane
a
-helical segments and intracellular termini.5 The crystal
structure of the P2X4 receptor in complex with ATP reveals three
equivalent intersubunit ATP-binding sites, which are 40 Å from
the extracellular boundary of the transmembrane domain and lo-
cate at each of the three pairs of subunit interfaces in the trimeric
receptor.6 ATP binding promotes an iris-like opening of the ion
However, only few P2X4 antagonists are known so far that
allow blockade of P2X4 receptor signaling, and all of them show
only moderate potency and/or are lacking selectivity.17–19 The
⇑
Corresponding author. Tel.: +49 228 73 2301; fax: +49 228 73 2567.
E-mail address: christa.mueller@uni-bonn.de (C.E. Müller).
A. E.-T. is on leave from the University of Al-Azhar, Assiut, Egypt.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.035

1078
M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
antidepressant drug paroxetine (I) represents one of the few re-
ported noncompetitive P2X4 antagonists, which displayed an IC50
of around 2 lM in calcium flux studies at both human and rat
reaction of dibenz[b,f]azepine with the corresponding chlorofor-
mates in the absence of solvent at 90–110 °C yielded the desired
carbamates 5–7 in moderate to good yields.
P2X4 receptors (Fig. 1).20 However, a subsequent investigation by
Khakh’s group suggested an indirect reduction of P2X4 responses
by paroxetine in a microglial cell line, which may be attributed
to interfering with lysosomal trafficking and a subsequent down-
regulation of P2X4 receptor expression.21 Recently, N-substituted
phenoxazine derivatives have been developed by our group as po-
tent P2X4 receptor antagonists.22 The published data represent the
only structure–activity relationship (SAR) study of P2X4 antago-
nists reported to date. N-(Benzyloxycarbonyl)phenoxazine (II),
The urea derivatives 9–31 were synthesized by the reaction of
dibenz[b,f]azepine-5-carbonyl chloride (8) with the corresponding
primary or secondary aliphatic amines in dichloromethane at room
temperature, or 40 °C, respectively, in good yields. Reactions with
aromatic amines were performed at 100–110 °C to achieve reason-
able reaction rates and yields.
In an attempt to synthesize urea derivative 34 with an N,N-
diisopropyl substituent, no desired product was obtained in the
reaction of dibenz[b,f]azepine-5-carbonyl chloride with diisopro-
pylamine, presumably due to the significant steric hindrance of
the bulky N,N-diisopropyl substituent. Notably, the reaction of
dibenz[b,f]azepine with diisopropylcarbamoyl chloride in the pres-
ence of LDA gave 4-substituted dibenz[b,f]azepine derivative 32 as
the main product, most likely via a dilithio intermediate, which
was subsequently trapped by diisopropylcarbamoyl chloride.24 It
should be noted that no desired product, namely the urea deriva-
tive 34, was observed under this reaction condition. The structure
of 32 was confirmed by 1H and 13C NMR spectroscopy, and mass
spectrometric data. Further reaction of 32 with triphosgene and
subsequent treatment with diethylamine yielded urea derivative
33. The substitution pattern of 33 was clearly confirmed by the
nuclear Overhauser effect (NOE) between the ethyl group and
iso-propyl group. In another approach, compound 34 was finally
obtained by the reaction of dibenz[b,f]azepine with diisopropylcar-
bamoyl chloride in refluxing toluene.
which exhibits an IC50 of 0.189 lM at the human P2X4 receptor,
is currently one of the most potent antagonists at human P2X4
receptors.22
With the goal to further explore the SARs of P2X4 receptor
antagonists, and considering the structural similarity of the tricy-
clic heterocycles of phenoxazines (II) and carbamazepine (III), we
planned to design and synthesize a series of carbamazepine deriv-
atives, and evaluate them as P2X4 receptor antagonists. Carbamaz-
epine is a well-known anticonvulsant drug used in the treatment of
epilepsy. It is also used for treating trigeminal neuralgia, as well as
bipolar disorder.23
In the present study, a broad range of functionalities, including
ureas, carbamates and amides, at the N5 position of the
dibenzo[b,f]azepine scaffold of III was explored. Additionally, pal-
ladium ligand-controlled cyclization was employed to synthesize
derivatives containing various substituents on the aryl rings. Fur-
ther derivatives were prepared by functionalization of the 10,11-
double bond and investigated for potential interactions with
P2X4 receptors.
2.1.2. Introduction of substituents on the phenyl ring and
replacement of phenyl by heteroaromatic rings
The introduction of various substituents on the phenyl rings of
the dibenzazepine core structure cannot be easily achieved. The
synthesis of dibenzazepine analogs usually requires harsh, func-
tional group-intolerant conditions, for example gas-phase dehy-
drogenation of iminodibenzyls at high temperatures.25 On the
other hand, functionalization of the tricyclic dibenzazepine core
is usually inefficient and unselective.26 Recently, Pd-ligand con-
trolled cyclization was developed for a general and efficient prep-
aration of functionalized dibenzazepines.27 Employing this
method, various substituents were introduced on the phenyl ring.
The palladium-catalyzed cyclization of 2-bromostyrene and substi-
tuted 2-chloroanilines was performed in refluxing 1,4-dioxane in
the presence of tris(dibenzylideneacetone)dipalladium [Pd2(dba)3],
2-dicyclohexylphosphino-20-(N,N-dimethylamino)biphenyl (Dave-
Phos), and sodium tert-butoxide (NaOt-Bu) to provide substituted
dibenzazepine derivatives in moderate yields (Scheme 2). The urea
derivatives were prepared by further reaction of dibenzazepine
derivatives with sodium isocyanate in good yields.28 However, in
the case of pyridobenzazepine 42, attempts to react it with sodium
isocyanate failed, presumably due to the significant electronic
2. Results and discussion
2.1. Chemistry
2.1.1. Functionalization at the N5 nitrogen atom of the
dibenzazepine core structure
Modification of the lead structure of carbamazepine was initi-
ated by variation of the functionalities at the N5-atom by preparing
ureas, carbamates, and amides, respectively. Amide derivatives 2
and 3 were synthesized by the reaction of dibenz[b,f]azepine (1)
with acid anhydrides, or acyl chlorides, respectively, in good yields
(Scheme 1). Further reduction of the obtained amide with lithium
aluminum hydride (LAH) gave the amine 4.
The first attempt to synthesize carbamate 5 was performed by
the reaction of dibenz[b,f]azepine-5-carbonyl chloride (8) with
methanol in the presence of triethylamine. However, only traces
of the product were observed by liquid chromatography-mass
spectrometry (LC–MS) analysis after the reaction had been per-
formed under reflux for 24 h. In an alternative approach, the
H
N
O
N
11
O
10
9
1
2
O
8
5
N
3
7
O
6
4
O
O
H2N
II
N-(benzyloxycarbonyl)-
phenoxazine
III Carbamazepine
I
Paroxetine
F
human P2X4: IC50 1.87 µM
rat P2X4: IC50 2.45 µM
human P2X4: IC50 0.189 µM
rat P2X4: IC50 2.10 µM
Figure 1. Paroxetine (I), N-(benzyloxycarbonyl)phenoxazine (II) and carbamazepine (III).

M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
1079
e
N
N
N
R1
N
O
R1
Cl
O
R2
4
8
9-31
b
d
a
c
N
N
H
1
N
R1
O
O
R1
2-3
O
5-7
h
f
g
N
H
N
N
O
O
N(i-Pr)2
(Et)2N
(i-Pr)2N
O
(i-Pr)2N
34
O
33
32
cpd.
2
3
4
5
6
7
9
10
11
12
13
14
15
16
17
R1
R2
-
-
-
-
cpd.
18
19
20
21
22
23
24
25
26
27
28
29
30
31
R1
R2
methyl
phenyl
phenyl
methyl
i-butyl
benzyl
ethyl
benzyl
methyl
-CH2CH2OCH2CH2-
dimethylaminopropyl
i-propyl
-CH2CH2CH2CH2CH2-
-CH2CH2CH2CH2-
mehyl
butyl
propyl
dimethylaminoethyl
t-butyl
1-methylpiperidin-4-yl
hydroxyethyl
-CH2CH2CH(OH)CH2CH2-
4-hydroxycyclohexyl
4-aminocyclohexyl
phenyl
methyl
H
propyl
methyl
H
H
-
-
ethyl
H
H
hydroxyethyl
H
H
H
H
H
H
H
H
H
4-methoxyphenylethyl
cyclohexyl
phenylethyl
pyridin-4-yl
Scheme 1. Substitution of dibenz[b,f]azepine (1) at N5: synthesis of amides, carbamates, ureas and amines. Reagents and conditions: (a) anhydride or acyl chloride, rt to
100 °C, 3 h, yields 71–81%; (b) lithium aluminum hydride (LAH), tetrahydrofuran, (THF), rt, 1 h, yield 26%; (c) chloroformates, 90–110 °C, 3 h, yields 57–91%; (d) triphosgene,
dichloromethane (DCM), 0 °C to rt; (e) R1R2NH, rt to 110 °C, dichloromethane or toluene, 1–4 h, yields 50–95%; (f) lithium diisopropylamide (LDA), diisopropylcarbamoyl
chloride, THF, yield 56%; (g) triphosgene, diethylamine, DCM, yield 76%; (h) diisopropylcarbamoyl chloride, toluene, reflux, yield 55%.
effects of the pyridine ring, which decreases the nucleophilicity of
the N5 atom. Gratifyingly, the urea derivative 50 could be obtained
by the reaction of 42 with diethylamine in the presence of lithium
bis(trimethylsilyl)amide (LHMDS). One of the metabolites of
carbamazepine, namely 3-hydroxy-5H-dibenz[b,f]azepine-5-car-
boxamide (51), was prepared by demethylation of the 3-meth-
oxy-substituted precursor 46 with boron tribromide in 37% yield.29
A starting material 4-O-substituted 2-chloroaniline is not read-
ily available. Therefore, 2-hydroxy-5H-dibenz[b,f]azepine-5-car-
boxamide (54), which is another metabolite of carbamazepine,
was synthesized by an alternative approach (Scheme 3). Oxidation
of 1 with Fremy’s salt followed by the reduction of 52 with sodium
dithionite gave 2-hydroxydibenzazepine 53 in moderate yield.30
Reaction of 53 with sodium isocyanate and subsequent selective
hydrolysis of the intermediate with sodium hydrogencarbonate
yielded 2-hydroycarbamazepine 54. It should be noted that
stronger bases for example sodium carbonate or sodium hydroxide
resulted in extensive hydrolysis of the urea functionality to
regenerate 53.
2.1.3. Functionalization at positions 10 and 11
It is well known that the 10,11-double bond of carbamazepine
is metabolically unstable resulting in several active metabolites.31
In order to get
a deeper insight into the structure–activity
relationships of carbamazepine derivatives at P2X4 receptors, a
few derivatives were to be prepared by functionalization of the
10,11-double bond (Scheme 4). The racemic alcohol 56 was
synthesized by epoxidation of carbamazepine and subsequent
palladium-catalyzed hydrogenation of the epoxide 55. In another
approach, oxidation of 56 gave oxcarbamazepine 57 in 90% yield.
In the reaction of 57 with amines in the presence of NaBH3CN,
Na(CH3COO)3BH, or Ti(OiPr)4/NaBH4, which are the reagents

1080
M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
R1
R1
R2
R3
e
c
R2
R3
N
N
a
N
H
35-41
OH
H2N
O
H2N
O
43-49
51
Br
b
R1
H
H
H
H
R2
R3
methyl
F
trifluoromethyl
methoxy
cpd.
d
35 43
H
H
H
H
,
N
N
N
N
H
42
36, 44
N
O
37 45
,
38 46
,
50
39 47
,
R1,R2
=
H
40 48
41 49
,
,
H
H
H
F
dimethylamino
H
Scheme 2. Synthesis of carbamazepine derivatives with substituents on the phenyl ring and aza-analogs. Reagents and conditions: (a) 2-chloroaniline derivatives, Pd2(dba)3,
DavePhos, NaOt-Bu, 1,4-dioxane, 115 °C, yields: 20–65%; (b) 4-chloro-3-aminopyridine, Pd2(dba)3, DavePhos, NaOt-Bu, 1,4-dioxane, 115 °C, yield 26%; (c) NaOCN, HOAc,
65 °C, 2 h, yields: 70–81%. (d) LHMDS, diethylamine, 0 °C to rt, 2 h, yield: 24%; (e) BBr3, DCM, 0 °C to rt, 24 h, yield: 37%.
b
c
OH
a
OH
O
N
N
H
N
N
H
1
H2N
O
52
53
54
Scheme 3. Synthesis of 2-hydroxycarbamazepine. Reagents and conditions: (a) (KSO3)2NO, Na2HPO4, H2O/acetone, rt 3 h, yield: 34%; (b) Na2S2O4, rt, 69%; (c) NaOCN, HOAc,
70 °C, 2 h; NaHCO3, H2O/MeOH, 0 °C, 30 min, yield: 29% over two steps.
O
R
NH
HO
O
N
c
d
a
b
N
N
N
N
H2N
O
H2N
O
H2N
O
H2N
55
O
H2N
O
57
56
58: R = Me
59 R
:
= H
Scheme 4. Synthesis of carbamazepine derivatives by functionalization of the 10,11-double bond. Reagents and conditions: (a) HOOAc, KMnO4/Al2O3, DCM, rt, 2 h, yield:
66%; (b) H2, Pd/C, N(Et)3, MeOH–H2O, 2 h, yield: 63%; (c) K2Cr2O7, CH3CO3H, yield: 90%; (d) RNH2, Ti(OiPr)4, EtOH.
frequently used for reductive amination reactions, the formation of
the corresponding amines could not be observed. Notably, the 10-
amino-substituted carbamazepine derivatives 58 and 59, resulting
from the aromatization of the imine intermediates, were isolated
as the main products.
Selected compounds were additionally investigated at rat and
mouse P2X4 receptors (Table 2). The most potent compounds were
also evaluated for their inhibitory potency at P2X1, P2X2, P2X3,
and P2X7 receptors (Table 2).
Overall, 47 carbamazepine derivatives, including 32 novel com-
pounds, were synthesized, and their structures and purities (>95%)
were firmly confirmed by 1H and 13C NMR spectroscopy, and
LC–MS analysis, respectively.
2.3. Structure–activity relationships of N5-substituted
dibenzazepines
Under the experimental conditions established in our labora-
tory, paroxetine (I) exhibited P2X4-antagonistic potency with an
2.2. Biological evaluation at P2X4 receptors
IC50 of 4.68 l
M, which is comparable to the reported results.20 In
preliminary experiments the commercially available anticonvul-
sant drug carbamazepine (III) showed some P2X4-antagonistic
activity at micromolar concentrations. Further testing revealed
that it was a relatively weak P2X4 receptor antagonist at human
P2X4 receptors (35% inhibition of ATP-induced calcium influx at
Inhibition of ATP-induced calcium influx in 1321N1 astrocy-
toma cells stably transfected with the human P2X4 receptor was
determined as previously described.22 The compounds were ini-
tially tested at a high concentration of 10–300 lM depending on
their solubility. Full concentration-inhibition curves were deter-
mined for potent compounds and IC50 values were calculated. Re-
sults are collected in Table 1.
100 lM, see Table 1).
The synthesized carbamazepine derivatives and analogs that
were modified at N5 of the dibenz[b,f]azepine core can be divided

M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
1081
Table 1
Potency of carbamazepine derivatives and analogs at human P2X4 receptors
N
N
N
N
N
R1
R1
N
O
O
O
H2N
O
R1
2-3
O
Ph
R2
III
4
5-7
9-31, 34
Compound
R1
R2
—
Human P2X4 IC50 SEMa
(l
M)
Paroxetine (I)
4.68 1.38
N-(Benzyloxy carbonyl)-phenoxazine (II)22
Carbamazepine (III)
0.189 0.059 (77%)b
>100 (35%)b
—
Amides
2
3
Methyl
Phenyl
—
—
>100 (39%)b
23.3 5.8 (75%)b
Amine
4
—
—
>100 (34%)b
Carbamates
5
6
7
Methyl
sec-Butyl
Benzyl
—
—
—
ca. 100 (51%)b
ca. 100 (58%)b
6.02 1.64 (73%)c
Ureas
9
Ethyl
Benzyl
Methyl
ethyl
H
H
11.4 1.5 (95%)b
78.9 19.6 (75%)b
P100 (47%)b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
34
–CH2CH2OCH2CH2–
P100 (50%)b
Dimethylaminopropyl
Isopropyl
4-Methoxyphenylethyl
Cyclohexyl
H
H
H
H
H
ca. 100 (54%)b
19.2 7.3 (47%)b
27.4 3.8 (83%)b
33.8 6.9 (100%)b
36.2 7.2 (107%)b
13.7 2.8 (72%)b
34.1 2.1 (98%)b
P100 (49%)b
2-Phenylethyl
–CH2CH2CH2CH2CH2–
–CH2CH2CH2CH2–
Methyl
Butyl
Propyl
Dimethylaminoethyl
t-Butyl
1-Methylpiperidin-4-yl
Hydroxyethyl
methyl
H
Propyl
Methyl
H
P100 (49%)b
7.78 1.60 (49%)c
P100 (42%)b
13.8 1.6 (92%)b
P100 (56%)b
H
Hydroxyethyl
>>100 (2%)b
–CH2CH2CH(OH)CH2CH2–
>>100 (ꢀ18%)b
4-Hydroxycyclohexyl
4-Aminocyclohexyl
Phenyl
Pyridin-4-yl
i-Propyl
H
H
H
H
>>20 (0%)d
>>10 (11%)c
ca. 100 (49%)b
>100 (30%)b
i-Propyl
3.44 1.15 (71%)c
11
10
9
1
R1
2
8
5
N
N
N
N
N
7
2
3
4
R
6
H2N
R1
N
O
O
H2N
O
O
(i-Pr)2N
32-33
43-46, 48, 49, 51, 54
50
47
Compound
R1
R2
Human P2X4 IC50 SEM (
l
M)a
32
33
43
44
45
46
47
48
49
50
51
54
H
—
—
30.6 13.1 (74%)b
52.5 16.3b
Diethylcarbamoyl
H
H
H
H
—
H
F
—
H
OH
Methyl
F
CF3
OMe
>100 (21%)b
>>100 (ꢀ2%)b
54.6 13.7 (72%)b
>100 (30%)b
—
51.4 11.4 (91%)b
15.3 5.9 (39%)b
>100 (11%)b
Dimethylamino
H
—
OH
H
ca. 100 (52%)b
72.8 13.2 (99%)e
146 19 (82%)e
(continued on next page)

1082
M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
Table 1 (continued)
R1
R2
R1
11
O
10
11
10
9
1
9
1
2
2
8
8
5
5
N
N
3
3
7
7
6
4
6
H2N
4
H2N
55, 56, 60-62
R1
O
57-59, 63
R2
Compound
Human P2X4 IC50 SEMa
>>10 (1%)c
(lM)
55
R1, R2
=
O
56
rac-OH
H
(S)-Acetoxy
rac-Ethoxy
OH
MeNH
NH2
Methoxy
H
H
H
H
—
—
—
—
>100 (28%)b
>100 (35%)b
>20 (25%)d
60f
61g
62f
57
>100 (17%)b
>10 (10%)c
58
36.9 8.7 (80%)b
>10 (12%)c
59
63f
>100 (34%)b
a
n = 3.
b
Percent inhibition at 100 lM of test compound.
c
Percent inhibition at 10
lM of test compound.
d
e
f
Percent inhibition at 20
lM of test compound.
Percent inhibition at 300
Purchased from Sigma–Aldrich.
Extracted from commercially available eslicarbazepine acetate 800 mg tablets (ZebinixÒ).
lM of test compound.
g
Table 2
Potency of selected carbamazepine derivatives at P2X4 orthologues and different P2X receptor subtypes
Compd.
Human P2X4
Mouse P2X4
Rat P2X4
Human P2X1
Human P2X2
Human P2X3
Human P2X7
9
11.4 1.5
33.8 6.9
13.7 2.8
34.1 2.1
7.78 1.60
3.44 1.15
>100 (27%)a
98.4 12.6
102 11
43.0 10.8
25.2 3.0
69.6 12.1
20.7 1.9
68.8 14.5
4.55 1.23
5.32 1.91
>100 (1%)a
>100 (11%)a
>100 (4%)a
>100 (6%)a
>100 (ꢀ2%)a
>100 (8%)a
14.2 0.9
28.0 3.9
7.98 1.22
29.8 4.3
15.2 0.5
7.18 2.86
69.8 12.6
40.8 5.8
16
18
19
22
34
109
2
95.8 9.8
69.3 17.4
35.9 4.6
54.6 13.9
>100 (ꢀ10%)a
>100 (33%)a
>100 (ꢀ11%)a
95.2 8.3
123 18
>100 (38%)a
14.9 2.2
a
Inhibition of calcium flux at 100 lM concentration. IC50 SEM (lM).
into four subgroups (Table 1): amide (2, 3), amine (4), carbaminic
acid (5–7), and urea (9–31 and 34) derivatives. Methylamide 2
showed similarly low activity as carbamazepine, while the phe-
nylamide 3 exhibited somewhat higher P2X4-antagonistic activity
29) were not well tolerated. Phenyl-substitution (30) or bioisos-
teric substitution with a 4-pyridyl residue (31) were also not tol-
erated. However, phenyl residues connected to the urea via a
methylene (benzyl derivative 10, IC50 78.9
so by an ethylene linker (phenethyl derivative 17, IC50
36.2 M; and p-methoxyphenethyl derivative 15, IC50 27.4 M)
led to somewhat higher potency.
lM) or even more
(IC50 of 23 lM). However, the benzyl-substituted tertiary amine 4
showed lower activity, and therefore no further amine derivatives
were explored. A carbonyl group attached to the ring nitrogen
atom appeared to be required. Subsequently, three carbamate
derivatives were investigated (5–7). The alkyl carbamates 5 and
l
l
Disubstitution of the urea NH2 function resulted in increased
potency. The rank order of potency of dialkyl-substituted urea
6 showed weak activity (estimated IC50 value around 100
whereas the benzyl carbamate 7 displayed significantly higher po-
tency with an IC50 of 6.02 M. A structurally related phenoxazine
l
M),
derivatives was: diisopropyl (34, IC50 3.44
IC50 7.78 M) > diethyl (9, IC50 11.4 M > dimethyl (20, IC50 ca.
100 M) = N-methyl-N-dimethylaminoethyl (23, ca. 100 M) >
dihydroxyethyl (26, >> 100 M). We also investigated sterically
lM) > dipropyl (22,
l
l
l
l
l
derivative had recently been developed as a potent P2X4 receptor
l
antagonist.22
fixed derivatives, in which both N-substituents were integrated
As a next step a series of urea derivatives was explored.
Investigation of monoalkyl-substituted urea derivatives showed
that small, branched substituents were best tolerated, namely
into a cyclic structure (compounds 12, 18, 19, 27). The rank order
of potency was as follows: piperidine (18, IC50 13.7
dine (19, IC50 34.1 M) > morpholine (12, IC50 ca. 100
p-hydroxypiperidine (27, >> 100 M). The SARs observed for the
l
M) > pyrroli-
l
lM) >>
the isopropyl derivative 14 (19.2
tive 24 (13.8 M), while smaller (methyl derivative 11) and lin-
ear alkyl substituents (butyl derivative 21) resulted in very
weakly active compounds (IC50 ca. 100 M). The cyclohexyl
derivative 16 was intermediate in potency (IC50 33.8 M). Polar
substituents on the alkyl substituent (compounds 13, 25, 28,
lM) and the tert-butyl deriva-
l
l
N,N-disubstituted urea derivatives confirmed the trend observed
with monosubstituted compounds: small, branched alkyl substitu-
ents were optimal while polar residues (e.g., in 12, 27) were not
tolerated at that position. The diisopropylurea derivative 34 was
the best compound of the present series.
l
l

M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
1083
2.4. Structure–activity relationships of ring-modified
carbamazepine derivatives and analogs
P2X4
P2X1
P2X3
P2X7
P2X2
125
100
75
Annelation of another benzene ring at the 1,2-position of the
dibenzazepine (compound 47) slightly increased potency of car-
bamazepine resulting in
a
moderately potent compound
(51.4 M). In the 2-position of the dibenzazepine ring only F and
l
50
OH were tested, both substituents resulting in inactive or very
weakly active compounds (49, 54). Introduction of a nitrogen atom
at the 3-position of the dibenzazepine ring to obtain aza-analog 50
resulted in a large drop in potency (compare 9 and 50). A hydroxyl
residue in the 3-position was well accepted (compound 51).
Substitution in position 4 of the dibenzazepine ring with a large
diisopropylaminocarbonyl residue was tolerated (32, 33; compare
32 with carbamazepine). Substitution at the 3-position of carbam-
azepine with fluoro (44), methyl (43), or methoxy (46) did not im-
prove the potency of the lead structure and resulted in virtually
25
0
-25
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2
[compound 34], M
Figure 2. Inhibition of ATP-induced calcium influx by compound 34 at different
P2X receptor subtypes. ATP concentrations correspond to the determined EC80
values. The corresponding IC50 values are shown in Table 2.
inactive compounds. Hydroxy-(51, 72.8
l
M), trifluoromethyl-(45,
M), and, even more so, dimethylamino-substitution (48,
M) increased the potency of carbamazepine. Substitution at
54.6
15.3
l
l
the 10-position of the dibenzazepine core of carbamazepine with
a hydroxy (57), methoxy (63) or amino (59) residue did not show
positive effects on potency. However, methylamino substitution
125
ATP without inhibitor
100
led to
36.9 M). Carbamazepine derivatives, in which the 10,11-double
bond of the dibenzazepine ring system was hydrogenated and the
resulting dihydrocarbamazepine (60) was substituted with
a moderately potent P2X4 receptor antagonist (58,
ATP + 3µM34
l
75
50
25
0
ATP + 10µM34
ATP + 30µM34
a
10,11-epoxy ring (55), an ethoxy (62) or an (S)-acetoxy residue
(61) were investigated. The latter compound corresponds to the
marketed antiepileptic prodrug eslicarbazepine acetate, which is
converted in vivo by ester hydrolysis to the corresponding alcohol.
All of these hydrogenated carbamazepine derivatives were inactive.
In conclusion, polar substituents appear to be well tolerated at
the dibenzazepine tricycle especially in positions 3, 4 and 10. The
best modification of the present series was the 3-dimethylamino-
-25
10-8
10-7
10-6
[ATP], M
10-5
10-4
10-3
Figure 3. ATP-induced intracellular calcium levels in 1321N1 astrocytoma cells
stably transfected with the human P2X4 receptors, measured in the absence and
presence of different concentrations of compound 34. Data were normalized to ATP
without inhibitor (100%) and buffer (0%). The corresponding EC50 values and
maximal effects are shown in Table 3.
substituted carbamazepine 48 (15.3 lM). Substitution of the dib-
enzazepine tricycle should be further explored. Since polar substit-
uents are tolerated in various positions of the ring system, such
modifications could not only enhance potency but may also lead
to increased water-solubility.
Table 3
EC50 values and maximal effects for ATP in the absence and presence of different
concentrations of compound 34
2.5. Inhibitory potency at P2X4 receptor orthologs and other
P2X receptor subtypes
ATP dose–response curve
Without inhibitor
EC50
(
l
M)
Emax (%)
In order to assess whether the identified P2X4 receptor antago-
nists are not only active at human, but also at the rat and mouse
receptor orthologs, selected potent compounds were additionally
tested in calcium flux assays at rat and mouse P2X4 receptors
(Table 2).
1.93 0.45
2.18 0.70
3.47 1.74
1.78 0.91
100
70 17
36 14
3
lM Compound 34
10
30
l
l
M Compound 34
M Compound 34
12
5
All of the selected compounds exhibited the highest potencies
at the human P2X4 receptor. Compounds 16, 18, 19, 22 and 34
showed a slight (3–7 fold) drop in potency at the mouse P2X4
receptor compared with the human P2X4 receptor, whereas
diethyl- and dipropyl-substituted urea derivatives 9 and 22 were
virtually inactive at the mouse P2X4 receptor. On the other
hand, compounds 9 and 22 exhibited only a 4–5 fold drop in po-
tency at the rat P2X4 receptor compared with the human ortho-
logue. Compounds 16, 18 and 19 were nearly equipotent at rat
and mouse P2X4-receptors. The bulky diisopropyl-substituted
urea 34 was 10-fold less active at the rat P2X4 receptor than
at the human orthologue. Overall, it is demonstrated that signif-
icant differences between potencies at human versus rodent
P2X4 receptors may be a common phenomenon. A similar result
had been observed for the previously reported N-substituted
phenoxazine derivatives with P2X4 receptor antagonistic
activity.22
Finally, selected P2X4 antagonists were investigated for their
potency to inhibit the related P2X receptor subtypes P2X1, P2X2,
P2X3 and P2X4. The inhibition of ATP-induced calcium influx by
one representative compound 34 is shown in Figure 2. None of
the compounds showed antagonistic activity at the human P2X2
receptor (Table 2). However, all of the selected compounds were
able to block some of the other P2X receptor subtypes in addition
to P2X4. The most potent compounds of the present series, 18, 22
and 34, were about equipotent at the human P2X1, P2X3, and P2X4
receptors, but selective versus P2X2 and P2X7. Our results indicate
that improved P2X4 selectivity may be achieved by further optimi-
zation, since it has already been obtained versus some of the P2X
receptor subtypes. On the other hand, antagonists blocking more
than one P2X receptor subtype may be advantageous, since several
P2X receptor subtypes are known to be involved in pain and
inflammation.32

1084
M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
2.6. Mechanism of action
(ESI) mass spectrometry (LC–MS) on an Applied Biosystems API
2000 LCMS/MS, HPLC Agilent 1100 system using a Phenomenex
Luna 3
C18 column (50 ꢁ 2.00 mm). UV absorption was detected
In order to assess whether the P2X4 receptor inhibition by the
new antagonists is via a competitive mechanism, that is, blockade
of the ATP binding site, or via an allosteric mechanism, concentra-
tion–response curves for ATP were determined in the absence and
l
from 200 to 950 nm using a diode array detector. The purity of the
compounds was determined at 220–400 nm. The purity of the
compounds was proven to be P95%. Compounds III, 60–63 were
purchased from Sigma–Aldrich. For identification of the synthe-
sized known compounds 2,26 3,35 4,36 5,37 9,38 10,39 11,40 19,41
30,42 35–37,27 38,43 39,44 41,45 42,27 49,46 51,46 52-53,30 54,47
and 55–57,48 the analytical data were compared with literature
data and provided as the Supplementary data.
in the presence of different concentrations (3, 10, and 30 lM) of
the selected, potent antagonist 34. As shown in Figure 3 and Ta-
ble 3, the concentration–response curves showed the characteristic
behaviour of a negative allosteric modulator, that is, depression of
the maximal response with essentially no alteration of the EC50
values.33,34 This result, is comparable to the previously reported
data obtained with structurally related phenoxazine derivatives,
which had also been identified as noncompetitive P2X4 receptor
antagonists.22
4.2. General procedure for the synthesis of amide derivatives 2
and 3 (GP-1)
In contrast to competitive antagonists, negative allosteric mod-
ulators may not lead to a complete blockade of the signal. In fact,
we observed that some of the potent antagonists only led to a par-
tial inhibition of ATP-mediated calcium influx even at high concen-
A solution of dibenzazepine (193 mg, 1.00 mmol), the appropri-
ate acid chloride (1.2 mmol), and triethylamine (101 mg,
1.0 mmol) in dry dichloromethane (10 mL) were stirred under re-
flux for 3 h (TLC control). After the mixture had been cooled down
to rt, dichloromethane (150 mL) was added. The mixture was
washed with an aqueous saturated solution of NaHCO3
(2 ꢁ 50 mL), HCl (1%, 100 mL), and subsequently with brine
(100 mL). The organic layer was separated and dried with MgSO4.
The solvent was evaporated in vacuo. The residue was submitted
to column chromatography (SiO2, eluent: petroleum ether/EtOAc,
5:1) to give the desired products. An analytically pure sample
was obtained by recrystallization from EtOH.
trations. For example, antagonist 48 (EC50 15.3
lM) showed only
39% maximal inhibition at a high concentration of 100
l
M at which
a maximal effect should be observed.
3. Conclusions
In conclusion, we have synthesized a series of mostly new car-
bamazepine derivatives, whose structural modification was system-
atically and extensively explored. These compounds were evaluated
and optimized as P2X4 receptor antagonists. The most potent com-
pound of the present series was N,N-diisopropyl-5H-dibenz[b,f]aze-
4.3. General procedure for the synthesis of carbamate
derivatives 5–7 (GP-2)
pine-5-carboxamide (34) with an IC50 of 3.44 lM at human P2X4
receptors. However, it should be noted that compound 34 is not
completely P2X4-selective, since it also shows considerable potency
at the P2X1 receptor subtype (IC50 = 5.32 lM). It was found to exhi-
bit an allosteric mechanism of action. This study represents one of
the very few reported SAR studies of P2X4 receptor antagonists,
which is complementary to the recent discovery of structurally re-
lated N-substituted phenoxazine derivatives as P2X4 receptor
antagonists.22 Further improvement of affinity and selectivity as
well as physicochemical properties will be required to obtain com-
pounds useful as pharmacological tools for in vivo studies.
A reaction mixture of dibenzazepine (193 mg, 1.00 mmol) and
the appropriate chloroformate (2 mL) was stirred under a N2 atmo-
sphere at 100–110 °C for 3 h. After it had been cooled down to rt,
dichloromethane (50 mL) was added. An aqueous solution of
NaHCO3 (5%, 10 mL) was added to the mixture at 0 °C. It was stir-
red at rt for 1 h, washed with water (150 mL) and subsequently
with brine (150 mL). The organic phase was separated and dried
with MgSO4. The volatile components were evaporated in vacuo.
Pure products were obtained by crystallization of the residues from
the appropriate solvent.
4. Experimental section
4.1. General
4.4. General procedure for the synthesis of urea derivatives 9–
29 (GP-3)
All commercially available materials were used as purchased
without further purification. Carbamazepine (III), compounds 60,
62 and 63 were purchased from Sigma–Aldrich. Compound 61 was
extracted from eslicarbazepine acetate 800 mg tablets (ZebinixÒ),
which were purchased from a German pharmacy. If not stated other-
wise, deionized water was used for the experiments. Thin-layer
chromatography (TLC) was performed using TLC aluminum sheets
with silica gel 60 F254 (Merck, Darmstadt, Germany). The compounds
were visualized under UV light (254 nm). Column chromatography
was carried out with silica gel 0.060–0.200 mm, pore diameter ca.
6 nm. 1H and 13C nuclear magnetic resonance (NMR) data were col-
lected on a BrukerAvance 500 MHz NMR spectrometer at 500 (1H) or
125 MHz (13C), respectively. DMSO-d6 or CDCl3 was used as a sol-
vent. Chemical shifts are reported in parts per million (ppm) relative
to the deuterated solvent, that is, DMSO, d 1H: 2.50 ppm, 13C:
39.5 ppm; CDCl3, d 1H: 7.26 ppm, 13C: 77.2 ppm; coupling constants
J are given in Hertz, and spin multiplicities are given as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), or br (broad).
The corresponding aliphatic amine (2.00 mmol) was added
dropwise to a solution of dibenz[b,f]azepine-5-carbonyl chloride
(256 mg, 1.00 mmol) in dichloromethane (10 mL) at 0 °C. The reac-
tion mixture was stirred at rt for 4 h. Dichloromethane (100 mL)
was added to the reaction mixture. It was washed with HCl (1%,
100 mL), an aqueous solution of NaHCO3 (5%, 100 mL), and
subsequently with brine (100 mL). The organic phase was dried
over MgSO4. After evaporation of the solvent, the product was
obtained by recrystallization of the residue from appropriate
solvents.
4.5. General procedure for the synthesis of urea derivatives 30
and 31 (GP-4)
A solution of the appropriate aromatic amine (2.00 mmol) and
dibenz[b,f]azepine-5-carbonyl chloride (256 mg, 1.00 mmol) in tol-
uene (10 mL) was stirred under reflux for 2 h. After it had been
cooled down to rt, the volatile components were evaporated in va-
cuo. The product was purified by column chromatography (silica
gel, eluent DCM/MeOH 20:1). After evaporation of the eluent, an
The purities of the isolated final products were determined by a
liquid chromatography system coupled to electrospray ionization

M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
1085
analytically pure sample was obtained by recrystallization from
5. 5H-Dibenz[b,f]azepin-5-yl-4-morpholinyl-methanone (12)
EtOAc.
GP-3, recrystallization from diethyl ether–petroleum ether;
yield 231 mg (75%), colorless prisms; 1H NMR (500 MHz, CDCl3):
d 7.49 (dd, J = 8, J = 1 Hz, 2H, Har), 7.28–7.35 (m, 2H, Har), 7.22–
7.12 (m, 4H, Har), 6.86 (s, 2H, Har), 3.33 (dd, J = 5 Hz, J = 5 Hz, 4H,
CH2), 3.03 (dd, J = 5 Hz, J = 5 Hz, 4H, CH2); 13C NMR (125 MHz,
CDCl3): d 159.3, 142.6, 134.2, 131.4, 129.2, 129.0, 127.7, 126.6,
66.4, 46.5; LC–MS (m/z): 307 [M+H]+. Purity by HPLC-UV (220–
400 nm)-ESI-MS: 100%.
4.6. General procedure for palladium-catalyzed synthesis of
dibenzazepine derivatives 35–41 (GP-5)
A mixture of 2-bromostyrene (549 mg, 3.00 mmol), the appro-
priate 2-chloroaniline derivative (3.00 mmol), Pd(dba)3 (68 mg,
0.075 mmol), DavePhos (79 mg, 0.20 mmol), and NaOtBu
(864 mg, 9.00 mmol) in dry 1,4-dioxane (10 mL) was stirred under
a N2 atmosphere at 115 °C for 6 h. After it had been cooled down to
rt, EtOAc (150 mL) was added. The mixture was washed with water
(5 ꢁ 150 mL) and subsequently with brine (150 mL). The organic
phase was separated and dried over MgSO4. After evaporation of
the solvent, the residue was submitted to column chromatography
(SiO2, eluent: petroleum ether/EtOAc 20:1). The yellow fraction
was collected, and evaporation of the eluent in vacuo gave the de-
sired product.
5.1. N-[3-(Dimethylamino)propyl]-5H-dibenz[b,f]azepine-5-
carboxamide (13)
GP-3, recrystallization from EtOAc; yield 202 mg (63%), color-
less prisms; 1H NMR (500 MHz, CDCl3): d 7.48–7.37 (m, 4H, Har),
7.37–7.27 (m, 4H, Har), 6.90 (s, 2H, Har), 6.03 (br s, 1H, NH),
3.28–3.21 (m, 2H, CH2), 2.20 (t, J = 6 Hz, 2H, CH2), 1.90 (s, 6H,
CH3), 1.54–1.46 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3): d
156.8, 140.7, 135.5, 130.6, 129.5, 129.4, 127.4, 59.2, 45.3, 41.2,
26.0; LC–MS (m/z): 322 [M+H]+. Purity by HPLC-UV (220–
400 nm)-ESI-MS: 100%.
4.7. General procedure for synthesis of urea derivatives 43–49
(GP-6)
NaOCN (98 mg, 1.5 mmol) was added in small portions to a solu-
tion of the appropriate azepine derivative (1.00 mmol) in HOAc
(10 mL) at 70 °C. The reaction mixture was stirred at this tempera-
ture for 2 h. After it had been cooled down to rt, the solvent was
evaporated in vacuo. Water was added to the residue. The resulting
colorless solid was collected and dried. The product was obtained
by recrystallization of the solid from appropriate solvents.
5.2. N-(iso-Propyl)-5H-dibenz[b,f]azepine-5-carboxamide (14)
GP-3, recrystallization from EtOAc; yield 187 mg (67%), color-
less prisms; 1H NMR (500 MHz, CDCl3): d 7.49–7.28 (m, 8H, Har),
6.91 (s, 2H, Har), 4.03 (d, J = 7 Hz, 1H, NH), 3.97–3.83 (m, 1H, CH),
1.01 (d, J = 7 Hz, 6H, CH3); 13C NMR (125 MHz, CDCl3): d 155.8,
140.4, 135.4, 130.6, 129.7, 129.4, 129.3, 127.6, 42.6, 23.3; LC–MS
(m/z): 279 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS:
100%.
4.8. General procedure for the synthesis of 10-
aminocarbamazepine derivatives 58 and 59 (GP-7)
5.3. N-(p-Methoxyphenylethyl)-5H-dibenz[b,f]azepine-5-
A reaction mixture of 57 (252 mg, 1.00 mmol), titanium(IV) iso-
propoxide (0.6 mL, 2.00 mmol) and the appropriate amine
(5.00 mmol) in absolute ethanol (10 mL) was stirred under an
argon atmosphere for 6 h. Sodium borohydride (60.0 mg,
1.50 mmol) was then added and the resulting mixture was
stirred at rt for 3 additional hours. The reaction was then quenched
by pouring it into an aq. ammonium hydroxide solution (2 M,
2.5 mL). The mixture was extracted with DCM (2 ꢁ 50 mL). The
combined organic phase was washed with brine, and dried with
MgSO4. The solvent was evaporated in vacuo. The product was ob-
tained by recrystallization of the residue from ethyl acetate.
carboxamide (15)
GP-3, recrystallization from EtOAc–EtOH; yield 305 mg (82%),
colorless powder; 1H NMR (500 MHz, CDCl3): d 7.41–7.28 (m, 8H,
Har), 6.98–6.94 (m, 2H, Har), 6.90 (s, 2H, Har), 6.80–6.75 (m, 2H,
Har), 4.29 (t, J = 6 Hz, 1H, NH), 3.79 (s, 3H, OCH3), 3.33 (dd,
J = 13 Hz, J = 7 Hz, 2H, CH2), 2.65 (t, J = 7 Hz, 2H, CH2); 13C NMR
(125 MHz, CDCl3): d 158.2, 156.3, 140.1, 135.4, 131.4, 130.6, 129.8,
129.7, 129.5, 129.3, 127.7, 114.0, 55.4, 42.1, 35.2; LC–MS (m/z):
371 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
5.4. N-Cyclohexyl-5H-dibenz[b,f]azepine-5-carboxamide (16)
4.9. N-Isobutyl-5H-dibenz[b,f]azepine-5-carbamate (6)
GP-3, recrystallization from EtOAc–petroleum ether; yield
247 mg (78%), colorless powder; 1H NMR (500 MHz, CDCl3): d
7.47–7.29 (m, 8H, Har), 6.91 (s, 2H, Har), 4.12 (d, J = 8 Hz, 1H, NH),
3.65–3.53 (m, 1H, CH), 1.87–1.76 (m, 2H, CH2), 1.58–1.46 (m, 3H,
CH2), 1.34–1.21 (m, 2H, CH2), 1.13–1.01 (m, 1H, CH2), 1.00–0.88
(m, 2H, CH2); 13C NMR (125 MHz, CDCl3): d 155.7, 140.4, 135.4,
130.6, 129.7, 129.4, 129.4, 127.6, 49.3, 33.5, 25.7, 24.8; LC–MS (m/
z): 319 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
GP-2, recrystallized from EtOH; yield 206 mg (70%), colorless
prisms; 1H NMR (500 MHz, CDCl3): d 7.51–7.27 (m, 8H, Har), 6.91
(s, 2H, Har), 3.92–3.70 (m, 2H, CH2), 1.85–1.70 (m, 1H, CH), 0.76
(d, J = 7 Hz, 6H, CH3); 13C NMR (125 MHz, CDCl3): d 155.3, 140.1,
134.4, 130.8, 130.2, 129.4, 129.1, 128.8, 127.4, 71.9, 28.0, 19.0;
LC–MS (m/z): 294 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-
MS: 100%.
5.5. N-Phenylethyl-5H-dibenz[b,f]azepine-5-carboxamide (17)
4.10. N-Benzyl-5H-dibenz[b,f]azepine-5-carbamate (7)
GP-3, recrystallization from diethyl ether-petroleum ether;
yield 264 mg (78%), colorless powder; 1H NMR (500 MHz, CDCl3):
d 7.41–7.16 (m, 11H, Har), 7.04 (d, J = 7 Hz, 2H, Har), 6.90 (s, 2H,
Har), 4.29 (t, J = 5, 1H, NH), 3.37 (td, J = 7 Hz, J = 7 Hz, 2H, Har),
2.71 (t, J = 7 Hz, 2H, Har); 13C NMR (125 MHz, CDCl3): d 156.3,
140.1, 139.4, 135.4, 130.6, 129.7, 129.5, 129.3, 129.0, 128.6,
127.7, 126.4, 41.9, 36.2; LC–MS (m/z): 341 [M+H]+. Purity by
HPLC-UV (220–400 nm)-ESI-MS: 100%.
GP-2, recrystallized from diethyl ether–petroleum ether; yield
187 mg (57%), colorless powder; 1H NMR (500 MHz, CDCl3): d
7.54–7.17 (m, 11H, Har), 7.18–7.06 (m, 2H, Har), 6.89 (d, J = 1 Hz,
2H, Har), 5.10 (d, J = 74 Hz, 2H, CH2); 13C NMR (125 MHz, CDCl3):
d 155.0, 140.2, 136.7, 134.4, 130.2, 129.4, 129.0, 128.7, 128.4,
127.8, 127.6, 127.3, 67.3; LC–MS (m/z): 328 [M+H]+. Purity by
HPLC-UV (220–400 nm)-ESI-MS: 100%.

1086
M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
6.91 (s, 2H, Har), 4.10 (d, J = 8 Hz, 1H, NH), 3.66–3.53 (m, 1H, CH),
2.56 (br s, 2H, CH2), 2.19 (s, 3H, CH3), 2.03 (t, J = 9 Hz, 2H, CH2),
1.89–1.78 (m, 2H, CH2), 1.31–1.16 (m, 2H, CH2); 13C NMR
(125 MHz, CDCl3): d 155.8, 140.2, 135.4, 130.6, 129.8, 129.5,
129.3, 127.8, 54.4, 47.1, 46.3, 32.6; LC–MS (m/z): 334 [M+H]+. Pur-
ity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
6. 5H-Dibenz[b,f]azepin-5-yl-1-piperidinyl-methanone (18)
GP-3, recrystallzation from EtOAc; yield 285 mg (94%), colorless
prisms; 1H NMR (500 MHz, CDCl3): d 7.58 (dd, J = 8, J = 1 Hz, 2H,
Har), 7.40–7.32(m, 2H, Har), 7.28–7.16 (m, 4H, Har), 6.93 (s, 2H,
Har), 3.10–3.06 (m, 4H, CH2), 1.46–1.34 (m, 2H, CH2), 1.29–1.14
(m, 4H, CH2); 13C NMR (125 MHz, CDCl3): d 159.7, 143.4, 134.3),
131.5, 129.0, 128.9, 127.7, 126.2, 47.3, 25.3, 24.7; LC–MS (m/z):
305 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
6.7. N,N-Dihydroxyethyl-5H-dibenz[b,f]azepine-5-carboxamide
(26)
GP-3, recrystallization from diethyl ether–petroleum ether;
yield 266 mg (82%), colorless prisms; 1H NMR (500 MHz, CDCl3):
d 7.53 (dd, J = 8, J = 1 Hz, 2H, Har), 7.40–7.35 (m, 2H, Har), 7.28–
7.20 (m, 4H, Har), 6.94 (s, 2H, Har), 3.37 (dd, J = 9, J = 5 Hz, 4H,
CH2), 3.30 (t, J = 5 Hz, 2H, OH), 3.20 (t, J = 5 Hz, 4H, CH2); 13C
NMR (125 MHz, CDCl3): d 161.7, 142.3, 134.3, 131.4, 129.5, 129.1,
127.6, 126.9, 61.1, 52.5; LC–MS (m/z): 325 [M+H]+. Purity by
HPLC-UV (220–400 nm)-ESI-MS: 100%.
6.1. N,N-Dimethyl-5H-dibenz[b,f]azepine-5-carboxamide (20)
GP-3, recrystallization from EtOAc; yield 231 mg (88%), color-
less prisms; 1H NMR (500 MHz, CDCl3): d 7.56 (dd, J = 8, J = 1 Hz,
2H, Har), 7.40–7.33 (m, 2H, Har), 7.28–7.15 (m, 4H, Har), 6.94 (s,
2H, Har), 2.58 (s, 6H, CH3); 13C NMR (125 MHz, CDCl3): d 160.1,
143.2, 134.4, 131.5, 129.1, 128.9, 127.6, 126.2, 38.4; LC–MS (m/
z): 265 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
6.2. N-n-Butyl-5H-dibenz[b,f]azepine-5-carboxamide (21)
7. 5H-Dibenz[b,f]azepin-5-yl-1-(4-hydroxy)piperidinyl-
methanone (27)
GP-3, recrystallization from EtOAc; yield 201 mg (69%), colorless
powder; 1H NMR (500 MHz, CDCl3): d 7.49–7.29 (m, 8H, Har), 6.91 (s,
2H, Har), 4.25 (br s, 1H, NH), 3.18–3.06 (m, 2H, CH2), 1.40–1.31 (m,
2H, CH2), 1.28–1.15 (m, 2H, CH2), 0.85 (t, J = 7 Hz, 3H, CH3); 13C
NMR (125 MHz, CDCl3): d 156.4, 140.3, 135.5, 130.6, 129.7, 129.5,
129.4, 127.7, 40.5, 32.3, 20.0, 13.9; LC–MS (m/z): 293 [M+H]+. Purity
by HPLC-UV (220–400 nm)-ESI-MS: 100%.
GP-3, recrystallization from diethyl ether–petroleum ether;
yield 244 mg (76%), colorless powder; 1H NMR (500 MHz, CDCl3):
d 7.55 (dd, J = 8, J = 1 Hz, 2H, Har), 7.40–7.34 (m, 2H, Har), 7.27–
7.18 (m, 4H, Har), 6.93 (s, 2H, Har), 3.69–3.59 (m, 1H, CH), 3.54–
3.42 (m, 2H, CH2), 2.81–2.70 (m, 2H, CH2), 1.62–1.53 (m, 2H,
CH2), , 1.46 (d, J = 4 Hz, 1H, OH), 1.22–1.09 (m, 2H, CH2); 13C
NMR (125 MHz, CDCl3): d 159.4, 143.0, 134.2, 131.4, 129.1, 129.0,
127.7, 126.4, 67.9, 43.7, 33.8; LC–MS (m/z): 321 [M+H]+. Purity
by HPLC-UV (220–400 nm)-ESI-MS: 100%.
6.3. N,N-Dipropyl-5H-dibenz[b,f]azepine-5-carboxamide (22)
GP-3, recrystallization from hexane; yield 236 mg (74%), color-
less solid; 1H NMR (500 MHz, CDCl3): d 7.56 (dd, J = 8, J = 1 Hz, 2H,
Har), 7.37–7.30 (m, 2H, Har), 7.23 (dd, J = 8 Hz, J = 2 Hz, 2H, Har),
7.20–7.14 (m, 2H, Har), 6.92 (s, 2H, Har), 3.01–2.73 (m, 4H, CH2),
1.28–1.13 (m, 4H, CH2), 0.69 (t, J = 7 Hz, 6H, CH3); 13C NMR
(125 MHz, CDCl3): d 160.2, 143.4, 134.5, 131.5, 129.2, 128.9,
127.7, 126.2, 50.6, 20.9, 11.5; LC–MS (m/z): 321 [M+H]+. Purity
by HPLC-UV (220–400 nm)-ESI-MS: 99.4%.
7.1. N-(4-Hydroxy-cyclohexyl)-5H-dibenz[b,f]azepine-5-
carboxamide (28)
GP-3, recrystallization from EtOAc; yield 235 mg (70%), color-
less prisms; 1H NMR (500 MHz, CDCl3): d 7.50–7.27 (m, 8H, Har),
6.90 (s, 2H, Har), 4.03 (d, J = 8 Hz, 1H, NH), 3.67–3.35 (m, 2H,
2CH), 1.97–1.78 (m, 4H, CH2), 1.45 (d, J = 4 Hz, 1H, OH), 1.37–
1.22 (m, 2H, CH2), 1.11–0.87 (m, 2H, CH2); 13C NMR (125 MHz,
CDCl3): d 155.8, 140.2, 135.4, 130.6, 129.7, 129.5, 129.2, 127.7,
70.0, 48.9, 34.1, 31.2; LC–MS (m/z): 335 [M+H]+. Purity by HPLC-
UV (220–400 nm)-ESI-MS: 100%.
6.4. N-[2-(Dimethylamino)ethyl]-N-methyl-5H-
dibenzo[b,f]azepine-5-carboxamide (23)
GP-3, recrystalli zation from diethyl ether–petroleum ether;
yield 255 mg (79%), colorless prisms; 1H NMR (500 MHz, CDCl3):
d 7.56 (dd, J = 8, J = 1 Hz, 2H, Har), 7.37–7.31 (m, 2H, Har), 7.25–
7.21 (m, 2H, Har), 7.20–7.15 (m, 2H, Har), 6.92 (s, 2H, Har), 3.26–
3.17 (m, 2H, CH2), 2.43 (s, 3H, CH3), 2.21–2.09 (m, 8H, CH3, CH2);
13C NMR (125 MHz, CDCl3): d 159.7, 142.1, 134.3, 131.4, 129.4,
129.2, 127.4, 126.9, 54.4, 46.0, 43.5, 37.0; LC–MS (m/z): 322
[M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
7.2. N-(4-Amino)cyclohexyl-5H-dibenz[b,f]azepine-5-
carboxamide (29)
GP-3, recrystallization from MeOH-H2O; yield 156 mg (54%), col-
orless powder; 1H NMR (500 MHz, CDCl3): d 7.48–7.27 (m, 8H, Har),
6.90 (s, 2H, Har), 4.02 (d, J = 8 Hz, 1H, NH), 3.60–3.46 (m, 1H, CH),
2.56–2.48 (m, 1H, CH), 1.95–1.84 (m, 2H, CH2), 1.83–1.69 (m, 2H,
CH2), 1.48 (br s, 2H, NH2), 1.20–1.05 (m, 2H, CH2), 1.03–0.87 (m,
2H, CH2); 13C NMR (125 MHz, CDCl3): d 155.8, 140.3, 135.4, 130.6,
129.7, 129.4, 129.3, 127.7, 50.0, 49.3, 35.5, 32.3; LC–MS (m/z): 291
[M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
6.5. N-tert-Butyl-5H-dibenz[b,f]azepine-5-carboxamide (24)
GP-3, recrystallization from EtOAc; yield 211 mg (72%), color-
less prisms; 1H NMR (500 MHz, CDCl3): d 7.46–7.29 (s, 8H, Har),
6.92 (s, 2H, Har), 4.14 (s, 1H, NH), 1.21 (s, 9H, CH3); 13C NMR
(125 MHz, CDCl3): d 155.6, 140.6, 135.4, 130.6, 129.8, 129.4,
129.4, 127.6, 51.0, 29.3; LC–MS (m/z): 293 [M+H]+. Purity by
HPLC-UV (220–400 nm)-ESI-MS: 99.7%.
7.3. N-(Pyridin-4-yl)-5H-dibenzo[b,f]azepine-5-carboxamide
(31)
GP-4, yield 131 mg (42%), colorless needles; 1H NMR (500 MHz,
CDCl3) d 8.35 (d, J = 6 Hz, 2H, Har), 7.59–7.38 (m, 8H, Har), 7.20 (dd,
J = 5 Hz, J = 2 Hz, 2H, Har), 6.98 (s, 2H, Har), 6.40 (s, 1H, NH); 13C
NMR (125 MHz, CDCl3) d 152.8, 150.5, 145.6, 138.9, 135.2, 130.6,
130.1, 129.9, 128.7, 128.5, 113.0. LC–MS (m/z): 314 [M+H]+. Purity
by HPLC-UV (220–400 nm)-ESI-MS: 100%.
6.6. N-(1-Methylpiperidin-4-yl)-5H-dibenzo[b,f]azepine-5-
carboxamide (25)
GP-3, recrystalli zation from EtOAc; yield 206 mg (62%), color-
less prisms; 1H NMR (500 MHz, CDCl3): d 7.48–7.29 (m, 8H, Har),

M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
1087
7.4. 3-Methyl-5H-dibenz[b,f]azepine-5-carboxamide (43)
111.4, 39.9; LC–MS (m/z): 280 [M+H]+. Purity by HPLC-UV (220–
400 nm)-ESI-MS: 100%.
GP-6, recrystallization from EtOAc; yield 202 mg (81%), color-
less powder; 1H NMR (500 MHz, CDCl3) d 7.50–7.23 (m, 6H, Har),
7.14 (dd, J = 8, J = 1 Hz, 1H, Har), 6.95–6.86 (m, 2H, Har), 4.55 (s,
2H, NH2), 2.40 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3): d 157.2,
140.1, 140.0, 135.3, 132.3, 130.5, 129.7, 129.5, 129.4, 129.2,
128.9, 128.8, 127.8, 21.2; LC–MS (m/z): 251 [M+H]+. Purity by
HPLC-UV (220–400 nm)-ESI-MS: 98.74%.
8.2. 10-(Methylamino)-5H-dibenzo[b,f]azepine-5-carboxamide
(58)
GP-7, yield 52%, colorless powder; 1H NMR (500 MHz, CDCl3) d
7.62 (d, J = 8 Hz, 1H, Har), 7.52–7.43 (m, 2H, Har), 7.40–7.15 (m, 5H,
Har), 5.72 (s, 1H, CH), 4.57 (s, 2H, NH2), 3.89 (s, 1H, NH), 2.93 (s, 3H,
CH3); 13C NMR (125 MHz, CDCl3) d 157.1, 147.0, 141.1, 138.0,
137.4, 134.7, 130.3, 129.2, 129.0, 128.1, 128.0, 127.4, 126.8,
125.9, 97.1, 31.1; LC–MS (m/z): 266 [M+H]+; Purity by HPLC-UV
(220–400 nm)-ESI-MS: 95.94%.
7.5. 3-Fluoro-5H-dibenz[b,f]azepine-5-carboxamide (44)
GP-6, recrystallization from EtOAc–EtOH; yield 178 mg (70%),
colorless powder; 1H NMR (500 MHz, CDCl3) d 7.49–7.41 (m, 2H,
Har), 7.39–7.29 (m, 3H, Har), 7.24–7.16 (m, 1H, Har), 7.08–7.02 (m,
1H, Har), 6.90 (s, 2H, Har), 4.65 (s, 2H, NH2); 13C NMR (125 MHz,
CDCl3): d 163.4 (d, J = 250 Hz), 156.9, 141.4 (d, J = 10 Hz), 139.4,
135.3, 131.3, 130.7 (d, J = 9 Hz), 130.0, 129.8 (d, J = 6 Hz), 129.7,
128.7, 128.3, 116.2 (d, J = 22 Hz), 115.4 (d, J = 22 Hz); LC–MS
(m/z): 255 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS:
99.68%.
8.3. 10-Amino-5H-dibenzo[b,f]azepine-5-carboxamide (59)
GP-7, yield 46%, colorless powder; 1H NMR (500 MHz, CDCl3) d
7.73 (d, J = 8 Hz, 1H, Har), 7.52–7.44 (m, 2H, Har), 7.41–7.32 (m, 2H,
Har), 7.25–7.14 (m, 3H, Har), 6.13 (s, 1H, CH), 4.57 (s, 2H, NH2), 3.77
(s, 2H, NH2); 13C NMR (125 MHz, CDCl3) d 157.1, 140.6, 138.2,
136.5, 134.1, 133.9, 130.6, 129.2, 128.6, 128.1, 128.0, 127.6,
126.8, 126.7, 103.4; LC–MS (m/z): 252 [M+H]+; Purity by HPLC-
UV (220–400 nm)-ESI-MS: 96.17%.
7.6. 3-Trifluoromethyl-5H-dibenz[b,f]azepine-5-carboxamide
(45)
8.4. 4-(N,N-Diisopropyl)-5H-dibenz[b,f]azepine-5-carboxamide
(32)
GP-6, recrystallization from diethyl ether; yield 215 mg (71%),
colorless powder; 1H NMR (500 MHz, CDCl3) d 7.77–7.72 (m, 1H,
Har), 7.58–7.53 (m, 1H, Har), 7.53–7.44 (m, 3H, Har), 7.43–7.35 (m,
2H, Har), 7.03 (d, J = 12 Hz, 1H, Har), 6.97 (d, J = 12 Hz, 1H, Har),
4.61 (s, 2H, NH2); 13C NMR (125 MHz, CDCl3): d 156.7, 140.4,
139.9, 138.4, 134.9, 132.2, 131.5 (q, J = 33 Hz), 130.3, 130.1,
130.1, 129.8, 128.7, 128.5, 126.5, 124.5 (q, J = 3 Hz), 123.9 (q,
J = 272 Hz); LC–MS (m/z): 305 [M+H]+. Purity by HPLC-UV (220–
400 nm)-ESI-MS: 99.54%.
LDA (2 M, 3 mL) was added dropwsie to the solution of imino-
stilbene (580 mg, 3.00 mmol) and diisopropylcarbamoyl chloride
(982 mg, 6.00 mmol) in dry THF (10 mL) at 0 °C. The reaction mix-
ture was stirred under reflux for 0.5 h. After cooled down to room
temperature, water (100 mL) was added. The mixture was ex-
tracted with dichloromethane (200 mL). The organic phase was
separated, washed with brine (200 mL), and dried with MgSO4.
After evaporation of the solvent in vacuo, the residue was submit-
ted to column chromatography (SiO2, EtOAc–petroleum ether,
1:10) to give the product as orange prisms (490 mg, 51%). 1H
NMR (500 MHz, CDCl3): d 7.06–6.99 (m, 1H, CHar), 6.94–6.77
(m, 5H, CHar), 6.47–6.34 (m, 3H, CHar), 6.14 (s, 1H, NH), 4.06–
3.47 (br d, 2H, CH), 1.85–0.80 (br d, 12 H, CH3); 13C NMR
(125 MHz, CDCl3): d 169.6, 149.0, 145.0, 133.0, 132.6, 132.0,
130.6, 130.5, 130.4, 129.7, 128.9, 126.1, 123.2, 122.3, 120.1,
51.2, 46.3, 20.9; LC–MS (m/z): 321 [M+H]+. Purity by HPLC-UV
(220–400 nm)-ESI-MS: 100%.
7.7. 3-Methoxy-5H-dibenz[b,f]azepine-5-carboxamide (46)
GP-6, recrystallization from EtOAc; yield 210 mg (79%), color-
less powder; 1H NMR (500 MHz, CDCl3) d 7.50–7.18 (m, 5H, Har),
6.92–6.74 (m, 3H, Har), 4.57 (s, 2H, NH2), 3.81 (s, 1H, OMe); 13C
NMR (125 MHz, CDCl3): d 161.1, 157.2, 141.2, 139.5, 135.5, 130.6,
130.3, 129.6, 129.3, 128.8, 128.4, 128.0, 114.7, 113.4, 55.7; LC–
MS (m/z): 267 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS:
100%.
8. 5H-Benzo[b]naphtho[4,3-f]azepin-5-carboxamide (47)
8.5. 4-(N,N-Diisopropylcarbamoyl)-N,N-diethyl-5H-
dibenz[b,f]azepine-5-carboxamide (33)
GP-6, recrystallization from EtOAc; yield 220 mg (77%), color-
less powder; 1H NMR (500 MHz, CDCl3) d 8.18 (d, J = 8 Hz, 1H,
Har), 7.91 (d, J = 9 Hz, 1H, Har), 7.88–7.83 (m, 1H, Har), 7.70 (d,
J = 12 Hz, 1H, Har), 7.60–7.49 (m, 4H, Har), 7.48–7.41 (m, 2H, Har),
7.37–7.32 (m, 1H, Har), 7.30–7.20 (m, 1H, Har), 4.59 (s, 2H, NH2);
13C NMR (125 MHz, CDCl3): d 157.0, 140.9, 135.4, 132.8, 131.6,
130.4, 129.8, 129.4, 128.7, 128.4, 127.9, 127.1, 126.6, 126.4,
124.3; LC–MS (m/z): 287 [M+H]+. Purity by HPLC-UV (220–
400 nm)-ESI-MS: 97.22%.
the solution of triphosgene (445 mg, 1.5 mmol) in DCM (5 mL)
was added to the solution of 4-(N,N-Diisopropyl)-5H-
dibenz[b,f]azepine-5-carboxamide (32, 320 mg, 1.00 mmol) and
DIEA (0.4 mL) in DCM (5 mL) at 0 °C. The reaction mixture was stir-
red at room temperature for 1.5 h (TLC control). Diethylamine
(1 mL) was added at 0 °C. It was stirred at room temperature for
another 2 h. DCM (150 mL) was added to the reaction mixture. It
was washed with HCl (1%, 200 mL), NaHCO3 (5%, 200 mL), and
brine (200 mL). The organic phase was dried with MgSO4. After
evaporation of the solvent in vacuo, the product was purified with
column chromatography (SiO2, EtOAc–petroleum ether, 1:5) as
colorless prisms (320 mg, 76%). 1H NMR (500 MHz, CDCl3): d 7.80
(dd, J = 8, J = 1 Hz, 1H), 7.44–7.38 (m, 1H, Har), 7.37–7.32 (m, 1H,
Har), 7.32–7.28 (m, 1H, Har), 7.25–7.19 (m, 3H, Har), 7.01 (d,
J = 12 Hz, 1H, Har), 6.92 (d, J = 12 Hz, 1H, Har), 4.02–3.90 (m, 1H,
CH), 3.58–3.50 (m, 1H, CH), 3.19–3.08 (m, 2H, CH2), 2.91–2.83
(m, 2H, CH2), 1.68 (d, J = 7 Hz, 3H, CH3), 1.60 (d, J = 7 Hz, 3H,
CH3), 1.13 (d, J = 5 Hz, 3H, CH3), 1.12 (d, J = 5 Hz, 3H, CH3), 0.64
8.1. 3-Dimethylamino-5H-dibenz[b,f]azepine-5-carboxamide
(48)
GP-6, recrystallization from diethyl ether; yield 190 mg (68%),
pale yellow powder; 1H NMR (500 MHz, CDCl3) d 7.38–7.32 (m,
3H, Har), 7.31–7.26 (m, 1H, Har), 7.24–7.19 (m, 1H), 6.85–6.78 (m,
1H), 6.75–6.63 (m, 3H, Har), 5.42 (s, 2H, NH2), 3.28 (s, 6H, Har);
13C NMR (125 MHz, CDCl3): d 156.4, 151.6, 141.3, 139.7, 135.5,
130.2, 129.9, 129.4, 128.6, 128.3, 126.8, 126.1, 122.8, 111.6,

1088
M. Tian et al. / Bioorg. Med. Chem. 22 (2014) 1077–1088
(t, J = 7 Hz, 6H, CH3); 13C NMR (125 MHz, CDCl3): d 169.2, 159.5,
140.9, 138.8, 137.6, 136.8, 135.8, 131.4, 130.9, 129.6, 129.1,
128.9, 128.3, 126.8, 126.3, 125.7, 51.0, 45.7, 43.0, 21.0, 20.9, 20.9,
20.6, 12.9; LC–MS (m/z): 420 [M+H]+. Purity by HPLC-UV (220–
400 nm)-ESI-MS: 100%.
References and notes
1. Burnstock, G. Front. Biosci. 2011, 3, 896.
2. Burnstock, G. BioEssays 2012, 34, 218.
3. Coddou, C.; Yan, Z.; Obsil, T.; Huidobro-Toro, J. P.; Stojilkovic, S. S. Pharmacol.
Rev. 2011, 63, 641.
4. Abbracchio, M. P.; Burnstock, G.; Boeynaems, J. M.; Barnard, E. A.; Boyer, J. L.;
Kennedy, C.; Knight, G. E.; Fumagalli, M.; Gachet, C.; Jacobson, K. A.; Weisman,
G. A. Pharmacol. Rev. 2006, 58, 281.
8.6. N,N-Diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide (34)
5. Kawate, T.; Michel, J. C.; Birdsong, W. B.; Gouaux, E. Nature 2009, 460, 592.
6. Hattori, M.; Gouaux, E. Nature 2012, 485, 207.
The reaction mixture of dibenz[b,f]azepine (193 mg,
1.00 mmol), diisopropyl carbamoyl chloride (326 mg, 2.00 mmol),
and DIEA (0.33 mL, 2.00 mmol) in toluene (15 mL) was stirred un-
der Ar atmosphere at 140 °C for 24 h. After cooled down to room
temperature, the volatile components were evaporated in vacuo.
The product was purified with the column chromatography (SiO2,
EtOAc–petroleum ether, 1:30). The analytically pure sample was
obtained by recrystallization from diethyl ether–petroleum ether
as colorless prisms (176 mg, 55%). 1H NMR (500 MHz, CDCl3): d
7.63 (dd, J = 8, J = 1 Hz, 2H, Har), 7.37–7.32 (m, 2H, Har), 7.25–7.15
(m, 4H, Har), 6.93 (s, 2H, Har), 3.59–3.45 (m, 2H, CH), 1.01 (d,
7. Nicke, A.; Bäumert, H. G.; Rettinger, J.; Eichele, A.; Lambrecht, G.; Mutschler, E.;
Schmalzing, G. EMBO J. 1998, 17, 3016.
8. Inoue, K.; Tsuda, M. Exp. Neurol. 2012, 234, 293.
9. Beggs, S.; Salter, M. W. Neurosci. Lett. 2013, 557, 37.
10. Inoue, K.; Tsuda, M. CNS Neurol. Disord.: Drug Targets 2012, 11, 699.
11. Beggs, S.; Trang, T.; Salter, M. W. Nat. Neurosci. 2012, 15, 1068.
12. Ulmann, L.; Hatcher, J. P.; Hughes, J. P.; Chaumont, S.; Green, P. J.; Conquet, F.;
Buell, G. N.; Reeve, A. J.; Chessell, I. P.; Rassendren, F. J. Neurosci. 2008, 28,
11263.
13. Chen, K.; Zhang, J.; Zhang, W.; Zhang, J.; Yang, J.; Li, K.; He, Y. Int. J. Biochem. Cell
Biol. 2013, 45, 932.
14. Li, F.; Guo, N.; Ma, Y.; Ning, B.; Wang, Y.; Kou, L. Inflammation 2013, in press,
doi: http://dx.doi.org/10.1007/s10753-013-9723-y.
15. Lu, W. H.; Wang, C. Y.; Chen, P. S.; Wang, J. W.; Chuang, D. M.; Yang, C. S.;
Tzeng, S. F. J. Neurosci. Res. 2013, 91, 694.
16. Ulmann, L.; Levavasseur, F.; Avignone, E.; Peyroutou, R.; Hirbec, H.; Audinat, E.;
Rassendren, F. Glia 2013, 61, 1306.
J = 6.5 Hz, 12H, CH3); 13C NMR (125 MHz, CDCl3):
d 159.0,
144.0, 134.8, 131.6, 129.2, 128.9, 127.5, 126.1, 48.1, 20.9; LC–MS
(m/z): 321 [M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS:
100%.
17. Müller, C. E. Purinergic Signal. 2010, 6, 145.
18. Gum, R. J.; Wakefield, B.; Jarvis, M. F. Purinergic Signal. 2012, 8, S41.
19. Gunosewoyo, H.; Kassiou, M. Expert Opin. Ther. Patents 2010, 20, 625.
20. Nagata, K.; Imai, T.; Yamashita, T.; Tsuda, M.; Tozaki-Saitoh, H.; Inoue, K. Mol.
Pain 2009, 5, 20.
21. Toulme, E.; Garcia, A.; Samways, D.; Egan, T. M.; Carson, M. J.; Khakh, B. S. J.
Gen. Physiol. 2010, 135, 333.
8.7. 11-Diethylcarbamyl-benzo[b]pyrido[4,3-f]azepine (50)
22. Hernandez-Olmos, V.; Abdelrahman, A.; El-Tayeb, A.; Freudendahl, D.;
Weinhausen, S.; Müller, C. E. J. Med. Chem. 2012, 55, 9576.
23. Ambrosio, A. F.; Soares-da-Silva, P.; Carvalho, C. M.; Carvalho, A. P. Neurochem.
Res. 2002, 27, 121.
24. Dahlgren, T.; Hallberg, A.; Helitzer, R.; Martin, A. R. J. Heterocycl. Chem. 1983,
20, 341.
25. (a) Kricka, L. J.; Ledwith, A. Chem. Rev. 1974, 74, 101; (b) Tokmakov, G. P.;
Grandberg, I. I. Tetrahedron 1995, 51, 2091; (c) Craig, P. N.; Lester, B. M.;
Saggiomo, A. J.; Kaiser, C.; Zirkle, C. L. J. Org. Chem. 1961, 26, 135; (d) Monti, K.
D.; Maciejewski, A. B. Appl. Catal. A 1995, 121, 139.
26. Singh, H.; Gupta, N.; Kumar, P.; Dubey, S. K.; Sharma, P. K. Org. Process Res. Dev.
2009, 13, 870.
27. Tsvelikhovsky, D.; Buchwald, S. L. J. Am. Chem. Soc. 2010, 132, 14048.
28. Eckardt, R.; Jaensch, H.-J. U.S. Patent 7015322, 2006.
29. Doyagüez, E. G. Synlett 2005, 1636.
30. Rosowsky, A.; Fu, H.; Chan, D. C.; Queener, S. F. J. Med. Chem. 2004, 47, 2475.
31. Maggs, J. L.; Pirmohamed, M.; Kitteringham, N. R.; Park, B. K. Drug Metab.
Dispos. 1997, 25, 275.
32. Kaczmarek-Hájek, K.; Lörinczi, E.; Hausmann, R.; Nicke, A. Purinergic Signal.
2012, 8, 375.
33. Gaddum, J. H.; Hameed, K. A.; Hathaway, D. E.; Stephens, F. F. Q. J. Exp. Physiol.
1955, 40, 49.
Lithium bis(trimethylsilyl)amide (1 M, 2.00 mL, 2.00 mmol)
was added dropwise to the solution of 11H-Benzo[b]pyrido[4,3-
f]azepine (42, 194 mg, 1.00 mmol) and N,N-diethylcarbamoyl chlo-
ride (2.00 mmol, 272 mg) in dry THF (5 mL) at 0 °C. The reaction
mixture was stirred under Ar atmosphere at room temperature
for 2 h. Methanol (1 mL) was added to the reaction mixture. The
volatile components were evaporated in vacuo. The residue was
submitted to column chromatography (SiO2, EtOAc–petroleum
ether, 1:1). The analytically pure sample was obtained by purifica-
tion with preparative reverse phase HPLC. Yield 70 mg (24%), pale
yellow solid. 1H NMR (500 MHz, CDCl3) d 8.89 (s, 1H, Har), 8.39 (d,
J = 5 Hz, 1H, Har), 7.61–7.53 (m, 1H, Har), 7.47–7.36 (m, 1H, Har),
7.32–7.20 (m, 2H, Har), 7.13 (d, J = 5 Hz, 1H, Har), 7.10 (d,
J = 12 Hz, 1H, Har), 6.87 (d, J = 12 Hz, 1H, Har), 3.06 (q, J = 7 Hz,
4H, CH2), 0.78 (t, J = 7 Hz, 6H, CH3); 13C NMR (125 MHz, CDCl3): d
159.5, 149.4, 146.6, 144.0, 141.3, 139.1, 135.6, 133.7, 130.2,
129.7, 129.3, 127.7, 126.7, 122.3, 42.7, 12.5; LC–MS (m/z): 294
[M+H]+. Purity by HPLC-UV (220–400 nm)-ESI-MS: 100%.
34. Vauquelin, G.; Van Liefde, I.; Birzbier, B. B.; Vanderheyden, P. M. Fundam. Clin.
Pharmacol. 2002, 16, 263.
35. Landek, I. O.; Pesic, D.; Mercep, M.; Stanic, B.; Mesic, M. J. Heterocycl. Chem.
2011, 48, 856.
36. Crawford, L. A.; McNab, H.; Mount, A. R.; Wharton, S. I. J. Org. Chem. 2008, 73,
6642.
9. Biological assays
37. Shieh, W.-C.; Dell, S.; Bach, A.; Repic, O.; Blacklock, T. J. J. Org. Chem. 1954, 2003,
68.
38. Schindler, W. U.S. Patent 2948718 19600809, 1960.
39. Kawashima, K.; Ishiguro, T. Ger. Offen. DE 2246842 A1 19730405, 1973.
40. Kulkarni, G. H.; Naik, R. H.; Tandel, S. K.; Rajappa, S. Tetrahedron 1991, 47,
1249.
All biological assays were performed as previously described.22
Acknowledgments
We thank Marion Schneider for LCMS analyses, Sabine Terhart-
Krabbe and Annette Reiner for NMR spectra, Ralf Hausmann and
Günter Schmalzing (University of Aachen, Germany) for the gift
of some P2X receptor clones, and Florence Moureau and col-
leagues, UCB Pharma, for helpful suggestions. This study was sup-
ported by the BMBF (German Federal Ministry for Education and
Research) within the BioPharma initiative ‘Neuroallianz’.
41. Balaure, P. C.; Costea, I.; Iordache, F.; Draghici, C.; Enache, C. Rev. Roum. Chim.
2009, 54, 935.
42. Aziz-Ur-Rahman; Fareedullah, Md.; Rizwan, S. H.; Abdul Mateen, S. Int. J.
Pharm. Technol. 2012, 4, 4423.
43. Lucini, V.; Pannacci, M.; Scaglione, F.; Fraschini, F.; Rivara, S.; Mor, M.; Bordi, F.;
Plazzi, P. V.; Spadoni, G.; Bedini, A.; Piersanti, G.; Diamantini, G.; Tarzia, G. J.
Med. Chem. 2004, 47, 4202.
44. Della Ca’, N.; Maestri, G.; Malacria, M.; Derat, E.; Catellani, M. Angew. Chem., Int.
Ed. 2011, 50, 12257.
45. Elliott, E.-C.; Bowkett, E. R.; Maggs, J. L.; Bacsa, J.; Park, B. K.; Regan, S. L.;
O’Neill, P. M.; Stachulski, A. V. Org. Lett. 2011, 13, 5592.
46. Elliott, E.-C.; Regan, S. L.; Maggs, J. L.; Bowkett, E. R.; Parry, L. J.; Williams, D. P.;
Park, B. K.; Stachulski, A. V. J. Med. Chem. 2012, 55, 9773.
47. Chang, V. H. T. J. Heterocycl. Chem. 1983, 20, 237.
48. Learmonth, David Alexander, U.S. Patent 7241886, 2007.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.035.