Design, synthesis, and structure-activity relationships of novel tetracyclic compounds as peripheral benzodiazepine receptor ligands

Article abstract of DOI:10.1016/j.bmc.2004.04.025

The peripheral benzodiazepine receptor (PBR) is pharmacologically distinct from the central benzodiazepine receptor (CBR) and has been identified in a wide range of peripheral tissues as well as in the central nervous system. Although numerous studies have been performed of it, the physiological roles and functions of the PBR are still unclear. In the present study, in exploring new types of ligands for PBR, we found that a new series of compounds having a tetracyclic ring system, which were designed from FGIN-1-27, exhibited high affinities for PBR. We prepared and evaluated them for PBR affinities. The results of binding tests showed that 12e and 12f were the most potent PBR ligands among them (12e: IC₅₀=0.44nM, 12f: IC₅₀=0.37nM). In this paper, we present the design, synthesis, and structure-activity relationships (SARs) of novel tetracyclic compounds.

Full text of DOI:10.1016/j.bmc.2004.04.025

Bioorganic & Medicinal Chemistry 12 (2004) 3569–3580
Design, synthesis, and structure–activity relationships of novel
tetracyclic compounds as peripheral benzodiazepine receptor
ligandsq
Taketoshi Okubo,a,* Ryoko Yoshikawa,b Shigeyuki Chaki,b Shigeru Okuyamac
and Atsuro Nakazatoa
aMedicinal Chemistry Laboratory, Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho,
Kita-ku, Saitama-shi, Saitama 331-9530, Japan
bMedicinal Pharmacology Laboratory, Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho,
Kita-ku, Saitama-shi, Saitama 331-9530, Japan
cEthical Business Strategy Division, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku,
Saitama-shi, Saitama 331-9530, Japan
Received 25 March 2004; revised 19 April 2004; accepted 19 April 2004
Available online 18 May 2004
Abstract—The peripheral benzodiazepine receptor (PBR) is pharmacologically distinct from the central benzodiazepine receptor
(CBR) and has been identified in a wide range of peripheral tissues as well as in the central nervous system. Although numerous
studies have been performed of it, the physiological roles and functions of the PBR are still unclear. In the present study, in
exploring new types of ligands for PBR, we found that a new series of compounds having a tetracyclic ring system, which were
designed from FGIN-1-27, exhibited high affinities for PBR. We prepared and evaluated them for PBR affinities. The results of
binding tests showed that 12e and 12f were the most potent PBR ligands among them (12e: IC50 ¼ 0.44 nM, 12f: IC50 ¼ 0.37 nM). In
this paper, we present the design, synthesis, and structure–activity relationships (SARs) of novel tetracyclic compounds.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
such as steroidogenic organs and blood cells.7 The later
study demonstrated PBR in the central nervous sys-
tem.8;9 It regulates translocation of cholesterol from the
outer to the inner mitochondrial membranes10;11 and is
involved in numerous functions including biosynthe-
sis of neurosteroids12;13 such as pregnenolone sulfate,
3a-hydroxy-5a-pregnan-20-one, and 3a,21-dihydroxy-
5a-pregnan-20-one. Neurosteroids modulate neurotrans-
mitter-gated ion channel activity at GABAA receptors14
Benzodiazepine receptors can be subdivided into two
types: central benzodiazepine receptors (CBR) and
peripheral benzodiazepine receptors (PBR). The CBR is
found only on neurons in the central nervous system,1;2
coupled with the c-aminobutyric acid (GABA)A recep-
tor,3 and mediates the classical pharmacological prop-
erties, that is, anxiolytic, anticonvulsant, sedative, and
muscle relaxant, of the widely used benzodiazepines.2
The PBR is a high-affinity binding site for diazepam in
rat kidney4 and is pharmacologically different from the
CBR. The PBR has been detected in the outer mito-
chondrial membranes in several peripheral tissues5;6
and N-methyl-D
-aspartate receptors,15 an event which
results in indirect modulation of GABAergic and
glutamatergic transmission.
Several specific or nonspecific PBR ligands have been
reported (Chart 1). Compound 1 (Ro5-4864),16 40-chlo-
rodiazepam, exhibits high affinity for the PBR and very
low affinity for the CBR. Compound 2 (PK11195)17;18 is
an isoquinoline derivative and currently the most widely
used specific probe for peripheral benzodiazepine
receptors. Compound 3 (FGIN-1-27)19;20 is a 2-aryl-3-
indoleacetamide derivative and exhibits high affinity for
the PBR with high selectivity over the CBR. Compound
Keywords: PBR (peripheral benzodiazepine receptor); Tetracyclic ring;
SAR; PBR ligands.
q Supplementary data associated with this article can be found, in the
online version, at doi: 10.1016/j.bmc.2004.04.025
* Corresponding author. Tel.: +81-48-663-1111; fax: +81-48-652-7254;
e-mail: t.okubo@po.rd.taisho.co.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.025

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T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
12a with NaH and alkylhalide provided N-alkylated 14a
and 14b. Compound 14c was prepared by addition of a
4-chlorotetramethylene group to 12a and followed by
the substitution of the chloro group with dimethyl-
amine. Carboxylic acid 15 was obtained from thiophe-
nol 7 and pent-2-enedioic acid using the same method as
for synthesis of 8 from 7. Condensation of the carboxyl
acid in 15 with amine and Fischer indolization under the
same condition as used in the synthesis of 12a from 11
gave acetamide derivative 16.
Me
N
O
O
Me
Me
N
N
N
Me
Cl
Cl
Cl
1 Ro 5-4864
2 PK11195
N
The method of synthesis of 19 is shown in Scheme 2.
Commercially available 2-benzylsuccinic acid 17 was
used as a starting material. Stirring 17 in concd H2SO4
at room temperature for 9 h provided 18 in 45% yield.25
Compound 19 was obtained from 18 using the same
method as for synthesis of 16 from 15 in Scheme 1.
O
N
O
Cl
F
N
N
H
Cl
N
4 Alpidem
3 FGIN-1-27
Synthesis of benzimidazolyl acetic amide 23a was
accomplished in three steps using benzene-1,2-diamine
20 as a starting material (Scheme 3). Heating 20 in
polyphosphoric acid at 250 ꢁC, followed by alkylation
with bromoacetic acid using KOH as a base in DMSO,
provided carboxylic acid 22. Coupling with the corre-
sponding amine via a mixed acid anhydride gave 23a.
OMe
OMe
O
N
O
N
F
O
O
Cl
O
Preparation of 28, a benzimidazolyl propionyl amide
derivative, is shown in Scheme 4. Substitution reaction
of 2-fluoronitrobenzene 24 with b-alaninamide 25 was
performed in the presence of K2CO3 in DMF. Treat-
ment of 26 with benzoyl chloride provided 27. Reduc-
tion of the nitro group in 27 with iron powder was
performed in acetic acid. Under the conditions, an
imidazole ring was formed to provide 28 in good yield.
5 DAA1106
6 DAA1097
Chart 1.
4 (alpidem)21 has an imidazopyridine skeleton in its
structure and binds with high affinity to both PBR and
CBR.
Synthesis of compound 34a, a benzoimidazole derivative
having a tetracyclic system, is shown in Scheme 5.
Treatment of 2-cyanobenzyl bromide 29 and Boc-ami-
nomalonic acid diethyl ester 30 with sodium ethoxide in
ethanol provided 31. Under the conditions for hydro-
lysis of both ester groups with NaOH, one of the
carboxyl groups was removed. The resulting monocar-
boxylic acid was coupled with amine to give 2-cyan-
ophenylalaninamide 32. After removal of the Boc group,
heating with 2-fluoronitrobenzene in the presence of
K2CO3 in DMF gave 33. Hydrogenation of the nitro
group with PtO2 as a catalyst followed by treatment
with HCl gas in EtOH provided compound 34a.
We have recently reported a series of potent and selec-
tive PBR ligands, for example, 5 (DAA1106)22–24 and 6
(DAA1097)22;23 designed from 1 (Ro5-4864) by opening
the diazepine ring. In exploring further diverse PBR
ligands, we have found novel potent PBR ligands having
tetracyclic systems, designed from 3 (FGIN-1-27).
In this paper, we present the design, synthesis, and
structure–activity relationships of the new tetracyclic
compounds.
2. Chemistry
Compounds 12, 13, 14, and 16, tetracyclic compounds
having a thioether, were synthesized using the methods
shown in Scheme 1. 4-Oxo-thiochroman-2-carboxylic
acid 8 was obtained by reacting benzenethiol with furan-
2,5-dione in the presence of Et3N and with subsequent
treatment with AlCl3. Next, Fischer indolization was
performed by H2SO4 in EtOH to yield ethylester 9.
Hydrolysis of the ester group and condensation with
amines provided 12a. Compound 12a was also obtained
by Fischer indolization of amide 11, which had been
synthesized by coupling carboxylic acid 8 with dihexyl-
amine. Conversion of the thioether group in 12a to the
sulfonyl group was achieved by mCPBA. Treatment of
3. Results and discussion
Compounds 1, 2, 3, 12a–12m, 13a–13c, 14a–14c, 16, 19,
23a, 23b, 28, and 34a–34c were evaluated for binding
affinities for PBR in mitochondria prepared from rat
cerebral cortex against radioligand [3H]PK11195,19 and
the obtained IC50 values are shown in Tables 1–3.
We designed a new tetracyclic compound from FGIN-1-
27 (3) by closing the ring and accomplished the synthesis
shown in Scheme 1. Compound 12a exhibited high

T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
3571
CO2Et
S
CO2H
S
d
N
H
N
H
c
O
S
9
10
SH
a, b
e
CO2H
f,g
CON(nHex)2
S
CON(nHex)2
SO2
7
8
O
h
i
N
H
N
H
CON(nHex)2
S
l
11
12a
13
j
CON(nHex)2
S
CON(nHex)2
S
O
m, h
N
CO2H
N
H
S
R3
15
16
14a: R3 = Me
14b: R3 = -(CH2)2NMe2
R3 = -(CH2)4Cl
k
14c: R3 = -(CH2)4NMe2
Scheme 1. Reagents and conditions: (a) furan-2,5-dione, Et3N, toluene; (b) AlCl3, CH2Cl2; (c) PhNHNH2, H2SO4, EtOH; (d) KOH, EtOH, H2O;
(e) n-Hex2NH, EDCÆHCl, HOBt, CH2Cl2; (f) SOCl2, PhH; (g) n-Hex2NH, Et3N, CH2Cl2; (h) PhNHNH2, ZnCl2, heat; (i) mCPBA, CH2Cl2; (j) R3Cl
or R3Br, NaH, DMF; (k) Me2NH, MeOH; (l) pent-2-enedioic acid, Et3N; (m) ClCO2Et, n-Hex2NH, Et3N, THF. Method A: a, b, c, d, e; method B:
a, b, f, g, h; method C: i; method D: j, k; method E: l, m, h.
O
H
N
NH2
NH2
p
CO2H
CO2H
o
n
F
N
CO2H
21
20
17
18
nHex
nHex
nHex
nHex
O
N
CO2H
O
N
CH2
N
CH2
N
m
m, h
F
F
N
H
N
N
19
22
23a
Scheme 2. Reagents and conditions: (n) H2SO4; (m) ClCO2Et, (n-
Hex)2NH, Et3N, THF; (h) PhNHNH2, ZnCl2, heat. Method F: n, m,
h.
Scheme 3. Reagents and conditions: (o) 4-F–Ph–CO2H, PPA; (p)
BrCH2CO2H, KOH, DMSO; (m) ClCO2Et, (n-Hex)2NH, Et3N, THF.
Method G: o, p, m.
affinity for PBR (IC50 ¼ 3.8 nM) and no affinity for CBR
(IC50>1000 nM) in [H3]flunitrazepam binding. This
finding encouraged us to explore a new type of PBR
ligand using 12a as a lead compound. Initially, conver-
sion from R1 and R2 to various alkyl groups was
examined. The results of the binding test for 12b–12i
demonstrated significant changes in affinities for PBR.
Compounds 12c (R1, R2 ¼ n-Pr, H) and 12d (R1,
R2 ¼ Me, Me) exhibited lower PBR affinities than 12a,
and 12b (R1, R2 ¼ H, H) had no affinity for PBR.
Introducing an amino group into the alkyl chain sig-
nificantly decreased PBR affinity (12g), while an ether
group was tolerated (12h). The results of structural
transformations at this site indicated that the most
suitable group for this site was a diethylamino (12e) or
dipropylamino (12f) group. Compounds 12e and 12f
demonstrated significant increase on PBR affinity over
12a, by about 10-fold. These findings suggested that R1

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T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
to the pyrrole resulted in a small decrease in PBR
NO2
F
NO2
O
r
q
affinity (12j–12m). For compounds having a dihexyl-
amino group or dipropylamino group, conversion of the
sulfide group to a sulfonyl group was studied, and the
changes obtained resulted in a slight increase in affinities
for PBR (13a vs 12a and 13c vs 12f). On the other hand,
for compounds having a mono-alkyl amino group,
oxidation of the sulfide group provided a slight decrease
in affinity (12c vs 13b). With introduction of an alkyl
group including a basic group onto the indol ring,
affinity for PBR disappeared completely (14b and 14c).
The conversion of thioether to methylene did not
influence the affinity for PBR (12a vs 19), suggesting
that this structural change does not cause an important
change in conformation of the molecule. Optical
resolution of 12a was performed by HPLC, and the
(+)-enantiomer exhibited higher affinity than the
())-enantiomer.
nHex
nHex
N
H
N
24
26
NO2
N
CON(nHex)2
O
nHex
nHex
(CH2)2
N
s
N
O
N
27
28
Scheme 4. Reagents and conditions: (q) NH2CH2CH2CON(n-Hex)2
(25), K2CO3, DMF; (r) PhCOCl, Et3N, CH2Cl2; (s) Fe, AcOH.
Method H: q, r, s.
These tetracyclic compounds were highly lipophilic
molecules, and they will probably show low solubility in
water. Thus, we studied to find other PBR ligands
having a basic part to improve the lipophilic property,
and we extended our exploration to new types of PBR
ligands having a different framework. Structural con-
version from the indol ring in the structure of 3 (FGIN-
1-27) to a benzimidazole ring was investigated, and
23a–23b and 28 were obtained using the methods shown
in Schemes 3 and 4, respectively. Contrary to our
expectations, both 23a (X3 ¼ F) and 23b (X3 ¼ H)
exhibited much lower affinities for PBR than compound
3 (23a: IC50 ¼ 310 nM, 23b: IC50 ¼ 230 nM), and propi-
onyl amide 28 exhibited no affinity for PBR. As a next
structural conversion, a new tetracyclic compound,
which was a hybrid compound between 23b and 12a,
was designed and the method of synthesis was studied
(Chart 2). Synthesis of 34a–34c was achieved using the
methods as shown in Scheme 5, and these compounds
were evaluated for PBR affinities. Compounds 34a (R1,
R2 ¼ hexyl) and 34c (R1, R2 ¼ propyl), a new type of
tetracyclic compounds having a basic benzimidazole
ring, exhibited higher affinities than 23b (34a:
IC50 ¼ 42 nM, 34c: IC50 ¼ 74 nM). Surprisingly, 34b (R1,
R2 ¼ ethyl) had no affinity for PBR. Taking into account
the fact that 12e (R1, R2 ¼ ethyl) was one of the most
potent PBR ligands among the aforementioned tetra-
cyclic compounds, it is notable that the two different
tetracyclic ring systems have different suitable alkyl
groups.
EtO2C
BocHN
CON(nHex)2
w, x
CO2Et
Br
u, v
BocHN
NC
t
NC
NC
29
31
32
CON(nHex)2
CON(nHex)2
y, z
N
N
H
N
NC
NO2
33
34a
Scheme 5. Reagents and conditions: (t) BocNHCH(CO2Et)2 30,
EtONa, EtOH; (u) NaOH, EtOH, H2O; (v) (n-Hex)2NH, EDCÆHCl,
HOBt, DMF; (w) TFA, CH2Cl2; (x) 2-fluoronitrobenzene, K2CO3,
DMF; (y) H2, PtO2, MeOH; (z) HCl gas, EtOH. Method H: t–z.
CONR1R2
CON(nHex)2
(CH2)n
X1
Y
F
N
H
N
H
X2
12 (Y = S), 19 (Y = CH2)
FGIN-1-27
CONR1R2
CONR1R2
(CH2)n
N
N
N
4. Conclusions
X3
N
In this paper, we have reported the synthesis and SARs
of novel PBR ligands. We designed a new tetracyclic
compound from FGIN-1-27 (3) and found that com-
23 (n = 1), 28 (n = 2)
34
Chart 2.
pound 12a exhibited
a
high affinity for PBR
(IC50 ¼ 3.8 nM). The SAR study led us to more potent
PBR ligands (12e: IC50 ¼ 0.44 nM, 12f: IC50 ¼ 0.37 nM).
In addition, we found that another type of PBR ligand,
34a and 34c, which have another basic tetracyclic ring
and R2 groups play an important role in favoring con-
formation for binding to PBR. Introducing halogen
groups or a methyl group to the benzene ring attached

T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
Table 1. Binding data (PBR) for 12a–12m, 13a–13c, 14a–14c, 16, 19
3573
CONR1R2
(CH2)n
X1
7
8
Y
9
X2
N
10
R3
Compd
Method
Y
R3
X1, X2
n
NR1R2
IC50 (nM)a
12a
())-12a
(+)-12a
A, B
A
A
B
S
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
8-F, H
8-Cl, H
8-Me, H
8-F, 10-F
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
NH2
3.8
42
S
S
2.2
12b
12c
S
>1000
56
39
B
S
N(H)n-Pr
NMe2
12d
12e
B
S
B
S
NEt2
N(n-Pr)2
0.44
0.37
110
1.4
12f
12g
B
S
A
A
B
S
N(n-Hex)((CH2)2N(H)n-Pr)
N(n-Hex)((CH2)2O-n-Pr)
N((CH2)2OMe)2
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
N(H)n-Pr
12h
12i
S
S
5.0
12j
12k
B
S
14
20
B
S
12l
12m
B
S
14
22
B
S
13a
13b
C
C
C
D
D
D
E
SO2
SO2
SO2
S
1.2
129
1.1
1.4
13c
14a
N(n-Pr)2
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
14b
14c
S
Me2N(CH2)2
Me2N(CH2)4
>1000
>1000
250
1.4
S
16
19
S
CH2
H
H
F
Ro-5-4864 (1)
PK11195 (2)
FGIN-1-27 (3)
3.1
1.1
5.5
a IC50 values from duplicate determinations.
Table 2. Binding data (PBR) for 23a, 23b, and 28
system, showed moderate affinities for PBR (34a:
IC50 ¼ 42 nM, 34c: IC50 ¼ 74 nM). We believe that the
SAR study will provide new opportunities to explore
new PBR ligands with improved features, and the PBR
ligands reported here will aid study of the physiological
and biological roles of PBR.
CONR1R2
(CH2)n
N
X3
N
Compd
Method
n
NR1R2
X3
IC50 (nM)a
23a
23b
28
G
G
H
1
1
2
N(n-Hex)2
N(n-Hex)2
N(n-Hex)2
F
310
230
H
H
>1000
5. Experimental section
a IC50 values from duplicate determinations.
Melting points were determined on a Yanaco MP-500D
melting point apparatus and are uncorrected. Proton
nuclear magnetic resonance (NMR) spectra were ob-
tained using a Varian Gemini 2000 (200 MHz). Chemi-
cal shifts are reported in parts per million relative to
tetramethylsilane as an internal standard. Mass spectra
(MS) were obtained on a Shimadzu Profile (EI and CI),
JEOL JMS-SX102 (FAB) or Micromass Platform LC
(IonSpray and ES). Elemental analyses were performed
by a Perkin–Elmer 2400 (carbon, hydrogen, and nitro-
gen) and were within 0.4% of the theoretical values.
Silica gel [C-200, 100–200 mesh (Wako Pure Chemical)]
was used for column chromatography, using the solvent
systems (volume ratios) indicated below.
Table 3. Binding data (PBR) for 34a–34c
CONR1R2
N
N
Compd
Method
NR1R2
PBR affinity IC50 (nM)a
34a
34b
34c
I
I
I
N(n-Hex)2
NEt2
N(n-Pr)2
42
>1000
74
a IC50 values from duplicate determinations.

3574
T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
5.1. General methods for the synthesis of 12a, 12g, and
12h (method A)
the mixture was extracted with EtOAc. The organic
phase was washed with brine, dried (Na2SO4), and
evaporated under reduced pressure. The residue was
purified by silica gel chromatography [hexane/EtOAc,
5:1 (v/v), as eluent] and crystallized from EtOAc/hexane
to obtain 12a (0.43 g, 54%) as a crystal. Mp 138.5–
140.5 ꢁC; 1H NMR (CDCl3) d 0.68–1.89 (22H, m), 3.17–
3.66 (4H, m), 5.33 (1H, s), 6.75–7.42 (8H, m), 8.99 (1H,
br s); MS (CI, Pos) m=z 449 (Mþ+1, 100%); Anal.
(C28H36N2OS) C, H, N. Each enantiomer was obtained
by using HPLC for optical resolution [Chiralpak AD
(Daicel Chemical Industries, Ltd), 2 · 25 cm, mobile
phase: hexane/EtOH ¼ 3:7, flow rate: 5 mL/min]. (+)-
5.1.1. ( )-4-Oxo-thiochroman-2-carboxylic acid (8). A
mixture of benzenethiol 7 (82.6 g, 0.75 mol) and furan-
2,5-dione (73.5 g, 0.75 mol) in toluene (180 mL) was
stirred at 50 ꢁC. After all materials were dissolved, Et3N
(10 drops) in toluene (10 mL) was added over 10 min
keeping the temperature below 70 ꢁC. After stirring at
70 ꢁC for 20 min, the solvent was concentrated under
reduced pressure to obtain crude 3-phenyl-
thiodihydrofuran-2,5-dione. The residue was dissolved
in CH2Cl2 (150 mL) and the mixture was cooled with an
ice-cooling bath. AlCl3 (150 g, 1.12 mol) was added and
the mixture was stirred at room temperature for 1.5 h.
The reaction mixture was diluted in CH2Cl2 (150 mL)
and poured into ice-cooling concd HCl. After adding
IPE into the mixture, a solid precipitated. The solid was
collected by filtration, dried, and recrystallized from
THF/IPE to obtain 8 (67.7 g, 43%) as a white crystal. 1H
NMR (DMSO-d6) d 2.95–3.22 (2H, m), 4.40 (1H, dd,
J ¼ 4.6, 5.9 Hz), 7.18–7.57 (3H, m), 7.94 (1H, dd,
J ¼ 7.9, 1.5 Hz); MS m=z (ESI) m=z 231 (Mþ+Na, 100%).
6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-carbox-
26
ylic acid dihexylamide [(+)-12a]: ½aꢀ +25.9 (c 0.207,
D
EtOH); HPLC retention time: 37 min. ())-6,11-Dihy-
dro-5-thia-11-aza-benzo[a]fluorene-6-carboxylic acid di-
26
D
HPLC retention time: 20 min.
hexylamide [())-12a]: ½aꢀ
)25.9 (c 0.180, EtOH);
5.1.5. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid hexyl-(2-propoxyethyl)amide (12h).
Starting from 10 and hexyl-(2-propoxyethyl)amine, 12h
was obtained as a crystal. Yield 55%; mp 165.0–167.0 ꢁC
(EtOAc); 1H NMR (CDCl3) d 0.70–1.91 (16H, m), 3.15–
4.02 (8H, m), 5.36 (0.5H, s), 5.56 (0.5H, s), 6.73–7.49
(8H, m), 8.82–8.96 (1H, m); MS (ESI, Pos) m=z 473
(Mþ+Na, 100%); Anal. (C27H34N2O2S) C, H, N.
5.1.2. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid ethyl ester (9). To a solution of 8 (22.5 g,
108 mmol) and phenylhydrazine (10.7 mL, 108 mmol) in
EtOH (100 mL), H2SO4 (15 mL) was added, and the
mixture was heated at reflux for 5 h. After cooling to
room temperature, the reaction mixture was poured into
ice water (500 mL) and extracted with Et2O. The organic
phase was washed with brine, dried (Na2SO4), and
evaporated under reduced pressure. The residue was
recrystallized from hexane/EtOAc to obtain 9 (21.8 g,
65%) as a solid. 1H NMR (CDCl3) d 1.16 (3H, t,
J ¼ 7.1 Hz), 4.10 (2H, q, J ¼ 7.1 Hz), 5.01 (1H, s), 6.97–
7.42 (7H, m), 7.47–7.62 (1H, m), 8.46 (1H, br s); MS
(EI) m=z 309 (Mþ), 236 (Mþ)73, 100%).
5.1.6. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid hexyl-(2-propylaminoethyl)amide (12g).
To a mixture of 10 (844 mg, 3.00 mmol) and (2-hexyl-
aminoethyl)propylcarbamic acid tert-butyl ester (1.72 g,
6.00 mmol) in CH2Cl2 (44 mL), HOBtÆ1H2O (552 mg,
3.60 mmol), and EDCÆHCl (863 mg, 4.50 mmol) were
added and the mixture was stirred at room temperature
overnight. After evaporation of the solvent under re-
duced pressure, the residue was poured into EtOAc, and
the organic phase was washed with H2O, 5% KHSO4
solution, satd NaHCO3 solution and brine, dried
(Na2SO4), and evaporated under reduced pressure. The
residue was purified by silica gel chromatography
[Chromatorex NHDM1020 (Fuji Silysia) as silica gel;
hexane/EtOAc, 3:2 (v/v), as eluent] to obtain ( )-{2-
[(6,11-dihydro-5-thia-11-aza-benzo[a]fluorene-6-carbon-
yl)hexylamino]ethyl}propylcarbamic acid tert-butyl
ester (1.35 g, 82%) as an amorphous. 1H NMR (CDCl3)
d 0.60–1.90 (25H, m), 2.88–3.86 (8H, m), 5.28 (1H, s),
6.65–7.43 (8H, m), 9.02 (1H, br s); MS (ESI, Neg) m=z
548 (Mþ)1, 100%).
5.1.3. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid (10). To a solution of 9 (15.0 g,
48.5 mmol) in EtOH (100 mL), a solution of KOH (85%,
12.8 g, 194 mmol) in water (40 mL) was added. The
mixture was heated at reflux for 2 h. The reaction mix-
ture was acidified with 3 M HCl and extracted with
EtOAc. The organic phase was washed with brine, dried
(Na2SO4), and evaporated under reduced pressure to
obtain a solid. The solid was recrystallized from hexane/
1
EtOH to obtain 10 (10.3 g, 75%) as a solid. H NMR
(DMSO-d6) d 5.20 (1H, s), 6.97–7.58 (7H, m), 7.72–7.83
(1H, m), 11.71 (1H, br s), 12.63 (1H, br s); MS (EI) m=z
281 (Mþ), 236 (Mþ)45, 100%).
( )-{2-[(6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carbonyl)hexylamino]ethyl}propylcarbamic acid tert-
butyl ester (600 mg) was dissolved in HCO2H (4.2 mL)
at room temperature for 5 h. HCO2H was removed
under reduced pressure and EtOAc was poured into the
residue. The solution was washed with satd NaHCO3
solution and brine, dried (Na2SO4), and evaporated
under reduced pressure to obtain a solid. The solid was
recrystallized from EtOAc to obtain 12g (406 mg, 83%)
5.1.4. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid dihexylamide (12a). A solution of
10 (501 mg, 1.78 mmol) in THF (8.0 mL) was cooled
to )15 ꢁC. N-Methylmorpholine (180 mg, 1.78 mmol),
isobutyl chloroformate (243 mg), and dihexylamine
(330 mg, 1.78 mmol) were added and stirred at )15 ꢁC
for 4 h. The reaction was quenched with 0.5 M HCl and
1
as a crystal. Mp 135.5–138.0 ꢁC; H NMR (CDCl3) d

T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
3575
0.62–1.95 (16H, m), 2.18–3.82 (8H, m), 5.33 (0.5H, s),
5.52 (0.5H, s), 6.80–7.52 (8H, m), 8.72–8.90 (1H, m); MS
(ESI, Pos) m=z 450 (Mþ+1, 100%); Anal.
(C27H35N3OSÆ0.1EtOAc) C, H, N.
5.2.5. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid dimethylamide (12d). Starting from 4-
oxo-thiochroman-2-carboxylic acid dimethylamide and
phenylhydrazine, 12d was obtained as a crystal. Yield
1
61%; mp 254.5–256.5 ꢁC (EtOAc); H NMR (CDCl3) d
2.80 (3H, s), 3.32 (3H, s), 5.69 (1H, s), 6.92–7.50 (7H,
m), 7.72–7.85 (1H, m), 11.64 (1H, br s); MS (CI, Pos)
m=z 309 (Mþ+1), 236 (Mþ)72, 100%); Anal.
(C18H16N2OS) C, H, N.
5.2. General methods for the synthesis of 12b–f and
12i–m (method B)
5.2.1. ( )-4-Oxo-thiochroman-2-carboxylic acid di-
hexylamide (11). To a solution of 8 (20.0 g, 96.0 mmol)
in benzene (200 mL), thionyl chloride (14.0 mL,
193 mmol) was added, and the mixture was heated at
reflux for 3 h. After evaporation of the solvent under
reduced pressure, the residue was dissolved in dry
CH2Cl2 (100 mL). The solution was added dropwise into
a solution of dihexylamine (24.6 mL, 106 mmol) and
Et3N (20.0 mL, 143 mmol) in CH2Cl2 (200 mL), and the
mixture was stirred at room temperature overnight. The
reaction mixture was diluted with EtOAc, washed with
water, 1 M HCl, satd NaHCO3 solution and brine, dried
(Na2SO4), and evaporated under reduced pressure. The
residue was purified by silica gel chromatography [hex-
ane/EtOAc, 3:1 (v/v), as eluent] and recrystallized from
5.2.6. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid diethylamide (12e). Starting from 4-oxo-
thiochroman-2-carboxylic acid diethylamide and phen-
ylhydrazine, 12e was obtained as a crystal. Yield 8%; mp
1
242.0–243.5 ꢁC (EtOAc); H NMR (CDCl3) d 1.14 (3H,
t, J ¼ 7.1 Hz), 1.40 (3H, t, J ¼ 7.1 Hz), 3.25–3.78 (4H,
m), 5.34 (1H, s), 6.56–6.70 (1H, m), 6.74–7.40 (7H, m),
9.29 (1H, s); MS (CI, Pos) m/z 337 (Mþ+1), 236
(Mþ)100, 100%); Anal. (C20H20N2OS) C, H, N.
5.2.7. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid dipropylamide (12f). Starting from 4-oxo-
thiochroman-2-carboxylic acid dipropylamide and
phenylhydrazine, 12f was obtained as a crystal. Yield
1
hexane to obtain 11 (29.2 g, 81%). H NMR (CDCl3) d
0.68–1.82 (22H, m), 2.94–3.57 (6H, m), 4.26 (1H, dd,
J ¼ 3.7, 7.6 Hz), 7.09–745 (3H, m), 8.14 (1H, dd, J ¼ 1.4,
8.2 Hz); MS (ESI, Neg) m=z 374 (Mþ)1, 100%).
1
25%; mp 196.0–197.0 ꢁC (EtOAc); H NMR (CDCl3) d
0.85 (3H, t, J ¼ 7.5 Hz), 1.05 (3H, t, J ¼ 7.5 Hz), 1.45–
1.96 (4H, m), 3.17–3.66 (4H, m), 5.34 (1H, s), 6.52–6.66
(1H, m), 6.76–7.39 (7H, m), 9.35 (1H, br s); MS (CI,
Pos) m=z 365 (Mþ+1), 236 (Mþ)128, 100%); Anal.
(C22H24N2OS) C, H, N.
5.2.2. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid dihexylamide (12a). A mixture of 11
(1.00 g, 2.67 mmol) and phenylhydrazine (0.26 mL,
2.64 mmol) was heated at 100 ꢁC for 30 min. After dry-
ing under reduced pressure at 50 ꢁC, ZnCl2 (1.44 g,
10.6 mmol) was added and the mixture was heated at
170 ꢁC for 5 min and then cooled to room temperature.
To the reaction mixture, iced water was added, extracted
with EtOAc, and the organic phase was washed with
1 M HCl, satd NaHCO3 solution and brine, dried
(Na2SO4), and evaporated under reduced pressure. The
residue was crystallized from hexane/EtOAc to obtain
12a (0.79 g, 66%) as a crystal.
5.2.8. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid bis-(2-methoxyethyl)amide (12i). Starting
from 4-oxo-thiochroman-2-carboxylic acid bis-(2-meth-
oxyethyl)amide and phenylhydrazine, 12i was obtained
as a crystal. Yield 10%; mp 206.0–208.0 ꢁC (EtOAc); 1H
NMR (DMSO-d6) d 3.12–4.01 (8H, m), 3.20 (3H, s),
3.41 (3H, s), 5.68 (1H, s), 6.92–7.48 (7H, m), 7.72–7.83
(1H, m), 11.64 (1H, br s); MS (CI, Pos) m=z 397 (Mþ+1),
236 (Mþ)160, 100%); Anal. (C22H24N2O3S) C, H, N.
5.2.3. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid amide (12b). Starting from 4-oxo-thio-
chroman-2-carboxylic acid amide and phenylhydrazine,
12b was obtained as a crystal. Yield 36%; mp 241.0–
242.5 ꢁC (EtOAc); 1H NMR (CDCl3) d 5.05 (1H, s),
6.87–7.58 (9H, m), 7.78 (1H, dd, J ¼ 7.5, 1.3 Hz), 11.7
(1H, br s); MS (CI, Pos) m=z 281 (Mþ+1), 236 (Mþ)44,
100%); Anal. (C16H12N2OS) C, H, N.
5.2.9. ( )-8-Fluoro-6,11-dihydro-5-thia-11-aza-benzo[a]-
fluorene-6-carboxylic acid dihexylamide (12j). Starting
from 4-oxo-thiochroman-2-carboxylic acid dihexyl-
amide and 4-fluorophenylhydrazine, 12j was obtained as
a crystal. Yield 47%; mp 110.5–112.5 ꢁC (EtOAc/hex-
1
ane); H NMR (CDCl3) d 0.75–1.09 (6H, m), 1.11–1.95
(16H, m), 3.25–3.70 (4H, m), 5.27 (1H, s), 6.52–6.77
(3H, m), 6.82–7.03 (3H, m), 7.10–7.22 (1H, m), 9.66 (1H,
br s); MS (CI, Pos) m=z 467 (Mþ+1), 254 (Mþ)212,
100%); Anal. (C28H35FN2OS) C, H, N.
5.2.4. ( )-6,11-Dihydro-5-thia-11-aza-benzo[a]fluorene-6-
carboxylic acid propylamide (12c). Starting from 4-oxo-
thiochroman-2-carboxylic acid propylamide and phen-
ylhydrazine, 12c was obtained as a crystal. Yield 71%;
5.2.10. ( )-8-Chloro-6,11-dihydro-5-thia-11-aza-benzo[a]-
fluorene-6-carboxylic acid dihexylamide (12k). Starting
from 4-oxo-thiochroman-2-carboxylic acid dihexyla-
mide and 4-chlorophenylhydrazine, 12k was obtained as
a crystal. Yield 43%; mp 123.0–124.5 ꢁC (EtOAc/hex-
1
mp 172.0–174.0 ꢁC (EtOAc/hexane); H NMR (CDCl3)
d 0.59 (3H, t, J ¼ 7.4 Hz), 1.10–1.40 (2H, m), 2.85–3.26
(2H, m), 4.96 (1H, s), 5.84–6.12 (1H, m), 7.05–7.64 (1H,
m), 8.96 (1H, br s); MS (CI, Pos) m=z 323 (Mþ+1), 236
(Mþ)86, 100%); Anal. (C19H18N2OS) C, H, N.
1
ane); H NMR (CDCl3) d 0.72–1.08 (6H, m), 1.10–1.99

3576
T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
(16H, m), 3.20–3.72 (4H, m), 5.27 (1H, s), 6.34–6.53
(2H, m), 6.62–6.95 (3H, m), 7.02–7.23 (2H, m), 10.11
(1H, br s); MS (FAB, Pos) m=z 483 (Mþ+1), 485
(Mþ+3), 270 (Mþ)212, 100%); Anal. (C28H35ClN2OS)
C, H, N.
397 (Mþ+1), 128 (Mþ)268, 100%); Anal. (C19H18-
N2O3S) C, H, N.
5.3.3. 5,5-Dioxo-6,11-dihydro-5H-5k6-thia-11-aza-benzo[a]-
fluorene-6-carboxylic acid propylamide (13c). Starting
from 12c, 13c was obtained as a crystal. Yield 31%; mp
266.0–267.0 ꢁC (EtOAc/hexane); 1H NMR (CDCl3) d
0.82 (3H, t, J ¼ 7.1 Hz), 1.26–1.52 (2H, m), 2.90–3.09
(2H, m), 5.69 (1H, s), 7.05–7.32 (2H, m), 7.43–7.65 (3H,
m), 7.73–8.06 (3H, m), 8.57–8.72 (1H, br t), 12.13 (1H,
br s); MS (CI, Pos) m/z 355 (Mþ+1), 205 (Mþ)149,
100%); Anal. (C22H24N2O3S) C, H, N.
5.2.11. ( )-8-Methyl-6,11-dihydro-5-thia-11-aza-benzo[a]-
fluorene-6-carboxylic acid dihexylamide (12l). Starting
from 4-oxo-thiochroman-2-carboxylic acid dihexyl-
amide and 4-methylphenylhydrazine, 12l was obtained
as a crystal. Yield 32%; mp 139.5–141.5 ꢁC (EtOAc/
1
hexane); H NMR (CDCl3) d 0.72–1.07 (6H, m), 1.10–
1.90 (16H, m), 2.41 (3H, s), 3.15–3.67 (4H, m), 5.32 (1H,
s), 6.74–7.27 (7H, m), 8.88 (1H, br s); MS (CI, Pos) m=z
463 (Mþ, 100%); Anal. (C29H38N2OS) C, H, N.
5.4. General methods for the synthesis of 14a–c
(method D)
5.2.12. ( )-8,10-Difluoro-6,11-dihydro-5-thia-11-aza-ben-
zo[a]fluorene-6-carboxylic acid dihexylamide (12m).
Starting from 4-oxo-thiochroman-2-carboxylic acid di-
hexylamide and 2,4-difluorophenylhydrazine, 12m was
obtained as a crystal. Yield 25%; mp 148.0–149.0 ꢁC
5.4.1. ( )-11-(2-Dimethylaminoethyl)-6,11-dihydro-5-
thia-11-aza-benzo[a]fluorene-6-carboxylic acid dihexyl-
amide hydrochloride (14b). To a suspension of NaH
(53 mg, 1.33 mmol) in dry DMF (5 mL), a solution of
12a in dry DMF (10 mL) dropwise was added and the
mixture was stirred at room temperature for 30 min. A
solution of 2-(dimethylamino)ethylchloride hydrochlo-
ride (96 mg, 0.67 mmol) was added and the mixture was
heated at 80 ꢁC for 3 h. The reaction was quenched with
H2O and extracted with EtOAc. The organic phase was
washed with H2O and brine, dried (Na2SO4), and
evaporated under reduced pressure. The residue was
purified by silica gel chromatography [CH2Cl2/EtOH,
15:1 (v/v), as eluent] to obtain the free base of 14b.
Treatment of the free base with 4 M HCl/EtOAc in
EtOAc to give 14b (10 mg, 2.7%) and crystallized from
Et2O/hexane to obtain a solid. Mp 89.5–90.5 ꢁC; 1H
NMR (CDCl3) d 0.60–1.90 (22H, m), 2.38–2.72 (6H, m),
2.95–3.68 (6H, m), 4.90–5.19 (3H, m), 7.08–7.82 (8H,
m), 13.20 (1H, br s); MS (CI, Pos) m=z 520 (Mþ+1), 307
(Mþ)212, 100%); HRMS calculated 520.3362, found
520.3373.
1
(EtOAc/hexane); H NMR (CDCl3) d 0.75–1.07 (6H,
m), 1.12–1.97 (16H, m), 3.25–3.70 (4H, m), 5.19 (1H, s),
6.28–6.45 (1H, m), 6.54–6.74 (2H, m), 6.84–6.98 (1H, m),
7.10–7.31 (2H, m), 10.01 (1H, br s); MS (CI, Pos) m=z
485 (Mþ+31, 100%); Anal. (C28H34F2N2OS) C, H, N.
5.3. General methods for the synthesis of 13a–c
(method C)
5.3.1. ( )-5,5-Dioxo-6,11-dihydro-5H-5k6-thia-11-aza-
benzo[a]fluorene-6-carboxylic acid dihexylamide (13a).
A solution of mCPBA (>70%, 346 mg, 1.40 mmol) in
CH2Cl2 (20 mL) was added to a solution of 12a (300 mg,
0.669 mmol) in CH2Cl2 (20 mL) dropwise with cooling
in an ice-water bath. The reaction mixture was stirred at
room temperature for 1 h and then the solvent was
evaporated under reduced pressure. The residue was
dissolved in EtOAc and the solution was washed with
satd NaHCO3 solution and brine, dried (Na2SO4), and
evaporated under reduced pressure. The residue was
purified by silica gel chromatography [hexane/EtOAc,
1:1 (v/v), as eluent] and crystallized from EtOAc/hexane
to obtain 13a (130 mg, 40%) as a crystal. Mp 156.0–
157.0 ꢁC (EtOAc/hexane); 1H NMR (CDCl3) d 0.78–
2.10 (22H, m), 3.24–4.02 (4H, m), 5.77 (1H, s), 5.52
(0.5H, s), 6.66–7.11 (6H, m), 7.22–7.36 (1H, m), 7.72–
7.83 (1H, m), 9.73 (1H, br s); MS (CI, Pos) m=z 481
(Mþ+1), 85 (Mþ)395, 100%); Anal. (C28H36N2O3S) C,
H, N.
5.4.2. ( )-11-Methyl-6,11-dihydro-5-thia-11-aza-benzo-
[a]fluorene-6-carboxylic acid dihexylamide (14a). Com-
pound 14a was synthesized using the same procedure as
14b using MeI instead of 2-(dimethylamino)ethylchlo-
ride hydrochloride. Yield 37%; mp 111.0–112.0 ꢁC
(hexane); 1H NMR (CDCl3) d 0.70–1.90 (22H, m), 3.11–
3.67 (4H, m), 4.01 (3H, s), 5.12 (1H, s), 7.03–7.49 (7H,
m), 7.73 (1H, dd, J ¼ 7.9, 1.3 Hz); MS (CI, Pos) m=z 463
(Mþ+1), 250 (Mþ)212, 100%); Anal. (C29H38N2OS)
C, H, N.
5.4.3. ( )-11-(4-Dimethylaminobutyl)-6,11-dihydro-5-
thia-11-aza-benzo[a]fluorene-6-carboxylic acid dihexyl-
5.3.2. ( )-5,5-Dioxo-6,11-dihydro-5H-5k6-thia-11-aza-
benzo[a]fluorene-6-carboxylic acid dipropylamide (13b).
Starting from 12f, 13b was obtained as a crystal. Yield
amide (14c). To
a suspension of NaH (37 mg,
0.93 mmol) in dry DMF (5 mL), a solution of 12a in dry
DMF (10 mL) dropwise was added and the mixture was
stirred at room temperature for 25 min. A solution of 1-
bromo-4-chlorobutane (0.11 mL, 0.96 mmol) in dry
DMF (5 mL) was added with a cooling ice-water bath
and the mixture was heated at room temperature for
1
31%; mp 296.5–298.0 ꢁC (EtOAc); H NMR (CDCl3) d
0.86 (3H, t, J ¼ 7.3 Hz), 1.18 (3H, t, J ¼ 7.3 Hz), 1.40–
1.78 (2H, m), 1.80–2.25 (2H, m), 3.20–3.98 (4H, m), 5.78
(1H, s), 6.60–7.12 (6H, m), 7.21–7.36 (1H, m), 7.78 (1H,
dd, J ¼ 7.7, 1.1 Hz), 9.72 (1H, br s); MS (CI, Pos) m=z

T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
3577
30 min. The reaction was quenched with H2O and ex-
5.6. Method F
5.6.1. ( )-4-Oxo-1,2,3,4-tetrahydronaphthalene-2-car-
tracted with EtOAc. The organic phase was washed with
H2O and brine, dried (Na2SO4), and evaporated under
reduced pressure. The residue was purified by silica gel
chromatography [CH2Cl2/EtOH, 15:1 (v/v), as eluent] to
obtain a solid. The solid was recrystallized from hexane
to provide 11-(4-chlorobutyl)-6,11-dihydro-5-thia-11-
aza-benzo[a]fluorene-6-carboxylic acid dihexylamide
boxylic acid (18). 2-Benzylsuccinic acid (5.00 g,
24.0 mmol) was added to concd H2SO4 and the mixture
was stirred at room temperature for 9 h. The reaction
mixture was poured into ice and the resulting precipitate
was collected by filtration and washed with H2O. The
precipitate was recrystallized from EtOH/H2O to obtain
1
(175 mg, 53%) as a crystal. H NMR (CDCl3) d 0.68–
1
2.25 (26H, m), 3.08–3.64 (4H, m), 3.53 (2H, t,
J ¼ 6.4 Hz), 4.25–4.53 (2H, m), 5.09 (1H, s), 7.02–7.52
(7H, m), 7.63 (1H, dd, J ¼ 1.2, 7.8 Hz); MS (CI, Pos)
m=z 539 (Mþ+1, 100%). A solution of 11-(4-chloro-bu-
tyl)-6,11-dihydro-5-thia-11-aza-benzo[a]fluorene-6-carb-
oxylic acid dihexylamide (0.150 g, 0.28 mmol) and 50%
aqueous dimethylamine solution (1.46 mL, 8.83 mmol)
in MeOH (20 mL) was heated at reflux for two days.
H2O was added into the reaction mixture and extracted
with EtOAc. The organic phase was washed with brine,
dried (Na2SO4), and evaporated under reduced pressure
to obtain a solid. The solid was recrystallized from
hexane to obtain 14c (0.12 g, 77%) as a crystal. Mp 50.0–
18 (2.1 g, 45%) as a crystal. H NMR (CDCl3) d 2.72–
3.39 (5H, m), 7.19–7.60 (3H, m), 7.95–8.13 (1H, m),
13.25 (1H, br s); MS (ESI, Neg) m=z 189 (Mþ)1), 145
(Mþ)45, 100%).
5.6.2. ( )-5,11-Dihydro-6H-benzo[a]carbazole-6-carbox-
ylic acid dihexylamide (19). Compound 19 was obtained
from 18 using the same method as for synthesis of 12a
from 11. Yield 56%; mp 152.0–153.0 ꢁC (EtOAc/hexane);
1H NMR (CDCl3) d 0.70–1.84 (22H, m), 3.01 (1H, dd,
J ¼ 15.5, 5.9 Hz), 3.25–3.67 (5H, m), 4.38 (1H, dd,
J ¼ 12.6, 5.9 Hz), 6.97–7.42 (8H, m), 8.40 (1H, br s); MS
(CI, Pos) m=z 431 (Mþ+1), 218 (Mþ)212, 100%); Anal.
(C29H38N2O) C, H, N.
1
52.0 ꢁC; H NMR (CDCl3) d 0.72–2.10 (26H, m), 2.21
(6H, s), 2.20–2.36 (2H, m), 3.08–3.65 (4H, m), 4.23–4.50
(2H, m), 5.10 (1H, s), 7.02–7.49 (7H, m), 7.61–7.72 (1H,
m); MS (EI) m=z 547 (Mþ), 335 (Mþ)212, 100%); Anal.
(C34H49N3OS) C, H, N.
5.7. General methods for the synthesis of 23a and 23b
(method G)
5.5. Method E
5.7.1. 2-(4-Fluorophenyl)-1H-benzoimidazole (21). A
mixture of benzene-1,2-diamine (7.72 g, 71 mmol), 4-
fluorobenzoic acid (10.0 g, 71 mmol), and polyphos-
phoric acid (150 g) was heated at 250 ꢁC for 4 h. After
cooling to room temperature, with addition of water to
the reaction mixture a solid precipitated. The solid was
collected by filtration, washed with H2O, 10% Na2CO3,
and H2O again, and purified by recrystallization to ob-
tain 21 (13.2 g, 87%). H NMR (DMSO-d6) d 7.13–7.28
(2H, m), 7.30–7.72 (4H, m), 8.13–8.32 (2H, m), 12.91
(1H, br s); MS (ESI) m=z 213 (Mþ+1, 100%).
5.5.1. ( )-2-(6,11-Dihydro-5-thia-11-aza-benzo[a]fluoren-
6-yl)-N,N-hexylacetamide (16). A solution of (4-oxo-
thiochroman-2-yl)acetic acid 15 (0.89 g, 4.0 mmol),
which was prepared from benzenethiol and glutaconic
acid using the same method as shown for preparation of
8, and Et3N (1.96 mL, 14.1 mmol) in dry THF (30 mL)
was cooled to )40 ꢁC and ethyl chloroformate (0.42 mL,
1
4.4 mmol), and then
a
solution of dihexylamine
(1.03 mL, 4.4 mmol) in dry THF (10 mL) was added.
The reaction mixture was stirred at room temperature
for 2.5 h, quenched with H2O, and extracted with
EtOAc. The organic phase was washed with 5% HCl
and brine, dried (Na2SO4), and evaporated under re-
duced pressure to obtain crude N,N-dihexyl-2-(4-oxo-
thiochroman-2-yl)acetamide. Phenylhydrazine (0.39 mL,
4.0 mmol) was added to the crude N,N-dihexyl-2-(4-oxo-
thiochroman-2-yl)acetamide and the mixture was heated
at 100 ꢁC, and then the water generated was removed
under reduced pressure. ZnCl2 (2.19 g, 16.0 mmol) was
added and the mixture was heated at 170 ꢁC for 5 min
and cooled to 80 ꢁC. After the reaction mixture was
dissolved in acetone (30 mL), Et2O and H2O were added
and separated. The organic phase was washed with 5%
HCl and brine, dried (Na2SO4), and evaporated under
reduced pressure to obtain a solid. The solid was
recrystallized from hexane/EtOAc to obtain 16 (0.56 g,
30%) as a crystal. Mp 155.0–157.0 ꢁC; 1H NMR
(CDCl3) d 0.68–1.61 (22H, m), 2.65–3.00 (4H, m), 3.12–
3.42 (2H, m), 5.06 (1H, dd, J ¼ 9.5, 5.1 Hz), 7.08–7.30
(4H, m), 7.37–7.49 (3H, m), 7.60–7.69 (1H, m), 8.33 (1H,
br s); MS (CI, Pos) m=z 463 (Mþ+1, 100%); Anal.
(C29H38N2OS) C, H, N.
5.7.2. [2-(4-Fluorophenyl)benzoimidazol-1-yl]acetic acid
(22). A mixture of KOH (5.57 g, 94 mmol) and DMSO
(100 mL) was stirred at room temperature for 15 min,
and then 21 (5.00 g, 24.0 mmol) was added to the mix-
ture. After stirring at room temperature for 1 h,
bromoacetic acid (3.60 g, 26 mmol) was added and the
mixture was stirred at room temperature for 15 h. The
reaction was quenched with H2O and washed with
EtOAc three times. The aqueous phase was acidified
with 3 M HCl and washed with EtOAc twice. Next, the
aqueous phase was neutralized with 10% NaOH and a
solid was precipitated. The solid was collected by fil-
tration and washed with H2O to provide 22 (4.3 g, 67%).
1H NMR (DMSO-d6) d 5.10 (2H, s), 7.17–7.86 (8H, m),
13.33 (1H, br s); MS (FAB, Pos) m=z 271 (Mþ+1).
5.7.3. 2-[2-(4-Fluorophenyl)benzoimidazol-1-yl]-N,N-di-
hexylacetamide (23a). To a solution of 22 (1.00 g,
3.70 mmol) and Et3N (1.81 mL, 13.0 mmol) in THF
(29 mL), ClCO2Et (0.39 mL, 4.1 mmol) was added, and

3578
T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
then a solution of dihexylamine (0.95 mL, 4.1 mmol) in
THF (10 mL) at )40 ꢁC, and the mixture was stirred at
room temperature for 1.5 h. The reaction was quenched
with H2O and extracted with EtOAc. The organic phase
was washed with H2O and brine, dried (Na2SO4), and
evaporated under reduced pressure to give a solid. The
solid was recrystallized from hexane to obtain 23a
(0.48g, 30%). Mp 90.0–91.5 ꢁC; 1H NMR (CDCl3) d
0.80–1.05 (6H, m), 1.14–1.73 (16H, m), 3.17–3.49 (4H,
m), 4.91 (2H, m), 7.10–7.39 (5H, m), 7.64–7.90 (3H, m);
MS (FAB, Pos) m=z 438 (Mþ+1, 100%); Anal.
(C27H36FN3O) C, H, N.
EtOAc, and the solution obtained was washed with H2O
and brine, dried (Na2SO4), and evaporated under
reduced pressure. The residue was purified by silica gel
chromatography [hexane/EtOAc, 3:1 (v/v), as eluent] to
1
obtain 26 (7.79 g) as an orange oil. H NMR (CDCl3) d
0.75–1.65 (22H, m), 2.69 (4H, t, J ¼ 6.9 Hz), 3.10–3.39
(4H, m), 3.61–3.79 (2H, m), 6.57–6.71 (1H, m), 6.87–
6.99 (1H, m), 7.36–7.52 (1H, m), 8.06–8.29 (2H, m); MS
(ESI, Pos) m=z 400 (Mþ+Na, 100%).
5.8.2. N-(2-Dihexylcarbamoylethyl)-N-(2-nitrophenyl)-
benzamide (27). To a solution of 26 (2.31 g, 6.12 mmol)
and Et3N (1.71 mL, 12.2 mmol) in CH2Cl2 (20 mL),
benzoyl chloride (0.75 mL, 6.43 mmol) was added, and
the mixture was heated at reflux for 5 h. The reaction
mixture was poured into EtOAc, washed with 0.5 M
HCl, satd NaHCO3 and brine, dried (MgSO4), and
evaporated under reduced pressure. The residue was
purified by silica gel chromatography [hexane/EtOAc,
3:1 (v/v), as eluent] to obtain 27 (1.38 g, 47%) as a yellow
5.7.4. N,N-Dihexyl-2-(2-phenylbenzoimidazol-1-yl)acet-
amide (23b). Starting from (2-phenylbenzoimidazol-1-
yl)acetic acid, 23b was obtained as a crystal. Yield 45%;
1
mp 83.5–85.0 ꢁC (hexane); H NMR (CDCl3) d 0.78–
1.02 (6H, m), 1.12–1.80 (16H, m), 3.22 (2H, t,
J ¼ 7.5 Hz), 3.40 (2H, t, J ¼ 7.5 Hz), 4.92 (2H, s), 7.18–
7.59 (6H, m), 7.65–7.91 (3H, m); MS (EI) m=z 419 (Mþ),
43 (Mþ)376, 100%); Anal. (C27H37N3O) C, H, N.
1
oil. H NMR (CDCl3) d 0.72–1.80 (22H, m), 2.65–3.50
(6H, m), 3.97–4.46 (2H, m), 6.99–7.90 (9H, m); MS
(FAB, Pos) m=z 482 (Mþ+1, 100%).
5.8. Method H
5.8.1. N,N-Dihexyl-3-(2-nitrophenylamino)propionamide
(26). b-Alanine (10.0 g, 112 mmol) was dissolved in a
mixture of 1,4-dioxane (220 mL) and 0.5 M NaOH
solution (220 mL). After cooling with an ice-cooling
bath, Boc2O (27.0 g, 123 mmol) was added to the solu-
tion. The solution was stirred at room temperature
overnight. The solvent was evaporated under reduced
pressure, and to the residue 5% KHSO4 solution was
added and extracted with CH2Cl2, dried (Na2SO4), and
evaporated under reduced pressure. The residue was
crystallized from EtOAc/hexane and the crystal was
collected by filtration to obtain 3-tert-butoxycarbon-
5.8.3. N,N-Dihexyl-3-(2-phenylbenzoimidazol-1-yl)prop-
ionamide hydrochloride (28). To a solution of 27
(433 mg, 0.899 mmol) in CH3CO2H (10.0 mL), iron
powder (151 mg, 2.70 mmol) was added, and the mixture
was heated at 120 ꢁC for 2 h. The insoluble material was
removed by filtration with a Celite filter, and the filtrate
was evaporated under reduced pressure. The residue was
dissolved in EtOAc and the solution was washed with
satd NaHCO3 solution, dried (Na2SO4), and evaporated
under reduced pressure. The residue was purified by
silica gel chromatography [hexane/EtOAc, 2:1 (v/v), as
eluent] to obtain a free base of 28 (0.38 g, 97%) as a
colorless oil. Treatment of a solution of the free base of
28 in EtOAc with 4 M HCl/EtOAc and crystallized from
diisopropylether to provide 28 (HCl salt) as a white
crystal. Mp 101.0–103.0 ꢁC; 1H NMR (DMSO-d6) d
0.75–0.91 (6H, m), 1.00–1.43 (16H, m), 2.92 (2H, t,
J ¼ 7.3 Hz), 2.95–3.20 (4H, m), 4.68 (2H, t, J ¼ 7.3 Hz),
7.53–7.97 (8H, m), 8.07–8.18 (1H, m); MS (CI, Pos) m=z
434 (Mþ+1, 100%); Anal. (C28H40ClN3O) C, H, N.
1
ylaminopropionic acid (17.7 g, 91%). H NMR (CDCl3)
d 1.37 (9H, m), 2.34 (2H, t, J ¼ 7.1 Hz), 2.95–3.22 (2H,
m), 6.65–6.90 (1H, m), 12.16 (1H, br s). To the solution
of 3-tert-butoxycarbonylaminopropionic acid (14.1 g,
81.5 mmol) and dihexylamine (19.0 mL, 81.5 mmol) in
CH2Cl2 (100 mL), EDCÆHCl (16.4 g, 85.6 mmol) was
added, and the mixture was stirred overnight. The
solution was washed with 5% KHSO4 solution, satd
NaHCO3 solution, and brine, dried (Na2SO4), and
evaporated under reduced pressure to obtain crude
3-tert-butoxycarbonylamino-N,N-dihexylpropionamide
(29.9 g, quant.). To a solution of crude 3-tert-butoxy-
carbonylamino-N,N-dihexylpropionamide (7.75 g, 21.8
mmol) in CH2Cl2 (17 mL), trifluoroacetic acid (17 mL)
was added. The mixture was stirred at room tempera-
ture for 3.5 h. The solvent was evaporated under re-
duced pressure and the residue was dissolved in CHCl3.
The solution was washed with satd NaHCO3 and brine,
dried (Na2SO4), and evaporated to obtain crude 3-
amino-N,N-dihexylpropionamide (25). The product was
used in the next step without further purification. A
mixture of 25, synthesized using the method described
above, 2-fluoronitrobenzene (3.07 g, 21.8 mmol) and
K2CO3 (3.61 g) in DMF (50 mL) was heated at 150 ꢁC
for 3 h. The resulting orange solution was diluted with
5.9. General methods for the synthesis of 12b–f and
12i–m (method I)
5.9.1. 2-tert-Butoxycarbonylamino-2-(2-cyanobenzyl)ma-
lonic acid diethyl ester (31) . Aminomalonic acid diethyl
ester hydrochloride (25.1 g, 119 mmol) was dissolved in a
mixture of 1 M NaOH solution (119 mL, 119 mmol) and
1,4-dioxane (100 mL). A solution of Boc2O (28.5 g,
130 mmol) in 1,4-dioxane (50 mL) was added dropwise
to the solution and the mixture was stirred at room
temperature overnight. The solvent was removed by
evaporation under reduced pressure and the residue was
dissolved in EtOAc. The solution was washed with 5%
KHSO4 solution, satd NaHCO3 solution and brine,
dried (MgSO4), and evaporated under reduced pressure

T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
3579
to provide crude 2-tert-butoxycarbonylaminomalonic
acid diethyl ester 30 (33.2 g, quant.).
diluted with EtOAc, washed with H2O and brine, dried
(MgSO4), and evaporated under reduced pressure. The
residue was purified by silica gel chromatography [hex-
ane/EtOAc, 5:1 (v/v), as eluent] to obtain 33 (1.01 g,
74%) as a yellow oil. 1H NMR (CDCl3) d 0.67–1.68
(22H, m), 2.76–3.65 (6H, m), 4.81–5.05 (1H, m), 6.51–
6.97 (2H, m), 7.10–7.75 (4H, m), 8.04–8.23 (1H, m),
8.50–8.72 (1H, m); MS (CI, Pos) m=z 479 (Mþ+1,
100%).
Na (492 mg, 21.4 mmol) was dissolved in EtOH (20 mL).
To the solution, crude 30 (5.90 g, 21.4 mmol) and 2-cy-
anobenzyl bromide (4.00 g, 20.4 mmol) were added. The
mixture was heated at reflux for 3.5 h. EtOH was
removed by evaporation under reduced pressure and the
residue was dissolved in EtOAc. The solution was
washed with 5% KHSO4 solution, satd NaHCO3 solu-
tion and brine, dried (MgSO4), and evaporated under
reduced pressure. The residue was purified by silica gel
chromatography [hexane/EtOAc, 5:1 (v/v), as eluent]
5.9.4. ( )-5,6-Dihydrobenzo[4,5]imidazo[2,1-a]isoquino-
line-6-carboxylic acid dihexylamide (34a). To a solution
of 33 (95 mg, 0.20 mmol) in MeOH (3.0 mL), PtO2
(10 mg) was added. Under an H2 atmosphere, the mix-
ture was stirred at room temperature for 2 h. The cata-
lyst was removed by filtration using a Celite filter, and
the filtrate was concentrated under reduced pressure.
The residue was passed through a silica gel short column
to obtain crude 2-(2-aminophenylamino)-3-(2-cyan-
ophenyl)-N,N-dihexylpropionamide (84 mg) as a dark
red oil. The crude 2-(2-aminophenylamino)-3-(2-cyan-
ophenyl)-N,N-dihexylpropionamide was dissolved in
EtOH (5 mL). HCl gas was passed through the solution
until it was saturated with HCl. The mixture was then
stirred at room temperature for 4 h. The reaction mix-
ture was poured into satd NaHCO3 solution and ex-
tracted with EtOAc. The organic phase was washed with
brine, dried (Na2SO4), and evaporated under reduced
pressure. The residue was purified by silica gel chro-
matography [hexane/EtOAc, 2:1 (v/v), as eluent] and
crystallized from EtOAc/hexane to obtain 34a (24 mg,
1
to obtain 31 (8.50 g, quant.) as a pale yellow oil. H
NMR (CDCl3) d 1.29 (6H, t, J ¼ 7.1 Hz), 1.47 (9H, s),
3.84 (2H, s), 4.02–4.46 (4H, m), 5.73 (1H, br s),
7.17–7.70 (4H, m); MS (ESI, Neg) m=z 389
(Mþ)1, 100%).
5.9.2. ( )-2-tert-Butoxycarbonylamino-3-(2-cyanophen-
yl)-N,N-dihexylpropionamide (32). To a solution of 31
(1.17 g, 3.00 mmol) in a mixture of EtOH (20 mL) and
H2O (0.5 mL), NaOH (360 mg, 9.00 mmol) was added,
and the solution was heated at reflux for 2 h. The solvent
was removed by evaporation under reduced pressure.
The residue was dissolved in EtOAc and the solution
was washed with 5% KHSO4 solution, satd NaHCO3
solution and brine, dried (MgSO4), and evaporated
under reduced pressure to provide crude 2-tert-butoxy-
carbonylamino-3-(2-cyanophenyl)propionic acid (0.81
g). To a mixture of crude 2-tert-butoxycarbonylamino-
3-(2-cyanophenyl)propionic acid and dihexylamine
(0.67 g, 3.6 mmol) in DMF (8.0 mL) were added HOBt
(0.55 g, 3.6 mmol) and EDCÆHCl (0.69 g, 3.6 mmol).
After stirring at room temperature overnight, the reac-
tion mixture was poured into EtOAc, washed with 5%
KHSO4 solution, satd NaHCO3 solution and brine,
dried (MgSO4), and evaporated under reduced pressure.
The residue was purified by silica gel chromatography
[hexane/EtOAc, 4:1 (v/v), as eluent] to obtain 32 (1.01 g,
74%). 1H NMR (CDCl3) d 0.72–1.68 (22H, m), 1.29
(9H, s), 2.79–3.70 (6H, m), 4.76–5.00 (1H, m), 5.27–5.48
(1H, m), 7.17–7.70 (4H, m); MS (FAB, Pos) m=z 458
(Mþ+1), 402 (Mþ)55, 100%).
1
24%) as a white crystal. Mp 134.5–136.5 ꢁC; H NMR
(CDCl3) d 0.72–1.90 (22H, m), 3.02–3.72 (6H, m), 5.40–
5.54 (1H, m), 7.07–7.50 (6H, m), 7.75–7.91 (1H, m),
8.27–8.42 (1H, m); MS (FAB, Pos) m=z 432 (Mþ+1,
100%); Anal. (C28H37N3O) C, H, N.
5.9.5. ( )-5,6-Dihydrobenzo[4,5]imidazo[2,1-a]isoquino-
line-6-carboxylic acid diethylamide (34b). Starting from
( )-3-(2-cyanophenyl)-N,N-diethyl-2-(2-nitrophenylamino)-
propionamide, 34b was obtained as a crystal. Yield 15%;
1
mp 206.5–207.5 ꢁC (EtOAc/hexane); H NMR (CDCl3)
d 1.12 (3H, t, J ¼ 7.0 Hz), 1.41 (3H, t, J ¼ 7.0 Hz), 3.17–
3.78 (6H, m), 5.47 (1H, dd, J ¼ 6.8, 4.8 Hz), 7.10–7.51
(6H, m), 7.75–7.90 (1H, m), 8.30–8.42 (1H, m); MS
(CI, Pos) m=z 320 (Mþ+1, 100%); Anal. (C20H21N3O) C,
H, N.
5.9.3. ( )-3-(2-Cyanophenyl)-N,N-dihexyl-2-(2-nitrophen-
ylamino)propionamide (33). To a solution of 32 (0.98 g,
2.14 mmol) in CH2Cl2 (1.65 mL), trifluoroacetic acid
(1.65 mL, 21.4 mmol) was added. The mixture was stir-
red at room temperature for 1.5 h. The solvent and tri-
fluoroacetic acid were removed by evaporation under
reduced pressure. The residue was dissolved in CHCl3
and the resulting solution was washed with satd
NaHCO3 solution and brine, dried (Na2SO4), and
evaporated under reduced pressure to obtain crude
2-amino-3-(2-cyanophenyl)-N,N-dihexylpropionamide
(0.88 g). The crude 2-amino-3-(2-cyanophenyl)-N,N-di-
hexylpropionamide was dissolved in DMF (8 mL). 2-
Fluoronitrobenzene (302 mg, 2.14 mmol) and K2CO3
(355 mg) were added to the solution. The mixture was
heated at 150 ꢁC for 2 h. The reaction mixture was
5.9.6. ( )-5,6-Dihydrobenzo[4,5]imidazo[2,1-a]isoquino-
line-6-carboxylic acid dipropylamide (34c). Starting
from
( )-3-(2-cyanophenyl)-N,N-dipropyl-2-(2-nitro-
phenylamino)propionamide, 34c was obtained as a
crystal. Yield 14%; mp 138.5–140.5 ꢁC (EtOAc/hexane);
1H NMR (CDCl3) d 0.82 (3H, t, J ¼ 7.4 Hz), 1.08
(3H, t, J ¼ 7.4 Hz), 1.35–1.97 (4H, m), 2.99–3.74
(6H, m), 5.48 (1H, dd, J ¼ 6.8, 4.4 Hz), 7.08–7.50
(6H, m), 7.75–7.91 (1H, m), 8.28–8.42 (1H, m); MS
(CI, Pos) m=z 348 (Mþ+1, 100%); Anal. (C22H25N3O)
C, H, N.

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T. Okubo et al. / Bioorg. Med. Chem. 12 (2004) 3569–3580
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Acknowledgements
A part of this synthetic research was carried out by M.
Nagamine, M. Gotoh, K. Kondoh and Dr. M. Yoshida
(Research Division of Nihon Nohyaku Co., Ltd). We
wish to express our special thanks to them.
23. Okubo, T.; Yoshikawa, R.; Chaki, S.; Okuyama, S.;
Nakazato, A. Bioorg. Med. Chem. 2004, 12, 423.
24. Chaki, S.; Funakoshi, T.; Yoshikawa, R.; Okuyama, S.;
Okubo, T.; Nakazato, A.; Nagamine, M.; Tomisawa, K.
Eur. J. Pharmacol. 1999, 371, 197.
References and notes
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25. Hathaway, B. A.; Nichols, D. E.; Nichols, M. B.; Yim, G.
K. J. Med. Chem. 1982, 25, 535.