Synthesis and structure-activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2013.10.010

To identify potent dual 5-HT_2B and 5-HT₇ receptor antagonists, we synthesized a series of novel carbonyl guanidine derivatives and examined their structure-activity relationships. Among these compounds, N-(9-hydroxy-9H-fluorene-2-car

Full text of DOI:10.1016/j.bmc.2013.10.010

Bioorganic & Medicinal Chemistry 21 (2013) 7841–7852
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of new carbonyl
guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor
antagonists
a
a
b
Ayako Moritomo a, , Hiroyoshi Yamada , Toshihiro Watanabe , Hirotsune Itahana ,
⇑
Shinobu Akuzawa a, Minoru Okada c, Mitsuaki Ohta a
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Development, Astellas Pharma Inc., 3-17-1 Hasune, Itabashi, Tokyo 174-8612, Japan
c Process Chemistry Laboratories, Astellas Pharma Inc., 160-2 Akahama, Takahagi, Ibaraki 318-0001, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify potent dual 5-HT2B and 5-HT7 receptor antagonists, we synthesized a series of novel carbonyl
guanidine derivatives and examined their structure–activity relationships. Among these compounds,
N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (10) had a good in vitro profile, that is, potent affinity
for human 5-HT2B and 5-HT7 receptor subtypes (Ki = 1.8 nM and Ki = 17.6 nM, respectively) and high
Received 8 August 2013
Revised 9 October 2013
Accepted 10 October 2013
Available online 21 October 2013
selectivity over 5-HT2A, 5-HT2C
on 5-HT-induced dural protein extravasation in guinea pigs when orally administered.
Ó 2013 Elsevier Ltd. All rights reserved.
, a1, D2 and M1 receptors. Compound 10 also showed a suppressing effect
Keywords:
Migraine
5-HT2B receptor
5-HT7 receptor
Dual antagonist
Carbonyl guanidine
1. Introduction
anti-migraine.3 Among the anti-migraine 5-HT receptor antago-
nists, Pizotifen (1; Fig. 1) was launched in Europe, but a high fre-
quency of adverse effects at its effective dose were reported,
including fatigue, sleepiness, giddiness and weight gain.4 These
side effects might be due to Pizotifen (1) having a high affinity
Migraine is a complex paroxysmal disorder resulting in a peri-
odically occurring pulsating headache in which a strong pain oc-
curs in one side or both sides of the head and continues from
several hours to 3 days. Although migraine affects a substantial
proportion of the population, the exact pathophysiology is not fully
understood. Recently, it was suggested that the morbid state of mi-
graine is advanced by the following hypothetical mechanism.1
Firstly, dural blood vessel is contracted by a rapid increase in blood
5-hydroxytryptamine (5-HT, serotonin) levels, followed by the
metabolism and depletion of 5-HT and the 5-HT depletion cause
vasodilatation of dural blood vessels. During this time, the trigem-
inal nerve is activated and releases plasma proteins, such as sub-
stance P (SubP), and vasoactive peptides, such as calcitonin gene-
related peptide (CGRP). The release of SubP and CGRP promote
inflammation around the dural blood vessels, which results in mi-
graine. The above findings therefore suggest that 5-HT plays an
important roles in the onset of migraine.2
for most 5-HT receptor subtypes and adrenergic
al, muscarine
M1, dopamine D2 and other receptors.5
In recent years, a number of pharmacological studies on 5-HT
receptor subtypes have been conducted. One study found that 5-
HT2B receptor antagonist inhibited m-chlorophenylpiperazine
(mCPP)-induced leakage of protein outside of the dural blood ves-
sel in guinea pigs,6 and that 5-HT2B receptor localized on vascular
smooth muscle caused nitric oxide (NO) release, which accelerated
the release of CGRP and SubP from trigeminal nerves.7 The selec-
tive 5-HT2B receptor antagonist RS-127445 (2)8 also showed a sup-
pressing effect on mCPP-induced plasma extravasations in the
dural blood vessels of anesthetized rats.9 5-HT7 receptor was also
reported to be distributed in the trigeminal nerve,10 contribute to
vasodilatation of the cerebrovascular smooth muscle,11 and accel-
erate protein leakage in the dural matar blood vessel.12 Adminis-
tration of the selective 5-HT7 antagonist SB-269970 (3)13 was
recently shown to partially inhibit CGRP release evoked by the tri-
geminal ganglion (TEGS) in a rat model,14 and 5-HT1D, 5-HT2B and
5-HT7 receptors have been shown to be expressed in the dural ma-
tar blood vessels.15 Taking these previous findings into account, we
Medicines for migraine are divided into two categories; preven-
tive medicines and treatment medicines. Agents exhibiting 5-HT
receptor antagonism have been reported effective in preventing
⇑
Corresponding author. Tel.: +81 29 863 6709; fax: +81 29 854 1519.
E-mail address: ayako.moritomo@astellas.com (A. Moritomo).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.010

7842
A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
NH2
N
N
NH2
NH2
N
N
N
N
S O
O
HO
O
S
F
4
1, Pizotifen
2, RS-127445
3, SB-269970
Figure 1. Structures of Pizotifen (1), RS-127445 (2, 5-HT2B antagonist), SB-269970 (3, 5-HT7 antagonist), and HTS hit (4).
250
200
OH
N=4
CHO
O
OTf
O
b, c
a
CO2Me
CO2Me
NH2
CO2Me
∗
150
100
50
21
22
23
e
d
N
CO2H
O
O
NH2
O
5
24
0
Scheme 2. Reagents and conditions: (a) 2-fluorobenzaldehyde, K2CO3, DMF, 70 °C;
(b) m-chloroperoxybenzoic acid, CHCl3, 30 °C then concd HCl (cat), MeOH; (c)
trifluoromethanesulfonic anhydride, pyridine, CH2Cl2; (d) Pd(PPh3)2Cl2 (cat), DBU,
LiCl, DMF, 145 °C; (e) CDI, DMF, rt then guanidine carbonate, rt.
control
5-HT
2
2 (3 mg/kg i.p.)
+3(10 mg/kg i.p.)
3 mg/kg i.p.
Figure 2. Suppressing effects of single dosings of compounds on the 5-HT induced
dural plasma protein extravasation in guinea pigs. Values are mean or SEM for four
⁄
animals in each group. <0.05: significant difference. Student’s t-test.
NH2
O
a
N
4
NH2
4
N
H
N
H
O
O
a
2
2
NH2
NH2
OH
N
25
6
1
1
O
O
Scheme 3. Reagent and condition: (a) guanidine hydrochloride, NaH, DMF, 70 °C.
15:2-CO H
4:2-CONC(NH2)2
2
16:1-CO H
11:1-CONC(NH2)2
13:4-CONC(NH2)2
2
(RS-127445 (2): 3 mg/kg ip, SB-269970 (3): 10 mg/kg ip, respec-
tively) were simultaneously administered, the amount of leaked
protein was suppressed nearly completely to the normal level in
the same model (Fig. 2). This result shows that when 5-HT2B and
5-HT7 receptors are simultaneously inhibited, a cumulative effect
is observed that cannot be attained by selective inhibition of only
one receptor.
17:4-CO H
2
3
3
2
a
2
NH2
NH2
OH
N
O
O
O
O
We next focused our attention on creating novel dual 5-HT2B
and 5-HT7 receptor antagonists as a potential new class of preven-
tive medicines for anti-migraines. Here, to identify novel dual
5-HT2B and 5-HT7 receptor antagonists, we conducted high-
throughput screening (HTS) of the Astellas chemical library, which
led to the discovery of N-(diaminomethylene)-9H-fluorene-2-
carboxamide (4). We now describe the synthesis and structure–
activity relationships (SAR) of carbonyl guanidine derivatives as
novel dual 5-HT2B and 5-HT7 receptor antagonists.
18:2-CO H
19:3-CO2H
9:2-CONC(NH)2
2
2
20:3-CONC(NH)2
2
Scheme 1. Reagent and condition: (a) CDI, DMF, rt then guanidine hydrochloride,
NaH, DMF, rt.
hypothesized that dual 5-HT2B and 5-HT7 receptor antagonists may
represent a new class of medicines that prevent migraine with a
reduction in the number and severity of side effects.
We previously reported a synergistic effect using the 5-HT2B
receptor selective antagonist RS-127445 (2) and the 5-HT7 receptor
selective antagonist SB-269970 (3).16 RS-127445 (2) showed an ef-
fect to reduce amount of leaked protein from dural blood vessel in
guinea pigs evoked by 5-HT at 3 mg/kg intraperitoneal administra-
tion (ip), but did not reduce to the normal level when the dose was
increased from 3 mg/kg to 10 mg/kg. SB-269970 (3) also showed a
similar effect at 10 mg/kg ip in the same model, but did not reduce
the amount of leaked protein to the reference value when the dose
was increased from 10 mg/kg to 30 mg/kg. When both compounds
2. Chemistry
Synthesis of fluorene carbonyl guanidine derivatives 4, 11, 13, 9
and 20 is depicted in Scheme 1. Fluorene carboxylic acids 15–18
were commercially available and fluorene carboxylic acid 19 was
prepared based on a protocol reported by Weisburger et al.17 Con-
densation with guanidine and fluorene carboxylic acids 15–19 in
the presence of 1,10-carbonyldiimidazole (CDI) gave fluorene car-
bonyl guanidine derivatives 4, 11, 13, 9 and 20.

A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
7843
F
3
3
O
OH
a
b
O
2
2
a
CN
N
H
NH2
R
CN
NH2
R
OH
26
27
28
O
9 : R = 2-CONC(NH2)2
10:R = 2-CONC(NH2)2
12:R = 3-CONC(NH2)2
O
O
c
d
N
NH2
NH2
20:R= 3-CONC(NH2)2
CO2H
N
H
N
H
O
36:R = 2-CH=CHCONC(NH2)2
14:R = 2-CH=CHCONC(NH2)2
29
7
Scheme 6. Reagent and condition: (a) NaBH4, MeOH, rt.
Cl
H
N
H
N
g
e,f
gave 3-cyanophenoxazine 31. Hydrolysis of the cyanophenoxa-
zines 28 and 31 under acidic conditions generated corresponding
carboxylic acids 29 and 32. Condensation of the carboxylic acids
29 and 32 with guanidine in the presence of CDI gave compounds
7 and 8, respectively.
Scheme 5 shows the synthesis of (2E)-N-(diaminomethylene)-
3-(9-oxo-9H-fluoren-2-yl)acrylamide (36). Ethyl 9-oxo-9H-
fluorene-2-carboxylate 33 was converted into aldehyde 34 by
reduction of the ester with lithium aluminum hydride (LiAlH4)
followed by oxidatation of the diol with manganese(IV) oxide
(MnO2). Wittig reaction of the resulting aldehyde 34 with methyl
(triphenylphosphoranylidene)acetate gave acrylic acid ester,
which was converted to acrylic acid 35 by treatment with NaOH.
Condensation of the acrylic acid 35 with guanidine in the presence
of CDI gave compound 36.
26
CN
O
CN
OH
30
31
H
N
H
N
c
d
N
NH2
NH2
O
CO2H
O
O
32
8
Scheme 4. Reagents and conditions: (a) 3,4-difluorobenzonitrile, NaH, DMF, 0 °C;
(b) DMF, 165 °C; (c) concd HCl, AcOH, 120 °C; (d) CDI, DMF, 50 °C then guanidine
hydrochloride, NaH, DMF, rt; (e) 3-chloro-4-fluorobenzonitrile, NaH, DMF, 0 °C; (f)
NaH, DMF, 100 °C; (g) K2CO3, DMF, 150 °C.
Preparation of dibenzo[b,d]furan carbonyl guanidine derivative
5 is outlined in Scheme 2. A base promoted reaction of 2-fluoro-
benzaldehyde with phenol 21 yielded the aryloxy benzaldehyde
derivative 22, followed by Bayer–Villiger oxidation with m-chloro-
perbenzoic acid (m-CPBA) to furnished phenol formate. The for-
mate was hydrolyzed to free phenol, which was converted to
triflate 23 by treatment with triflic anhydride. Palladium-catalyzed
cyclization of the triflate 23 provided caroboxylic acid 24. Conden-
sation with 24 and guanidine in the presence of CDI gave com-
pound 5.
Conversion of 9-oxo-fluorene derivatives 9, 20 and 36 to
9-hydroxy-9H-fluorene derivatives 10, 12 and 14 is shown in
Scheme 6. Reduction of the 9-oxo-fluorenone derivatives 9, 20
and 36 with sodium borohydride (NaBH4) gave the target
compounds 10, 12 and 14.
3. Results and discussion
5-HT2B and 5-HT7 receptor binding affinities of the synthesized
compounds 4–14 were determined using the displacement of
antagonist ([3H]Mesulergine) radioligand binding to human
5-HT2B receptor sites in HEK293 cell membranes, and using agonist
([3H]5-HT) radioligand binding to human 5-HT7 receptor sites in
CHO cell membranes, respectively.
Compound 4 was found to have extremely potent affinity for
5-HT2B and 5-HT7 receptors (Ki = 1.8 nM and Ki = 12.4 nM, respec-
tively), whereas its aqueous solubility was poor (solubility at pH
Scheme 3 shows the synthesis of carbazole carbonyl guanidine
derivative 6. Condensation with 2518 and guanidine under thermal
condition gave target compound 6.
10H-Phenoxazine carbonyl guanidine derivative 7, 8 were syn-
thesized as shown in Scheme 4. 10H-Phenoxazine carboxylic acids
29 and 32 were prepared by cyano-activated fluoro displacement
reactions based on a protocol by Eastmond et al.19 A fluoro dis-
placement reaction between 3,4-difluorobenzonitrile and 2-ami-
nophenol 26 gave aminoether 27, followed by thermal reaction
in the absence of base yielded 2-cyanophenoxazine 28. On the
other hand, a reaction between 3-chloro-4-fluorobenzonitrile and
2-aminophenol 26 gave aminoether, followed by Smiles rearrange-
ment with base to furnish phenol 30. Cyclization of the phenol 30
6.8 <1 lg/mL). We carried out an SAR study with compound 4 as
the lead compound to improve both affinity and aqueous solubil-
ity. To improve aqueous solubility, reducing lipophilicity20 or
increasing topographical polar surface area (TPSA)21 have been
proven effective strategies. Lipophilicities were estimated by
c, d
a, b
CO2H
CO2Et
CHO
O
O
O
35
33
34
NH2
N
e
NH2
O
O
36
Scheme 5. Reagents and conditions: (a) LiAlH4, THF, rt; (b) MnO2, CHCl3, rt; (c) Ph3P = CHCO2Me, toluene, 90 °C; (d) 1 M NaOH aq, MeOH–THF, rt; (e) CDI, DMF, rt then
guanidine hydrochloride, NaH, DMF, rt.

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A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
Table 1
5-HT2B and 5-HT7 receptor binding affinities of tricyclic ring derivatives 4–8
O
NH2
NH2
HCl
N
R
Compound
–R
5-HT2B Kia,b (nM)
5-HT7 Kia,c (nM)
Solubility pH6.8 (
lg/mL)
CLogPf
TPSAg
81.5
4d
1.8
2.1
12.4
<1
2.61
*
*
5
6
>100
42
NTe
NTe
2.65
2.13
94.6
97.3
O
*
10
N
H
O
7
8
3.6
2.4
144
NTe
2.22
2.22
102.7
102.7
N
H
*
H
N
>1000
NTe
O
*
a
b
c
d
e
f
The data were obtained from at least two independent experiments.
Ki for [3H]Mesulergine binding; human 5-HT2B receptor expressed in HEK293 cells.
Ki for [3H]5-HT binding; human 5-HT7 receptor expressed in CHO cells.
Free form.
Not tested.
CLogP value was calculated by ACD/Labs Software, version 12.01.
TPSA value was calculated by MOE2011.10.
g
Figure 3. Atomic charge distributions of fluorene ring (40), dibenzofuran ring (50), and carbazole ring (60). Compounds are colored blue for nitrogen, red for oxygen and green
for carbon. Connolly surfaces24 around the core scaffolds are colored red for negative charge and blue for positive charge.
CLogP values, calculated using ACD LogP prediction software22 and
TPSA values were calculated using Molecular Operating Environ-
ment (MOE23) software.
loss of affinity for 5-HT7 receptor, although almost equal affinity
for 5-HT2B receptor (Ki = 1.8 nM). Replacement of the fluorene moi-
ety with a carbazole moiety (6) caused a slight decrease in affinity
for 5-HT2B and 5-HT7 receptors (Ki = 10 nM and Ki = 42 nM, respec-
tively). Conversion of the fluorene moiety to 2-phenoxazine moiety
(7) and 3-phenoxazine moiety (8) resulted in significantly de-
creased affinity for 5-HT7 receptor, although almost equally potent
to 5-HT2B receptor (Ki = 3.6 nM and Ki = 2.4 nM, respectively).
These results suggested that the 6,5,6-membered ring systems
were preferable to the 6,6,6-membered ring systems.
At the initiation of our SAR studies, we hypothesized that the
carbonyl guanidine moiety of compound 4 would be a structure
mimicking a basic amino moiety in 5-HT. We subsequently synthe-
sized derivatives containing modifications of the fluorene ring. We
first studied the SAR of compounds containing with a dibenzofu-
ran, carbazole, and phenoxazine (Table 1). The ClogP values
(2.13–2.65) of compounds 5–8 were equal to or lower than that
of compound 4 (2.61), and compounds 5–8 exhibited higher TPSA
values (94.6–102.7) than that of compound 4 (81.5). Replacement
of the fluorene moiety with a dibenzofuran moiety (5) resulted in a
We calculated the atomic charge distributions of the fluorene
moiety of compound 4, the dibenzofuran moiety of compound 5
and the carbazole moiety of compound 6 to examine these results

A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
7845
Table 2
5-HT2B and 5-HT7 receptor binding affinities of 9-position substituent fluorene derivatives 4, 9 and 10
O
NH2
NH2
N
R
Compound
–R
5-HT2B Kia,b (nM)
5-HT7 Kia,c (nM)
CLogPe
TPSAf
*
4
1.8
12.4
2.61
81.5
98.5
101.7
9
*
9
0.4
1.8
185
2.09
0.98
O
*
10d
17.6
OH
a
b
c
d
e
f
The data were obtained from at least two independent experiments.
Ki for [3H]Mesulergine binding; human 5-HT2B receptor expressed in HEK293 cells.
Ki for [3H]5-HT binding; human 5-HT7 receptor expressed in CHO cells.
HCl salt.
CLogP value was calculated by ACD/Labs Software, version 12.01.
TPSA value was calculated by MOE2011.10.
to the 9-position of fluorene ring maintained the affinity towards
5-HT2B and 5-HT7 receptors (Ki = 1.8 nM and Ki = 17.6 nM, respec-
tively), and indicated a possible improvement in both affinity and
aqueous solubility. Additionally, as shown in Figure 3, the calculated
atomic charge distributions of the fluorene moiety of compound 10
were consistent with the results shown in Figure 4.
We then examined the effects of substitution patterns of a car-
bonyl guanidine moiety on the fluorene ring (Table 3). Compound
11 showed a 20-fold loss of affinity for 5-HT7 receptor (Ki = 239
nM) compared to compound 4, while affinities of both compounds
for 5-HT2B receptor were maintained. Compounds 12 and 13 exhib-
ited a loss of affinity for 5-HT7 receptor, and a significantly de-
creased affinity for 5-HT2B receptor was observed (Ki = 86.8 nM
and Ki = 27.4 nM, respectively). Moreover, insertion of an olefin lin-
ker between the fluorene ring and a carbonyl guanidine (14) re-
sulted in a decrease in affinity for 5-HT2B and 5-HT7 receptors (Ki
= 52 nM and Ki = >100 nM, respectively). These results indicated
that direct substitution of the carbonyl guanidine moiety at the
2-position on fluorene ring was an essential structure.
Figure 4. Atomic charge distributions of 9-hydoxy fluorene (100). Compound is
colored gray for polar-hydrogen, red for oxygen and green for carbon. Connolly
surfaces24 around the core scaffolds are colored red for negative charge and blue for
positive charge.
further (Fig. 3). For the fluorene ring 40 and the carbazole ring 60,
the positive charge (blue) was distributed on the center-bottom
side of the 6,5,6-membered ring, whereas the dibenzofuran ring
50 had a negative charge (red) in the corresponding region. These
results suggested that a positive charge was localized at the cen-
ter–bottom side of the 6,5,6-membered ring, resulting in affinity
for 5-HT7 receptor. On the other hands, the atomic charge distribu-
tions were not related to 5-HT2B receptor affinity. On the basis of
these results, we selected compound 4 as the lead compound for
further optimizations.
Next, we investigated the effects of substitution on the 9-position
of fluorene ring of compound 4 (Table 2). The ClogP values of com-
pounds 9 (2.09) and 10 (0.98) were lower than that of compound 4
(2.61), and the TPSA values of compounds 9 (98.5) and 10 (101.7)
were higher than that of compound 4 (81.5). The introduction of a
carbonyl group (9) to the 9-position of fluorene ring resulted in an
increased affinity for 5-HT2B receptor (Ki = 0.4 nM), while a signifi-
cantly decreased affinity for 5-HT7 receptor was observed (Ki = 185
nM). On the other hands, the introduction of a hydroxyl group (10)
Having identified 10 as a promising compound, we investigated
other off-target activities (5-HT2A, 5-HT2C, adrenergic a1, dopamine
D2 and muscarine M1 receptors) and aqueous solubility. The results
are summarized in Table 4. While Pizotifen (1) has no selectivity to
other off-target receptors, both 4 and 10 showed low affinity for 5-
HT2A, D2 and M1 receptors. The HTS hit compound 4 showed mod-
erate affinities for 5-HT2c and a1 receptors (Ki = 200 nM and Ki =
790 nM, respectively), however, compound 10 showed consider-
ably weaker affinities for 5-HT2c and a1 receptors (Ki = >1000
nM) than compound 4. Furthermore, as predicted with the ClogP
value or the TPSA value, compound 10 showed higher solubility
than compound 4 in a pH 6.8 aqueous solution. These results dem-
onstrated that compound 10 had an excellent 5-HT2B/7 selectivity
versus other off-target receptors, with significantly-improved
aqueous solubility over compound 4.
The functional activities of compounds 4 and 10 on human 5-
HT2B and 5-HT7 receptors were determined by measuring inhibi-

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A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
Table 3
5-HT2B and 5-HT7 receptor binding affinities of carbonyl guanidine derivatives 4, 10–14
NH2
NH2
O
4
3
N
2
1
HCl
R
Compound
–R
Carbonyl guanidine
5-HT2B Kia,b (nM)
5-HT7 Kia,c (nM)
4
H
–OH
H
–OH
–H
2
2
1
3
4
1.8
1.8
1.1
86.8
27.4
12.4
17.6
239
>1000
>1000
10
11
12
13
NH2
N
NH2
O
14
52
>100
OH
a
b
c
The data were obtained from at least two independent experiments.
Ki for [3H]Mesulergine binding; human 5-HT2B receptor expressed in HEK293 cells.
Ki for [3H]5-HT binding; human 5-HT7 receptor expressed in CHO cells.
Table 4
Affinity for other receptors, and aqueous solubility of compounds 4 and 10
Compound
Ki (nM)
5-HT2C
Solubility
a,b
a,c
d
d
d
d
d
5-HT2B
5-HT7
5-HT2A
a1
D2
M1
pH 6.8 (lg/mL)
Pizotifen (1)
4
10
2.0
1.8
1.8
25
12.4
17.6
7.5
1000
>1000
1.4
200
>1000
75
790
>1000
2.4
1400
>1000
2.0
>1000
>1000
NTe
<1
10<
a
b
c
The data were obtained from at least two independent experiments.
Ki for [3H]Mesulergine binding; human 5-HT2B receptor expressed in HEK293 cells.
Ki for [3H]5-HT binding; human 5-HT7 receptor expressed in CHO cells.
Ki value was measured in accordance with the established method.28–31
Not tested.
d
e
Table 5
Table 6
Human 5-HT2B and 5-HT7 receptor antagonistic activity of 4 and 10
Suppressing effects of 10, RS-127445 (2, 5-HT2B receptor antagonist) and SB-269970
(3, 5-HT7 receptor antagonist) on 5-HT induced dural plasma protein extravasation in
guinea pigs
5-HT7 % inhibitiona,c (%)
a,b
Compound
5-HT2B IC50 (nM)
4
10
36.4
19.6
98
97
Compound
% Changea (%)
RS-127445 (2)b
60
91
93
a
The data were obtained from at least three independent experiments.
RS-127445 (2) and SB-269970 (3)c
b
IC50 against [3H]PI metabolism; human 5-HT2B receptor expressed in HEK293
10d
cells.
c
%
Inhibition at 10
l
M
against cAMP production; human 5-HT7 receptor
a
% Change from 5-HT group. The following formula was used in this calculation:
expressed in CHO cells.
%
change = [compound group ꢀ control]/[5-HT group ꢀ control] ꢁ100%. See
Section 5.
b
At 3 mg/kg ip.
tory activity of PI metabolism in HEK 293 cells or cAMP production
in CHO cells. Compounds 4 and 10 both showed antagonistic activ-
ity towards human 5-HT2B and 5-HT7 receptors (Table 5).
c
Simulutaneously administration of 2 (3 mg/kg ip) and 3 (10 mg/kg ip).
At 3 mg/kg ip.
d
Given its potent inhibitory activity against 5-HT2B/7 receptors
and high selectivity against other off-target receptors, compound
10 was submitted for in vivo studies. The results are presented
with % change values [that is, suppressing effects compared with
control and how the compounds suppressed the protein extravasa-
tion] in Table 6. As shown in Figure 5A, single intraperitoneal doses
(0.3–3 mg/kg) of compound 10 suppressed 5-HT-induced dural
protein extravasation in guinea pigs in a dose-dependent manner.
When compound 10 was administered at 3 mg/kg ip, the amount
of leaked protein was suppressed nearly completely to the normal
level (% change = 93%). This was a comparable effect when 5-HT2B
receptor selective antagonist RS-127445 (2) and 5-HT7 receptor
selective antagonist SB-269970 (3) were simultaneously
administered in the same model (% change = 91%). When only
RS-127445 (2) was administered, the% change value was 60%. That
is, a dual 5-HT2B and 5-HT7 receptor antagonist compound 10 can
inhibit leakage of the inflammatory protein nearly complete by its
cumulative effect that cannot be attained by selective inhibition of
only one receptor. In addition, compound 10 showed a suppressive
effect in the same model when orally administered (3–30 mg/kg)
in a dose-dependent manner (Fig. 5B). Taken together, these

A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
7847
for the signal patterns are as follows: s, singlet; br s, broad singlet;
d, doublet; t, triplet; dd, double doublet; dt, double triplet; td, tri-
ple doublet; m, multiplet. Mass spectra were recorded on a Hitachi
M-80 or JEOL JMS-DX300 mass spectrometer, and the ionization
method was chosen from ESI and FAB. The elemental analyses
were performed with a Yanaco MT-5 microanalyzer (C, H, N) and
a Yokogawa IC-7000S ion chromatographic analyzer (Cl). Prepara-
tive column chromatography was performed with Wakogel C-200
or Merck silica gel 60 or Fuji silisia NH2 Chromatorex.
(A)
(N=4-5) Dunnettís test (* p<0.05, **p<0.01; vs. 5-HT)
5.1.1. N-(Diaminomethylene)-9H-fluorene-2-carboxamide (4)
To a solution of 9H-fluorene-2-carboxylic acid (15, 520 mg,
2.47 mmol) in DMF (6 mL) was added 1,10-carbonyldiimidazole
(CDI) (440 mg, 2.71 mmol) at room temperature, the reaction mix-
ture was stirred at 50 °C for 1 h, then allowed to cool. This mixture
was added under ice-cooling to a solution which had been pre-
pared by addition of NaH (55% dispersion in mineral oil; 500 mg,
12.5 mmol) to DMF (6 mL) solution of guanidine hydrochloride
(1.18 g, 12.3 mmol) and stirring at room temperature for 1 h, and
the reaction mixture was stirred at room temperature for 2 h.
The reaction mixture was concentrated in vacuo and added CHCl3
and water. The aqueous layer was extracted with CHCl3, and the
combined organic layer was washed with 1 M NaOH aq, dried over
MgSO4. After evaporation in vacuo, the resultant residue was puri-
fied by silica gel column chromatography (EtOAc), recrystalization
from MeOH to give the title compound 4 (140 mg, 23%) as a pale
yellow solid.
10 i.p.
(B)
200
150
100
50
(N=5) Dunnettís test (* p<0.05, **p<0.01; vs. 5 -HT)
*
**
0
control
5-HT
3 mg/kg
10 mg/kg
30 mg/kg
10 p.o.
1H NMR (DMSO-d6) d: 3.96 (2H, s), 7.31–7.42 (2H, m), 7.60 (1H,
d, J = 6.9 Hz), 7.88 (1H, d, J = 8.6 Hz), 7.93 (1H, d, J = 8.3 Hz), 8.12
(1H, dd, J = 1.4, 8.3 Hz), 8.31 (1H, s). MS (FAB) m/z: 252 (M++H).
Mp: 210–212 °C. Anal. Calcd for C15H13N3O: C, 71.70; H, 5.21; N,
16.72. Found: C, 71.61; H, 5.18; N, 16.63.
Figure 5. Suppressing effects of single dosings of compounds on the 5-HT induced
dural plasma protein extravasation in guinea pigs. (A) intraperitoneal dosing of 10.
(B) oral dosings of 10. Values are mean or SEM for five animals in each group. ⁄p
<0.05, ⁄⁄p <0.01 versus 5-HT. Dunnett’s multiple comparison test.
5.1.2. Methyl 3-(2-formylphenoxy)benzoate (22)
findings strongly supported the potential of dual 5-HT2B and 5-HT7
receptor antagonists in preventive medicine for anti-migraines.
To a solution of methyl 3-hydroxybenzoate (2 g, 13.1 mmol)
and 2-fluorobenzaldehyde (1.38 mL, 13.1 mmol) in DMF (15 mL)
was added potassium carbonate (2.72 g, 19.6 mmol) and the mix-
ture was stirred at 70 °C for 18 h, and then allowed to cool. The
reaction mixture was concentrated in vacuo and added EtOAc
and water. The aqueous layer was extracted with EtOAc, and the
combined organic layer was washed with brine, dried over MgSO4.
After evaporation in vacuo, the resultant residue was purified by
silica gel column chromatography (EtOAc–hexane) to give the title
compound 22 (2.54 g, 76%) as a white solid.
4. Conclusion
In this investigation of novel dual 5-HT2B and 5-HT7 receptor
antagonists, a series of carbonyl guanidines with various tricyclic
aromatic rings were synthesized and evaluated. Structure–activity
relationship (SAR) studies of this novel class of compounds revealed
that fluorene 2-carbonyl guanidine played an important structures
in showing potent inhibitory activity against both 5-HT2B and 5-HT7
receptors. The atomic charge distributions of 6,5,6-membered rings
suggested that a positive charge is localized at the 9-position of flu-
orene ring, endowing affinity for 5-HT7 receptor. Of this series, N-
(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (10) exerts potent
affinity for human 5-HT2B and 5-HT7 receptor subtype (Ki = 1.8
nM and Ki = 17.6 nM, respectively) with high selectivity over 5-
1H NMR (CDCl3) d: 3.91 (3H, s), 6.90 (1H, d, J = 8.4 Hz), 7.21–
7.30 (2H, m), 7.47 (1H, t, J = 8.0 Hz), 7.53–7.56 (1H, m), 7.71–7.72
(1H, m), 7.86 (1H, dt, J = 1.2, 8.0 Hz), 7.96 (1H, dd, J = 1.2, 8.4 Hz).
MS (ESI) m/z: 257 (M++H).
5.1.3. Methyl 3-(2-tifluoromethanesulofonate)benzoate (23)
To a solution of 22 (2.54 g, 9.9 mmol) in chloloform (40 mL)
was add m-chloroperoxybenzoic acid (4.28 g, 24.7 mmol) and
the mixture was stirred at 30 °C for 3 h, then cooled down to
room temperature. The reaction mixture was quenched by addi-
tion of dilute aqueous sodium hydrogen sulfate solution. The or-
ganic layer was washed with saturated aqueous sodium
bicarbonate solution and water, dried over Na2SO4 and evapo-
rated in vacuo to give the corresponding formate as a pale yellow
oil, which was used for the next step without further purification.
To a solution of the formate obtained above in MeOH (50 mL) was
added 2 drops conc. HCl aq, and the solution was stirred at room
temperature for 1.5 h. After addition of sodium carbonate (1 g),
the solution was filtered and evaporated in vacuo, the resultant
residue was purified by silica gel column chromatography
(MeOH–CHCl3) to give the phenol (2.25 g, 93%) as a white solid.
To a cold (0 °C) solution of the phenol obtained above (2.18 g,
HT2A, 5-HT2C, a1, D2 and M1 receptors. Compound 10 also showed
a suppressing effect on 5-HT-induced dural protein extravasation
in gunea pigs when orally administered. Further investigation will
be carried out to improve the potency of this series of compounds
and the results will be reported in due course.
5. Experimental
5.1. Chemistry
Uncorrected melting points (Mps) were determined using
BÜCHI B-545 or Yanaco MP-500D micro melting apparatuses. 1H
NMR spectra were recorded on a JEOL JNM-LA300, JEOL JNM-
EX400 or JEOL JNM-A500 spectrometer and were referenced to
an internal standard, tetramethylsilane. The abbreviations used

7848
A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
8.92 mmol) and pyridine (3.60 mL, 44.6 mmol) in CH2Cl2 (25 mL)
was added triflic anhydride (2.0 mL, 11.5 mmol), then the mixture
was warmed up to room temperature and stirred for 30 min.
After addition of water (100 mL), the aqueous layer was extracted
with CHCl3. The organic layer was washed with 1 M HCl aq,
saturated aqueous sodium bicarbonate solution and brine,
dried over MgSO4. After evaporation in vacuo, the resultant
residue was purified by silica gel column chromatography (CHCl3)
to give the title compound 23 (3.35 g, quantitative) as a colorless
oil.
5.1.7. N-(Diaminomethylene)-9H-carbazole-2-carboxamide
monohydrochloride (6)
To a cold (0 °C) solution of guanidine hydrochloride (573 mg,
6 mmol) in DMF (6.5 mL) was added portion wise NaH (60% disper-
sion in mineral oil; 192 mg, 4.8 mmol) and the mixture was stirred
at room temperature for 1 h. To the mixture was added dropwise a
solution of 25 (271 mg, 1.2 mmol) in DMF (6.5 mL) and the mixture
was stirred at 70 °C for 2 days. After cooling, the reaction mixture
was concentrated in vacuo. To the resultant residue was added
water and stirred at room temperature for 3 h. The precipitate
was collected by filtration, washed with water, dried in vacuo at
50 °C. The resultant residue was purified by silica gel column chro-
matography (CHCl3–MeOH) to give N-(diaminomethylene)-9H-
carbazole-2-carboxamide (236 mg) as a pale yellow solid. The
compound was converted to its monohydrochloride salt by treat-
ing it with 4 M HCl–EtOAc (0.45 mL, 1.8 mmol) in EtOH (9.0 mL).
The crude salt was suspended with EtOH and filtered to give the ti-
tle compound 6 (266 mg, 77%) as a pale yellow solid.
1H NMR (CDCl3) d: 3.90 (3H, s), 6.98 (1H, dd, J = 1.2, 8.0 Hz), 7.17
(1H, dt, J = 1.6, 8.0 Hz), 7.26–7.33 (2H, m), 7.36 (1H, dd, J = 1.2,
8.0 Hz), 7.45 (1H, t, J = 8.0 Hz), 7.70–7.72 (1H, m), 7.86 (1H, dt,
J = 1.2, 8.0 Hz). MS (ESI) m/z: 377 (M++H).
5.1.4. Dibenzo[b,d]furan-3-carboxylic acid (24)
A solution of 23 (1.01 g, 2.7 mmol), Pd(PPh3)2Cl2 (192 mg,
0.27 mmol), LiCl (347 mg, 8.2 mmol), and DBU (0.5 mL, 3.3 mmol)
in DMF (30 mL) was stirred at 145 °C for 22.5 h. After cooling,
Et2O and water were added and the aqueous layer was extracted
with Et2O. The aqueous layer was acidified (pH 2.0) with 1 M HCl
aq and the resulting solid was filtered. The combined organic layer
was washed with brine, dry over MgSO4 and evaporated in vacuo
and the resulting residue was added water and acidified (pH2.0)
with 1 M HCl aq and the resulting solid was filtered. These two sol-
ids were combined and purified by silica gel column chromatogra-
phy (CHCl3–MeOH) and recrystallization from EtOH to give the
title compound 24 (102 mg, 17.8%) as a white solid.
1H NMR (DMSO-d6) d: 7.24 (1H, td, J = 1.0, 7.3 Hz), 7.51 (1H, td,
J = 1.0, 7.3 Hz), 7.58 (1H, d, J = 8.3 Hz), 7.97 (1H, dd, J = 1.5, 8.3 Hz),
8.24 (1H, d, J = 7.3 Hz), 8.32 (1H, d, J = 6.4 Hz), 8.33 (1H, s), 8.56
(2H, br s), 8.83 (2H, br s), 11.80 (1H, s), 12.04 (1H, s). MS (FAB)
m/z: 253 (M++H). Mp: >250 °C decompose. Anal. Calcd for C14H12-
N4OꢂHClꢂ0.3C2H6Oꢂ0.1H2O: C, 57.62; H, 4.97; N, 18.41; Cl, 11.65.
Found; C, 57.67; H, 4.97; N, 18.21; Cl, 11.81.
5.1.8. 4-(2-Aminophenoxy)-3-fluorobenzonitrile (27)
To a cold (0 °C) solution of 2-aminophenol (3.0 g, 27.4 mmol) in
DMF (30 mL) was added portion wise NaH (55% dispersion in min-
eral oil; 1.26 g, 28.7 mmol) and the mixture was stirred at 0 °C for
30 min. To this cold mixture was added 3,4-difluorobenzonitrile
(4.02 g, 28.7 mmol) and the mixture was stirred at 0 °C for 3 days.
Reaction was quenched by addition of water, and the aqueous
layer was extracted with EtOAc. The organic layer was washed
with water and brine, dried over Na2SO4. After evaporation in va-
cuo, the resultant residue was purified by silica gel column chro-
matography (EtOAc–hexane) to give the title compound 27
(4.88 g, 78%) as an orange oil.
1H NMR (DMSO-d6) d: 7.46 (1H, t, J = 7.6 Hz), 7.61 (1H, td, J = 0.8,
7.6 Hz), 7.77 (1H, d, J = 8.0 Hz), 8.01 (1H, dd, J = 0.8, 8.0 Hz), 8.20
(1H, s), 8.24 (1H, d, J = 8.4 Hz), 8.27 (1H, d, J = 8.4 Hz), 13.15 (1H,
br s). MS (FAB) m/z: 211 (M+ꢀH).
5.1.5. N-(Diaminomethylene)dibenzo[b,d]furan-3-carboxamide
monohydrochloride (5)
To
a solution of dibenzo[b,d]furan-3-carboxylic acid (24,
100 mg, 0.47 mmol) in DMF (3 mL) was added CDI (115 mg,
0.70 mmol) and the mixture was stirred at room temperature
for 1 h. To the mixture was added guanidine carbonate
(203 mg, 1.12 mmol) and the mixture was stirred at room tem-
perature for 2 h. The reaction mixture was concentrated in vacuo.
To the resultant residue was added water and stirred at room
temperature for 30 min. The precipitate was collected by filtra-
tion, washed with water, dried in vacuo at 50 °C to give N-
1H NMR (DMSO-d6) d: 5.12 (2H, s), 6.58 (1H, td, J = 1.5, 7.4 Hz),
6.75 (1H, t, J = 7.3 Hz), 6.85 (1H, dd, J = 1.4, 8.3 Hz), 6.90 (1H, dd,
J = 1.0, 7.8 Hz), 7.01 (1H, td, J = 1.4, 7.9 Hz), 7.58 (1H, dt, J = 1.4,
8.3 Hz), 7.99 (1H, dd, J = 2.0, 11.3 Hz). MS (FAB) m/z: 227 (M+ꢀH).
5.1.9. 10H-Phenoxazine-2-carbonitrile (28)
A solution of 27 (4.88 g, 21.3 mmol) in DMF (25 mL) was stirred
at 145 °C for 4 days. After cooling, water was added to the mixture
and the aqueous layer was extracted with EtOAc. The organic layer
was washed with water, dried over Na2SO4. After evaporation in
vacuo, the resultant residue was purified by silica gel column chro-
matography (EtOAc–hexane) to give the title compound 28
(494 mg, 11%) as a brown solid.
(diaminomethylene)dibenzo[b,d]furan-3-carboxamide
(102 mg)
as a white solid. The compound was converted to its monohydro-
chloride salt by treating it with 4 M HCl–EtOAc (0.2 mL,
0.8 mmol) in EtOH (3.5 mL). The crude salt was suspended with
EtOH and filtered to give the title compound 5 (102 mg, 75%)
as a white solid.
1H NMR (DMSO-d6) d: 7.50 (1H, t, J = 7.8 Hz), 7.66 (1H, t,
J = 7.8 Hz), 7.81 (1H, d, J = 8.3 Hz), 8.21 (1H, d, J = 7.8 Hz), 8.29
(1H, d, J = 7.3 Hz), 8.37 (1H, d, J = 7.8 Hz), 8.59 (1H, s), 8.62 (2H,
br s), 8.85 (2H, br s), 12.27 (1H, s). MS (FAB) m/z: 254 (M++H).
Mp: 299–300 °C. Anal. Calcd for C14H11N3O2ꢂ0.95HClꢂ0.3H2O: C,
57.33; H, 4.31; N, 14.33; Cl, 11.48. Found: C, 57.14; H, 4.15; N,
14.23; Cl, 11.68.
1H NMR (DMSO-d6) d: 6.47 (1H, dd, J = 0.9, 7.8 Hz), 6.58–6.65
(2H, m), 6.68 (1H, d, J = 2.0 Hz), 6.72 (1H, d, J = 8.3 Hz), 6.77 (1H,
td, J = 1.9, 7.8 Hz), 7.02 (1H, dd, J = 1.9, 8.3 Hz), 8.55 (1H, s). MS
(FAB) m/z: 207 (M+ꢀH).
5.1.10. 10H-Phenoxazine-2-carboxylic acid (29)
A solution of 28 (287 mg, 1.37 mmol) and conc. HCl (14 mL) in
AcOH (14 mL) was stirred at 120 °C for 18 h, then allowed to cool.
The reaction mixture was concentrated in vacuo. To the resultant
residue was added water and stirred at room temperature for
30 min. The precipitate was collected by filtration, washed with
water, dried in vacuo at 50 °C to give the title compound 29
(297 mg, 95%) as a gray solid.
5.1.6. Methyl 9H-carbazole-2-carboxylate (25)
The title compound was prepared according to a literature
protocol.18
1H NMR (DMSO-d6) d: 3.30 (3H, s), 7.21 (1H, td, J = 1.2, 8.0 Hz),
7.47 (1H, td, J = 8.0, 1.2 Hz), 7.56 (1H, d, J = 8.0 Hz), 7.78 (1H, dd,
J = 1.2, 8.0 Hz), 8.11 (1H, s), 8.20 (1H, d, J = 8.0 Hz), 8.23 (1H, d,
J = 8.0 Hz), 11.53 (1H, s). MS (FAB) m/z: 226 (M++H).
1H NMR (DMSO-d6) d: 6.44 (1H, dd, J = 1.4, 7.8 Hz), 6.56–6.66
(3H, m), 6.75 (1H, td, J = 1.4, 7.3 Hz), 7.03 (1H, d, J = 2.0 Hz), 7.17

A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
7849
(1H, dd, J = 1.9, 8.3 Hz), 8.36 (1H, s), 12.63 (1H, br s). MS (FAB) m/z:
226 (M+ꢀH).
1H NMR (DMSO-d6) d: 7.42 (1H, t, J = 8.3 Hz), 7.62–7.67 (2H, m),
7.80–7.85 (2H, m), 8.28 (1H, dd, J = 1.4, 7.8 Hz), 8.31 (1H, s). MS
(FAB) m/z: 266 (M++H). Anal. Calcd for C15H11N3O2ꢂ0.1H2O: C,
67.46; H, 4.23; N, 15.73. Found: C, 67.38; H, 4.17; N, 15.77.
5.1.11. N-(Diaminomethylene)-10H-phenoxazine-2-
carboxamide monohydrochloride (7)
The title compound was prepared in the same manner as de-
scribed for 4 using 29 instead of 15 in 53% yield.
5.1.17. N-(9-Hydroxy-9H-fluorene-2-carbonyl)guanidine
monohydrochloride (10)
1H NMR (DMSO-d6) d: 6.49 (1H, dd, J = 1.5, 7.9 Hz), 6.58–6.66
(2H, m), 6.73–6.79 (2H, m), 7.04 (1H, d, J = 2.4 Hz), 7.48 (1H, dd,
J = 2.4, 8.3 Hz), 8.45 (2H, br s), 8.59 (1H, s), 8.64 (2H, br s), 11.69
(1H, s). MS (FAB) m/z: 269 (M++H) Mp: 264–269 °C Anal. Calcd
for C14H12N4O2ꢂHClꢂ1.2H2O: C, 51.52; H, 4.76; N, 17.17; Cl, 10.86.
Found: C, 51.42, H, 4.84; N, 17.10; Cl, 10.95.
To a solution of 9 (400 mg, 1.51 mmol) in MeOH (10 mL) was
added sodium borohydride (NaBH4) (110 mg, 3.02 mmol) and the
mixture was stirred at room temperature for 1 h. Reaction was
quenched by addition of water, and the solution was evaporated
in vacuo. To the resultant residue was added CHCl3 and 1 M NaOH
aq and the precipitate was collected by filtration to give
N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine. The compound
was converted to its monohydrochloride salt by treating it with
4 M HCl–EtOAc (0.2 mL, 0.8 mmol) in EtOH (30 mL). The crude salt
was suspended with EtOH and filtered to give the title compound
10 (380 mg, 83%) as a colorless solid.
5.1.12. 3-Chloro-4-[(2-hydroxyphenyl)amino]benzonitrile (30)
4-(2-Aminophenoxy)-3-chlorobenzonitrile was prepared in the
same manner as described for 27 using 3-chloro-4-fluorobenzoni-
trile instead of 3,4-difluorobenzonitrile in 75% yield as a pale orange
solid. To a hot (100 °C) solution of 4-(2-aminophenoxy)-3-chloro-
benzonitrile (5.0 g, 20.5 mmol) in DMF (25 mL) was added portion
wise NaH (55% dispersion in mineral oil; 938 mg, 21.5 mmol) and
the mixture was stirred at 100 °C for 2 h. After cooling, water was
added to the mixture and the aqueous layer was extracted with
EtOAc. The organic layer was washed with water and brine, dried
over Na2SO4. After evaporation in vacuo, the resultant residue was
purified by silica gel column chromatography (EtOAc–hexane) to
give the title compound 30 (3.52 g, 70%) as a beige solid.
1H NMR (DMSO-d6) d: 5.59 (1H, s), 6.07 (1H, br s), 7.40–7.49
(2H, m), 7.66 (1H, dd, J = 1.5, 8.3 Hz), 7.93–7.95 (1H, m), 8.01
(1H, d, J = 8.8 Hz), 8.23–8.28 (2H, m), 8.55 (2H, br s), 8.70 (2H, br
s), 11.87 (1H, s). MS (FAB) m/z: 268 (M++H) Mp: 236–238 °C Anal.
Calcd for C15H13N3O2ꢂHClꢂH2O: C, 55.99; H, 5.01; N, 13.06; Cl,
11.02. Found: C, 55.65; H, 4.87; N, 12.99; Cl, 11.30.
5.1.18. N-(9H-Fluorene-1-carbonyl)guanidine
monohydrochloride (11)
The title compound was prepared in the same manner as de-
scribed for 4 using 9H-fluorene-1-carboxylic acid (16) instead of
15 in 51% yield.
1H NMR (DMSO-d6) d: 6.58 (1H, d, J = 8.8 Hz), 6.85 (1H, td, J = 1.0,
7.3 Hz), 6.96 (1H, dd, J = 1.5, 7.8 Hz), 7.10 (1H, td, J = 1.5, 7.3 Hz), 7.18
(1H, dd, J = 2.0, 7.8 Hz), 7.48 (1H, dd, J = 2.0, 8.8 Hz), 7.74 (1H, s), 7.85
(1H, d, J = 2.0 Hz), 9.72 (1H, br s). MS (FAB) m/z: 243 (M+ꢀH).
1H NMR (DMSO-d6) d: 4.26 (2H, s), 7.37–7.46 (2H, m), 7.62–7.68
(2H, m), 8.00 (1H, d, J = 6.8 Hz), 8.03 (1H, d, J = 7.8 Hz), 8.25 (1H, d,
J = 7.8 Hz), 8.54 (2H, br s), 8.67 (2H, br s), 11.87 (1H, s). MS (ESI) m/
z: 252 (M++H). Mp: 261–263 °C Anal. Calcd for C15H11N3O2ꢂHCl: C,
62.61; H, 4.90; N, 14.60; Cl, 12.32. Found: C, 62.59; H, 4.85; N,
14.60; Cl, 12.36.
5.1.13. 10H-Phenoxazine-3-carbonitrile (31)
A solution of 30 (3.0 g, 12.3 mmol), K2CO3 (5.1 g, 36.9 mmol) in
DMF (50 mL) was stirred at 150 °C for 4 h. After cooling, water was
added and the precipitate was collected by filtration, washed with
water, dried in vacuo at 50 °C. The resultant residue was purified
by silica gel column chromatography (EtOAc–hexane) to give the
title compound 31 (1.49 g, 58%) as a yellow solid.
5.1.19. 9-Oxo-9H-fluorene-3-carboxylic acid (19)
The title compound was prepared according to a literature
protocol.17
1H NMR (DMSO-d6) d: 6.48 (1H, d, J = 8.3 Hz), 6.60–6.66 (2H, m),
6.73–6.78 (1H, m), 6.97 (1H, d, J = 2.0 Hz), 7.16 (1H, dd, J = 1.9,
8.3 Hz), 8.90 (1H, s). MS (FAB) m/z: 207 (M+ꢀH).
1H NMR (DMSO-d6) d: 7.42 (1H, t, J = 7.6 Hz), 7.64–7.67 (2H, m),
7.70 (1H, d, J = 7.6 Hz), 7.94 (1H, dd, J = 2.4, 7.6 Hz), 8.29 (1H, s),
13.43 (1H, br s). MS (FAB) m/z: 225 (M++H).
5.1.14. 10H-Phenoxazine-3-carboxylic acid (32)
The title compound was prepared in the same manner as de-
scribed for 29 using 31 instead of 28 in 14% yield.
5.1.20. N-(9-Oxo-9H-fluorene-3-carbonyl) guanidine
monohydrochloride (20)
1H NMR (DMSO-d6) d: 6.45–6.49 (2H, m), 6.60–6.64 (2H, m),
6.72–6.76 (1H, m), 7.02 (1H, d, J = 1.9 Hz), 7.34 (1H, dd, J = 1.5,
8.3 Hz), 8.73 (1H, s), 12.43 (1H, s). MS (FAB) m/z: 226 (M+ꢀH).
The title compound was prepared in the same manner as de-
scribed for 4 using 19 instead of 15 in 97% yield.
1H NMR (DMSO-d6) d: 7.47 (1H, t, J = 7.4 Hz), 7.68–7.73 (2H, m),
7.80 (1H, d, J = 7.6 Hz), 7.91 (1H, d, J = 7.6 Hz), 8.06 (1H, d,
J = 7.6 Hz), 8.57 (2H, br s), 8.67 (1H, s), 8.72 (2H, br s), 12.22 (1H,
s). MS (FAB) m/z: 266 (M++H). Anal. Calcd for C15H11N3O2ꢂHCl: C,
59.31; H, 4.65; N, 13.83; Cl, 11.67. Found: C, 58.95; H, 4.59; N,
13.54; Cl, 11.32.
5.1.15. N-(Diaminomethylene)-10H-phenoxazine-3-
carboxamide monohydrochloride (8)
The title compound was prepared in the same manner as de-
scribed for 4 using 32 instead of 15 in 77% yield.
1H NMR (DMSO-d6) d: 6.51–6.54 (2H, m), 6.64–6.69 (2H, m),
6.75–6.79 (1H, m), 7.29 (1H, d, J = 1.9 Hz), 7.59 (1H, dd, J = 2.0,
8.3 Hz), 8.35 (2H, br s), 8.57 (2H, br s), 9.06 (1H, s), 11.43 (1H, s).
MS (FAB) m/z: 269 (M++H) Mp: 286–293 °C Anal. Calcd for C14H12-
N4O2ꢂHClꢂ0.4H2O: C, 53.90; H, 4.46; N, 17.96; Cl, 11.37. Found: C,
53.92; H, 4.48; N, 17.86; Cl, 11.44.
5.1.21. N-(9-Hydroxy-9H-fluorene-3-carbonyl)guanidine
monohydrochloride (12)
The title compound was prepared in the same manner as de-
scribed for 10 using 20 instead of 9 in 78% yield.
1H NMR (DMSO-d6) d: 5.59 (1H, s), 6.06 (1H, br s), 7.39 (1H, t,
J = 7.4 Hz), 7.45 (1H, t, J = 7.6 Hz), 7.63 (1H, d, J = 8.0 Hz), 7.78
(1H, d, J = 8.0 Hz), 7.90 (1H, d, J = 7.2 Hz), 8.07 (1H, dd, J = 1.2,
8.0 Hz), 8.55 (2H, br s), 8.74 (1H, s), 8.82 (2H, br s), 12.14 (1H, s).
MS (FAB) m/z: 268 (M++H). Anal. Calcd for C15H13N3O2ꢂHCl: C,
59.31, H, 4.65; N, 13.83; Cl, 11.67. Found: C, 59.15; H, 4.69; N,
13.66; Cl, 11.62.
5.1.16. N-(Diaminomethylene)-9-oxo-9H-fluorene-2-
carboxamide (9)
The title compound was prepared in the same manner as de-
scribed for 4 using 9-oxo-9H-fluorene-2-carboxylic acid (18) in-
stead of 15 in 58% yield.

7850
A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
5.1.22. N-(9H-Fluorene-4-carbonyl)guanidine
monohydrochloride (13)
The title compound was prepared in the same manner as de-
scribed for 4 using 9H-fluorene-4-carboxylic acid (17) instead of
15 in 3% yield.
7.95 (3H, m), 8.41 (4H, br s), 12.21 (1H, s). MS (FAB) m/z: 294
(M++H) Mp: 280–283 °C Anal. Calcd for C17H15N3O2ꢂHClꢂ0.1H2O:
C, 61.58; H, 4.92; N, 12.67; Cl, 10.69. Found: C, 61.40; H, 5.00; N,
12.41; Cl, 10.40.
1H NMR (DMSO-d6) d: 4.01 (2H, s), 7.36–7.44(2H, m), 7.47 (1H,
t, J = 7.6 Hz), 7.64–7.66 (1H, m), 7.69 (1H, d, J = 7.6 Hz), 7.84 (1H, d,
J = 7.2 Hz), 7.87–7.91 (1H, m), 8.57 (2H, br s), 8.74 (2H, br s), 12.18
(1H, s). MS (FAB) m/z: 252 (M++H). Anal. Calcd for C15H13N3OꢂHCl:
C, 62.41; H, 4.90; N, 14.60; Cl, 12.32. Found: C, 62.44; H, 4.79; N,
14.53; Cl, 12.37.
5.2. Molecular modeling
5.2.1. Calculation of atomic charge distribution
This caluculation was performed using the 6,5,6-membered
ring structures for the sake of shorthand. The structures of 40, 50,
60 and 100 were sketched in MOE.23 The molecular conformations
were optimized to the semiempirical AM1 level and electrostatic
potential-fitted atomic partial charges were calculated using MO-
PAC 7.1 module in MOE. Analytic Connolly surfaces24 that sur-
rounded the van der Waals surfaces of the core scaffold are
generated and colored with red for negative charge and blue for
positive charge, respectively, using the program MOE.
5.1.23. 9-Oxo-9H-fluorene-2-carbaldehyde (34)
To a cold (0 °C) solution of ethyl 9-oxo-9H-fluorene-2-carboxyl-
ate (1.50 g, 5.95 mmol) in THF (15 mL) was added portion wise
LiAlH4 (451 mg, 11.9 mmol), and the mixture was warmed up to
room temperature and stirred for 30 min at that temperature.
Reaction was quenched with Na2SO4ꢂ10H2O, NaCl and brine. The
mixture was filtered and the residue was washed with THF. After
evaporated in vacuo to give 2-(hydroxymethyl)-9H-fluoren-9-ol
as a yellow solid, which was used for the next step without further
purification. A solution of the alcohol compound obtained above,
MnO2 (12.8 g, 147 mmol) in CHCl3 (130 mL) was stirred at room
temperature overnight. Celite was added to the reaction mixture,
then filtered and washed with CHCl3. The filtrate was evaporated
in vacuo and the resulting residue was purified by silica gel column
chromatography (CHCl3–hexane) to give the title compound 34
(871 mg, 70% in two steps) as a yellow solid.
5.3. Pharmacology
5.3.1. Binding assay of 5-HT2B receptor
5.3.1.1. Membrane preparation.
A human 5-HT2B receptor
expressing cell was prepared in accordance with a reference.25
HEK293-EBNA cell was used as the gene transferring cell. Cultured
HEK293-EBNA cells expressing human 5-HT2B receptor were
washed with PBS(ꢀ). The cells were scraped in the presence of
PBS(ꢀ), and the cells were recovered by centrifugation
(1000 rpm, 10 min, 4 °C). They were homogenized using Polytron
(PTA 10-TS) in the presence of 5 mM Tris-HCl (pH 7.4) buffer and
centrifuged (40,000 xg. 10 min, 4 °C). They were suspended using
a homogenizer in the presence of 50 mM Tris–HCl (pH 7.4) buffer.
They were subjected to centrifugation (40,000 xg, 10 min, 4 °C),
suspended in 50 mM Tris–HCl (pH 7.4) and stored at ꢀ80 °C.
1H NMR (DMSO-d6) d: 7.50 (1H, t, J = 7.6 Hz), 7.68–7.72 (2H, m),
7.96 (1H, d, J = 7.6 Hz), 8.06 (2H, s), 8.18 (1H, dd, J = 1.4, 7.6 Hz),
10.04 (1H, s). MS (EI) m/z: 208 (M++H).
5.1.24. (2E)-3-(9-Oxo-9H-fluoren-2-yl)acrylic acid (35)
A solution of 34 (871 mg, 4.18 mmol), methyl (triphenylphos-
phoranylidene)acetate (1.40 g, 4.18 mmol) in toluene (15 mL) was
stirred at 90 °C for 5 h, then allowed to cool. The reaction mixture
was concentrated in vacuo, the resultant residue was purified by sil-
ica gel column chromatography (CHCl3–EtOAc–hexane) to give
methyl (2E)-3-(9-oxo-9H-fluoren-2-yl)acrylate (931 mg, 84%) as a
yellow solid. To a mixture of the ester compound obtained above
in MeOH (5 mL) and THF (5 mL) was added 1 M NaOH aq (1.7 mL)
and the mixture was stirred at room temperature overnight. The
reaction mixture was acidified (pH 1.0) with 1 M HCl aq. The precip-
itate was collected by filtration, washed with water, dried in vacuo
to give the title compound 35 (357 mg, 94%) as a yellow solid.
1H NMR (DMSO-d6) d: 6.65 (1H, d, J = 16.1 Hz), 7.42 (1H, t,
J = 7.3 Hz), 7.63–7.67 (3H, m), 7.86 (1H, d, J = 7.8 Hz), 7.93–7.96
(2H, m), 12.46 (1H, br s). MS (FAB) m/z: 250 (M+ꢀH).
5.3.1.2. Receptor binding assay.
A total volume of 500 lL
containing 50 mM Tris–HCl–4 mM CaCl2 (pH 7.4) buffer, the hu-
man 5-HT2B receptor expressing HEK293-EBNA cell membrane
preparation and a radio ligand [3H] Mesulergine (3.1 TBq/mmol)
was incubated at 25 °C for 1 h. The compound was dissolved in
100% DMSO and diluted to respective concentrations. Nonspecific
binding was defined as the binding quantity in the presence of
1 lM ritanserin, and the result of subtracting the nonspecific bind-
ing quantity from the total binding quantity was defined as the
specific binding quantity. This was mixed with 4 mL of 50 mM
Tris–HCl buffer (pH 7.4) and filtered under a reduced pressure
using a GF/B glass filter, and the filter was washed (4 mL ꢁ 3) with
the same buffer. The glass filter was soaked in 5 mL of a liquid scin-
tillator (Aquasol-2) and the radioactivity was measured using a li-
quid scintillation counter. Concentration of the compound which
inhibits 50% of the receptor binding, IC50 value, was obtained by
nonlinear regression analysis using SAS (ver. 6.11), and the Ki value
which represents its affinity for the receptor was calculated using
the formula of Cheng & Prussoff;26 Ki = IC50/(1 + [L]/[Kd]) ([L]: li-
gand concentration, [Kd]: dissociation constant).
5.1.25. (2E)-N-(Diaminomethylene)-3-(9-oxo-9H-fluoren-2-
yl)acrylamide (36)
The title compound was prepared in the same manner as de-
scribed for 4 using 35 instead of 15 in 78% yield.
1H NMR (DMSO-d6) d: 6.60 (2H, d, J = 15.6 Hz), 7.39 (1H, t,
J = 7.3 Hz), 7.45 (1H, s), 7.49 (1H, s), 7.61–7.65 (3H, m), 7.76 (1H,
s), 7.82–7.84 (4H, m). MS (FAB) m/z: 292 (M++H). Mp: 217–
220 °C Anal. Calcd for C17H13N3O2ꢂH2O: C, 66.01; H, 4.89; N,
13.58. Found: C, 66.20; H, 4.92; N, 13.52.
5.3.2. Binding assay of 5-HT7 receptor
5.3.2.1. Membrane preparation.
A human 5-HT7 receptor
expressing cell was prepared in accordance with references.27
CHO cell was used as the gene transferring cell.
5.1.26. (2E)-N-(Diaminomethylene)-3-(9-hydroxy-9H-fluoren-2-
yl)acrylamide monohydrochloride (14)
Cultured CHO cells expressing human 5-HT7 receptor were
washed with PBS(ꢀ). The cells were scraped in the presence of
PBS(ꢀ), and the cells were recovered by centrifugation
(1000 rpm, 10 min, 4 °C). They were homogenized using Polytron
(PTA 10-TS) in the presence of 5 mM Tris–HCl (pH 7.4) buffer
and centrifuged (40,000 xg 10 min, 4 °C). They were suspended
using a homogenizer in the presence of 50 mM Tris–HCl (pH 7.4)
The title compound was prepared in the same manner as de-
scribed for 10 using 36 instead of 9 in 82% yield.
1H NMR (DMSO-d6) d: 5.55 (1H, d, J = 5.9 Hz), 5.99 (1H, d,
J = 7.3 Hz), 6.79 (1H, d, J = 15.6 Hz), 7.35–7.44 (2H, m), 7.62 (1H,
d, J = 7.4 Hz), 7.72 (1H, d, J = 8.7 Hz), 7.85 (1H, d, J = 6.8 Hz), 7.88–

A. Moritomo et al. / Bioorg. Med. Chem. 21 (2013) 7841–7852
7851
buffer. They were subjected to centrifugation (40,000 xg 10 min,
4 °C), suspended in 50 mM Tris–HCl (pH 7.4) and stored at ꢀ80 °C.
approximately 3). After incubation (10 min at 37 °C), reaction
was terminated by exchanging the reaction mixture to 50 L of
ice-cold 0.1 M HCl. After standing for 1 h at 4 °C, 5 L of 1 M NaOH
l
l
5.3.2.2. Receptor binding assay.
A total volume of 500
l
L
was added into wells and mix. Cyclic AMP level was determined
using Biotrak cAMP EIA System. The IC50 and maximal inhibition
(Imax) values were calculated from the non-linear regression anal-
ysis using SAS ver.8.2. Final values were represented as the means
of three separate experiments.
containing 50 mM Tris–HCl–4 mM CaCl2 (pH 7.4) buffer, the hu-
man 5-HT7 receptor expressing CHO cell membrane preparation
and a radio ligand [3H]5-HT (3.40 TBq/mmol) was incubated at
25 °C for 1 h. The compound was dissolved in 100% DMSO and di-
luted to respective concentrations. Nonspecific binding was de-
fined as the binding quantity in the presence of 10
l
M
5.3.6. Suppressing effect on 5-HT-induced dural plasma protein
extravasation in guinea pigs
In this test system, suppressing effect was evaluated by measur-
ing leaked protein in the presence of a compound to be tested, and
this was carried out by partially modifying the method reported by
Rachel et al.32
The male Hartley guinea pigs (250–350 g) were anesthetized by
peritoneal administration (ip) of urethane (1.5 g/kg). By applying
simple canulation to femoral vein, 50 mg/kg of a fluorescent pro-
tein (FITC-BSA) was intravenously administered (iv) and 5 min
metergoline, and the result of subtracting the nonspecific binding
quantity from the total binding quantity was defined as the specific
binding quantity. This was mixed with 4 mL of 50 mM Tris–HCl
buffer (pH 7.4) and filtered under at educed pressure using a GF/
B glass filter, and the filter was washed (4 mL ꢁ 3) with the same
buffer. The glass filter was soaked in 5 mL of a liquid scintillator
(Aquasol-2) and the radioactivity was measured using a liquid
scintillation counter. Concentration of the compound which inhib-
its 50% of the receptor binding, IC50 value, was obtained by nonlin-
ear regression analysis using SAS (ver. 6.11), and the Ki value which
represents its affinity for the receptor was calculated using the for-
mula of Cheng & Prussoff;26 Ki = IC50/(1 + [L]/[Kd]) ([L]: ligand con-
centration, [Kd]:dissociation constant).
thereafter, physiological saline or 1 lM of 5-HT was intravenously
administered. By carrying out perfusion with physiological saline
15 min thereafter, blood was washed out.
Compound 2, 3 and 10 were intraperitoneally administered, or
compound 10 was orally administered, 30 min before administra-
tion of the fluorescent protein. By detaching the skull, dura mater
was took out and incubated at 37 °C for 16 h in a tube in the pres-
ence of physiological saline of pH 11. Centrifugation was carried
out, and the supernatant was dispensed into a plate. Fluorescence
intensity was measured using an fluorescence plate reader (excita-
tion wavelength 485 nm, absorption wavelength 530 nm). By mea-
suring dura mater weight, fluorescence intensity per mg dural
protein was calculated.
5.3.3. Binding test of 5-HT2A, 5-HT2C
,
al, M1 and D2 receptors
Affinity of the compounds for 5-HT2A, 5-HT2C
,
al, M1 and D2
receptors were verified using conventionally known tech-
niques.28–31
5.3.4. PI metabolism assay
HEK293-EBNA cells expressing human 5HT2B receptors were
seeded on collagen I-coated 24-well plates at the density of 1 ꢁ
105 cells/well and cultured overnight at CO2 incubator (37 °C, 5%
CO2). At the next day, wells were labeled with [3H]-myo-inositol
5.3.7. Aqueous solubility
(3
l
Ci/mL) and cultured for overnight. At the third day, labeled
To 13 lL of a 10 mM DMSO solution of a test compound that
cells were washed with PBS (137 mM NaCl, 2.68 mM KCl,
1.05 mM MgCl2, 0.9 mM CaCl2, 8.1 mM Na2HPO4, 1.47 mM KH2-
PO4) and incubated for 20 min at 37 °C. After incubation, cells were
further incubated with test compounds (0.3–10,000 nM, geometric
ratio: approximately 3) in PBS including 10 mM LiCl (PBS-Li) for
20 min at 37 °C. After incubation, reaction was started by exchang-
ing to 5-HT and test compound including PBS-Li. After incubation
(20 min at 37 °C), Reaction was terminated by exchanging reaction
mixture to 1 mL of ice-cold 0.2 M perchlolic acid. After standing
had been prepared in advance was added exactly 1 mL of a second
liquid (pH 6.8) for a disintegration test of Japanese Pharmacopoeia,
followed by shaking at 25 °C for 20 h, thereby giving a sample stock
solution. Next, using a filter impregnated with 200
ple stock solution, 200 L of a fresh sample stock solution was
added for filtration to obtain a liquid, which was taken as a sample
solution. Separately to this, to 10 L of the 10 mM DMSO solution
of the test compound was added accurately 1 mL of methanol, fol-
lowed by stirring, thereby giving a standard solution. 10 L por-
lL of the sam-
l
l
l
(1–3 h at 4 °C), 100
l
L of 2 N KOH and 50
l
L of 100 mM EDTA-
tions each of the sample solution and standard solution were
tested by liquid chromatography, and the ratio of the peak area
of the sample solution to the peak area of the standard solution
was determined, thereby calculating the solubility.
Na were added into each well and mix. One mL of reaction extract
was added to AG1 8 columns (BIO-RAD, CA, USA), and washed with
7 mL of wash solution (5 mM sodium tetraborate, 60 mM sodium
formate). IPs were extracted by addition of 4 mL of elution solution
(0.1 M formate, 1 M ammonium formate), and whole elute were
collected into liquid scintillation vials. Radioactivity of the elute
were counted using liquid scintillation counter. IC50 values against
5-HT-induced IPs accumulation were calculated from the non-lin-
ear regression analysis using SAS ver.8.2. Final values were repre-
sented as the means of three separate experiments.
Acknowledgments
We are grateful to Drs. Susumu Watanuki and Hirofumi Omura
for their useful advice. We also thank to Mr. Yukihiro Takemoto
and Dr. Tomonari Watabiki for performing pharmacological exper-
iments, and the staff of the Division of Analytical & Pharmacokinet-
ics Research Laboratories for the elemental analysis and spectral
measurements.
5.3.5. cAMP production assay
CHO cells expressing human 5HT7 receptors were seeded at the
density of 5 ꢁ 104 cells/well on 96-well plates and cultured over-
night in a CO2 incubator (37 °C, 5% CO2). At the next day, wells
were washed twice with PBS-IBMX-ascorbate (137 mM NaCl,
2.68 mM KCl, 1 mM MgCl2, 0.85 mM CaCl2, 8.1 mM Na2HPO4,
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