Design, synthesis, and biological evaluation of novel (1-thioxo-1,2,3,4- tetrahydro-β-carbolin-9-yl)acetic acids as selective inhibitors for AKR1B1

Article abstract of DOI:10.1016/j.bmc.2011.10.073

New substituted (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids were designed as the inhibitor of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa, and twenty derivatives were synthesized and evaluated. The most active compound of the series was (2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl) acetic acid (7m), which showed comparable inhibitory activity for AKR1B1 (IC₅₀ = 0.15 μM) with clinically used epalrestat (IC₅₀ = 0.1 μM). In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC₅₀ = 0.17 μM) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared with epalrestat.

Full text of DOI:10.1016/j.bmc.2011.10.073

Bioorganic & Medicinal Chemistry 20 (2012) 356–367
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel (1-thioxo-1,2,3,4-tetrahy-
dro-b-carbolin-9-yl)acetic acids as selective inhibitors for AKR1B1
Daisuke Minehira a, Daisuke Takeda a, Hirokazu Urata a, Atsushi Kato a, , Isao Adachi a, Xu Wang b,
⇑
Yuji Matsuya b, Kenji Sugimoto b, Mayuko Takemura c, Satoshi Endo c, Toshiyuki Matsunaga c,
Akira Hara c, Jun Koseki d, Kayo Narukawa d, Shuichi Hirono d, Naoki Toyooka b,e,f,
⇑
a Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan
b Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
c Gifu Pharmaceutical University, Gifu 502-8585, Japan
d School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan
e Graduate School of Science and Technology for Research, University of Toyama, Toyama 930-8555, Japan
f Graduate School of Innovative Life Science, University of Toyama, Toyama 930-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
New substituted (1-thioxo-1,2,3,4-tetrahydro-b-carbolin-9-yl)acetic acids were designed as the inhibitor
of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa,
and twenty derivatives were synthesized and evaluated. The most active compound of the series was
(2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-b-carbolin-9-yl)acetic acid (7m), which showed
Received 5 September 2011
Revised 23 October 2011
Accepted 25 October 2011
Available online 3 November 2011
comparable inhibitory activity for AKR1B1 (IC50 = 0.15
lM) with clinically used epalrestat (IC50 = 0.1 lM).
In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-
Keywords:
AKR1B1
Diabetes mellitus
Aldose reductase
Molecular docking
Selectivity index
1,2,3,4-tetrahydro-b-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC50
0.17
with epalrestat.
=
l
M) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
metabolized through the polyol pathway, and the product, sorbitol,
cannot cross the cell membrane easily and therefore causes swell-
ing and cell dysfunction in a number of tissues. In addition, fructose
can become phosphorylated to fructose-3-phosphate, which is
broken down to 3-deoxyglucosone, ultimately forming advanced
glycation end products that are capable of cellular damage.2–4
These abnormal metabolic results have been reported to be factors
responsible for diabetic complications such as cataracts,5 retinopa-
thy,6 neuropathy,7 and nephropathy.8
Mammalian aldose reductases belong to the aldo–keto reduc-
tase (AKR) 1B subfamily of the AKR superfamily, in which the
human enzyme is named AKR1B1.9 The inhibition of AKR1B1 is
an important potential therapeutic mechanism in treating diabetic
complications. At present, the inhibitors are divided into two
different chemical classes. One is the spiro-hydantoin or -imido
derivatives, such as sorbinil, fidarestat and ranirestat, and the other
is the carboxylic acid derivatives, such as epalrestat and tolrestat.
Although many laboratories have synthesized various structures
of AKR1B1 inhibitors which appear to be promising during
in vitro studies and in trials with animal models, they often failed
to proceed any further because of their unexpected side effects
and lack of selectivity towards the target enzyme AKR1B1. This
lack of specificity is a major concern and has been attributed to
the high degree of structural and sequence similarity of AKR1B1
Diabetes mellitus is one of the most common chronic metabolic
diseases, which occurs when the pancreas does not produce enough
insulin, or when the body cannot effectively use the insulin it pro-
duces. Today, the worldwide prevalence of diabetes is taking
pandemic dimensions, as changing lifestyles lead to reduced phys-
ical activity, and increased obesity. In 2010, 285 million people
were suffering from diabetes and this number is estimated to
increase to 439 million by 2030.1 Prolonged hyperglycemia is a pri-
mary causal factor of several diabetic complications. Many studies
have revealed a correlation between glucose metabolism via the
polyol pathway and long-term complications. Aldose reductase
(EC 1.1.1.21) is the first and rate-limiting enzyme in this pathway
responsible for converting glucose into sorbitol, which is
further metabolized into fructose by sorbitol dehydrogenase (EC
1.1.1.14). The enzyme has a low affinity for glucose and the polyol
pathway plays a minor role in glucose metabolism because it
competes with the glucose hexokinase of the glycolytic pathway.
Under hyperglycaemic conditions, however, glucose is rapidly
⇑
Corresponding authors.
E-mail addresses: kato@med.u-toyama.ac.jp (A. Kato), toyooka@eng.u-toyama.
ac.jp (N. Toyooka).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.073

D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
357
nitrogen on the indole nuclei gave rise to the methyl ester (3),
which was converted to desired acid (4) (Scheme 1).
The inhibitory activity of 4 was not improved compared with
benzoyl derivatives23 (see Table 1). Next, we designed the thiolac-
tam-type of tricyclic carboxylic acids (7b–7s). Reaction of lactams
(2b–r), derived from lactones (1b–1k) same as in Scheme 1, with
Lawesson’s reagent proceeded smoothly to provide the corre-
sponding thiolactams (5b–5r) in high yield, which were trans-
formed into the esters (6b–6r). Finally, deprotection of t-butyl or
methyl esters with TMSI or LiOH furnished desired acids (7b–7r).
The OH (7s) and COOH (7t) derivatives were also synthesized from
(6m) or (6l) by treatment with BBr3 or Pd-catalyzed CO insertion
reaction (6t) followed by hydrolysis, respectively, as shown in
Scheme 2.
Figure 1. Structures of rhetsinine and (1-thioxo-1,2,3,4-tetrahydro-b-carbolin-9-
yl)acetic acids.
with human aldehyde reductase (AKR1A1, EC 1.1.1.2).9 AKR1A1 is
identical to D-glucuronate reductase in the glucuronic acid/uronate
cycle of glucose metabolism,10 and also plays an important role in
the detoxification of 3-deoxyglucosone, a precursor of advanced
glycation end products.11 In addition to AKR1A1, AKR1B10 with
amino acid sequence identity of 71% with AKR1B112 is inhibited
by AKR1B1 inhibitors.13 AKR1B10 exhibits board substrate speci-
ficity for the AKR1B1 substrates other than aldoses that are specif-
ically reduced by AKR1B1.12,14 AKR1B10 has specialized roles in the
controlling levels of signaling molecules, all-trans-, 9-cis and 13-
cis-retinoic acids,13 and regulating isoprenoid metabolism14 and
fatty acid synthesis.15 Therefore, it is necessary to elucidate the
structural requirements for the design of selective AKR1B1 inhibi-
tors without affecting to AKR1A1 and AKR1B10.
2.2. Biological evaluation
Many of newly synthesized compounds showed potent inhibi-
tion for AKR1B1 compared with the lead compound (4) (Table 1).
The inhibitory potency was not affected in the introduction of
halogen(s), alkyl, alkoxy, OH, or COOH substituent on 6- or 5,7-
positions on the parent nuclei except for 5,7-difluoro-derivative
(7o). Introduction of halogen (7e, 7g, and 7j) or trifluoromethyl
group (7d and 7f) on the benzene ring of benzyl moiety at the
2-position much decreased the inhibitory effect on AKR1B1, and
these compounds were more selective on AKR1B10 rather than
AKR1B1. Substitution on the nitrogen at the 2-position with phenyl
(7q) or phenylethyl (7r) group instead of simple benzyl substituent
did not improve the inhibitory potency for AKR1B1. Accordingly,
the best structure on this position would be simple benzyl group.
The IC50 values for several derivatives (7l–7p, 7s and 7t) were com-
parable to that of the known inhibitor, epalrestat that was deter-
mined under the same conditions. In addition, these synthesized
compounds showed higher selectivity to AKR1B1 over both
AKR1A1 and AKR1B10 than epalrestat. In particular, the COOH
derivative (7t) showed the highest selectivity indexes of 312 and
253 in AKR1A1/AKR1B1 and AKR1B10/AKR1B1, respectively.
Many reports on the selectivity of inhibitors to AKR1B1 over
AKR1A1 have been published,16–19 however, no report has been
made on the inhibitory selectivity to AKR1B1 over AKR1B10. In re-
cent years, many kinds of AKR1B1 inhibitors have been found from
natural sources, such as flavones, flavonoids, coumarins and so
on.20 In our search for AKR1B1 inhibitors, we found that a hot
water extract of Evodia rutaecarpa exhibited significant inhibitory
activity and reported that rhetsinine, which is a minor alkaloidal
component of E. rutaecarpa, significantly suppressed sorbitol accu-
mulation in human erythrocytes by 79.3% at 100 l
M.21 Here, we
report the design, synthesis, and evaluation of novel (1-thioxo-
1,2,3,4-tetrahydro-b-carbolin-9-yl)acetic acids, based upon the
structure of rhetsinine, as selective inhibitors of AKR1B1 (Fig. 1).
2.3. Kinetic analysis
The inhibition patterns of the potent derivatives (7m and 7t)
were non-competitive with respect to the substrate pyridine-
3-aldehyde, showing the two inhibition constants (Kis = 79 1 nM
and Kii = 180 20 nM for 7m, and Kis = 55 7 nM and Kii = 220
57 nM for 7t). The inhibition patterns are similar to those of known
AKR1B1 inhibitors, which are demonstrated to bind to the active
site of the enzyme–coenzyme binary complex by crystallographic
2. Results and discussion
2.1. Chemistry
The synthesis began with tricyclic lactam (2a), which was syn-
thesized according to the known procedure by treatment with cor-
responding lactone (1a)22 with 4-fluorobenzyl amine. Alkylation of
Scheme 1.

358
D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
Table 1
Inhibitory potency and selectivity of tricyclic carboxylic acids for AKR1A1, AKR1Bl and AKR1B10
Compound
IC50
(l
M)
Selectivity index
AKR1A1
AKR1B1
AKR1B10
(AKR1A1/AKR1B1)
(AKR1B10/AKR1B1)
4
ND
48.6
ND
—
—
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
71
7m
7n
7p
7o
7q
7r
9.5 0.2
0.22 0.03
0.23 0.02
10 1.6
1.7 0.3
14 0.1
5.1 0.8
3.3 0.4
1.6 0.2
0.28 0.02
3.8 0.1
0.85 0.1
1.4 0.3
1.4 0.1
0.68 0.07
1.4 0.2
1.4 0.1
2.9 0.1
3.4 0.1
6.1 0.5
0.48 0.04
21 0.3
43
83
—
—
—
2.4
34
88
—
43
58
80
80
53
3.6
—
23
14
0.2
0.2
0.3
0.1
4.4
5.8
0.2
4
7.4
19
17
32
0.4
0.6
0.2
10
19
1
(27%)
(45%)
(45%)
14
11
21
2
1
1
5.9 0.9
0.32 0.03
0.24 0.01
4.2 0.2
0.35 0.02
0.19 0.01
0.15 0.01
0.20 0.01
0.19 0.02
1.3 0.1
(45%)
15
11
12
16
10
1
1
1
1
1
4.7 0.4
(2%)
(39%)
(28%)
31
2
1.8 0.2
0.40 0.04
1.9 0.2
—
—
312
26
7s
7t
0.20 0.01
0.17 0.02
0.10 0.01
53
6
40
1
253
3.3
Epalrestat
2.6 0.2
0.33 0.04
ND: Not determined. The values in parentheses are inhibition percentages by 20 lM.
studies.24 The inhibition constants for 7m and 7t are comparable or
exhibited strong inhibitory potency (IC50 = 0.17 lM) with the high-
superior to those for known inhibitors including epalrestat.25,26
est selectivity for AKR1B1 (selectivity ratios to AKR1A1 and
AKR1B10 were 311 and 253, respectively). According to the struc-
ture–activity relationship (SAR), non-substituted simple benzyl
moiety on the 2-nitrogen was essential for high inhibitory activity
(7b vs 7q), and addition of substituent(s) such as halogen or trifluo-
romethyl on the phenyl ring of this moiety was not effected for the
improvement of inhibitory activity (7b vs 7f, 7g or 7j, 7c vs 7d or 7e).
2.4. Molecular modeling
The docking simulations of 7t were performed for AKR1B1 and
AKR1B10. Figure 2A and B shows the binding conformation of 7t
for AKR1B1 and for AKR1B10, respectively. In this figure, we can
find that the binding orientation and conformation of 7t for
AKR1B1 differ from those for AKR1B10. In AKR1B1, the benzyl
group of 7t is located in the inner part of the ligand-binding pocket
4. Experimental section
4.1. Chemistry
where is hydrophobic. The p–p stacking and CH–p interactions are
formed between tricyclic group of 7t and W219. In addition, the
strong hydrogen bonding is formed between carboxyl group con-
nected the nitrogen on the tricyclic group and S302.
On the other hand, 7t binds to AKR1B10 with different orienta-
tion and conformation from in AKR1B1. In AKR1B10, the benzyl
group of 7t interacts with F123 in T-shape and with V301 through
Typical procedure for lactones (1a–1j): The known lactones (1a,
1b, 1c, 1d, 1e, 1f, and 1h) and other lactones (1g, 1i, 1j, and 1k)
were prepared by the literature procedure.27
4.1.1. 6-Iodo-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (1g)
Yield: 52%; mp: 228–230 °C; IR (KBr): 3283, 1705 cmÀ1 1H
;
CH-p interaction. The poor hydrogen bonding is formed between
carboxyl group (6-position) and Y49. As a whole, the interaction
of 7t with AKR1B10 (Glide Score: À7.76 kcal/mol) is weaker than
that with AKR1B1 (Glide Score: À8.60 kcal/mol). The difference be-
tween the binding conformations of 7t in AKR1B1 and in AKR1B10
seems to come from the difference between the binding pocket
shape of AKR1B1 and that of AKR1B10. Figure 2C shows the bind-
ing pocket of AKR1B1 (green solid surface) and that of AKR1B10
(red wireframe). In Figure 2 we can find that the inner part of
the binding pocket of AKR1B10 is narrower than that of AKR1B1.
Therefore, the benzyl group of 7t cannot be located in the inner
part of the binding pocket of AKR1B10, and so it fails to form favor-
able hydrophobic and hydrogen bonding interactions with residues
of AKR1B10.
NMR (300 MHz, CDCl3): d 9.13 (1H, br), 8.00 (1H, s), 7.63 (1H, dd,
J = 1.5, 8.4 Hz), 7.27 (1H, d, J = 8.4 Hz), 4.71 (2H, t, J = 6.3 Hz), 3.13
(2H, t, J = 6.3 Hz); 13C NMR (75 MHz, CDCl3): d 160.93, 137.01,
134.99, 129.84, 128.99, 126.95, 121.90, 114.66, 84.16, 69.44,
21.27; MS (EI) m/z 313 (M+); HRMS (EI) calcd for C11H8O2NI:
312.9600 (M+), found: 312.9620.
4.1.2. 6-Iso-propyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one
(1i)
Yield: 62%; brown oil; IR (neat): 3276, 1689 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 9.64 (1H, br), 7.45 (2H, d, J = 8.8 Hz), 7.31–
7.28 (1H, m), 4.71 (2H, t, J = 6.3 Hz), 3.17 (2H, t, J = 6.3 Hz), 3.03
(1H, sept, J = 6.9 Hz), 1.32 (6H, d, J = 6.9 Hz); 13C NMR (75 MHz,
CDCl3): d 161.72, 141.28, 137.08, 126.33, 124.21, 122.79, 122.00,
116.87, 112.79, 69.48, 34.08, 24.37, 21.46; MS (EI) m/z 229 (M+);
HRMS (EI) calcd for C14H15O2N: 229.1103 (M+), found: 229.1096.
3. Conclusion
In conclusion, a series of novel (1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acids (7b–7t) were designed, synthesized,
and inhibitory activity for AKR1B1 was evaluated in vitro. Among
the carboxylic acids, the MeO derivative (7m) showed the highest
4.1.3. 5,7-Difluoro-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one
(1j)
Yield: 40%; mp: 186–187 °C; IR (KBr): 3275, 1697 cmÀ1 1H
;
inhibitory activity for AKR1B1 (IC50 = 0.15
l
M) comparable to that
NMR (500 MHz, DMSO-d6): d 7.03 (1H, dd, J = 2.1, 9.4 Hz), 7.00–
of epalrestat (IC50 = 0.10 M), and the COOH derivative (7t)
l
6.96 (1H, m), 4.63 (2H, t, J = 6.2 Hz), 3.19 (2H, t, J = 6.2 Hz); 13C

D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
359
Scheme 2.
NMR (125 MHz, CDCl3): d 162.11 (dd, J = 12.5, 246.3 Hz), 160.60,
157.70 (dd, J = 15.3, 253.0 Hz), 139.25 (dd, J = 12.5, 15.3 Hz),
122.77 (d, J = 3.8 Hz), 121.47 (d, J = 1.9 Hz), 111.36 (d,
J = 21.1 Hz), 97.03 (dd, J = 24.0, 29.7 Hz), 94.98 (dd, J = 4.8,
26.8 Hz), 69.48, 22.28; MS (EI) m/z 223 (M+); HRMS (EI) calcd for
C
11H7O2NF2: 223.0445 (M+), found: 223.0417.

360
D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
Y49
W21
F123
W219
V301
S302
Figure 2. The binding conformation of the 7t (blue stick) for AKR1B1 (A) and for AKR1B10 (B), and the difference of binding pocket (C) between AKR1B1 (green solid surface)
and AKR1B10 (red wireframe).
4.1.4. 5,7-Dichloro-4,9-dihydro-3H-pyrano[3,4-b]indol-1-one
3.71 (2H, t, J = 6.9 Hz), 3.01 (2H, t, J = 6.9 Hz); 13C NMR
(100 MHz, CDCl3): d 161.50, 160.86 (d, J = 251.2 Hz), 157.95 (d,
J = 236.8 Hz), 134.14, 131.53 (d, J = 4.8 Hz), 128.12, 127.83 (d,
J = 3.8 Hz), 125.28 (d, J = 9.6 Hz), 123.58 (d, J = 14.4 Hz), 121.61
(d, J = 9.6 Hz), 119.14 (d, J = 24.9 Hz), 118.27 (d, J = 4.8 Hz),
113.93 (d, J = 26.8 Hz), 113.40 (d, J = 9.6 Hz), 104.64 (d,
J = 24.0 Hz), 47.97, 43.08 (d, J = 2.9 Hz), 20.64; MS (EI) m/z 390
(M+); HRMS (EI) calcd for C18H13ON2F2Br: 390.0179 (M+), found:
390.0175.
(1k)
Yield: 42%; mp: 137–139 °C; IR (KBr): 3267, 1712 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.17 (1H, br), 7.38 (1H, d, J = 1.6 Hz),
7.17 (1H, d, J = 1.6 Hz), 4.71 (2H, t, J = 6.3 Hz), 3.44 (2H, t,
J = 6.3 Hz); 13C NMR (75 MHz, CDCl3): d 168.47, 139.60, 135.20,
128.49, 124.21, 122.08, 117.91, 111.32, 69.51, 24.60, 22.64; MS
(EI) m/z 255 (M+); HRMS (EI) calcd for C11H7O2NCl2: 254.9854
(M+), found: 254.9856.
4.1.5. Typical procedure for lactams (2a–2s)
The known lactams (2b, 2c, 2h, 2i, 2k, 2m, 2q, 2s) and other lac-
tams (2a, 2d, 2e, 2f, 2g, 2j, 2l, 2n, 2o, 2p, 2r) were prepared by the
literature procedure.24
4.1.9. 2-(4-Trifluoromethylbenzyl)-2,3,4,9-tetrahydro-b-
carbolin-1-one (2f)
Yield: 37%; mp: 224–227 °C; IR (KBr): 3230, 1636 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.15 (1H, br), 7.62–7.57 (3H, m), 7.48
(2H, d, J = 7.7 Hz), 7.44 (1H, d, J = 8.2 Hz), 7.31 (1H, t, J = 7.1 Hz),
7.16 (1H, t, J = 7.1 Hz), 4.85 (2H, s), 3.66 (2H, t, J = 7.1 Hz), 3.06
(2H, t, J = 7.1 Hz); 13C NMR (100 MHz, CDCl3): d 161.61, 141.72,
137.50, 129.83 (q, J = 32.6 Hz), 128.10, 126.55, 125.67 (q,
J = 3.8 Hz), 125.24, 125.10, 122.74, 120.34, 120.20, 118.50,
112.45, 49.29, 47.76, 20.71; MS (EI) m/z 344 (M+); HRMS (EI) calcd
for C19H15ON2F3: 344.1137 (M+), found: 344.1156.
4.1.6. 6-Fluoro-2-(4-fluorobenzyl)-2,3,4,9-tetrahydro-b-
carbolin-1-one (2a)
Yield: 34%; mp: 211–213 °C; IR (KBr) 3216, 1635 cmÀ1; 1H NMR
(300 MHz, DMSO-d6): d 11.8 (1H, br s), 7.40–7.36 (4H, m), 7.18 (2H,
t-like, J = 9.0 Hz), 7.08 (1H, td, J = 2.6, 9.2 Hz), 4.68 (2H, s), 3.60 (2H,
t, J = 7.0 Hz), 2.95 (2H, t, J = 7.0 Hz); 13C NMR (75 MHz, DMSO-d6): d
160.1, 133.9, 129.5 & 129.4, 128.4, 124.7, 117.4 & 117.3, 115.3,
115.0, 113.7 & 113.5, 112.8 & 112.4, 104.6 & 104.3, 48.0, 47.2,
20.0; MS 312 (M+), 312 (100); HRMS calcd for C18H14ON2F2:
312.1074, found: 312.1046.
4.1.10. 2-(4-Bromobenzyl)-2,3,4,9-tetrahydro-b-carbolin-1-one
(2g)
Yield: 40%; mp: 249–252 °C; IR (KBr): 3218, 1635 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 8.96 (1H, br), 7.57 (1H, d, J = 7.7 Hz),
7.48–7.42 (3H, m), 7.34–7.22 (3H, m), 7.15 (1H, t, J = 7.7 Hz), 4.73
(2H, s), 3.63 (2H, t, J = 7.0 Hz), 3.03 (2H, t, J = 7.0 Hz); 13C NMR
4.1.7. 6-Fluoro-2-(4-trifluoromethylbenzyl)-2,3,4,9-tetrahydro-
b-carbolin-1-one (2d)
Yield: 35%; mp: 237–240 °C; IR (KBr): 3223, 1635 cmÀ1
;
1H
(75 MHz, CDCl3): d 161.25, 137.22, 136.49, 131.70, 129.57,
NMR (400 MHz, CDCl3): d 9.74 (1H, br), 7.61 (2H, d, J = 8.0 Hz),
7.48 (2H, d, J = 8.0 Hz), 7.35–7.32 (1H, m), 7.19 (1H, dd, J = 1.4,
9.2 Hz), 7.04 (1H, td, J = 2.3, 9.2 Hz), 4.86 (2H, s), 3.67 (2H, t,
J = 6.9 Hz), 3.02 (2H, t, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3): d
161.37, 157.97 (d, J = 236.7 Hz), 141.52, 134.07, 129.92 (q,
J = 32.6 Hz), 128.09, 127.77, 125.71 (q, J = 3.8 Hz), 125.39, 125.28,
118.25 (d, J = 4.8 Hz), 113.96 (d, J = 26.8 Hz), 113.35 (d,
J = 9.6 Hz), 104.67 (d, J = 24.0 Hz), 49.36, 47.72, 20.61; MS (EI) m/z
362 (M+); HRMS (EI) calcd for C19H14ON2F4: 362.1042 (M+), found:
362.1027.
126.59, 125.22, 124.99, 121.30, 120.28, 120.12, 118.32, 112.29,
49.04, 47.54, 20.78; MS (EI) m/z 354 (M+); HRMS (EI) calcd for
C18H15ON2Br: 354.0368 (M+), found: 354.0323.
4.1.11. 2-(4-Bromo-2-fluorobenzyl)-2,3,4,9-tetrahydro-
b-carbolin-1-one (2j)
Yield: 33%; mp: 217–219 °C; IR (KBr): 3223, 1635 cmÀ1 1H
;
NMR (500 MHz, DMSO-d6): d 11.66 (1H, s), 7.60–7.56 (2H, m),
7.43–7.32 (3H, m), 7.22 (1H, t, J = 8.1 Hz), 7.06 (1H, t, J = 8.1 Hz),
4.71 (2H, s), 3.65 (2H, t, J = 7.0 Hz), 3.00 (2H, t, J = 7.0 Hz); 13C
NMR (75 MHz, DMSO-d6):
d 160.09 (d, J = 249.0 Hz), 160.46,
4.1.8. 2-(4-Bromo-2-fluorobenzyl)-6-fluoro-2,3,4,9-tetrahydro-
137.21, 131.21 (d, J = 4.9 Hz), 127.57 (d, J = 3.7 Hz), 126.53,
124.56, 124.33, 124.10 (d, J = 4.9 Hz), 120.30 (d, J = 9.8 Hz),
120.01, 119.39, 118.59 (d, J = 25.6 Hz), 117.66, 112.44, 47.64,
42.61, 20.15; MS (EI) m/z 371 (M+); HRMS (EI) calcd for
b-carbolin-1-one (2e)
Yield: 33%; mp: 235–238 °C; IR (KBr): 3217, 1637 cmÀ1 1H
;
NMR (400 MHz, CDCl3): d 9.53 (1H, br), 7.36–7.26 (4H, m), 7.19
(1H, dd, J = 2.3, 9.2 Hz), 7.05 (1H, td, J = 2.3, 9.2 Hz), 4.79 (2H, s),
C
18H14ON2FBr: 372.0274 (M+), found: 372.0299.

D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
361
4.1.12. 2-Benzyl-6-iodo-2,3,4,9-tetrahydro-b-carbolin-1-one (2l)
Yield: 45%; mp: 227–229 °C; IR (KBr): 3202, 1631 cmÀ1 1H
extracted with Et2O (10 mL Â 3). The organic extracts were com-
bined, dried over MgSO4, and evaporated. The residue was chro-
matographed on SiO2 (Hexane–Acetone = 8: 1) to give 3 (173 mg,
;
NMR (400 MHz, CDCl3): d 10.57 (1H, br), 7.78 (1H, s), 7.35 (1H,
dd, J = 1.4 Hz, 8.7 Hz), 7.27–7.15 (5H, m), 7.08 (1H, d. J = 8.7 Hz),
4.73 (2H, s), 3.55 (2H, t, J = 7.1 Hz), 2.87 (2H, t, J = 7.1 Hz); 13C
NMR (100 MHz, CDCl3): d 161.43, 137.30, 136.73, 132.91, 128.97,
128.77, 127.88, 127.84, 127.60, 127.44, 117.07, 114.70, 83.29,
49.74, 47.47, 20.51; MS (EI) m/z 402 (M+); HRMS (EI) calcd for
73%). Mp: 87–89 °C; IR (KBr) 1737, 1650 cmÀ1
;
1H NMR
(300 MHz, CDCl3): d 7.31–7.28 (2H, m), 7.22–7.19 (2H, m), 7.10
(1H, td, J = 2.6, 9.0 Hz), 7.02 (2H, t, J = 8.5 Hz), 5.44 (2H, s), 4.70
(2H, s), 3.77 (3H, s), 3.61 (2H, t, J = 7.0 Hz), 2.97 (2H, t, J = 7.0 Hz);
13C NMR (75 MHz, CDCl3): d 169.3, 160.9, 135.3, 133.0 & 132.9,
129.4 & 129.2, 126.9, 124.3 & 124.2, 118.9 & 118.8, 115.4 &
115.1, 113.8 & 113.5, 110.6 & 110.4, 105.0 & 104.7, 52.3, 48.6,
46.9, 45.7, 20.4; MS (EI) m/z 384 (M+); HRMS (EI) calcd for
C
18H15ON2I: 402.0230 (M+), found: 402.0228.
4.1.13. 2-Benzyl-6-iso-propyl-2,3,4,9-tetrahydro-b-carbolin-1-
one (2n)
C
21H18O3N2F2: 384.1286 (M+), found: 384.1260.
Yield: 40%; mp: 205–207 °C; IR (KBr): 3214, 1635 cmÀ1 1H
;
NMR (400 MHz, CDCl3): d 9.42 (1H, br), 7.38–7.26 (7H, m), 7.19
(1H, dd, J = 1.6, 8.5 Hz), 4.82 (2H, s), 3.64 (2H, t, J = 6.9 Hz), 3.03–
2.97 (3H, m), 1.30 (6H, d, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3):
d 161.64, 140.91, 137.64, 136.14, 128.66, 127.92, 127.42, 127.01,
125.31, 124.49, 118.00, 116.59, 112.28, 49.48, 47.44, 34.17, 24.49,
20.71; IR (KBr): 3214, 1635 cmÀ1; MS (EI) m/z 318 (M+); HRMS
(EI) calcd for C21H22ON2: 318.1732 (M+), found: 318.1780.
4.1.18. [2-(4-Fluorobenzyl)-6-fluoro-1-oxo-1,2,3,4-tetrahydro-b-
carbolin-9-yl]acetic acid (4)
To a stirred solution of 3 (173 mg, 0.45 mmol) in MeOH (3 mL)
and H2O (1 mL) was added LiOHÁH2O (37.2 mg, 0.90 mmol), and
the resulting mixture was refluxed for 1 h. After cooling, the reac-
tion was quenched with 10% HCl aq, and the aqueous mixture was
extracted with AcOEt (10 mL Â 3). The organic extracts were com-
bined, dried over MgSO4, and evaporated. The residue was chro-
matographed on SiO2 (Hexane–Acetone = 2: 1) to 4 (0.44 mmol,
4.1.14. 2-Benzyl-5,7-difluoro-2,3,4,9-tetrahydro-b-carbolin-1-
one (2o)
162.9 mg, 98%). Mp: 148–150 °C; IR (KBr) 2929, 1724, 1647 cm
À1
Yield: 40%; mp: 167–169 °C; IR (KBr): 3193, 1626 cmÀ1
;
1H
;
1H NMR (300 MHz, DMSO-d6): d 7.61–7.58 (1H, m), 7.44–7.42
NMR (400 MHz, CDCl3): d 10.62 (1H, br), 7.37–7.28 (5H, m), 6.89
(1H, dd, J = 1.8, 8.7 Hz), 6.58 (1H, dt, J = 1.8, 10.5 Hz), 4.83 (2H, s),
3.67 (2H, t, J = 7.1 Hz), 3.15 (2H, t, J = 7.1 Hz); 13C NMR (100 MHz,
CDCl3): d161.17 (dd, J = 11.5, 242.5 Hz), 159.96, 157.49 (dd,
J = 15.3, 251.1 Hz), 139.19 (dd, J = 13.4, 15.3 Hz), 137.51, 137.07,
129.12 (d, J = 3.8 Hz), 128.16, 127.54 (d, J = 3.8 Hz), 116.86,
111.94 (d, J = 21.1 Hz), 96.32 (dd, J = 22.5, 29.2 Hz), 95.13 (dd,
J = 4.8, 25.9 Hz), 49.98, 47.78, 21.85; MS (EI) m/z 312 (M+); HRMS
(EI) calcd for C18H14ON2F2: 312.1074 (M+), found: 312.1069.
(1H, dd, J = 2.6, 9.4 Hz), 7.37–7.34 (2H, m), 7.19–7.14 (3H, m),
5.35 (2H, s), 4.65 (2H, s), 3.59 (2H, t, J = 7.0 Hz), 2.97 (2H, t,
J = 7.0 Hz); 13C NMR (75 MHz, DMSO-d6): d 170.2, 160.2, 158.4,
155.7, 135.3, 133.9, 129.5 & 129.4, 126.9, 123.7, 118.6, 115.3 &
115.0, 113.2 & 112.9, 112.1 & 112.0, 104.8 & 104.5, 48.0, 47.0,
45.8, 19.9; MS 370 (M+), 370 (100); HRMS calcd for C20H16O3N2F2:
370.1129, found: 370.1124.
4.1.19. Typical procedure for thiolactams (5b–5r)
To a stirred solution of lactam (1 mmol) in toluene (5 mL) was
added Lawesson’s reagent (0.55 equiv), and the resulting mixture
was refluxed for 20–24 h. After cooling, the solvent was removed,
and the residue was chromatographed on SiO2 (Hexane–Ace-
tone = 25:1) to give thiolactam (5b–5r).
4.1.15. 2-Benzyl-5,7-dichloro-2,3,4,9-tetrahydro-b-carbolin-1-
one (2p)
Yield: 30%; mp: 248–250 °C; IR (KBr): 3188, 1633 cmÀ1 1H
;
NMR (400 MHz, CDCl3): d 10.04 (1H, br), 7.37–7.28 (6H, m), 7.09
(1H, d, J = 1.4 Hz), 4.81 (2H, s), 3.66 (2H, t, J = 7.1 Hz), 3.31 (2H, t,
J = 7.1 Hz); 13C NMR (100 MHz, CDCl3): d 160.96, 138.36, 137.04,
130.25, 128.81, 128.18, 128.00, 127.88, 127.68, 121.90, 121.21,
118.07, 111.23, 49.67, 47.38, 21.83; MS (EI) m/z 344 (M+); HRMS
(EI) calcd for C18H14ON2Cl2: 344.0483 (M+), found: 344.0474.
4.1.20. 2-Benzyl-2,3,4,9-tetrahydro-b-carboline-1-thione (5b)
Yield: 98%; mp: 197–198 °C; IR (KBr): 3319, 1556 cmÀ1 1H
;
NMR (300 MHz, DMSO-d6): d11.32 (1H, br), 7.60–7.22 (8H, m),
7.05 (1H, t, J = 7.5 Hz), 5.38 (2H, s), 3.77 (2H, t, J = 7.0 Hz), 3.00
(2H, t, J = 7.0 Hz); 13C NMR (75 MHz, DMSO-d6): d182.90, 138.31,
136.50, 132.32, 128.44, 127.39, 127.23, 124.52, 124.47, 120.61,
119.70, 112.66, 111.47, 54.94, 49.55, 19.65; MS (EI) m/z 292
(M+); HRMS (EI) calcd for C18H16N2S: 292.1034 (M+), found:
292.1054.
4.1.16. 5,7-Difluoro-2-phenylethyl-2,3,4,9-tetrahydro-b-
carbolin-1-one (2r)
Yield: 33%; mp: 220–222 °C; IR (KBr): 3161, 1629 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 10.51 (1H, br), 7.35–7.22 (5H, m), 6.98
(1H, dd, J = 1.9, 9.1 Hz), 6.60 (1H, dd, J = 1.9, 10.2 Hz), 3.84 (2H, t,
J = 7.4 Hz), 3.58 (2H, t, J = 7.0 Hz), 3.07 (2H, t, J = 7.0 Hz), 3.01 (2H,
t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): d 160.95 160.76 (dd,
J = 11.5, 241.5 Hz), 157.23 (dd, J = 15.3, 251.1 Hz), 139.17 (dd,
J = 12.9, 14.9 Hz), 138.92, 128.85, 128.66, 127.49 (d, J = 1.9 Hz),
126.57, 116.37 (d, J = 3.8 Hz), 111.52 (d, J = 1.9, 21.1 Hz), 95.82
(dd, J = 22.5, 29.2 Hz), 94.90 (dd, J = 4.8, 25.9 Hz), 49.03, 48.86,
34.64, 21.56; MS (EI) m/z 326 (M+); HRMS (EI) calcd for
4.1.21. 2-Benzyl-6-fluoro-2,3,4,9-tetrahydro-b-carboline-1-
thione (5c)
Yield: 53%; mp: 127–130 °C; IR (KBr): 3320, 1555 cmÀ1
;
1H
NMR (300 MHz, DMSO-d6):
d 11.43 (1H, br), 7.47 (1H, m),
7.41À7.26 (6H, m), 7.11 (1H, dt, J = 2.5, 9.4 Hz), 5.37 (2H, s), 3.77
(2H, t, J = 7.2 Hz), 2.97 (2H, t, J = 7.2 Hz); 13C NMR (75 MHz,
DMSO-d6): d 182.70, 156.84 (d, J = 233.2 Hz), 136.35, 135.01,
133.75, 128.44, 127.39, 127.26, 124.47 (d, J = 9.8 Hz), 113.96 (d,
J = 9.8 Hz), 113.31 (d, J = 26.9 Hz), 111.43 (d, J = 6.1 Hz), 104.84
(d, J = 23.2 Hz), 55.03, 49.60, 19.55; MS (EI) m/z 310 (M+); HRMS
(EI) calcd for C18H15N2FS: 310.0940 (M+), found: 310.0914.
C
19H16ON2F2: 326.1231 (M+), found: 326.1241.
4.1.17. [6-Fluoro-2-(4-fluorobenzyl)-1-oxo-1,2,3,4-tetrahydro-b-
carbolin-9-yl]acetic acid methyl ester (3)
To a stirred solution of 2a (193 mg, 0.62 mmol) in DMF (5 mL)
was added NaH (60%, 37 mg, 0.93 mmol) at 0 °C, and the reaction
mixture was stirred at 0 °C for 30 min. To the mixture was added
BrCH2COOMe (0.087 mL, 0.93 mmol) at 0 °C, and the resulting mix-
ture was stirred at room temperature for 24 h. The reaction was
quenched with H2O (10 mL), and the aqueous mixture was
4.1.22. 6-Fluoro-2-(4-trifluoromethylbenzyl)-2,3,4,9-
tetrahydro-b-carboline-1-thione (5d)
Yield: 90%; mp: 180–182 °C; IR (KBr): 3309, 1558 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.08 (1H, br), 7.62 (2H, d, J = 8.1 Hz),
7.51 (2H, d, J = 8.1 Hz), 7.38–7.34 (1H, m), 7.19 (1H, d, J = 9.1 Hz),

362
D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
7.08 (1H, td, J = 2.5, 9.1 Hz), 5.45 (2H, s), 3.78 (2H, t, J = 7.4 Hz), 3.02
(2H, t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): d 184.11, 157.88 (d,
J = 236.8 Hz), 140.10, 134.68, 133.48, 130.03 (q, J = 33.0 Hz),
127.92, 125.69 (q, J = 3.7 Hz), 122.11, 118.50, 114.60 (d,
J = 26.9 Hz), 113.10 (d, J = 9.8 Hz), 111.43 (d, J = 4.9 Hz), 105.19 (d,
J = 23.2 Hz), 55.56, 49.80, 20.36; MS (EI) m/z 378 (M+); HRMS (EI)
calcd for C19H14N2F4S: 378.0814 (M+), found: 378.0838.
121.45, 111.90, 111.35, 55.87, 49.33, 20.21; MS (EI) m/z 326
(M+); HRMS (EI) calcd for C18H15N2SCl: 326.0644 (M+), found:
326.0645.
4.1.28. 2-(4-Bromo-2-fluorombenzyl)-2,3,4,9-tetrahydro-b-
carboline-1-thione (5j)
Yield: 68%; mp: 160–162 °C; IR (KBr): 3321, 1555 cmÀ1 1H
;
NMR (300 MHz, DMSO-d6): d 11.32 (1H, br s), 7.62–7.57 (2H, m),
7.49 (1H, d, J = 8.2 Hz), 7.42–7.21 (3H, m), 7.06 (1H, t, J = 7.4 Hz),
5.35 (2H, s), 3.86 (2H, t, J = 7.4 Hz), 3.05 (2H, t, J = 7.4 Hz); 13C
NMR (75 MHz, CDCl3): d 184.18, 160.42 (d, J = 250.3 Hz), 137.98,
132.11, 131.15 (d, J = 3.7 Hz), 127.62 (d, J = 3.7 Hz), 127.48,
125.27, 122.31 (d, J = 14.6 Hz), 121.62 (d, J = 8.5 Hz), 120.72,
120.49, 118.97 (d, J = 24.4 Hz), 112.00, 111.59, 49.98, 48.87 (d,
J = 3.7 Hz), 20.36; MS (EI) m/z 389 (M+); HRMS (EI) calcd for
4.1.23. 2-(4-Bromo-2-fluorobenzyl)-6-fluoro-2,3,4,9-tetrahydro-
b-carboline-1-thione (5e)
Yield: 90%; mp: 128–131 °C; IR (KBr): 3310, 1554 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.07 (1H, br), 7.43 (1H, t, J = 8.1 Hz),
7.36–7.24 (3H, m), 7.19 (1H, dd, J = 1.6, 9.1 Hz), 7.07 (1H, td,
J = 2.5, 9.1 Hz), 5.38 (2H, s), 3.82 (2H, t, J = 7.4 Hz), 3.01 (2H, t,
J = 7.4 Hz); 13C NMR (100 MHz, CDCl3): d 184.23, 160.51 (d,
J = 278.0 Hz), 158.08 (d, J = 264.6 Hz), 134.70, 133.60, 131.34 (d,
J = 3.8 Hz), 127.78 (d, J = 3.8 Hz), 125.55 (d, J = 9.6 Hz), 122.29 (d,
J = 15.3 Hz), 121.86 (d, J = 9.6 Hz), 119.15 (d, J = 24.9 Hz), 114.55
(d, J = 26.8 Hz), 113.13 (d, J = 9.6 Hz), 111.48 (d, J = 5.8 Hz),
105.17 (d, J = 23.0 Hz), 49.99, 49.02 (d, J = 2.9 Hz), 20.19; MS (EI)
m/z 406 (M+); HRMS (EI) calcd for C18H13N2F2SBr: 405.9951 (M+),
found: 405.9924.
C
18H14N2FSBr: 388.0045 (M+), found: 388.0075.
4.1.29. 2-Benzyl-6-chloro-2,3,4,9-tetrahydro-b-carboline-1-
thione (5k)
Yield: 99%; mp: 194–196 °C; IR (KBr): 3317, 1551 cmÀ1 1H
;
NMR (300 MHz, DMSO-d6): d 11.53 (1H, br s), 7.69 (1H, s), 7.49
(1H, d, J = 9.4 Hz), 7.40–7.31 (4H, m), 7.30 (1H, t, J = 9.4 Hz), 7.23
(1H, d, J = 9.4 Hz), 5.37 (2H, s), 3.78 (2H, t, J = 7.3 Hz), 2.99 (2H, t,
4.1.24. 2-(4-Trifluoromethylbenzyl)-2,3,4,9-tetrahydro-b-
J = 7.3 Hz); 13C NMR (75 MHz, DMSO-d6):
d 182.61, 136.58,
carboline-1-thione (5f)
136.30, 133.42, 128.44, 127.39, 127.26, 125.45, 124.52, 124.12,
119.78, 114.27, 110.97, 55.03, 49.55, 19.42; MS (EI) 326 m/z
(M+); HRMS (EI) calcd for C18H15N2SCl: 326.0645 (M+), found:
326.0678.
Yield: 95%; mp: 187–190 °C; IR (KBr): 3340, 1557 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.08 (1H, br), 7.62 (2H, d, J = 8.5 Hz),
7.58 (1H, d, J = 8.2 Hz), 7.51 (2H, d, J = 8.5 Hz), 7.43 (1H, d,
J = 8.2 Hz), 7.33 (1H, t, J = 7.5 Hz), 7.14 (1H, t, J = 7.5 Hz), 5.47
(2H, s), 3.79 (2H, t, J = 7.4 Hz), 3.06 (2H, t, J = 7.4 Hz); 13C NMR
(100 MHz, CDCl3): d 184.54, 140.40, 138.26, 132.27, 130.08 (q,
J = 32.6 Hz), 128.00, 125.77 (q, J = 3.8 Hz), 125.50, 125.37, 122.66,
120.93, 120.74, 112.20, 118.78, 55.45, 49.78, 20.36; MS (EI) m/z
360 (M+); HRMS (EI) calcd for C19H15N2F3S: 360.0908 (M+), found:
360.0861.
4.1.30. 2-Benzyl-6-iodo-2,3,4,9-tetrahydro-b-carboline-1-thione
(5l)
Yield: 99%; mp: 185–187 °C; IR (KBr): 3318, 1551 cmÀ1 1H
;
NMR (300 MHz, DMSO-d6): d 11.50 (1H, s), 8.01 (1H, s), 7.50–
7.29 (7H, m), 5.36 (2H, s), 3.77 (2H, t, J = 7.6 Hz), 2.98 (2H, t,
J = 7.6 Hz); 13C NMR (75 MHz, DMSO-d6):
d 182.56, 137.13,
136.33, 132.76, 132.31, 129.13, 128.46, 127.42, 127.27, 127.08,
115.04, 110.51, 83.40, 55.03, 49.56, 19.42; MS (EI) m/z 418 (M+);
HRMS (EI) calcd for C18H15N2SI: 418.0001 (M+), found: 418.0062.
4.1.25. 2-(4-Bromobenzyl)-2,3,4,9-tetrahydro-b-carboline-1-
thione (5g)
Yield: 86%; mp: 171–174 °C; IR (KBr): 3334, 1558 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.08 (1H, br), 7.57 (1H, d, J = 8.0 Hz),
7.48 (2H, d, J = 8.2 Hz), 7.42 (1H, d, J = 8.5 Hz), 7.33 (2H, d,
J = 8.2 Hz), 7.29 (1H, d, J = 8.5 Hz), 7.14 (1H, m), 5.35 (2H, s), 3.76
(2H, t, J = 7.4 Hz), 3.03 (2H, t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3):
d 184.03, 138.09, 135.28, 132.21, 131.80, 129.47, 125.56, 125.41,
121.66, 120.81, 120.59, 112.11, 111.61, 55.29, 49.59, 20.44; MS
(EI) m/z 370 (M+); HRMS (EI) calcd for C18H15N2SBr: 370.0139
(M+), found: 370.0139.
4.1.31. 2-Benzyl-6-methoxy-2,3,4,9-tetrahydro-b-carboline-1-
thione (5m)
Yield: 94%; mp: 173–174 °C; IR (KBr): 3318, 1548 cmÀ1 1H
;
NMR (300 MHz, DMSO-d6): d 11.18 (1H, br), 7.41–7.28 (6H, m),
7.04 (1H, s), 6.90 (1H, dd, J = 2.6, 9.0 Hz), 5.37 (2H, s), 3.75 (3H,
s), 3.76 (2H, t, J = 7.5 Hz), 2.97 (2H, t, J = 7.3 Hz); 13C NMR
(75 MHz, DMSO-d6): d 182.82, 153.57, 136.54, 133.76, 132.74,
128.42, 127.40, 127.23, 124.60, 116.08, 113.62, 111.14, 100.63,
55.23, 54.90, 49.60, 19.73; MS (EI) m/z 322 (M+); HRMS (EI) calcd
for C19H18ON2S: 322.1140 (M+), found: 322.1131.
4.1.26. 2-Benzyl-6-bromo-2,3,4,9-tetrahydro-b-carboline-1-
thione (5h)
Yield: 85%; mp: 176–178 °C; IR (KBr): 3220, 1550 cmÀ1
;
1H
4.1.32. 2-Benzyl-6-iso-propyl-2,3,4,9-tetrahydro-b-carboline-1-
NMR (300 MHz, DMSO-d6): d 11.94 (1H, br s), 7.82 (1H, s),
7.42À7.27 (7H, m), 5.36 (2H, s), 3.76 (2H, t, J = 7.2 Hz), 2.98 (2H,
t, J = 7.2 Hz); 13C NMR (75 MHz, DMSO-d6): d 182.59, 136.79,
136.29, 133.23, 128.41, 127.39, 127.24, 126.97, 126.17, 122.87,
114.65, 112.05, 110.82, 55.03, 49.53, 19.44; MS (EI) 370 m/z
(M+); HRMS (EI) calcd for C18H15N2SBr: 370.0139 (M+), found:
370.0178.
thione (5n)
Yield: 86%; mp: 161–164 °C; IR (KBr): 3326, 1558 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.03 (1H, br), 7.42–7.20 (8H, m), 5.41
(2H, s), 3.76 (2H, t, J = 7.4 Hz), 3.04–2.95 (3H, m), 1.30 (6H, d,
J = 6.9 Hz); 13C NMR (75 MHz, CDCl3): d 184.06, 141.35, 136.90,
136.41, 132.61, 128.77, 127.83, 127.75, 125.59, 125.27, 117.26,
111.96, 111.46, 55.80, 49.49, 34.16, 24.41, 20.36; MS (EI) m/z 334
(M+); HRMS (EI) calcd for C21H22N2S: 334.1504 (M+), found:
334.1500.
4.1.27. 2-Benzyl-7-chloro-2,3,4,9-tetrahydro-b-carboline-1-
thione (5i)
Yield: 99%; mp: 162–163 °C; IR (KBr): 3318, 1551, cmÀ1
;
1H
4.1.33. 2-Benzyl-5,7-difluoro-2,3,4,9-tetrahydro-b-carboline-1-
NMR (300 MHz, DMSO-d6): d 11.45 (1H, br s), 7.62–7.30 (6H, m),
7.06 (2H, d-like, J = 6.6 Hz), 5.37 (2H, s), 3.79 (2H, t, J = 6.9 Hz),
3.00 (2H, t, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3): d 183.40,
138.09, 136.02, 132.79, 131.19, 128.69, 127.76, 124.02, 121.66,
thione (5o)
Yield: 93%; mp: 109–111 °C; IR (KBr): 3264, 1578 cmÀ1 1H
;
NMR (300 MHz, DMSO-d6): d 11.20 (1H, br), 7.39–7.30 (4H, m),
7.07 (1H, dd, J = 2.2, 9.6 Hz), 6.89 (1H, t, J = 10.6 Hz), 5.36 (2H, s),

D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
363
3.79 (2H, t, J = 7.4 Hz), 3.09 (2H, t, J = 7.4 Hz); 13C NMR (75 MHz,
4.1.39. (2-Benzyl-6-fluoro-1-thioxo-1,2,3,4-tetrahydro-b-
CDCl3): d 183.13, 161.30 (dd, J = 11.5, 244.4 Hz), 157.97 (dd,
J = 15.3 Hz, 252.1 Hz), 139.05 (d, J = 1.9 Hz), 136.07, 132.70 (d,
J = 3.8 Hz), 128.84, 127.91, 127.87, 109.74 (d, J = 1.9 Hz), 96.55
(dd, J = 22.5, 29.2 Hz), 94.45 (d, J = 4.8 Hz, 26.8 Hz), 55.84, 49.29,
21.07; MS (EI) m/z 328 (M+); HRMS (EI) calcd for C18H14N2F2S:
328.0846 (M+), found: 328.0829.
carbolin-9-yl)acetic acid t-butyl ester (6c)
Yield: 93%; mp: 54–56 °C; IR (KBr): 1742 cmÀ1
;
1H NMR
(300 MHz, CDCl3): d 7.37–7.18 (7H, m), 7.10 (1H, dt, J = 2.5,
9.4 Hz), 5.71 (2H, br), 5.46 (2H, s), 3.75 (2H, t, J = 7.2 Hz), 2.92
(2H, t, J = 7.2 Hz), 1.47 (9H, s); 13C NMR (75 MHz, CDCl3): d
183.17, 167.96, 157.98 (d, J = 237.5 Hz), 137.07, 136.19, 133.27,
128.49, 127.46, 127.41, 123.09 (d, J = 10.1 Hz), 114.70 (d,
J = 5.5 Hz), 114.19 (d, J = 26.7 Hz), 111.04 (d, J = 9.2 Hz), 104.90 (d,
J = 23.9 Hz), 81.72, 55.31, 49.07, 47.37, 27.92, 20.22; MS (EI) m/z
424 (M+); HRMS (EI) calcd for C24H25O2N2FS: 424.1621 (M+),
found: 424.1602.
4.1.34. 2-Benzyl-5,7-dichloro-2,3,4,9-tetrahydro-b-carboline-1-
thione (5p)
Yield: 98%; mp: 185–187 °C; IR (KBr): 3272, 1557 cmÀ1 1H
;
NMR (300 MHz, CDCl3): d 9.19 (1H, br), 7.39–7.30 (6H, m), 7.09
(1H, d, J = 1.6 Hz), 5.38 (2H, s), 3.76 (2H, t, J = 7.4 Hz), 3.30 (2H, t,
J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): d183.13, 138.56, 135.95,
133.29, 130.93, 128.86, 128.79, 127.95, 127.89, 122.26, 121.66,
111.30, 110.73, 55.92, 49.32, 21.37; MS (EI) m/z 360 (M+); HRMS
(EI) calcd for C18H14N2Cl2S: 360.0255 (M+), found: 360.0273.
4.1.40. [6-Fluoro-1-thioxo-2-(4-trifluoromethylbenzyl)-1,2,3,4-
tetrahydro-b-carbolin-9-yl]acetic acid t-butyl ester (6d)
Yield: 76%; mp: 53–55 °C; IR (KBr): 1740 cmÀ1
;
1H NMR
(400 MHz, CDCl3): d 7.59 (2H, d, J = 8.0 Hz), 7.47 (2H, d, J = 8.0 Hz),
7.24–7.05 (3H, m), 5.68 (2H, br), 5.51 (2H, s), 3.76 (2H, t,
J = 6.9 Hz), 2.96 (2H, t, J = 6.9 Hz), 1.47 (9H, s); 13C NMR (100 MHz,
CDCl3): d 184.00, 168.16, 158.30 (d, J = 237.7 Hz), 140.46, 137.37,
133.39, 129.89 (q, J = 32.6 Hz), 128.11, 128.00, 125.66 (q,
J = 3.8 Hz), 114.99 (d, J = 4.8 Hz), 114.74 (d, J = 26.8 Hz), 113.15 (d,
J = 9.6 Hz), 111.28 (d, J = 9.6 Hz), 105.17 (d, J = 23.0 Hz), 82.14,
55.32, 49.65, 47.58, 28.10, 20.58; MS (EI) m/z 492 (M+); HRMS (EI)
calcd for C25H24O2N2F4S: 492.1495 (M+), found: 492.1518.
4.1.35. 2-Phenyl-2,3,4,9-tetrahydro-b-carboline-1-thione (5q)
Yield: 99%; mp: 175–176 °C; IR (KBr): 3395, 1556 cmÀ1 1H
;
NMR (300 MHz, DMSO-d6): d 11.31 (1H, br s), 7.66 (1H, d,
J = 8.0 Hz), 7.52–7.32 (6H, m), 7.26 (1H, t, J = 8.0 Hz), 7.08 (1H, t,
J = 8.0 Hz), 4.11 (2H, t, J = 7.3 Hz), 3.18 (2H, t, J = 7.3 Hz); 13C NMR
(75 MHz, DMSO-d6): d 184.11, 146.04, 138.41, 132.95, 128.99,
127.14, 126.90, 125.36, 124.80, 120.85, 119.85, 113.33, 112.73,
54.01, 20.23; MS (EI) m/z 277 (M+); HRMS (EI) calcd for
C
17H14N2S: 278.0878 (M+), found: 278.0890.
4.1.41. [2-(4-Bromo-2-fluorobenzyl)-6-fluoro-1-thioxo-1,2,3,4-
tetrahydro-b-carbolin-9-yl]acetic acid t-butyl ester (6e)
4.1.36. 5,7-Difluoro-2-phenylethyl-2,3,4,9-tetrahydro-b-
carboline-1-thione (5r)
Yield: 95%; pale yellow oil; IR (neat): 1742 cmÀ1 1H NMR
;
Yield: 99%; mp: 150–153 °C; IR (KBr): 3336, 1551 cmÀ1 1H
;
(300 MHz, CDCl3): d 7.45–7.07 (6H, m), 5.65 (2H, br), 5.43 (2H, s),
3.79 (2H, t, J = 7.1 Hz), 2.96 (2H, t, J = 7.1 Hz), 1.46 (9H, s); 13C
NMR (75 MHz, CDCl3): d 183.85, 167.98, 160.48 (d, J = 250.3 Hz),
158.15 (d, J = 238.1 Hz), 137.24, 133.32, 130.96 (d, J = 4.9 Hz),
127.60 (d, J = 3.7 Hz), 123.18 (d, J = 9.8 Hz), 121.49 (d, J = 9.8 Hz),
119.10 (d, J = 24.4 Hz), 119.02 (d, J = 24.4 Hz), 114.95 (d,
J = 6.1 Hz), 114.65 (d, J = 26.9 Hz), 111.20 (d, J = 8.5 Hz), 105.13 (d,
J = 23.2 Hz), 82.11, 49.03 (d, J = 3.6 Hz), 47.63, 31.24, 28.20, 20.71;
MS (EI) m/z 520 (M+); HRMS (EI) calcd for C24H23O2FN2SBr:
520.0632 (M+), found: 520.0612.
NMR (300 MHz, CDCl3): d 9.18 (1H, br), 7.36–7.22 (5H, m), 6.87
(1H, dd, J = 1.9, 9.1 Hz), 6.58 (1H, dd, J = 1.9, 10.4 Hz), 4.29 (2H, t,
J = 7.7 Hz), 3.61 (2H, t, J = 7.4 Hz), 3.12 (2H, t, J = 7.7 Hz), 3.00 (2H,
t, J = 7.4 Hz); 13C NMR (75 MHz, CDCl3): d 181.95, 161.07 (dd,
J = 11.5, 243.5 Hz), 157.84 (dd, J = 15.3, 252.1 Hz), 138.85 (dd,
J = 12.9, 14.9 Hz), 138.46, 132.88 (d, J = 2.9 Hz), 128.90, 128.63,
126.68, 112.00 (d, J = 21.1 Hz), 109.52, 96.38 (dd, J = 22.5,
29.2 Hz), 94.45 (dd, J = 4.8, 24.9 Hz), 55.61, 51.35, 33.06, 20.87;
MS (EI) m/z 342 (M+); HRMS (EI) calcd for C19H16N2F2S: 342.1002
(M+), found: 342.0992.
4.1.42. [2-(4-Trifluoromethylbenzyl)-1-thioxo-1,2,3,4-
4.1.37. Typical procedure for esters (6b–6r)
tetrahydro-b-carbolin-9-yl]acetic acid t-butyl ester (6f)
To a stirred solution of thiolactam (1 mmol) in DMF (5 mL) was
added NaH (60%, 1.2 equiv) at 0 °C, and the reaction mixture was
stirred at 0 °C for 30 min. To the mixture was added BrCH2COOt-
Bu or BrCH2COOMe (1.2 equiv) at 0 °C, and the resulting mixture
was stirred at room temperature for 20–24 h. The reaction was
quenched with H2O (10 mL), and the aqueous mixture was ex-
tracted with Et2O (10 mL Â 3). The organic extracts were com-
bined, dried over MgSO4, and evaporated. The residue was
chromatographed on SiO2 (Hexane–Acetone = 20:1) to give t-butyl
ester (6b–6i, 6k, 6n, 6o, and 6q) or methyl ester (6j, 6l, 6m, 6p, and
6r).
Yield: 86%; pale yellow oil; IR (neat): 1735 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.61–7.57 (3H, m), 7.48 (2H, dd, J = 8.7 Hz),
7.41–7.30 (2H, m), 7.19–7.15 (1H, m), 5.70 (2H, br), 5.52 (2H, s),
3.77 (2H, t, J = 6.9 Hz), 3.01 (2H, t, J = 6.9 Hz), 1.47 (9H, s); 13C
NMR (75 MHz, CDCl3):
d
184.25, 168.33, 132.34, 129.86
(q, J = 32.6 Hz), 128.01, 127.82, 125.94, 125.80, 125.77, 125.66
(q, J = 3.8 Hz), 123.20, 121.09, 120.87, 115.46, 110.25, 81.94,
55.29, 49.67, 47.46, 28.14, 20.72; MS (EI) m/z 474 (M+); HRMS
(EI) calcd for C25H25O2N2F3S: 474.1589 (M+), found: 474.1571.
4.1.43. [2-(4-Bromobenzyl)-1-thioxo-1,2,3,4-tetrahydro-
4.1.38. (2-Benzyl-1-thioxo-1,2,3,4-tetrahydro-b-carbolin-9-
yl)acetic acid t-butyl ester (6b)
b-carbolin-9-yl]acetic acid t-butyl ester (6g)
Yield: 92%; pale yellow oil; IR (neat): 1744 cmÀ1 1H NMR
;
Yield: 66%; mp: 63–64 °C; IR (KBr): 1742 cmÀ1
;
1H NMR
(300 MHz, CDCl3): d 7.57 (1H, d, J = 8.0 Hz), 7.49–7.44 (3H, m),
7.40–7.24 (3H, m), 7.19–7.14 (1H, m), 5.70 (2H, br), 5.41 (2H, s),
3.74 (2H, t, J = 7.1 Hz), 2.98 (2H, t, J = 7.1 Hz), 1.47 (9H, s); 13C
NMR (75 MHz, CDCl3): d 183.74, 168.14, 140.73, 135.55, 131.80,
131.69, 129.47, 129.31, 125.75, 123.11, 120.93, 120.73, 115.31,
110.15, 81.87, 50.01, 49.43, 47.52, 28.24, 20.78; MS (EI) m/z 484
(M+); HRMS (EI) calcd for C24H25O2N2SBr: 484.0820 (M+), found:
484.0832.
(300 MHz, CDCl3): d 7.56 (1H, d, J = 9.0 Hz), 7.27–7.39 (7H, m),
7.16 (1H, t. J = 7.5 Hz), 5.73 (2H, br), 5.47 (2H, s), 3.75 (2H, t,
J = 7.3 Hz), 2.97 (2H, t, J = 7.3 Hz), 1.47 (9H, s); 13C NMR (75 MHz,
CDCl3): d 183.11, 167.91, 140.41, 136.22, 132.03, 128.31, 127.32,
127.19, 125.33, 122.89, 120.59, 120.51, 115.02, 109.88, 81.47,
55.22, 49.07, 47.28, 28.01, 20.37; MS (EI) m/z 406 (M+); HRMS
(EI) calcd for C24H26O2N2S: 406.1715 (M+), found: 406.1745.

364
D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
4.1.44. (2-Benzyl-6-bromo-1-thioxo-1,2,3,4-tetrahydro-
4.1.50. (2-Benzyl-6-iso-propyl-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid t-butyl ester (6h)
b-carbolin-9-yl)acetic acid t-butyl ester (6n)
Yield: 53%; mp: 157–158 °C; IR (KBr): 1742 cmÀ1
;
1H NMR
Yield: 80%; pale yellow oil; IR (neat):1743 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.69 (1H, s), 7.43–7.31 (6H, brm), 7.15 (1H,
d, J = 8.7 Hz), 5.70 (2H, br s), 5.45 (2H, s), 3.74 (2H, t, J = 7.2 Hz),
2.92 (2H, t, J = 7.2 Hz), 1.47 (9H, s); 13C NMR (75 MHz, CDCl3): d
183.11, 167.77, 139.05, 136.17, 128.65, 128.56, 128.21, 127.72,
127.55, 124.62, 122.98, 114.15, 113.71, 111.64, 82.00, 55.55,
49.20, 47.51, 28.15, 20.45; MS (EI) m/z 484 (M+); HRMS (EI) calcd
for C24H25O2N2SBr: 484.0820 (M+), found: 484.0800.
(300 MHz, CDCl3): d 7.36–7.18 (8H, m), 5.70 (2H, br), 5.47 (2H, s),
3.74 (2H, t, J = 7.0 Hz), 3.04–2.94 (3H, m), 1.48 (9H, s), 1.29 (6H,
d, J = 7.1 Hz); 13C NMR (75 MHz, CDCl3): d 183.61, 168.30, 141.54,
139.53, 136.59, 128.58, 127.74, 127.59, 127.45, 125.34, 117.24,
115.12, 109.96, 81.74, 55.55, 49.37, 47.57, 38.67, 34.19, 28.25,
24.51, 20.81; MS (EI) m/z 448 (M+); HRMS (EI) calcd for
C
27H32O2N2S: 448.2185 (M+), found: 448.2164.
4.1.45. (2-Benzyl-7-chloro-1-thioxo-1,2,3,4-tetrahydro-
4.1.51. (2-Benzyl-5,7-difluoro-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid t-butyl ester (6i)
b-carbolin-9-yl)acetic acid t-butyl ester (6o)
Yield: 52%; mp: 156–158 °C; IR (KBr): 1741 cmÀ1
;
1H NMR
Yield: 99%; mp: 118–121 °C; IR (KBr): 1743 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.49–7.46 (1H, m), 7.38–7.27 (6H, m), 7.14–
7.10 (1H, m), 5.67 (2H, s), 5.45 (2H, s), 3.74 (2H, t, J = 6.9 Hz),
2.94 (2H, t, J = 6.9 Hz), 1.49 (9H, s); 13C NMR (75 MHz, CDCl3): d
183.24, 167.85, 140.93, 136.33, 132.84, 131.56, 128.63, 127.61,
121.83, 121.75, 121.62 115.09, 110.20, 82.11, 55.58, 49.22, 47.63,
28.24, 20.61; MS (EI) m/z 440 (M+); HRMS (EI) calcd for
(300 MHz, CDCl3): d 7.37–7.28 (5H, m), 6.75 (1H, dd, J = 1.7,
9.4 Hz), 6.61 (1H, d, J = 10.9 Hz), 5.70 (2H, br), 5.44 (2H, s), 3.73
(2H, t, J = 7.0 Hz), 3.08 (2H, t, J = 7.0 Hz), 1.48 (9H, s); 13C NMR
(75 MHz, CDCl3): d 182.81, 167.76, 161.41 (dd, J = 12.5, 244.4 Hz),
158.00 (dd, J = 15.3, 252.1 Hz), 142.09 (dd, J = 12.0, 13.9 Hz),
136.31, 132.78 (d, J = 4.8 Hz), 128.81, 128.72, 127.88, 127.85,
127.68, 96.85 (dd, J = 23.0, 37.4 Hz), 92.92 (d, J = 4.8, 26.8 Hz),
82.30, 55.50, 49.40, 47.99, 28.10, 21.49; MS (EI) m/z 442 (M+); HRMS
(EI) calcd for C24H24O2N2F2S: 442.1523 (M+), found: 442.1546.
C
24H25O2SN2Cl: 440.1325 (M+), found: 440.1358.
4.1.46. [2-(4-Bromo-2-fluorobenzyl)-1-thioxo-1,2,3,4-
tetrahydro-b-carbolin-9-yl]acetic acid methyl ester (6j)
Yield: 58%; mp: 130–132 °C; IR (KBr): 1739 cmÀ1
;
1H NMR
4.1.52. (2-Benzyl-5,7-dichloro-1-thioxo-1,2,3,4-tetrahydro-
(500 MHz, CDCl3): d 7.59 (1H, d, J = 8.1 Hz), 7.40–7.36 (2H, m),
7.30–7.24 (3H, m), 7.18 (1H, t, J = 7.5 Hz), 5.81 (2H, br s), 5.44 (2H,
s), 3.81 (2H, t, J = 6.8 Hz), 3.76 (3H, s), 3.02 (2H, t, J = 6.8 Hz); 13C
NMR (75 MHz, CDCl3): d 183.82, 169.53, 160.44 (d, J = 249.0 Hz),
140.63, 131.96, 131.00 (d, J = 3.7 Hz), 127.60 (d, J = 3.7 Hz), 125.94,
123.10, 122.59 (d, J = 14.7 Hz), 121.45 (d, J = 9.8 Hz), 121.11,
120.83, 118.96 (d, J = 25.6 Hz), 115.62, 110.04, 52.31, 49.87, 48.90
(d, J = 3.7 Hz), 46.84, 20.74; MS (EI) m/z 460 (M+); HRMS (EI) calcd
for C21H18O2N2FSBr: 460.0256 (M+), found: 460.0245.
b-carbolin-9-yl)acetic acid t-butyl ester (6p)
Yield: 67%; pale yellow oil; IR (neat): 1754 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.36–7.32 (5H, m), 7.16–7.13 (2H, m), 5.80
(2H, br), 5.42 (2H, s), 3.79 (3H, s), 3.74 (2H, t, J = 6.9 Hz), 3.27
(2H, t, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3): d 182.57, 169.11,
141.53, 136.25, 133.34, 131.32, 129.04, 128.79, 127.80, 127.76,
122.31, 119.77, 115.54, 109.05, 55.60, 52.52, 48.96, 47.16, 21.73;
MS (EI) m/z 432 (M+); HRMS (EI) calcd for C21H18O2N2SCl2:
432.0466 (M+), found: 432.0420.
4.1.47. (2-Benzyl-6-chloro-1-thioxo-1,2,3,4-tetrahydro-
4.1.53. (2-Phenyl-1-thioxo-1,2,3,4-tetrahydro-b-carbolin-9-yl)-
acetic acid t-butyl ester (6q)
b-carbolin-9-yl)acetic acid t-butyl ester (6k)
Yield: 65%; mp: 34–35 °C; IR (KBr): 1743 cmÀ1
;
1H NMR
Yield: 30%; mp: 165–166 °C; IR (KBr): 1741 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.53 (1H, d, J = 9.0 Hz), 7.31–7.28 (7H, m),
5.70 (2H, br), 5.46 (2H, s), 3.74 (2H, t, J = 7.1 Hz), 2.92 (2H, t,
J = 7.1 Hz), 1.47 (9H, s); 13C NMR (75 MHz, CDCl3): d 183.07,
167.78, 136.14, 132.98, 128.60, 128.52, 127.67, 127.50, 126.19,
125.68, 123.90, 119.78, 114.26, 111.24, 81.95, 55.50, 49.19, 47.51,
28.12, 20.40; MS (EI) m/z 440 (M+); HRMS (EI) calcd for
(500 MHz, CDCl3): d 7.64 (1H, d, J = 7.7 Hz), 7.50–7.25 (7H, m),
7.19 (1H, t, J = 7.5 Hz), 5.66 (2H, br), 4.09 (2H, t, J = 6.9 Hz), 3.21
(2H, t, J = 6.9 Hz), 1.45 (9H, s); 13C NMR (75 MHz, CDCl3): d
184.37, 167.91, 146.17, 140.60, 132.24, 129.24, 127.38, 126.96,
125.68, 123.00, 120.78, 120.73, 115.49, 110.02, 81.56, 53.52,
47.36, 28.06, 21.05; MS (EI) m/z 392 (M+); HRMS (EI) calcd for
C
24H25O2N2SCl: 440.1325 (M+), found: 440.1313.
C
23H24O2N2S: 392.1559 (M+), found: 392.1546.
4.1.48. (2-Benzyl-6-iodo-1-thioxo-1,2,3,4-tetrahydro-
4.1.54. (5,7-Difluoro-2-phenylethyl-1-thioxo-1,2,3,4-tetra-
hydro-b-carbolin-9-yl)-acetic acid methyl ester (6r)
b-carbolin-9-yl)acetic acid methyl ester (6l)
Yield: 61%; mp: 70–72 °C; IR (KBr): 1749 cmÀ1
;
1H NMR
Yield: 60%; mp: 38–40 °C; 1H NMR (300 MHz, CDCl3): d 7.36–
7.22 (5H, m), 6.72 (1H, dd, J = 1.9, 9.3 Hz), 6.62 (1H, dd, J = 1.9,
10.2 Hz), 5.77 (2H, br s), 4.32 (2H, t, J = 7.3 Hz), 3.78 (3H, br s),
3.57 (2H, t, J = 7.1 Hz), 3.10 (2H, t, J = 7.3 Hz), 2.96 (2H, t,
J = 7.1 Hz); 13C NMR (75 MHz, CDCl3): d 181.47, 169.18, 161.36
(dd, J = 11.5, 244.4 Hz), 159.18 (dd, J = 15.3, 253.0 Hz), 141.87 (dd,
J = 12.5, 13.4 Hz), 138.56, 132.64 (d, J = 3.8 Hz), 128.91, 128.58,
126.60, 113.68, 109.51 (d, J = 21.1 Hz), 96.90 (dd, J = 22.5,
29.2 Hz), 92.82 (dd, J = 4.8, 24.0 Hz), 55.76, 52.39, 51.09, 47.19,
32.94, 21.22; IR (KBr): 1749 cmÀ1; MS (EI) m/z 414 (M+); HRMS
(EI) calcd for C22H20O2N2F2S: 414.1214 (M+), found: 414.1241.
(500 MHz, CDCl3): d 7.92 (1H, s), 7.59 (1H, d, J = 9.0 Hz), 7.37–
7.29 (5H, m), 7.05 (1H, d, J = 9.0 Hz), 5.83 (2H, br s), 5.44 (2H, s),
3.77 (3H, s), 3.74 (2H, t, J = 6.8 Hz), 2.92 (2H, t, J = 6.8 Hz); 13C
NMR (75 MHz, CDCl3): d 182.85, 169.18, 139.39, 136.14, 133.65,
132.16, 129.45, 128.56, 127.55, 125.49, 114.08, 111.96, 84.07,
55.50, 52.34, 49.06, 46.76, 20.42; MS (EI) m/z 490 (M+); HRMS
(EI) calcd for C21H19O2N2SI: 490.0212 (M+), found: 490.0204.
4.1.49. (2-Benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid methyl ester (6m)
Yield: 63%; mp: 117–118 °C; IR (KBr) 1751 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.37–6.93 (8H, m), 5.83 (2H, br s), 5.45 (2H, s),
3.85 (3H, s), 3.75 (2H, t, J = 5.6 Hz), 2.93 (2H, t, J = 7.1 Hz),; 13C NMR
(75 MHz, CDCl3): d 182.8, 169.4, 154.5, 137.3, 136.2, 135.8, 132.1,
128.3, 127.5, 127.3, 127.2, 127.0, 126.9, 123.0, 116.7, 114.7, 110.8,
100.8, 55.4, 55.1, 52.0, 48.9, 46.6, 20.3; MS 394 (M+), 394 (100);
HRMS calcd for C22H22O3N2S: 394.1351, found: 394.1355.
4.1.55. (2-Benzyl-6-methoxycarbonyl-1-thioxo-1,2,3,4-tetra-
hydro-b-carbolin-9-yl)-acetic acid methyl ester (6t)
To a stirred solution of 6l (105 mg, 0.21 mmol) in DMF (5 mL)
was added Pd(PPh3)4 (25.5 mg, 0.021 mmol), and the resulting
solution was stirred at room temperature under CO balloon pres-
sure for 30 min. To the reaction mixture were added NEt3

D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
365
(0.06 mL, 0.84 mmol) and MeOH (0.21 mL, 8.4 mmol), and then the
mixture was stirred at 70 °C under CO balloon pressure for 20 h.
After cooling, the reaction mixture was diluted with H2O (10 mL)
and brine (5 mL), and the aqueous mixture was extracted with
Et2O (10 mL Â 5). The organic extracts were combined, dried over
MgSO4, and evaporated to give pale yellow oil, which was chro-
matographed on SiO2 (Hexane–Acetone = 8:1) to give 6t
(2H, t, J = 6.9 Hz), 2.96 (2H, t, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3):
d 183.51, 173.17, 158.53 (d, J = 238.7 Hz), 140.27, 137.44, 133.04,
130.07 (q, J = 3.8 Hz), 128.10, 127.88, 125.78 (q, J = 3.8 Hz),
115.64 (d, J = 4.8 Hz), 115.29 (d, J = 26.8 Hz), 111.48 (d,
J = 9.6 Hz), 111.17 (d, J = 9.6 Hz), 105.39 (d, J = 24.0 Hz), 55.39,
49.48, 47.13, 20.56; MS (EI) m/z 436 (M+); HRMS (EI) calcd for
C
21H16O2N2SF4: 436.0869 (M+), found: 436.0916.
(72.6 mg, 81%). Mp: 63–65 °C; IR (KBr): 1750, 1702 cmÀ1 1H
;
NMR (500 MHz, CDCl3): d 8.36 (1H, d, J = 1.3 Hz), 8.03 (1H, dd,
J = 1.3, 9.0 Hz), 7.37–7.27 (6H, m), 5.87 (2H, br s), 5.45 (2H, s),
3.94 (3H, s), 3.79–3.76 (5H, m), 3.01 (2H, t, J = 7.2 Hz); 13C NMR
4.1.60. [2-(4-Bromo-2-fluorobenzyl)-6-fluoro-1-thioxo-1,2,3,4-
tetrahydro-b-carbolin-9-yl]acetic acid (7e)
Yield: 70%; mp: 173–175 °C; IR (KBr): 1712 cmÀ1 1H NMR
;
(125 MHz, CDCl3):
d
182.86, 169.11, 167.09, 142.58, 136.14,
(300 MHz, CDCl3): d 7.41–7.13 (6H, m), 5.74 (2H, br), 5.42 (2H, s),
3.80 (2H, t, J = 7.1 Hz), 2.97 (2H, t, J = 7.1 Hz); 13C NMR (75 MHz,
CDCl3): d 183.51, 173.74, 160.66 (d, J = 251.2 Hz), 158.51 (d,
J = 238.7 Hz), 137.37, 133.06, 131.24 (d, J = 4.8 Hz), 127.83 (d,
J = 3.8 Hz), 123.36 (d, J = 9.6 Hz), 122.35 (d, J = 14.4 Hz), 121.82
(d, J = 9.6 Hz), 119.19 (d, J = 24.9 Hz), 115.67 (d, J = 4.8 Hz),
115.25 (d, J = 26.8 Hz), 111.42 (d, J = 9.6 Hz), 105.40 (d,
J = 23.0 Hz), 49.83, 49.09 (d, J = 3.8 Hz), 47.09, 20.56; MS (EI) m/z
464 (M+); HRMS (EI) calcd for C20H15O2F2N2SBr: 464.0006 (M+),
found: 464.0016.
133.05, 128.56, 127.55, 126.38, 123.79, 123.02, 122.82, 116.32,
109.78, 55.50, 52.34, 51.95, 49.04, 47.00, 20.45; MS (EI) m/z 422
(M+); HRMS (EI) calcd for C23H22O4N2S: 422.1300 (M+), found:
422.1311.
4.1.56. Typical procedure for carboxylic acids (7b–7t)
To a stirred solution of methyl ester (1 mmol) in MeOH (3 mL)
and H2O (1 mL) was added LiOHÁH2O (4 equiv), and the resulting
mixture was refluxed for 1–4 h. After cooling, the reaction was
quenched with 10% HCl aq, and the aqueous mixture was extracted
with CHCl3 (10 mL Â 3). The organic extracts were combined, dried
over MgSO4, and evaporated. The residue was chromatographed on
SiO2 (Hexane–Acetone = 2:1) to give carboxylic acid (7l, 7m, and
7p). To a stirred solution of NaI (4 equiv) in ClCH2CH2Cl (3 mL)
was added TMSCl (4 equiv), and the resulting mixture was stirred
at room temperature for 15 min. To a solution of t-butyl ester
(1 mmol) in ClCH2CH2Cl (5 mL) was transferred a solution of TMSI,
prepared above, via a cannula, and then the resulting mixture was
refluxed for 1–2 days. After cooling, the reaction was quenched
with 10% HCl aq, and the aqueous mixture was extracted with
CHCl3 (10 mL Â 3). The organic extracts were combined, dried over
MgSO4, and evaporated. The residue was chromatographed on SiO2
(Hexane–Acetone = 2:1) to give carboxylic acid (7b–7k, 7n–7s, and
7u).
4.1.61. [1-Thioxo-2-(4-trifluoromethylbenzyl)-1,2,3,4-tetra-
hydro-b-carbolin-9-yl]acetic acid (7f)
Yield: 63%; mp: 148–151 °C; IR (KBr): 1723 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.61 (3H, m), 7.49 (2H, d, J = 8.0 Hz), 7.41
(2H, d, J = 6.0 Hz), 7.24–7.19 (1H, m), 5.77 (2H, br), 5.51 (2H, s),
3.78 (2H, t, J = 7.1 Hz), 3.02 (2H, t, J = 7.1 Hz); 13C NMR (75 MHz,
CDCl3): d 183.63, 173.50, 140.98, 140.47, 131.98, 129.99 (q,
J = 31.6 Hz), 128.09, 127.88, 126.47, 125.75 (q, J = 3.8 Hz), 123.19,
121.55, 120.98, 116.18, 110.40, 55.32, 49.46, 47.06, 20.64; MS
(EI) m/z 418 (M+); HRMS (EI) calcd for C21H17O2N2F3SBr:
418.0963 (M+), found: 418.0929.
4.1.62. [2-(4-Bromobenzyl)-1-thioxo-1,2,3,4-tetrahydro-b-
carbolin-9-yl]acetic acid (7g)
Yield: 57%; mp: 183–185 °C; IR (KBr): 1710 cmÀ1 1H NMR
;
4.1.57. (2-Benzyl-1-thioxo-1,2,3,4-tetrahydro-b-carbolin-9-yl)-
(300 MHz, CDCl3): d 7.58 (1H, d, J = 8.0 Hz), 7.48 (2H, d, J = 8.5 Hz),
7.40 (2H, d, J = 7.1 Hz), 7.28–7.18 (3H, m), 5.77 (2H, br), 5.39 (2H,
s), 3.74 (2H, t, J = 7.0 Hz), 2.98 (2H, t, J = 7.0 Hz); 13C NMR (75 MHz,
CDCl3): d 183.09, 173.30, 140.84, 135.33, 131.91, 131.78, 129.37,
126.32, 123.10, 121.59, 121.43, 120.88, 116.07, 110.35, 55.11,
49.27, 47.20, 20.73; MS (EI) m/z 428 (M+); HRMS (EI) calcd for
acetic acid (7b)
Yield: 78%; mp: 207–209 °C; IR (KBr): 1722 cmÀ1 1H NMR
;
(300 MHz, DMSO-d6): d 7.66 (1H, d, J = 7.5 Hz), 7.52 (1H, d,
J = 7.5 Hz), 7.37–7.28 (6H, m), 7.15 (1H, t, J = 7.5 Hz), 5.77 (2H,
br), 5.43 (2H, s), 3.76 (2H, t, J = 7.2 Hz), 2.99 (2H, t, J = 7.2 Hz);
13C NMR (75 MHz, DMSO-d6): d 182.14, 170.12, 140.17, 136.46,
131.51, 128.41, 127.19, 125.24, 122.48, 120.69, 120.54, 115.17,
110.90, 54.71, 49.48, 46.49, 19.81; MS (EI) m/z 350 (M+); HRMS
(EI) calcd for C20H18O2N2S: 350.1089 (M+), found: 350.1085.
C
20H17O2N2SBr: 428.0194 (M+), found: 428.0189.
4.1.63. (2-Benzyl-6-bromo-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7h)
Yield: 65%; mp: 227–229 °C; IR (KBr): 1727 cmÀ1 1H NMR
;
4.1.58. (2-Benzyl-6-fluoro-1-thioxo-1,2,3,4-tetrahydro-
(300 MHz, DMSO-d6): d 7.91 (1H, d, J = 2.1 Hz), 7.54 (1H, d,
J = 9.0 Hz), 7.43 (1H, dd, J = 2.1, 9.0 Hz), 7.36 (4H, m), 7.32–7.27
(1H, m), 5.76 (2H, br), 5.42 (2H, s), 3.75 (2H, t, J = 7.2 Hz), 2.98
(2H, t, J = 7.2 Hz); 13C NMR (75 MHz, DMSO-d6): d 181.86, 169.89,
138.76, 136.30, 132.24, 128.44, 127.57, 127.24, 124.22, 122.97,
114.48, 113.17, 112.84, 54.82, 49.52, 46.67, 19.63; MS (EI) m/z
428 (M+); HRMS (EI) calcd for C20H17O2N2SBr: 428.0194 (M+),
found: 428.0145.
b-carbolin-9-yl)acetic acid (7c)
Yield: 85%; mp: 190–191 °C; IR (KBr): 1722 cmÀ1 1H NMR
;
(300 MHz, DMSO-d6): d 7.58 (1H, dd, J = 4.3, 9.0 Hz), 7.48 (1H, dd,
J = 2.5, 9.4 Hz), 7.36 (4H, m), 7.30 (1H, m), 7.20 (1H, dt, J = 2.5,
9.4 Hz), 5.76 (2H, br), 5.42 (2H, s), 3.76 (2H, t, J = 7.3 Hz), 2.98
(2H, t, J = 7.3 Hz); 13C NMR (75 MHz, DMSO-d6): d 181.96, 170.00,
157.30 (d, J = 234.4 Hz), 136.84, 136.32, 132.69, 128.41, 127.19,
122.58 (d, J = 9.8 Hz), 114.94 (d, J = 4.9 Hz), 113.81 (d,
J = 26.9 Hz), 112.49 (d, J = 9.8 Hz), 105.00 (d, J = 23.2 Hz), 54.81,
49.53, 46.68, 19.75; MS (EI) m/z 368 (M+); HRMS (EI) calcd for
C20H17O2N2FS: 368.0995 (M+), found: 368.0952.
4.1.64. (2-Benzyl-7-chloro-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (6i)
Yield: 50%; mp: 184–186 °C; IR (KBr): 1717 cmÀ1 1H NMR
;
(300 MHz, DMSO-d6): d 7.74 (1H, d, J = 1.8 Hz), 7.69 (1H, d,
J = 8.5 Hz), 7.36 (4H, m), 7.32–7.27 (1H, m), 7.16 (1H, dd, J = 1.8,
8.5 Hz), 5.74 (2H, br), 5.42 (2H, s), 3.76 (2H, t, J = 7.3 Hz), 2.98
(2H, t, J = 7.3 Hz); 13C NMR (75 MHz, DMSO-d6): d 181.81, 169.91,
141.22, 140.48, 136.32, 132.16, 129.96, 128.41, 127.19, 122.21,
121.26, 121.06, 115.22, 110.93, 54.74, 49.42, 46.73, 19.65; MS
4.1.59. [6-Fluoro-1-thioxo-2-(4-trifluoromethylbenzyl)-1,2,3,4-
tetrahydro-b-carbolin-9-yl]acetic acid (7d)
Yield: 98%; mp: 84–86 °C; IR (KBr): 1723 cmÀ1
;
1H NMR
(300 MHz, CDCl3):
d 7.61 (2H, d, J = 8.0 Hz), 7.47 (2H, d,
J = 8.0 Hz), 7.32–7.13 (3H, m), 5.78 (2H, br), 5.49 (2H, s), 3.76

366
D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
(EI) m/z 384 (M+); HRMS (EI) calcd for C20H17O2N2SCl: 384.0699
m), 6.99 (1H, t, J = 10.2 Hz), 5.76 (2H, br), 5.41 (2H, s), 3.76 (2H, t,
J = 7.3 Hz), 3.08 (2H, t, J = 7.3 Hz); 13C NMR (75 MHz, DMSO-d6): d
181.62, 169.92, 160.48 (dd, J = 12.5, 240.6 Hz), 157.10 (dd,
J = 15.8, 250.6 Hz), 141.72 (d, J = 14.4 Hz), 136.43, 132.37 (d,
J = 2.9 Hz), 128.67, 127.44, 127.41, 113.15, 108.86 (d, J = 21.1 Hz),
96.60 (dd, J = 23.0, 29.7 Hz), 94.45 (dd, J = 4.3, 27.3 Hz), 54.84,
49.42, 47.36, 20.75; MS (EI) m/z 386 (M+); HRMS (EI) calcd for
(M+), found: 384.0709.
4.1.65. [2-(4-Bromo2-fluorobenzyl)-1-thioxo-1,2,3,4-tetra-
hydro-b-carbolin-9-yl]acetic acid (7j)
Yield: 93%; mp: 163–165 °C; IR (KBr): 1712 cmÀ1 1H NMR
;
(500 MHz, DMSO-d6): d 7.69 (1H, d, J = 8.1 Hz), 7.60 (1H, dd,
J = 1.7, 9.8 Hz), 7.51 (1H, d, J = 8.5 Hz), 7.39 (1H, dd, J = 1.7,
8.5 Hz), 7.34 (1H, t, J = 7.5 Hz), 7.26 (1H, t, J = 8.3 Hz), 7.16 (1H, t,
J = 7.5 Hz), 5.71 (2H, br), 5.38 (2H, s), 3.85 (2H, t, J = 7.0 Hz), 3.06
(2H, t, J = 7.0 Hz); 13C NMR (75 MHz, DMSO-d6): d 182.70, 170.07,
159.92 (d, J = 249.0 Hz), 140.27, 131.48, 130.36 (d, J = 6.1 Hz),
127.50, 125.43, 122.91 (d, J = 14.6 Hz), 122.50, 120.72 (d,
J = 9.8 Hz), 120.32 (d, J = 9.8 Hz), 118.69 (d, J = 25.6 Hz), 115.53,
110.93, 50.20, 49.29, 46.51, 19.89; MS (EI) m/z 446 (M+); HRMS
(EI) calcd for C20H16O2N2FSBr: 446.0100 (M+), found: 446.0099.
C
20H16O2N2SF2: 386.0901 (M+), found: 386.0902.
4.1.71. (2-Benzyl-5,7-dichloro-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7p)
Yield: 76%; mp: 208–210 °C; IR (KBr): 1727 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.37–7.28 (6H, m), 7.15 (1H, s), 5.75 (2H, br),
5.42 (2H, s), 3.73 (2H, t, J = 6.9 Hz), 3.27 (2H, t, J = 6.9 Hz); 13C
NMR (75 MHz, CD3OD): d 183.72, 172.28, 142.85, 137.78, 136.47,
134.82, 131.74, 129.59, 129.51, 128.69, 128.45, 122.58, 116.21,
110.62, 56.06, 50.09, 48.92 22.42; MS (EI) m/z 418 (M+); HRMS
(EI) calcd for C20H16O2N2SCl2: 418.0310 (M+), found: 418.0351.
4.1.66. (2-Benzyl-6-chloro-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7k)
Yield: 56%; mp: 228–229 °C; IR (KBr): 1728 cmÀ1
;
1H NMR
4.1.72. (2-Phenyl-1-thioxo-1,2,3,4-tetrahydro-b-carbolin-9-yl)-
(300 MHz, DMSO-d6): d 7.77 (1H, s), 7.59 (1H, d, J = 9.0 Hz),
7.38À7.28 (6H, m), 5.76 (2H, br), 5.42 (2H, s), 3.76 (2H, t,
J = 7.3 Hz), 2.98 (2H, t, J = 7.3 Hz); 13C NMR (75 MHz, DMSO-d6): d
181.88, 169.91, 138.52, 136.29, 132.44, 128.42, 127.23, 125.07,
124.96, 123.50, 119.85, 114.57, 112.78, 54.81, 49.52, 46.68,
19.63; MS (EI) m/z 384 (M+); HRMS (EI) calcd for C20H17O2N2SCl:
384.0699 (M+), found: 384.0662.
acetic acid (7q)
Yield: 78%; mp: 200–202 °C; IR (KBr): 1721 cmÀ1 1H NMR
;
(500 MHz, DMSO-d6): d 7.74 (1H, d, J = 8.1 Hz), 7.53–7.46 (3H,
m), 7.39–7.34 (4H, m), 7.19 (1H, t, J = 7.8 Hz), 5.75 (2H, br s),
4.04 (2H, t, J = 7.0 Hz), 3.20 (2H, t, J = 7.0 Hz); 13C NMR (75 MHz,
DMSO-d6): d 183.38, 170.02, 146.22, 140.28, 131.58, 129.13,
127.14, 125.51, 122.53, 120.90, 120.66, 115.71, 110.95, 53.67,
46.46, 20.34; IR (KBr): 2914, 1721 cmÀ1; MS (EI) m/z 336 (M+);
HRMS (EI) calcd for C19H16O2N2S: 336.0933 (M+), found: 336.0926.
4.1.67. (2-Benzyl-6-iodo-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7l)
Yield: 99%; mp: 216–218 °C; IR (KBr): 1726 cmÀ1
;
1H NMR
4.1.73. (5,7-Difluoro-2-phenylethyl-1-thioxo-1,2,3,4-tetra-
(300 MHz, DMSO-d6): d 8.32 (1H, s), 8.06 (1H, d, J = 1.4 Hz), 7.56
(1H, d, J = 8.8 Hz), 7.39–7.28 (5H, m), 5.73 (2H, s), 5.42 (2H, s),
3.74 (2H, t, J = 6.9 Hz), 2.96 (2H, t, J = 6.9 Hz); 13C NMR (75 MHz,
DMSO-d6): d 181.91, 170.31, 139.18, 136.37, 132.81, 131.93,
129.09, 128.44, 127.26, 127.21, 125.03, 114.06, 113.54, 84.09,
54.79, 49.50, 46.91, 19.66; MS (EI) m/z 476 (M+); HRMS (EI) calcd
for C20H17O2N2SI: 476.0056 (M+), found: 476.0044.
hydro-b-carbolin-9-yl)acetic acid (7r)
Yield: 73%; mp: 199–201 °C; IR (KBr): 1718 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.35–7.22 (5H, m), 6.84 (1H, dd, J = 1.9,
9.2 Hz), 6.63 (1H, dd, J = 1.9, 10.1 Hz), 5.72 (2H, br), 4.32 (2H, t,
J = 7.4 Hz), 3.55 (2H, t, J = 7.1 Hz), 3.11 (2H, t, J = 7.4 Hz), 2.95 (2H,
t, J = 7.1 Hz); 13C NMR (75 MHz, CDCl3): d 181.09, 172.47, 161.64
(dd, J = 12.5, 245.4 Hz), 157.95 (dd, J = 14.9, 253.5 Hz), 142.07 (dd,
J = 12.0, 13.9 Hz), 138.45, 132.58 (d, J = 1.9 Hz), 128.96, 128.68,
126.73, 114.25 (d, J = 2.9 Hz), 109.57 (d, J = 21.1 Hz), 97.32 (dd,
J = 22.5, 29.2 Hz), 93.19 (dd, J = 4.8, 26.8 Hz), 55.98, 51.17, 47.59,
33.00, 21.23; MS (EI) m/z 400 (M+); HRMS (EI) calcd for
C21H18O2N2F2S: 400.1057 (M+), found: 400.1030.
4.1.68. (2-Benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7m)
Yield: 81%; mp: 126–128 °C; IR (KBr): 1711 cmÀ1 1H NMR
;
(300 MHz, DMSO-d6): d 7.44 (1H, d, J = 9.0 Hz), 7.35 (4H, m),
7.34–7.27 (1H, m), 7.12 (1H, d, J = 2.6 Hz), 6.98 (1H, dd, J = 2.6,
9.0 Hz), 5.73 (2H, br), 5.42 (2H, s), 3.78 (3H, s), 3.74 (2H, t,
J = 7.3 Hz), 2.96 (2H, t, J = 7.3 Hz); 13C NMR (75 MHz, DMSO-d6): d
182.11, 170.18, 154.18, 136.53, 135.67, 131.85, 128.42, 127.23,
127.16, 122.68, 116.47, 114.77, 111.95, 100.90, 55.40, 54.69,
49.55, 46.55, 19.91; MS (EI) m/z 380 (M+); HRMS (EI) calcd for
C21H20O3N2S: 380.1195 (M+), found: 380.1188.
4.1.74. (2-Benzyl-6-hydoxy-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7s)
To a stirred solution of 6m (485 mg, 1.19 mmol) in CHCl3 (3 mL)
was added BBr3 (1 M in CHCl3, 3.56 mL, 3.56 mmol), and the result-
ing mixture was stirred at room temperature for 4 h. The reaction
was quenched with 10% HCl aq, and the aqueous mixture was ex-
tracted with CHCl3 (10 mL Â 3). The organic extracts were com-
bined, dried over MgSO4, and evaporated. The residue was
chromatographed on SiO2 (Hexane–Acetone = 3:1) to give 7s
4.1.69. (2-Benzyl-6-iso-propyl-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7n)
Yield: 63%; mp: 188–190 °C; IR (KBr): 1723 cmÀ1
;
1H NMR
(343.3 mg, 73%). Mp: 200–202 °C; IR (KBr): 1721 cmÀ1 1H NMR
;
(300 MHz, CDCl3): d 7.38–7.28 (8H, m), 5.76 (2H, br), 5.45 (2H, s),
3.74 (2H, t, J = 7.0 Hz), 3.05–2.93 (3H, m), 1.29 (6H, d, J = 6.9 Hz);
13C NMR (75 MHz, CDCl3): d 183.04, 173.55, 142.22, 139.78,
136.42, 135.49, 132.24, 128.75, 127.70, 126.02, 123.19, 117.39,
116.00, 110.30, 55.59, 49.14, 47.28, 34.08, 24.35, 20.61; MS (EI) m/
z 392 (M+); HRMS (EI) calcd for C23H24O2N2S: 392.1559 (M+), found:
392.1533.
(500 MHz, DMSO-d6): d 9.07 (1H, s), 7.36–7.29 (6H, m), 6.87–6.84
(2H, m), 5.66 (2H, br), 5.42 (2H, s), 3.72 (2H, t, J = 7.0 Hz), 2.89
(2H, t, J = 7.0 Hz); 13C NMR (75 MHz, DMSO-d6): d 179.61, 170.26,
160.78, 151.69, 136.58, 135.17, 131.80, 128.42, 127.21, 123.08,
116.50, 114.18, 111.60, 103.22, 54.66, 49.53, 46.64, 19.86; MS
(EI) m/z 366 (M+); HRMS (EI) calcd for C20H18O3N2S: 366.1038
(M+), found: 336.1057.
4.1.70. (2-Benzyl-5,7-difluoro-1-thioxo-1,2,3,4-tetrahydro-
4.1.75. (2-Benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-
b-carbolin-9-yl)acetic acid (7o)
b-carbolin-9-yl)acetic acid (7t)
Yield: 47%; mp: 219–220 °C; IR (KBr): 1709 cmÀ1
;
1H NMR
Yield: 94%; mp: 208–210 °C; IR (KBr): 1704 cmÀ1 1H NMR
;
(300 MHz, DMSO-d6): d 7.41 (1H, d, J = 10.2 Hz), 7.36–7.26 (5H,
(400 MHz, DMSO-d6): d 8.33 (1H, s), 7.88 (1H, d, J = 7.3 Hz), 7.60

D. Minehira et al. / Bioorg. Med. Chem. 20 (2012) 356–367
367
(1H, d, J = 8.7 Hz), 7.35–7.27 (5H, m), 5.78 (2H, br), 5.41 (2H, s),
3.77 (2H, t, J = 7.0 Hz), 3.04 (2H, t, J = 7.0 Hz); 13C NMR (75 MHz,
DMSO-d6): d 181.83, 169.89, 167.51, 142.21, 136.33, 132.69,
127.47, 127.27, 125.79, 123.47, 123.21, 122.21, 116.35, 110.97,
54.82, 49.50, 46.86, 19.63; MS (EI) m/z 394 (M+); HRMS (EI) calcd
for C21H18O4N2S: 394.0987 (M+), found: 394.0957.
was docked into AKR1B1s (2PEV, 1ADS) and Tolrestat was docked
into AKR1B10 (1ZUA) using Glide XP method. The binding confor-
mations obtained from the X-ray crystallography were reproduced
within 2.0 Å RMSD errors for both compounds. In addition, we per-
formed the docking simulations of epalrestat for AKR1A1, AKR1B1,
and AKR1B10. We also confirmed that there is a good linear corre-
lation between the Glide Docking Scores and pIC50 values as shown
in Figure 3. The Glide Score seems to be good estimation of the
binding affinities of ligands for AKR1A1, AKR1B1, and AKR1B10.
We have performed the docking simulations for 7t, after careful
validations of the computational ligand-docking method. As previ-
ously mentioned, we selected one pose with the best Glide score as
the binding pose of the compound 7t. The best Glide scores for
AKR1B1 (1ADS) and AKR1B10 (1ZUA) were À8.60 and À7.76 kcal/
mol, respectively. Then, we analyzed the difference between the
binding affinities of 7t to AKR1B1 and to AKR1B10, comparing
the binding modes of 7t to them.
4.2. Biological assays
4.2.1. Preparation of recombinant AKR1B1, AKR1B10 and
AKR1A1
AKR1B10 with N-terminal 6-His tag, AKR1B1 and AKR1A1 with-
out any additional amino acid were expressed in Escherichia coli
BL21(DE3) pLysS cells transformed with the expression plasmids
harboring their cDNAs, and purified to homogeneity as described
previously.14,28
4.2.2. Inhibition assays
The reductase activities of AKRs (1B1, 1B10 and 1A1) were as-
sayed by measuring the rate of change in NADPH absorbance (at
340 nm) accompanying the substrate reduction at 25 °C. The reac-
tion mixture consisted of 0.1 M potassium phosphate buffer, pH
7.4, 0.1 mM NADPH, substrate and enzyme, in a total volume of
2.0 ml. The substrate in the assay of both AKR1B1 and AKR1B10
activities was 0.2 mM pyridine-3-aldehyde, and that of AKR1A1
Acknowledgment
This work was supported in part by Research for Promoting
Technological Seeds, Japan Science and Technology Agency (JST).
We gratefully acknowledge Dr. Ossama El-Kabbani, Monash Insti-
tute of Pharmaceutical Sciences, Medicinal Chemistry and Drug Ac-
tion, for his helpful discussion.
was 10 mM
D-glucuronate. In the determination of IC50 values,
the inhibitors were dissolved into methanol, and added into the
reaction mixture, in which the final concentration of methanol
was less than 2.5%. Kinetic studies in the presence of inhibitors
were carried out in pyridine-3-aldehyde reduction over a range
of five substrate concentrations (0.2–5 Â Km) at the saturating con-
centration of NADPH. The inhibition pattern was analyzed by fit-
ting the initial velocities in the Lineweaver–Burk plot, in which a
noncompetitive inhibitor gave two inhibition constants, Kis (slope
effect) and Kii (intercept effect). The IC50 and Ki values are ex-
pressed as the means standard errors of at least three
determinations.
References and notes
1. Shaw, J. E.; Sicree, R. A.; Zimmet, P. Z. Diabetes Res. Clin. Pract. 2010, 87, 4.
2. Szwergold, B. S.; Kappler, F.; Brown, T. R. Science 1990, 247, 451.
3. Gonzalez, A. M.; Sochor, M.; Hothersall, J. S.; McLean, P. Diabetes 1986, 35, 1200.
4. Wells-Knecht, K. J.; Brinkmann, E.; Wells-Knecht, M. C.; Litchfield, J. E.; Ahmed,
M. U.; Reddy, S.; Zyzak, D. V.; Thorpe, S. R.; Baynes, J. W. Nephrol. Dial.
Transplant. 1996, 11, 41.
5. Robinson, W. G., Jr.; Kador, P. F.; Kinoshita, J. H. Science 1983, 221, 1177.
6. Engerman, R. L. Diabetes 1989, 38, 1203.
7. Young, R. J.; Ewing, D. J.; Clarke, B. F. Diabetes 1983, 32, 938.
8. Dunlop, M. Kidney Inter. 2000, 58, S-3.
9. Jez, J. M.; Flynn, T. G.; Penning, T. M. Biochem. Pharmacol. 1997, 54, 639.
10. Linster, C. L.; Van Schaftingen, E. FEBS J. 2007, 274, 1.
11. Kanazu, T.; Shinoda, M.; Nakayama, T.; Deyashiki, Y.; Hara, A.; Awada, H.
Biochem. J. 1991, 279, 903.
12. Cao, D.; Fan, S. T.; Chung, S. S. J. Biol. Chem. 1998, 273, 11429.
13. Gallego, O.; Ruiz, F. X.; Ardévol, A.; Dominguez, M.; Alvarez, S.; de Lera, A. R.;
Rovira, C.; Farrés, J.; Fita, I.; Parés, X. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
20764.
14. Endo, S.; Matsunaga, T.; Mamiya, H.; Ohta, C.; Soda, M.; Kitade, Y.; Tajima, K.;
Zhao, H. T.; El-Kabbani, O.; Hara, A. Arch. Biochem. Biophys. 2009, 487, 1.
15. Ma, J.; Yan, R.; Zu, X.; Cheng, J. M.; Rao, K.; Liao, D. F.; Cao, D. J. Biol. Chem. 2008,
283, 3418.
16. Van Zandt, M. C.; Doan, B.; Sawicki, D. R.; Sredy, J.; Podjarny, A. D. Bioorg. Med.
Chem. Lett. 2009, 19, 2006.
4.2.3. Molecular docking
The docking simulations were performed with Glide in Schrö-
dinger Suite 2009 (Schrödinger, LLC). The docking poses of each li-
gand were generated with Glide extra-precision (XP) method, and
we selected the pose with the best Glide score as its binding poses
for each ligand. The protein coordinates were taken from the Pro-
tein Data Bank whose entry code is 1ADS and 2PEV for AKR1B1 and
1ZUA for AKR1B10. Before the docking study of 7t, we validated the
application of Glide XP method using Fidarestat and Tolrestat,
whose X-ray crystal structures complexed with AKR1B1 (2PEV)
and AKR1B10 (1ZUA), respectively, had been known. Fidarestat
17. Chen, X.; Zhu, C.; Guo, F.; Qiu, X.; Yang, Y.; Zhang, S.; He, M.; Parveen, S.; Jing,
C.; Li, Y.; Ma, B. J. Med. Chem. 2010, 53, 8330.
18. Maccari, R.; Ciurleo, R.; Giglio, M.; Cappiello, M.; Moschini, R.; Del Corso, A.;
Mura, U.; Ottanà, R. Bioorg. Med. Chem. 2010, 18, 4049.
19. Maccari, R.; Del Corso, A.; Giglio, M.; Moschini, R.; Mura, U.; Ottanà, R. Bioorg.
Med. Chem. Lett. 2011, 21, 200.
20. Kato, A.; Kobayashi, K.; Narukawa, K.; Minoshima, Y.; Adachi, I.; Hirono, S.;
Nash, R. J. Bioorg. Med. Chem. 2010, 20, 5630.
21. Kato, A.; Yasuko, H.; Goto, H.; Hollinshesd, J.; Nash, R. J. Phytomedicine 2009, 16,
258.
22. Lehmann, J.; Pohl, U. Arch. Pharm. 1987, 320, 1202.
23. Toyooka, N.; Takeda, D.; Minoshima, Y.; Kato, A.; Adachi, I. Heterocycles 2009,
79, 1073.
24. Petrash, J. M.; Tarle, I.; Wilson, D. K.; Quiocho, F. A. Diabetes 1994, 143, 955.
25. Grimshaw, C. E.; Shahbaz, M.; Jahangiri, G.; Putney, C. G.; McKercher, S. R.;
Mathur Eur. J. Biochem. 1989, 28, 5343.
26. Ehrig, T.; Bohren, K. M.; Prendergast, F. G.; Gabbay, K. H. Biochemistry 1994, 33,
7157.
27. Lehmann, J.; Ghoneim, K. H.; El-Gendy, A. A. Arch. Pharm. 1987, 320, 22.
28. Endo, S.; Matsunaga, T.; Kuwata, K.; Zhao, H.-T.; El-Kabbani, O.; Kitade, Y.;
Hara, A. Bioorg. Med. Chem. 2010, 18, 2485.
Figure 3. The correlation between Glide Score and pIC50 values for AKR1A1,
AKR1B1, and AKR1B10.