Discovery of tetrahydro-β-carbolines as inhibitors of the mitotic kinesin KSP

Article abstract of DOI:10.1016/j.bmc.2010.05.024

Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the synthesis and biological evaluation of a novel series of tetrahydro-β-carboline analogs based on the structure of the known KSP inhibitor HR22C16. Preferred compounds 11b, 12a and 19b were identified as potent inhibitors in a KSP ATPase assay with good anti-proliferative activity in A549 cells.

Full text of DOI:10.1016/j.bmc.2010.05.024

Bioorganic & Medicinal Chemistry 18 (2010) 4167–4177
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of tetrahydro-b-carbolines as inhibitors of the mitotic kinesin KSP
Fei Liu a,b, Li-Qin Yu a,b, Cheng Jiang a,b, Lei Yang c, Wu-Tong Wu d, Qi-Dong You a,b,
*
a Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
b Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
c Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
d School of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic
arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the synthesis
and biological evaluation of a novel series of tetrahydro-b-carboline analogs based on the structure of
the known KSP inhibitor HR22C16. Preferred compounds 11b, 12a and 19b were identified as potent
inhibitors in a KSP ATPase assay with good anti-proliferative activity in A549 cells.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 25 March 2010
Revised 5 May 2010
Accepted 6 May 2010
Available online 11 May 2010
Keywords:
Mitotic kinesin
Inhibitor
Kinesin spindle protein
KSP
Tetrahydro-b-carboline
Anti-proliferation
1. Introduction
In 1999, Mayer et al. discovered monastrol, the first small mol-
ecule able to inhibit the mitotic kinesin Eg5 (KSP) using a pheno-
Antimitotic agents, such as vinca alkaloids, taxol and epothil-
ones, are a major class of cytotoxic drugs in the treatment of can-
cer. By interfering with the tubulins’ polymerization and
depolymerization, antimitotics inhibit mitotic spindle and arrest
dividing cells in metaphase.1,2 However, the use of these agents
is associated with undesired side effects, such as neurotoxicity, re-
lated to the central role tubulin plays in cellular transport pro-
cesses.3 The mechanism-related toxicities and acquired resistance
have stimulated considerable interest in developing antimitotics
which do not act on microtubule directly.
Members of the kinesin superfamily play important roles in car-
go transport, spindle and chromosome movement, and regulation
of microtubule dynamics.4 KSP (kinesin spindle protein, also
known as Hs Eg5) is a plus-end-directed motor of the BimC kinesin
subfamily which is responsible for the formation of the bipolar
spindle.5 KSP plays an important role in the early stage of mitosis
and mediates centrosome separation. Inhibition of KSP leads to a
stable mitotic block with monoastral microtubule arrays.6 The
function of KSP provides a novel route for the manipulation of
the cell cycle and the induction of apoptosis. Therefore, KSP inhib-
itors have become attractive and promising anti-proliferative
agents for cancer chemotherapy.7,8
type-based assay.9 Subsequently, a series of second-generation of
monastrol analogs with improved cellular potency and solubility
was developed. Moreover, several inhibitors that exhibit great
chemical diversity have also been reported including CK0106023,
dihydropyrazoles, tetrahydroquinolines, HR22C16, tetrahydroiso-
quinolines, and many more (Fig. 1).10,11 HR22C16 possesses a tet-
rahydro-b-carboline based structure and several laboratories
have reported efforts around it.12,13 Recently, a co-crystal structure
of an analog of HR22C16 in the KSP motor domain has confirmed
that this series of inhibitors binds in the allosteric loop L5 binding
pocket.14 The X-ray crystal structure also suggests that the alkyl
O
N
OH
O
N
O
N
Cl
O
O
NH
O
N
N
N
H
OH
H3C
N
H
S
Br
CK0106023
Monastrol
HR22C16
* Corresponding author.
E-mail address: youqidong@gmail.com (Q.-D. You).
Figure 1. The KSP inhibitors monastrol, CK0106023 and HR22C16.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.024

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F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
side chain along with the hydantoin ring point towards the solvent
exposed areas of the binding pocket, and what’s more, this
HR22C16 analog forms a hydrogen bond to the KSP backbone
through its phenol. During the course of preparing this paper, the
X-ray structure of a terahydro-b-carboline analogs bound to Eg5
in an allosteric site of Eg5 (PDB code: 3K3B) was reported,15 which
depicted that there was a strong hydrogen bond between the phe-
nolic –OH and the backbone carbonyl of Glu118, as well as a hydro-
gen bond between the carboline –NH and amide carbonyl of
Glu116. A series of N-acyl-tetrahydro-b-carbolines were studied
afterward and SAR was also discussed in due course.
measured by the MT-activated ATPase activity and A549 cell
proliferation.
2. Chemistry
Pictet–Spengler cyclization was one of the most powerful meth-
ods to synthesize the tetrahydro-b-carboline ring system. Utilizing
diverse 3-indolylethylamines and benzaldehyde as substrates, the
core scaffold of tetrahydro-b-carbolines was constructed favorably.
The following compounds were all synthesized as racemic
mixtures.
In the present paper, guided by the results of the co-crystal struc-
ture of HR22C16 analog, we describe our effort on designing more
potent terahydro-b-carboline analogs with the speculation that
retaining tetrahydro-b-carboline core with a pendant 3-hydroxy-
phenyl, removal of hydantoin ring of HR22C16 and varying the mod-
ifications at the N2, C3, C4 and C6/8 positions could lead to potent
KSP inhibitors (Fig. 2). Alternate to the pendent 1-(3-hydroxy-
phenyl) group was also investigated, giving rise to 1-(3-indolyl)-2-
acyl tetrahydro-b-carbolines in which the 3-indolyl ring substituent
was supposed to act in a similar fashion as the 3-hydroxyphenyl
moiety. Following above ideas, four structural series of compounds
were synthesized and tested for there ability to inhibit KSP
The synthetic pathways used to prepare 2,3-dicarbonyl tetrahy-
dro-b-carboline derivatives are shown in Schemes 1. Commercially
available tryptophan underwent Pictet–Spengler cyclization with
3-hydroxy benzaldehyde to give 3-carboxylic acid intermediate,
which was further esterified to 3-ethyl carboxylate 5. Treatment
of compound 5 with diverse acyl chloride led to the bis-acylated
compounds which underwent selective single hydrolysis when
treated with NaHCO3 to generate the desired compound 6a–d.
Compounds 6b and 6c were further hydrolyzed to 3-carboxylic
acid 7a and 7b (Scheme 1).
3-Amide tetrahydro-b-carboline analogs, 8a and 8b, were pre-
pared from 3-ethyl carboxylate intermediate 5 under amination
O
O
R'
NH
R
-COOR
R'
O
-CONHR
N
-H
R
-H,-Cl,-CH3
N
H
R2
-COR
-CH2R
-H
OH
N
H
3
OH
2N R3
R16
2
1
1
N
H
R'
O
R4
8
N
R'
N
R
R
N
H
N
H
NH
H
OH
OH
N
3
4
Figure 2. Design of novel series of tetrahydro-b-carboline analogs: series 1—N2,C3-dicarbonyl-1-(3-hydroxylphenyl) tetrahydro-b-carboline analogs; series 2—C3-acyl-1-
(3-hydroxylphenyl) tetrahydro-b-carboline analogs; series 3—N2-substituted-1-(3-hydroxylphenyl) tetrahydro-b-carboline analogs; series 4—N2-acyl-1-(3-indolyl) tetra-
hydro-b-carboline analogs.
O
COOC2H5
NH
NH
R2
NH
COOH
CHO
f
a, b
OH
NH2
+
N
H
N
H
OH
OH
N
H
5
c, d
8a R2=-CH2CH2OH
8b R2=-CH2CH2NH2
COOH
COOC2H5
O
O
e
N
N
R1
R1
N
H
N
H
OH
OH
6a R1=-CH3
7a R1=-CH2CH3
7b R1=-CH2CH2CH3
6b R1=-CH2CH3
6c R1=-CH2CH2CH3
6d R1=-CH(CH3)2
Scheme 1. Synthesis of 2,3-dicarbonyl and 3-amide tetrahydro-b-carboline derivatives. Reagents and condition: (a) acetic acid, 95 °C; (b) SOCl2, C2H5OH; (c) TEA, THF, acyl
chloride; (d) NaHCO3; (e) 10% NaOH, then HCl; (f) 2-aminoethanol or 1,2-diaminethane.

F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
4169
condition using ethanolamine and 1,2-diaminethane, respectively
(Scheme 1).
In order to obtain 3-unsubstituted tetrahydro-b-carboline ana-
logs, tryptamine and substituted tryptamine were first prepared
according the procedure reported by our group.16 Utilizing the Pic-
tet–Spengler cyclization conditions described in Scheme 1, the tet-
dehalogenated byproduct was observed when 6-chrolo-30OBn tet-
rahydro-b-carbolines was subjected to the deprotection condition
of Pt–C/HCOONH4, isopropyl is a good alternation for benzyl group
at this manner, which was favorably deprotected with AlCl3.
Different from one step Pictet–Spengler cyclization, the building
of 1-(3-indolyl) tetrahydro-b-carboline core 18 involves two steps:
a condensation reaction between tryptamine and 1H-indole-3-
carbaldehyde to generate the corresponding imine, and then an
intramolecular cyclization catalyzed by TFA to finish the cyclized
intermediate 18 (Scheme 4). Further treatment of 18 with diverse
acyl chloride provided desired compounds 19a–f. Conversion of 18
to the final 2-(b-aminopropionyl) 19g was achieved by coupling
with N-Boc b-alanine, followed by Boc cleavage with HCl.
rahydro-b-carboline core
9 were prepared from substituted
tryptamine and 3-hydroxy benzaldehyde. N2-Aminoalkylacyl com-
pounds 11a–b were obtained via standard amide coupling reaction
with Boc protected amino acid followed by Boc deprotection. p-
Aminobenzoic acid condensed directly with 9 providing desired
p-Aminobenzoyl 12a–b (Scheme 2).
2-Alkyl compounds 13a–e were synthesized utilizing Leuckart
reaction conditions (Scheme 3). In order to generate the homolo-
gated 2-aminoalkyl analogs 17, C6 substituted tryptamine was cy-
clized with 3-hydroxy-protected benzaldehyde to generate
intermediate 14, followed by standard coupling, hydrazinolysis
and deprotection, to furnish 2-aminoalkyl analogs 17a–f. Since
3. Result and discussions
All synthesized tetrahydro-b-carbolines were tested for their
inhibitory activities against KSP determined by measuring the
O
CHO
HCl
NH
b
HCl
+
a
NH2
d
N
R1
NH2
R1
R1
N
H
N
H
N
H
OH
OH
OH
9a R1= 6-Cl
9b R1= 6-CH3
9c R1= 8-Cl
12a R1= 6-Cl
12b R1= 6-CH3
NH2 HCl
NHBoc
OH
X
X
N
N
c
O
O
R1
R1
N
H
N
H
OH
10a
10b
11a R1= 6-Cl, X=-CH(CH3)-
11b R1= 8-Cl, X=-CH2CH2-
Scheme 2. Synthesis of N2-acyl tetrahydro-b-carboline derivatives. Reagents and condition: (a) acetic acid, reflux, then HCl; (b) PyBOP, TEA, THF, BocNHCH(CH3)COOH or
BocNHCH2CH2COOH; (c) HCl, ethyl acetate; (d) PyBOP, TEA, THF, 4-aminobenzoic acid.
CH2R2
N
NHHCl
a
R1
R1
N
H
N
H
OH
OH
9
13a R1= 6-Cl, R2=H
13d R1= 6-CH3, R2=H
13b R1= 6-Cl, R2=-C2H5 13e R1= 6-CH3, R2=-Ph
13c R1= 6-Cl, R2=-Ph
O
CHO
NHHCl
c
(CH2)n
N
N
b
NH2HCl
+
R1
R1
R1
N
H
O
N
H
N
OR2
OR2
OR2
H
14a R1= 6-CH3 R2=Bn
15a-f
14b R1=6-Cl R2= Isopropyl
(CH2)n NH2
OH
(CH2)n NH2
OR1
N
N
R1
R1
e
d
N
H
N
H
16d R1=Cl, R2= Isopropyl, n=2
16e R1=Cl, R2= Isopropyl, n=3
16f R1=Cl, R2= Isopropyl, n=4
16a R1=-CH3, R2=Bn, n=2
16b R1=-CH3, R2=Bn, n=3
16c R1=-CH3, R2=Bn, n=4
17a R1=-CH3, n=2 17d R1=Cl, n=2
17b R1=-CH3, n=3 17e R1=Cl, n=3
17f R1=Cl, n=4
17c R1=-CH3, n=4
Scheme 3. Synthesis of N2-alkly tetrahydro-b-carboline derivatives. Reagents and conditions: (a) R2CHO, K2CO3, HCOOH, DMF; (b) CH3COOH, 95 °C, then HCl; (c) KOH, DMF,
O
reflux; (d) CH3CH2OH, NH2–NH2ꢀH2O
, reflux; (e) 10% Pd–C, HCOONH or AlCl3,CH2Cl2.
4
N (CH2)nBr
O

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F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
NH
CHO
NH2
+
R1
c or d
a, b
N
H
R1
NH
N
H
N
H
O
18
N
R2
R1
19a R1= 6-CH3 R2= -CH3
19d R1= 6-Cl R2= -CH3
N
H
19b R1= 6-CH3 R2= -CH(CH3)2
19c R1= 6-CH3 R2= -CH2CH2CH3
19g R1= 6-Cl R2= -CH2CH2NH2HCl
19e R1= 6-Cl R2= -CH(CH3)2
19f R1= 6-Cl R2= -CH2CH2CH3
NH
Scheme 4. Synthesis of 1-(3-indolyl) tetrahydro-b-carboline derivatives. Reagents and conditions: (a) toluene, reflux; (b) TFA; (c) R2COCl, TEA, THF; (d) PyBOP, TEA,
BocNHCH2CH2COOH, THF, then HCl, EtOAc.
MT-activated ATPase activity. The anti-proliferative cytotoxic
activity was also evaluated by using A549 tumor cell line.
CK0106023 was synthesized and used as positive control.17
The initial series tested were the 2,3-dicarbonyl-1-(3-hydroxyl-
phenyl) tetrahydro-b-carboline analogs (6 and 7) represented in
Table 1. There appears to be an optimal chain length of the substi-
tutes at the N2 while an ethyl carboxylate group was substituted at
C3 position. The maximal activity of the propionyl 6b is reduced
neither as the length increases to the butyryl 6c nor shorten to
acetyl analog 6a. Additional branching did not improve activity,
as seen with isopropyl analog 6d. The hydrolysis of 3-ethyl carbox-
ylate into 3-carboxylic acid analogs leads to a twofold (7a vs 6b)
and 15-fold (7b vs 6c) loss of KSP activity. 3-(N-(2-Aminoethyl))
amide 8b exhibited sub-micromolar KSP activity (65 nM). As the
amino group of 8b changed to hydroxyl (8a), fourfold of decrease
in activity was observed, indicating a strict special restriction close
to the solvent exposed region of enzyme pockets. However, all of
these compounds were relatively ineffective at inhibiting cell pro-
liferation in the A549 assay. The poor cellular activity of these com-
pounds prompted the research for alterative substitutions.
The next investigation of tetrahydro-b-carboline core was fo-
cused on C6/8 substitution along with N2 acylation (Table 2).
Table 1
Activities of 1-(3-hydroxyphenyl) tetrahydro-b-carboline compounds
R2
N
R3
R1
N
OH
H
Compound
R1
R2
R3
–COCH3
–COCH2CH3
–COCH2CH2CH3
–COCH(CH3)2
–COCH2CH3
–COCH2CH2CH3
H
KSPa IC50
(
lM)
A549b IC50
(lM)
6a
6b
6c
6d
7a
7b
8a
8b
11a
11b
H
H
H
H
H
H
H
H
–COOC2H5
–COOC2H5
–COOC2H5
–COOC2H5
–COOH
0.724
0.075
0.224
0.380
0.118
3.453
0.290
0.065
0.483
0.040
>10
>10
>10
>10
>10
>10
>10
>10
5.65
0.94
–COOH
–CONHCH2CH2OH
–CONHCH2CH2NH2
H
H
6-Cl
8-Cl
–COCH(CH3)NH2HCl
–COCH2CH2NH2HCl
H
O
12a
12b
6-Cl
H
0.076
6.66
NH2
O
6-CH3
H
0.050
7.93
NH2
13a
13b
13c
13d
13e
17a
17b
17c
17d
17e
17f
CK0106023
6-Cl
6-Cl
6-Cl
6-CH3
6-CH3
6-CH3
6-CH3
6-CH3
6-Cl
H
H
H
H
H
H
H
H
H
H
H
—
–CH3
–CH2CH2CH3
–CH2Ph
–CH3
–CH2Ph
–CH2CH2NH2HCl
–CH2CH2CH2NH2HCl
–CH2CH2CH2CH2NH2HCl
–CH2CH2NH2HCl
–CH2CH2CH2NH2HCl
–CH2CH2CH2CH2NH2HCl
—
0.664
0.169
0.710
0.462
12.378
0.237
0.083
0.454
0.007
0.204
0.347
0.049
1.38
1.55
5.70
8.73
6.86
7.79
6.86
9.19
8.70
6.73
9.06
7.02
6-Cl
6-Cl
—
a
The inhibitory activities against KSP were determined by measuring the MT-activated ATPase activity.
Data represent mean values of at least three separate experiments.
b

F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
4171
Table 2
Table 3
Activities of 1-(3-indolyl) tetrahydro-b-carboline compounds
Cellular IC50 (lM) of selected compounds
Compound
A549
AGS
HepG2
HT-29
PC-3
N R2
11b
12a
12b
13a
16d
19f
19g
CK0106023
0.94
6.66
7.93
1.38
8.70
6.83
1.79
7.02
1.31
6.07
8.86
0.75
8.39
7.06
6.16
5.10
7.47
8.52
>10
>10
>10
7.64
>10
>10
5.75
6.30
7.47
7.40
7.44
5.86
6.28
3.33
1.28
7.30
>10
>10
>10
7.29
1.55
>10
R1
N
H
NH
Compound
R1
R2 IC50
(
lM)
KSPa IC50
M)
A549b IC50
M)
(
l
(l
19a
19b
19c
19d
19e
19f
19g
CK0106023
6-CH3
6-CH3
6-CH3
6-Cl
6-Cl
6-Cl
–COCH3
0.051
0.216
>10
>10
>10
9.80
6.83
9.11
1.79
7.02
–COCH2CH2CH3
–COCH(CH3)2
–COCH3
–COCH2CH2CH3
–COCH(CH3)2
–COCH2CH2NH2HCl
—
1-(3-Indolyl)-2-acyl-6-methyl tetrahydro-b-carbolines (Table
2), 19a–c, exhibited KSP enzymatic inhibition, but lacked potent
cellular activity. However, optimal KSP activity (IC50 < 1 nM) is ob-
served with the 2-isobutyryl 19c, relative to the corresponding 2-
acetyl 19a and 2-butyryl 19b, suggesting branched 3-atom chain
is optimal. The KSP activity trend is similar for the 6-chloro ana-
logs; however, they are 7- to 160-fold less potent than the corre-
sponding 6-methyl analogs. Interestingly, their loss in KSP
potency leads to an increase in cellular activity. With an amino
group at the terminal of 2-propionyl, 1-(3-indolyl)-6-chloro tetra-
hydro-b-carboline 19g displayed ꢁ40- and ꢁ4-fold of improve-
ment in KSP and cellular inhibitory activity, respectively,
compared with 2-isobutyryl 19e. The 3-hydroxyl along with the
pendant phenyl is alternative with 3-indolyl, suggesting the latter
group offer the same function as the former.
The most promising compounds representing different struc-
tural series were selected for further evaluation for their anti-pro-
liferative cytotoxic activity against other human tumor cell lines,
such as AGS (stomach), HepG2 (liver), HT-29 (colon) and PC-3
(prostate) (Table 3). The majority of the compounds displayed
excellent anti-proliferative profiles, showing that removal of
hydantoin ring of HR22C16 could maintain biological effects of
the molecules. In general, these compounds showed lower anti-
proliferative activities against HepG2 and PC-3 than against
A549, AGS and HT-29 cells.
<0.001
0.337
1.703
0.163
0.043
0.049
6-Cl
—
a
The inhibitory activities against KSP were determined by measuring the MT-
activated ATPase activity.
b
Data represent mean values of at least three separate experiments.
Within the two 2-aminopropionyl derivatives, optimal KSP and
cellular activity (IC50 = 40 and 930 nM, respectively) is observed
with the b-amino analog 11b, relative to the corresponding a-ami-
no analog 11a. Both 2-(p-aminobenzoyl) analogs 12a and 12b are
highly potent KSP inhibitors with good cellular activity. The im-
proved A549 activity and sub-micromolar KSP potency seen with
11 and 12 prompted analysis of the N2-alkyl tetrahydro-b-
carbolines.
To further investigate the enzymatic and cellular inhibitory
activity of 2,6-substituted KSP inhibitors, N2-alkyl tetrahydro-b-
carbolines were synthesized. The initial analogs tested, compounds
13a–e exhibit moderate KSP and cellular activity. Appending a
phenyl group at 2-methyl gave a little increase (cf. 13c with 13a)
or significantly reduced (cf. 1e with 1d) of KSP inhibiting capacity.
Interestingly the 6-methyl-2-benzyl 13e although showing weak
enzyme inhibition displayed moderate micromolar anti-prolifera-
tive activity.
4. Conclusion
Incorporation of an amino group at the terminal region of
hydrophobic chain was proved to be preferred in the solvent ex-
posed region, exemplified by 8b and 11b. As seen with the 2-ami-
noalkyl analogs 17a–f, a trend of an optimal length of chain
between the terminal amino and tetrahydro-b-carboline core was
observed. As the aminoalkyl chain of 6-methly analogs is extended
form the aminoethyl 17a, aminopropyl 17b, to aminobutyl 17c,
KSP activity is maximal with an optimal three carbon atom linker
17b. However, when it referred to 2-alkyl-6-chloro tetrahydro-b-
carbolines, aminoethyl 17d with two carbon atom chain is the
most potent (APTase IC50 = 7 nM). In general, the cellular activities
tested were proximately identical and displayed no obvious depen-
dence on KSP potency.
Above all, among the most potent compounds with sub-micro-
molar KSP activity, 3-(N-(2-aminoethyl)) amide 8b, for example,
possesses similar potency compared to 8-chloro-2-(b-aminopropi-
onyl) 11b, 6-chloro-2-(p-aminobenzoyl) 12a and 6-methyl-2-ami-
nopropyl 17b (65 vs 40, 76 and 83 nM, respectively), suggesting
the length of chain between the terminal amino and tetrahydro-
b-carboline core is an important driver of potency and not neces-
sarily the functionality contained in the linker or the substitution
position of N2 or C3. As seen in the first three structural series,
there is a preference for a 2- to 4-atom length of chain between
the terminal amino group and the tetrahydro-b-carboline core,
which were diverged between different structural series. More-
over, the substitution at C3 position seems to be detrimental to
cytotoxicity.
We have identified a series of novel tetrahydro-b-carboline KSP
inhibitors that are characterized by high potency in KSP and cellular
proliferation assays. Four structural series of tetrahydro-b-carbo-
lines were synthesized and the most active compounds were 8-
chloro-2-(b-aminopropionyl) 11b, 6-chloro-2-(p-aminobenzoyl)
12a, 1-(3-indolyl)-2-(b-aminopropionyl) 19g. Substitution of the
tetrahydro-b-carboline core at the C6- or C8-positions was required
for good cellular potency. The KSP potency maintained by incorpo-
rating C1-(3-indolyl) groups in stead of the phenol, coupled with
introduction of acyl moiety at C2 position. Moreover, all the four
structural series described herein were found to exhibit the similar
SAR with regard to the length of the side chain. Modification the ter-
minal of side chain with an amino group led to an increase in KSP
inhibitory activity. Further studies of these agents and related ana-
logs will be reported in due course.
5. Experimental
5.1. General
Melting points were determined on a Mel-TEMP II melting point
apparatus and are uncorrected. Infrared (IR) spectra (KBr) were re-
corded on a Nicolet Impact 410 instrument (KBr pellet). 1H NMR
spectra were recorded with a Bruker Avance 300 MHz spectrome-
ter at 300 K, using TMS as an internal standard. MS spectra were
recorded on a Shimadzu GC–MS 2010 (EI) or a Mariner Mass Spec-

4172
F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
trum (ESI), or a LC/MSD TOF HR-MS Spectrum. All compounds were
routinely checked by TLC and 1H NMR. TLCs and preparative thin-
layer chromatography were performed on silica gel GF/UV 254, and
the chromatograms were performed on silica gel (200–300 mesh)
visualized under UV light at 254 and 365 nm. All solvents were re-
agent grade and, when necessary, were purified and dried by stan-
dards methods. Concentration of solutions after reactions and
extractions involved the use of a rotary evaporator operating at a
reduced pressure of ca. 20 Torr. Organic solutions were dried over
anhydrous sodium sulfate. Analytical results are within (0.40% of
the theoretical values).
142 °C. IR (cmꢃ1): 3396, 2966, 1743, 1619, 1455. 1H NMR
(300 MHz, DMSO-d6): d 0.73 (m, 3H, –OCH2CH3), 0.93 (m, 3H,
–CH2CH2CH3), 1.62 (m, 2H, –CH2CH2CH3), 2.54 (m, 2H,
–CH2CH2CH3), 2.98 (m, 2H, –OCH2CH3), 3.44 (d, 1H, CH2, J = 16.8),
3.60 (m, 1H, CH2), 3.85 (m, 1H, CH), 5.25 (d, 1H, CH-Ar, J = 6.6),
6.55–7.53 (m, 8H, Ar-H), 9.24 (s, 1H, OH), 10.89 (s, 1H, NH). EI-
MS (m/z): 406 (M+). Anal. Calcd for (C24H26N2O4): C, 70.92; H,
6.45; N, 6.89. Found: C, 70.60; H, 6.49; N, 6.88.
5.1.5. Ethyl 1-(3-hydroxyphenyl)-2-isobutyryl-1,2,3,4-
tetrahydro-b-carboline-3-carboxylate (6d)
Compound 6d (0.21 g) was synthesized from 5 (0.34 g, 1 mmol)
and isobutyryl chloride (0.21 mL, 2 mmol) according to the proce-
dure used to synthesize 6a as an off-white solid in 51.5% yield. Mp:
129–131 °C. IR (cmꢃ1): 3394, 2930, 1721, 1632, 1457. 1H NMR
(300 MHz, DMSO-d6): d 0.80 (m, 9H, –OCH2CH3, –CH(CH3)2), 2.68
(m, 2H, –OCH2CH3), 2.91 (m, 1H, CH2), 3.21 (m, 1H, CH2), 3.35
(m, 1H, –CH(CH3)2), 3.70 (m, 1H, CH), 5.13 (d, 1H, CH-Ar, J = 6.6),
6.32–7.30 (m, 8H, Ar-H), 9.02 (s, 1H, OH), 10.68 (s, 1H, NH). EI-
MS (m/z): 406 (M+). Anal. Calcd for (C24H26N2O4): C, 70.92; H,
6.45; N, 6.89. Found: C, 70.61; H, 6.64; N, 6.99.
5.1.1. Ethyl 1-(3-hydroxyphenyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylate (5)
A mixture of tryptophan (1.02 g, 5.0 mmol) and 3-hydroxybenz-
aldehyde (0.67 g, 5.5 mmol) was dissolved in glacial acetic acid
(15 mL) under N2 protection. After stirring at 95 °C for 2 h, the mix-
ture was cooled to room temperature. The resulted precipitate was
filtered and dissolved in ethanol (50 mL). Thionyl chloride
(0.57 mL, 8.0 mmol) was added slowly over a period of 10 min.
After stirring at 80 °C for 2 h, the solvent was concentrated and
the pH of the mixture was adjusted to 8 with saturate NaHCO3
aqueous solution. Afterward, the solution was extracted with ace-
tic ether (3ꢂ 20 mL). The organic phase was concentrated and puri-
fied by column chromatography on silica gel by eluting with a
mixture of petroleum ether/ethyl acetate (1:1) to give the desired
product (1.14 g) as off-white solid in 67.8% yield; mp 198–200 °C.
5.1.6. 1-(3-Hydroxyphenyl)-2-propionyl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (7a)
Compound 6b (0.20 g, 0.53 mmol) was dissolved in a mixture of
ethanol (20 mL) and 10% NaOH aqueous solution (20 mL) and the
solutionwas allowed to stir at room temperature for 1 h. The ethanol
was then removed in vacuo. Afterward, the remaining residue was
acidified to pH ꢁ4 with 10% HCl and extracted with acetic ether
(3ꢂ 30 mL). The organic phase was dried and evaporated to dryness
to furnish crude product which was purified by column chromatog-
raphy on silica gel by eluting with a mixture of dichloromethane/
methanol (20:1) to give the desired product 7a (0.12 g, 63.0%) as a
white solid. Mp: 143–147 °C. IR (cmꢃ1): 3340, 1720, 1622, 1454,
1272. 1H NMR (300 MHz, DMSO-d6): d 0.81 (m, 3H, –COCH2CH3),
2.08 (m, 1H, –COCH2CH3), 3.27 (m, 2H, CH2), 5.20 (m, 1H, CH), 5.99
(d, 1H, CH-Ar, J = 35.7), 6.50–7.45 (m, 8H, Ar-H), 9.40 (d, 1H, OH,
J = 50), 10.93 (d, 1H, NH, J = 18.6), 12.50 (s, 1H, –COOH). EI-MS (m/
z): 364 (M+). Anal. Calcd for (C21H20N2O4ꢀH2O): C, 65.96; H, 5.80; N,
7.33. Found: C, 66.42; H, 6.02; N, 6.73.
5.1.2. Ethyl 1-(3-hydroxyphenyl)-2-acetyl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylate (6a)
Acetyl chloride (0.14 mL, 2 mmol) in THF (5 mL) was added to a
mixture of compound 5 (0.34 g, 1 mmol) in THF (10 mL) over a per-
iod of 20 min. After stirring at room temperature for 2 h, the mix-
ture was concentrated and added saturate NaHCO3 aqueous
solution (20 mL) and acetone (20 mL). After stirring for additional
3 h, the acetone was evaporated and the water layer was extracted
with acetic ether (3ꢂ 30 mL). Removal of the solvent in vacuo
afforded the crude product which was purified by column chroma-
tography on silica gel by eluting with a mixture of petroleum
ether/ethyl acetate (2:1) to give the desired product (0.21 g,
54.5%) as an off-white solid; mp 245 °C (dec); 1H NMR (300 MHz,
DMSO-d6): d 0.78 (m, 3H, –OCH2CH3), 2.25 (s, 3H, –COCH3), 2.98
(m, 3H, CH, –OCH2CH3), 3.44 (m, 2H, CH2), 3.68 (m, 1H, CH), 5.17
(d, 1H, CH-Ar, J = 6.6), 6.55–7.54 (m, 8H, Ar-H), 7.25 (s, 1H, OH),
10.89 (s, 1H, NH). IR (cmꢃ1): 3357, 1705, 1646, 1585, 1236. EI-
MS (m/z): 378 (M+). Anal. Calcd for (C22H22N2O4ꢀ0.3H2O): C,
68.84; H, 5.93; N, 7.30. Found: C, 68.71; H, 5.96; N, 6.96.
5.1.7. 1-(3-Hydroxyphenyl)-2-butyryl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (7b)
Compound 7b (0.11 g) was synthesized from 6c (0.20 g,
0.51 mmol) according to the procedure used to synthesize 7a as
an off-white solid in 58.0% yield. Mp: 196–198 °C. IR (cmꢃ1):
3420, 2966, 1719, 1618, 1454. 1H NMR (300 MHz, DMSO-d6): d
0.77 (m, 3H, –COCH2CH2CH3), 1.45 (m, 2H, –COCH2CH2CH3) d
2.17 (m, 2H, –COCH2CH2CH3), 3.34 (m, 2H, CH2), 5.18 (m, 1H,
CH), 6.00 (d, 1H, CH-Ar, J = 38.4), 6.49–7.45 (m, 8H, Ar-H), 9.32
(d, 1H, OH, J = 50), 10.92 (d, 1H, NH, J = 20.4), 12.50 (s, 1H, –COOH).
EI-MS (m/z): 378 (M+). Anal. Calcd for (C22H22N2O4ꢀ0.5H2O): C,
68.20; H, 5.98; N, 7.23. Found: C, 68.36; H, 6.17; N, 6.74.
5.1.3. Ethyl 1-(3-hydroxyphenyl)-2-propionyl-1,2,3,4-
tetrahydro-b-carboline-3-carboxylate (6b)
Compound 6b (0.21 g) was synthesized from 5 (0.34 g, 1 mmol)
and propionyl chloride (0.17 mL, 2 mmol) according to the proce-
dure used to synthesize 6a as an off-white solid in 49.2% yield.
Mp: 240 °C (dec). IR (cmꢃ1): 3400, 2946, 1704, 1653, 1218. 1H
NMR (300 MHz, DMSO-d6): d 0.73 (m, 3H, –COCH2CH3), 1.13 (m,
3H, –OCH2CH3), 2.62 (m, 2H, –COCH2CH3), 2.95 (m, 2H, –OCH2CH3),
3.31 (m, 1H, CH2), 3.44 (m, 1H, CH2), 3.62 (m, 1H, CH), 5.23 (d, 1H,
CH-Ar, J = 6.6), 6.56–7.53 (m, 8H, Ar-H), 9.24 (s, 1H, OH), 10.89 (s,
1H, NH). EI-MS (m/z): 392 (M+). Anal. Calcd for (C23H24N2O4): C,
70.39; H, 6.16; N, 7.14. Found: C, 69.94; H, 6.31; N, 6.76.
5.1.8. 1-(3-Hydroxyphenyl)-3-carboxy-1,2,3,4-tetrahydro-b-
carboline-3-(N-(2-hydroxyethyl)) formamide (8a)
A mixture of 5 (0.5 g, 1.6 mmol) and 2-aminoethanol (5 mL) was
stirred at 50 °C for 2 h. The surplus ethanolamine was then evapo-
rated. The resulting residue was purified by column chromatogra-
phy on silica gel by eluting with a mixture of dichloromethane/
methanol (20:1) to yield 8a as a white solid (0.43 g, 78.9%). Mp:
253 °C dec. IR (cmꢃ1): 3395, 3263, 1644, 1585, 1265. 1H NMR
(300 MHz, DMSO-d6): d 2.44 (m, 1H, NH), 2.72 (m, 1H, OH), 3.02 (d,
1H, CH2), 3.19 (m, 2H, NHCH2CH2OH), 3.32 (m, 1H, CH2), 3.45 (m,
2H, NHCH2CH2OH), 3.58 (m, 1H, CH), 5.09 (d, 1H, CH-Ar, J = 4.2),
6.72–7.80 (m, 9H, Ar-H, –OH), 9.31 (s, 1H, NH), 10.32 (s, 1H, NH).
5.1.4. Ethyl 1-(3-hydroxyphenyl)-2-butyryl-1,2,3,4-tetrahydro-
b-carboline-3-carboxylate (6c)
Compound 6c (0.24 g) was synthesized from 5 (0.34 g, 1 mmol)
and butyryl chloride (0.21 mL, 2 mmol) according to the procedure
used to synthesize 6a as an off-white solid in 58.0% yield. Mp: 140–

F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
4173
EI-MS(m/z):351(M+). Anal. Calcdfor(C20H21N3O3ꢀ0.5H2O): C, 66.65;
5.1.15. 1-(3-Hydroxyphenyl)-2-(b-(tert-
butoxycarbonylamino)propionyl)-8-chloro-2,3,4,9-
tetrahydrocarboline (10b)
H, 6.65; N, 11.66. Found: C, 67.08; H, 6.18; N, 12.08.
5.1.9. 1-(3-Hydroxyphenyl)-3-carboxy-1,2,3,4-tetrahydro-b-
carboline-3-(N-(2-aminoethyl)) formamide (8b)
Compound 10b (0.68 g) was synthesized from 9c (0.67 g,
2 mmol), N-Boc-alanine (0.42 g, 2.2 mmol) according to the
procedure used to synthesize 10a as a white solid in 72.7% yield.
Mp 230–232 °C. 1H NMR (300 MHz, DMSO-d6): d 1.35 (s, 9H,
–C(CH3)3), 2.61 (m, 2H, –COCH2CH2NH–), 2.82 (m, 2H, CH2), 3.20
(m, 2H, CH2), 4.00 (m, 2H, –CH2), 6.60 (s, 1H, CH-Ar), 6.63–7.44
(m, 8H, Ar-H, –NH–), 7.47 (s, 1H, –OH), 8.33 (s, 1H, NH). EI-MS
(m/z): 469 (M+).
Compound 8b (0.40 g) was synthesized from
5 (0.5 g,
1.6 mmol), 1,2-diaminethane (5 mL) according to the procedure
used to synthesize 8b as a white solid in 73.5% yield. Mp: 185–
197 °C. IR (cmꢃ1): 3400, 2946, 2676, 1708, 1648. 1H NMR
(300 MHz, DMSO-d6): d 0.93 (m, 2H, –NH2), 1.23 (m, 1H, NH),
2.43 (m, 4H, NHCH2CH2NH2), 2.57–3.12 (m, 3H, CH2, CH), 5.08 (s,
1H, CH-Ar), 6.74–8.00 (m, 10H, Ar-H, –OH, CONH), 10.33 (s, 1H,
NH). EI-MS (m/z): 350 (M+). Anal. Calcd for (C20H22N4O2): C,
68.55; H, 6.33; N, 15.99. Found: C, 68.58; N, 6.18; H, 16.08.
5.1.16. 1-(3-Hydroxyphenyl)-2-(b-aminopropionyl)-8-chloro-
1,2,3,4-tetrahydro-b-carboline hydrochloride (11b)
Compound 11b (0.27 g) was synthesized from 10a (0.47 g,
2.2 mmol) according to the procedure used to synthesize 11a as
a white solid in 67.0% yield. Mp: 235 °C dec. IR (cmꢃ1): 3384,
3229, 1650, 1446, 1277. 1H NMR (300 MHz, DMSO-d6): d 2.95
(m, 7H, –COCH2CH2NH2, CH2, CH2), 3.92 (m, 1H, CH2), 6.62 (s, 1H,
CH-Ar), 6.64–7.49 (m, 7H, Ar-H), 8.01 (s, 2H, NH2), 9.41 (s, 1H,
OH), 11.39 (s, 1H, NH). EI-MS (m/z): 369 (M+). Anal. Calcd for
(C20H20ClN3O2ꢀHClꢀ2.5H2O): C, 53.22; H, 5.81; N, 9.31. Found: C,
53.38; H, 5.56; N, 9.43.
5.1.10. 1-(3-Hydroxyphenyl)-6-chloro-1,2,3,4-tetrahydro-b-
carboline hydrochloride (9a)
A
mixture of 5-chlorotryptamine hydrochloride (2.31 g,
10 mmol) and 3-hydroxybenzaldehyde (1.34 g, 11 mmol) was dis-
solved in glacial acetic acid (25 mL) under N2 protection. After stir-
ring at 95 °C for 2 h, the mixture was cooled to room temperature.
The desired compound 9a (2.85 g, 85.1%) was abstained by filtra-
tion as a white solid. Mp: 262–264 °C dec.
5.1.11. 1-(3-Hydroxyphenyl)-6-methyl-1,2,3,4-tetrahydro-b-
carboline hydrochloride (9b)
5.1.17. 1-(3-Hydroxyphenyl)-2-(p-aminobenzoyl)-6-chloro-
1,2,3,4-tetrahydro-b-carboline (12a)
Compound 9b (2.58 g) was synthesized from 5-methyltrypta-
mine hydrochloride (2.12 g, 10 mmol), 3-hydroxybenzaldehyde
(1.34 g, 11 mmol) according to the procedure used to synthesize
9a as a white solid in 82.0% yield. Mp: 285 °C dec.
Compound 12a (0.52 g) was synthesized from 9a (0.67 g,
2 mmol), PyBOP (1.14 g, 2.2 mmol), 4-aminobenzoic acid (0.30 g,
2.2 mmol) according to the procedure used to synthesize 10a as
a white solid in 62.0% yield. Mp: 220–222 °C. IR (cmꢃ1): 3365,
2606, 1623, 1441, 1268. 1H NMR (300 MHz, DMSO-d6): d 2.83
(m, 2H, CH2), 3.28 (m, 2H, CH2), 3.95 (m, 2H, NH2), 5.53 (s, 1H,
CH-Ar), d 6.56–7.51 (m, 11H, Ar-H), 9.41 (s, 1H, OH), 11.24 (s,
1H, NH). EI-MS (m/z): 417 (M+). Anal. Calcd for (C24H20ClN3O2ꢀ
0.5H2O): C, 67.52; H, 4.96; N, 9.84. Found: C, 67.71; H, 4.95: N,
9.99.
5.1.12. 1-(3-Hydroxyphenyl)-8-chloro-1,2,3,4-tetrahydro-b-
carboline hydrochloride (9c)
Compound 9c (2.84 g) was synthesized from 7-chlorotrypta-
mine hydrochloride (2.31 g, 10 mmol), 3-hydroxybenzaldehyde
(1.34 g, 11 mmol) according to the procedure used to synthesize
9a as a white solid in 84.7% yield. Mp: 282 °C dec.
5.1.18. 1-(3-Hydroxyphenyl)-2-(p-aminobenzoyl)-6-methyl-
1,2,3,4-tetrahydro-b-carboline (12b)
5.1.13. 1-(3-Hydroxyphenyl)-2-(a-(tert-
butoxycarbonylamino)propionyl)-6-chloro-2,3,4,9-
tetrahydrocarboline (10a)
Compound 12b (0.46 g) was synthesized from 9b (0.63 g,
2 mmol), PyBOP (1.14 g, 2.2 mmol), 4-aminobenzoic acid
(0.30 g, 2.2 mmol) according to the procedure used to synthesize
10a as a white solid in 58.5% yield. Mp: 136–139 °C. IR (cmꢃ1):
3380, 2921, 1728, 1608, 1458. 1H NMR (300 MHz, DMSO-d6): d
2.43 (s, 3H, –CH3), 2.76 (m, 1H, CH2), 2.76 (m, 1H, CH2), 2.88
(m, 1H, CH2), 3.38 (m, 1H, CH2), 3.95 (s, 2H, NH2), 5.57 (s, 1H,
CH-Ar), 6.55–7.33 (m, 11H, Ar-H), 9.44 (s, 1H, OH), 10.91 (s,
A mixture of compound 9a (0.67 g, 2 mmol), N-Boc b-alanine
(0.42 g, 2.2 mmol), PyBOP (1.14 g, 2.2 mmol), TEA (0.5 mL) in THF
(30 mL) were stirred at room temperature for 2 h. Then the mix-
ture was filtered under vacuum. The filtrate was concentrated
and purified by column chromatography on silica gel by eluting
with a mixture of ethyl acetate/petroleum (1:3) to yield 10a as a
white solid (0.65 g, 69.3%). Mp: 160–162 °C. 1H NMR (300 MHz,
CDCl3): d 1.33 (m, 12H, –C(CH3)3, –CH3), d 2.85 (m, 2H, CH2),
1H, NH). EI-MS (m/z): 397 (M+). Anal. Calcd for (C25H23
-
N3O2ꢀH2O): C, 72.27; H, 6.06; N, 10.11. Found: C, 72.77; H,
d
3.23 (m, 1H, CH2),
d
3.85 (m, 1H, CH2), 4.63 (m, 1H,
6.32; N, 10.17.
–COCH(CH3)NH–), 5.57 (d, 1H, CH-Ar, J = 7.8), d 6.68–7.36 (m, 8H,
Ar-H, –OH), d 7.47 (s, 1H, –COCH(CH3)NH–), d 8.33 (s, 1H, NH).
EI-MS (m/z): 469 (M+).
5.1.19. 6-Chloro-1-(3-hydroxyphenyl)-2-methyl-1,2,3,4-
tetrahydro-b-carboline (13a)
A mixture of compound 9a (0.67 g, 2.0 mmol), K2CO3 (0.2 g,
2.0 mmol), formaldehyde (37%, 0.65 mL, 4.0 mmol), formic acid
(0.37 g, 4.0 mmol) in DMF (10 mL) was stirred at 150 °C for 3 h.
The mixture was then poured into water (30 mL) and filtered to ob-
tain white solid which was purified by column chromatography on
silica gel by eluting with a mixture of ethyl acetate/petroleum (1:1)
to yield 13a as an amber solid (0.38 g, 61.5%). Mp: 252–256 °C. IR
(cmꢃ1): 3293, 2961, 1581, 1261, 1097. 1H NMR (300 MHz, DMSO-
d6): d 2.27 (s, 3H, –CH3), 2.65 (m, 1H, CH2), 2.78 (m, 1H, CH2), 2.85
(m, 1H, CH2), 3.14 (m, 1H, CH2), 4.33 (s, 1H, CH-Ar), 6.74–7.50 (m,
7H, Ar-H), 9.35 (s, 1H, –OH), 10.40 (s, 1H, NH). EI-MS (m/z): 312
(M+). Anal. Calcd for (C18H17ClN2O): C, 69.12; H, 5.48; N, 8.96.
Found: C, 69.19; H, 5.68; N, 8.70.
5.1.14. 1-(3-Hydroxyphenyl)-2-(a-aminopropionyl)-6-chloro-
1,2,3,4-tetrahydro-b-carboline hydrochloride (11a)
Compound 10a (0.47 g, 1 mmol) was dissolved in ethyl acetate
(20 mL), treated with HCl gas flow and stirred at room temperature
for 2 h. The resulting mixture was then filtered under vacuum to ob-
tain the pure product 11a (0.22 g, 54.1%) as a white solid. Mp: 280 °C
dec. IR (cmꢃ1): 3323, 2974, 1625, 1480, 1272. 1H NMR (300 MHz,
DMSO-d6): d 2.86 (m, 3H, –CH3), 3.06 (m, 2H, CH2), 3.22 (m, 2H,
CH2), 3.96 (m, 1H, CH), 6.63 (s, 1H, CH-Ar), 6.66–7.45 (m, 7H, Ar-
H), 7.93 (s, 2H, NH2), 9.42 (s, 1H, OH), 11.26 (s, 1H, NH). EI-MS (m/
z): 369 (M+). Anal. Calcd for (C20H20ClN3O2ꢀHClꢀ0.5H2O): C, 57.84;
H, 5.34; N, 10.12. Found: C, 57.98; H, 5.18: N, 9.92.

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F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
5.1.20. 6-Chloro-1-(3-hydroxyphenyl)-2-propyl-1,2,3,4-
tetrahydro-b-carboline (13b)
5.1.26. 6-Methyl-1-(3-benzyloxyphenyl)-2-(2-phthalimide-
ethyl)-1,2,3,4-tetrahydro-b-carboline (15a)
Compound 13b (0.43 g) was synthesized from 9a (0.67 g,
2.0 mmol), propionaldehyde (0.23 g, 4.0 mmol) according to the
procedure used to synthesize 13a as a white solid in 63.4% yield.
Mp: 99–101 °C. IR (cmꢃ1): 3415, 2959, 1598, 1454, 1304. 1H
NMR (300 MHz, DMSO-d6): d 0.85 (m, 3H, –CH2CH2CH3), 1.47 (m,
2H, –CH2CH2CH3), 2.43 (m, 2H, –CH2CH2CH3), 2.73 (m, 1H, CH2),
2.89 (m, 2H, CH2), 3.12 (m, 1H, CH2), 4.54 (s, 1H, CH-Ar), 6.65–
7.43 (m, 7H, Ar-H), 9.27 (s, 1H, OH), 10.44 (s, 1H, NH). EI-MS (m/
z): 340 (M+). Anal. Calcd for (C20H21ClN2O): C, 70.48; H, 6.21; N,
8.22. Found: C, 70.12; H, 6.68; N, 8.00.
A mixture of 14a (1.98 g, 4.9 mmol), N-(2-bromoethyl)phthali-
mide (1.37 g, 5.4 mmol), K2CO3 (0.68 g, 4.9 mmol) in DMF
(30 mL) was stirred at 150 °C for 10 h under the protection of N2.
The mixture was then poured into water (30 mL) and filtered to ob-
tain yellow solid which was purified by column chromatography
on silica gel by eluting with a mixture of ethyl acetate/petroleum
(1:3) to yield 15a as a white solid (1.12 g, 41.9%). Mp: 183–
186 °C. 1H NMR (300 MHz, DMSO-d6): d 2.34 (s, 3H, ArCH3), 2.56
(m, 2H, CH2), 2.72 (m, 2H, CH2), 3.55 (m, 2H, CH2), 3.90 (m, 2H,
CH2), 4.54 (s, 1H, CH-Ar), 4.86 (s, 2H, CH2–O), 6.53–8.40 (m, 16H,
Ar-H), 11.38 (s, 1H, NH). EI-MS (m/z): 541 (M+).
5.1.21. 6-Chloro-1-(3-hydroxyphenyl)-2-benzyl-1,2,3,4-
tetrahydro-b-carboline (13c)
5.1.27. 6-Methyl-1-(3-benzyloxyphenyl)-2-(3-phthalimide-
propyl)-1,2,3,4-tetrahydro-b-carboline (15b)
Compound 13c (0.59 g) was synthesized from 9a (0.67 g,
2.0 mmol) and benzaldehyde (0.42 g, 4.0 mmol) according to the
procedure used to synthesize 13a as a white solid in 75.5% yield.
Mp: 148–150 °C. IR (cmꢃ1): 3410, 3299, 1653, 1452, 1284. 1H
NMR (300 MHz, CDCl3): d 2.67 (m, 2H, CH2), 2.9 (m, 1H, CH2),
3.24 (m, 1H, CH2), 3.39 (d, 1H, Ph-CH2, J = 133), 3.92 (d, 1H, Ph-
CH2, J = 127), 4.61 (s, 1H, CH-Ar), 5.45 (s, 1H, –OH), 6.83–7.45 (m,
12H, Ar-H), 8.00 (s, 1H, NH). EI-MS (m/z): 388 (M+). Anal. Calcd
for (C24H21ClN2O): C, 74.12; H, 5.44; N, 7.20. Found: C, 74.32; H,
5.40; N, 7.02.
Compound 15b (0.92 g) was synthesized from 14a (1.5 g,
3.7 mmol), N-(2-bromopropyl)phthalimide (1.07 g, 4 mmol)
according to the procedure used to synthesize 15a as a white solid
in 45.0% yield. Mp: 103–105 °C. 1H NMR (300 MHz, DMSO-d6): d
1.24 (m, 2H, CH2), 2.19 (s, 3H, ArCH3), 2.65 (m, 2H, CH2), 2.85 (m,
2H, CH2), 3.71 (m, 1H, CH2), 3.87 (m, 2H, CH2), 4.11 (m, 1H, CH2),
5.05 (s, 1H, CH-Ar), 5.19 (s, 2H, CH2–O), 6.89–7.83 (m, 16H, Ar-
H), 10.98 (s, 1H, NH). EI-MS (m/z): 555 (M+).
5.1.28. 6-Methyl-1-(3-benzyloxyphenyl)-2-(4-phthalimide-
butyl)-1,2,3,4-tetrahydro-b-carboline (15c)
5.1.22. 6-Methyl-1-(3-hydroxyphenyl)-2-methyl-1,2,3,4-
tetrahydro-b-carboline (13d)
Compound 15c (0.79 g) was synthesized from 14a (1.5 g,
3.7 mmol), N-(4-bromobutyl)phthalimide (1.13 g, 4 mmol) accord-
ing to the procedure used to synthesize 14a as a white solid in
37.5% yield. Mp: 133–136 °C. 1H NMR (300 MHz, DMSO-d6): d
1.20 (m, 4H, CH2CH2), 2.39 (s, 3H, ArCH3), 2.72 (m, 2H, CH2), 2.83
(m, 2H, CH2), 3.87 (m, 4H, 2ꢂ CH2), 5.02 (s, 1H, CH-Ar), 5.19 (s,
2H, CH2–O), 7.02–7.834 (m, 16H, Ar-H), 10.97 (s, 1H, NH). EI-MS
(m/z): 569 (M+).
Compound 13d (0.42 g) was synthesized from 9b (0.63 g,
2.0 mmol), formaldehyde (37%, 0.65 mL, 4.0 mmol) according to
the procedure used to synthesize 13a as a white solid in 72.0%
yield. Mp: 217–219 °C. IR (cmꢃ1): 3331, 2937, 1601, 1451, 1254.
1H NMR (300 MHz, DMSO-d6): d 2.21 (s, 3H, –NCH3), 2.35 (s, 3H,
Ar-CH3), 2.60 (m, 1H, CH2), 2.78 (m, 1H, CH2), 2.85 (m, 1H, CH2),
3.08 (m, 1H, CH2), 4.25 (s, 1H, CH-Ar), 6.67–7.18 (m, 7H, Ar-H),
9.27 (s, 1H, –OH), 9.96 (s, 1H, NH). EI-MS (m/z): 292 (M+). Anal.
Calcd for (C19H20N2O): C, 78.05; H, 6.89; N, 9.58. Found: C, 78.37;
H, 6.65; N, 9.49.
5.1.29. 6-Chloro-1-(3-isopropyloxyphenyl)-2-(2-phthalimide-
ethyl)-1,2,3,4-tetrahydro-b-carboline (15d)
Compound 15d (0.70 g) was synthesized from 14b (1.5 g,
4 mmol), N-(2-bromoethyl)phthalimide (1.02 g, 4 mmol) according
to the procedure used to synthesize 15a as a white solid in 34.0%
yield. Mp: 172–173 °C. 1H NMR (300 MHz, DMSO-d6): d 1.32 (m,
6H, –CH(CH3)2), 2.49 (m, 4H, CH2, –NCH2CH2N–), 3.30 (m, 2H,
CH2), 3.63 (m, 2H, –NCH2CH2N–), 4.75 (m, 1H, –CH(CH3)2), 4.95
(s, 1H, CH-Ar,), 7.06–8.48 (m, 11H, Ar-H), 11.63 (s, 1H, NH). EI-
MS (m/z): 513 (M+).
5.1.23. 6-Methyl-1-(3-hydroxyphenyl)-2-benzyl-1,2,3,4-
tetrahydro-b-carboline (13e)
Compound 13e (0.57 g) was synthesized from 9b (0.63 g,
2.0 mmol), benzaldehyde (0.42 g, 4.0 mmol) according to the pro-
cedure used to synthesize 13a as a white solid in 77.4% yield.
Mp: 144–145 °C. IR (cmꢃ1): 3324, 1653, 1599, 1460, 1386. 1H
NMR (300 MHz, CDCl3): d 2.43 (s, 3H, –CH3), 2.79 (m, 3H, CH2,
CH2), 3.21 (m, 1H, CH2), 3.38 (d, 1H, Ph-CH2, J = 150), 3.92 (d, 1H,
Ph-CH2, J = 150), 4.59 (s, 1H, CH-Ar), 5.80 (s, 1H, –OH), 6.81–7.34
(m, 12H, Ar-H), 7.99 (s, 1H, NH). EI-MS (m/z): 368 (M+). Anal. Calcd
for (C25H24N2O): C, 81.49; H, 6.57; N, 7.60. Found: C, 81.56; H, 6.83;
N, 7.62.
5.1.30. 6-Chloro-1-(3-isopropyloxyphenyl)-2-(3-phthalimide-
propyl)-1,2,3,4-tetrahydro-b-carboline (15e)
Compound 15e (0.71 g) was synthesized from 14b (1.5 g,
4 mmol), N-(2-bromopropyl)phthalimide (1.07 g, 4 mmol) accord-
ing to the procedure used to synthesize 15a as a white solid in
42.6% yield. Mp: 190–192 °C. 1H NMR (300 MHz, DMSO-d6): d
0.82 (m, 6H, –CH(CH3)2), 1.81 (m, 2H, –NCH2CH2CH2N–), 2.69 (m,
4H, CH2, –NCH2CH2CH2N–), 3.08 (m, 2H, CH2), 3.63 (m, 2H,
–NCH2CH2CH2N–), 4.53 (m, 1H, –CH(CH3)2), 4.65 (s, 1H, CH-Ar),
6.73–7.84 (m, 11H, Ar-H), 10.53 (s, 1H, NH). EI-MS (m/z): 527 (M+).
5.1.24. 6-Methyl-1-(3-benzyloxyphenyl)-1,2,3,4-tetrahydro-b-
carboline hydrochloride (14a)
Compound 14a (3.1 g) was synthesized from 5-methyltrypta-
mine hydrochloride (2.1 g, 10 mmol), 3-(benzyloxy)benzaldehyde
(2.1 g, 10 mmol) according to the procedure used to synthesize
9a as a white solid in 76.5% yield. Mp: 260–262 °C.
5.1.31. 6-Chloro-1-(3-isopropyloxyphenyl)-2-(4-phthalimide-
butyl)-1,2,3,4-tetrahydro-b-carboline (15f)
5.1.25. 6-Chloro-1-(3-isopropyloxyphenyl)-1,2,3,4-tetrahydro-b-
carboline hydrochloride (14b)
Compound 14b (0.28 g) was synthesized from 3-isopropoxy-
benzaldehyde (3.28 g, 20 mmol), 5-methyltryptamine hydrochlo-
ride (4.6 g, 20 mmol) according to the procedure used to
synthesize 9a as a white solid in 85.5% yield. Mp: 264–265 °C.
Compound 15f (0.81 g) was synthesized from 14b (1.5 g,
4 mmol), N-(4-bromobutyl)phthalimide (1.13 g, 4 mmol) accord-
ing to the procedure used to synthesize 15a as a white solid in
37.5% yield. Mp: 150–152 °C. 1H NMR (300 MHz, DMSO-d6): d
1.19 (m, 6H, –CH(CH3)2), 1.55 (m, 4H, –NCH2CH2CH2CH2N–), 2.65
(m, 4H, CH2, –NCH2CH2CH2CH2N–), 3.12 (m, 2H, CH2), 3.52 (m,

F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
4175
2H, –NCH2CH2CH2CH2N–), 4.49 (m, 1H, –CH(CH3)2), 4.59 (s, 1H, CH-
Ar,), 6.76–7.88 (m, 11H, Ar-H), 10.45 (s, 1H, NH). EI-MS (m/z): 541
(M+).
150 °C. 1H NMR (300 MHz, DMSO-d6): d 1.25 (m, 6H, –CH(CH3)2),
1.33 (m, 4H, –NCH2CH2CH2CH2N–), 1.51 (m, 2H, NH2), 2.43 (m,
2H, CH2), 2.62 (m, 2H, CH2), 2.73 (m, 2H, CH2), 3.11 (m, 2H, CH2),
4.55 (m, 1H, –CH(CH3)2), 4.60 (s, 1H, CH-Ar), 6.78–7.44 (m, 7H,
Ar-H), 10.48 (s, 1H, NH). EI-MS (m/z): 411 (M+).
5.1.32. 6-Methyl-1-(3-benzyloxyphenyl)-2-(2-aminoethyl)-
1,2,3,4-tetrahydro-b-carboline (16a)
To a solution of 15a (1.0 g, 1.8 mmol) in alcohol (50 mL) was
added NH2–NH2ꢀH2O (0.5 mL). The solution was stirred at 80 °C
for 3 h, then cooled to room temperature and filtered under vac-
uum. The filtrate was added sodium hydroxide aqueous (10%,
2 mL). The resulting mixture was evaporated to dryness and puri-
fied by column chromatography on silica gel by eluting with a mix-
ture of dichloromethane/methanol (20:1) to yield 16a as a white
solid (0.55 g, 72.4%). Mp: 166–170 °C. 1H NMR (300 MHz, DMSO-
d6): d 1.20 (m, 2H, –NH2), 2.22 (s, 3H, ArCH3), 2.70 (m, 4H, –
CH2CH2NH2), 3.15 (m, 4H, 2ꢂ CH2), 5.05 (s, 1H, CH-Ar), 5.25 (s,
2H, ArCH2-O), 6.81–8.50 (m, 12H, Ar-H), 10.12 (s, 1H, NH). EI-MS
(m/z): 411 (M+).
5.1.38. 6-Methyl-1-(3-hydroxyphenyl)-2-(2-aminoethyl)-
1,2,3,4-tetrahydro-b-carboline (17a)
To a solution of compound 16a (0.2 g, 3.7 ꢂ 10ꢃ4 mol) in meth-
anol (50 mL) was added Pd–C (10%, 0.05 g), HCOONH4 (0.2 g). The
mixture was stirred at room temperature for 3 h, then the Pd–C
was filtered and the solvent was concentrated to dryness and par-
titioned between water (20 mL) and acetyl acetate (20 mL). The
aqueous layer was extracted with acetyl acetate (3ꢂ 30 mL). The
combined organic phase was evaporated and purified by column
chromatography on silica gel by eluting with a mixture of dichlo-
romethane: methanol (20:1) to yield 17a as a white solid (0.14 g,
84.0%). Mp: 115–117 °C. IR (cmꢃ1): 3410, 2926, 1595, 1459,
1308. 1H NMR (300 MHz, DMSO-d6): d 1.74 (m, 2H, NH2), 2.27 (s,
3H, Ar-CH3), 2.66 (m, 6H, N–CH2CH2NH2, CH2), 3.17 (m, 2H, CH2),
4.57 (d, 1H, CH-Ar, J = 14.4), 6.64–7.18 (m, 8H, Ar-H, –OH), 10.11
(s, 1H, –NH). EI-MS (m/z): 321 (M+). Anal. Calcd for
(C20H23N3Oꢀ0.5H2O): C, 72.69; H, 7.32; N, 12.72. Found: C, 72.41;
H, 7.68; N, 12.56.
5.1.33. 6-Methyl-1-(3-benzyloxyphenyl)-2-(3-aminopropyl)-
1,2,3,4-tetrahydro-b-carboline (16b)
Compound 16b (0.48 g) was synthesized from 15b (1.0 g,
1.8 mmol)), NH2–NH2ꢀH2O (0.5 mL) according to the procedure
used to synthesize 16a as a white solid in 62.7% yield. Mp: 89–
91 °C. 1H NMR (300 MHz, DMSO-d6):
d 1.54 (m, 2H, –
CH2CH2CH2NH2), 1.60 (m, 2H, –NH2), 2.35 (s, 3H, ArCH3), 2.72 (m,
4H, CH2CH2CH2NH2), 3.17 (m, 4H, 2CH2), 4.48 (s, 1H, CH-Ar), 5.05
(s, 2H, ArCH2-O), 6.80–7.43 (m, 12H, Ar-H), 10.07 (s, 1H, NH). EI-
MS (m/z): 425 (M+).
5.1.39. 6-Methyl-1-(3-hydroxyphenyl)-2-(3-aminopropyl)-
1,2,3,4-tetrahydro-b-carboline (17b)
Compound 17b (0.27 g) was synthesized from 16b (0.4 g,
0.94 mmol) according to the procedure used to synthesize 17a as
a white solid in 87.0% yield. Mp: 101–103 °C. IR (cmꢃ1): 3408,
2931, 1597, 1460, 1313. 1H NMR (300 MHz, CDCl3): d 1.04 (m,
2H, N–CH2CH2CH2NH2), 1.40 (m, 2H, NH2), 2.41 (s, 3H, Ar-CH3),
2.65 (m, 4H, N–CH2CH2CH2NH2), 2.90 (m, 2H, CH2), 3.36 (m, 2H,
CH2), 4.34 (s, 1H, CH-Ar), 6.73–7.25 (m, 8H, Ar-H, OH). EI-MS (m/
z): 335 (M+). Anal. Calcd for (C21H25N3O.0.75H2O): C, 72.28; H,
7.65; N, 12.04. Found: C, 72.16; H, 7.87; N, 11.99.
5.1.34. 6-methyl-1-(3-benzyloxyphenyl)-2-(4-aminobutyl)-
1,2,3,4-tetrahydro-b-carboline (16c)
Compound 16c (0.50 g) was synthesized from 15c (1 g,
1.8 mmol), NH2–NH2ꢀH2O (0.5 mL) according to the procedure used
to synthesize 16a as a white solid in 64.8% yield. Mp: 108–110 °C. 1H
NMR (300 MHz, DMSO-d6): d 1.18 (m, 2H, –NH2), 2.36 (m, 7H, Ar-
CH3, N–CH2CH2CH2CH2NH2), 2.47 (m, 6H, N–CH2CH2CH2CH2NH2,
CH2), 2.90 (m, 2H, CH2), 5.19 (s, 1H, CH-Ar), 5.25 (s, 2H, ArCH2-O),
6.81–8.48 (m, 12H, Ar-H), 10.14 (s, 1H, NH). EI-MS (m/z): 439 (M+).
5.1.40. 6-Methyl-1-(3-hydroxyphenyl)-2-(4-aminobutyl)-
1,2,3,4-tetrahydro-b-carboline (17c)
Compound 17c (0.28 g) was synthesized from 16c (0.4 g,
0.91 mmol) according to the procedure used to synthesize 17a as
a white solid in 89.2% yield. Mp: 200 °C dec. IR (cmꢃ1): 3410,
2941, 1598, 1452, 1278. 1H NMR (300 MHz, DMSO-d6): d 1.63
(m, 2H, NH2), 2.30 (m, 7H, Ar-CH3, N–CH2CH2CH2CH2NH2), 2.47
(m, 6H, N–CH2CH2CH2CH2NH2, CH2), 2.90 (m, 2H, CH2), 4.41 (s,
1H, CH-Ar), 6.46–7.24 (m, 7H, Ar-H), 7.25 (s, 1H, –OH), 10.32 (s,
1H, NH). EI-MS (m/z): 349 (M+). Anal. Calcd for
(C22H27N3Oꢀ0.75H2O): C, 72.80; H, 7.91; N, 11.57. Found: C,
72.57; H, 7.83; N, 12.17.
5.1.35. 6-Chloro-1-(3-isopropyloxyphenyl)-2-(2-aminoethyl)-
1,2,3,4-tetrahydro-b-carboline (16d)
Compound 16d (0.61 g) was synthesized from 15d (1 g,
1.95 mmol), NH2–NH2ꢀH2O (0.5 mL) according to the procedure
used to synthesize 16a as a white solid in 82.0% yield. Mp: 171–
173 °C. 1H NMR (300 MHz, DMSO-d6): d 0.94 (m, 6H, –CH(CH3)2),
1.83 (s, 2H, NH2), 2.72 (m, 6H, 2ꢂ CH2, –NCH2CH2NH2), 3.12 (m,
2H, –NCH2CH2NH2), 4.54 (m, 1H, –CH(CH3)2), 4.65 (s, 1H, CH-Ar),
6.77–7.45 (m, 7H, Ar-H), 10.52 (s, 1H, NH). EI-MS (m/z): 383 (M+).
5.1.36. 6-Chloro-1-(3-isopropyloxyphenyl)-2-(3-aminopropyl)-
1,2,3,4-tetrahydro-b-carboline (16e)
5.1.41. 6-Chloro-1-(3-hydroxyphenyl)-2-(2-aminoethyl)-1,2,3,4-
tetrahydro-b-carboline (17d)
Compound 16e (0.61 g) was synthesized from 15e (1 g,
1.90 mmol), NH2–NH2ꢀH2O (0.5 mL) according to the procedure
used to synthesize 16a as a white solid in 71.0% yield. Mp: 190–
192 °C. 1H NMR (300 MHz, DMSO-d6): d 1.04 (m, 6H, –CH(CH3)2),
1.56 (m, 2H, –NCH2CH2CH2N–), 1.83 (s, 2H, NH2), 2.55 (m, 4H,
To solution of 17d (0.4 g, 1.04 mmol) in CH2Cl2 cooled in an ice
bath (50 mL) was added AlCl3 (0.2 g). After stirring at room tem-
perature for 2 h, the solvent was concentrated to dryness and par-
titioned between saturate NaHCO3 aqueous solution (20 mL) and
acetyl acetate (20 mL). The insoluble substance was removed by
filtration and the aqueous layer was extracted with acetyl acetate
(3ꢂ 30 mL). The combined organic phase was evaporated and puri-
fied by column chromatography on silica gel by eluting with a mix-
ture of dichloromethane/methanol (20:1) to yield 17d as a white
solid (0.26 g, 70.5%). Mp: 125–128 °C. IR (cmꢃ1): 3413, 1603,
1453, 1313, 799. 1H NMR (300 MHz, CDCl3): d 1.72 (m, 2H, NH2),
2.21 (m, 1H, CH2), 2.73 (m, 4H, N–CH2CH2NH2), 3.17 (m, 2H,
CH2), 4.42 (s, 1H, CH-Ar), 6.69–7.60 (m, 7H, Ar-H), 7.61 (s, 1H,
CH2, –NCH2CH2CH2N–), 2.74 (m, 2H, CH2), 3.12 (m, 2H,
–
NCH2CH2CH2N–), 4.54 (m, 1H, –CH(CH3)2), 4.59 (s, 1H, CH-Ar),
6.78–7.44 (m, 7H, Ar-H), 10.49 (s, 1H, NH). EI-MS (m/z): 397 (M+).
5.1.37. 6-Chloro-1-(3-isopropyloxyphenyl)-2-(4-aminobutyl)-
1,2,3,4-tetrahydro-b-carboline (16f)
Compound 16f (0.50 g) was synthesized from 15f (1 g,
1.85 mmol), NH2–NH2.H2O (0.5 mL) according to the procedure
used to synthesize 16a as a white solid in 65.5% yield. Mp: 148–

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F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
–OH). EI-MS (m/z): 341 (M+). Anal. Calcd for (C19H20ClN3Oꢀ0.5H2O):
for (C22H21N3O): C, 76.94; H, 6.16; N, 12.24. Found: C, 76.89; H,
6.34; N, 12.50.
C, 65.04; H, 6.03; N, 11.98. Found: C, 65.23; H, 6.05; N, 12.13.
5.1.42. 6-Chloro-1-(3-hydroxyphenyl)-2-(3-aminopropyl)-
1,2,3,4-tetrahydro-b-carboline (17e)
5.1.47. 6-Methyl-1-(indol-3-yl)-2-butyryl-1,2,3,4-tetrahydro-b-
carboline (19b)
Compound 17e (0.50 g) was synthesized from 16e (0.4 g,
1.00 mmol) according to the procedure used to synthesize 17d as
a white solid in 68.0% yield. Mp: 113–115 °C. IR (cmꢃ1): 3417,
1650, 1605, 1465, 1267. 1H NMR (300 MHz, CDCl3): d 1.05 (m,
2H, N–CH2CH2CH2NH2), 1.45 (s, 2H, NH2), 2.55 (m, 6H, N–
CH2CH2CH2NH2, CH2), 3.39 (m, 2H, CH2), 4.39 (s, 1H, CH-Ar),
6.71–7.40 (m, 8H, Ar-H, OH). EI-MS (m/z): 355 (M+). Anal. Calcd
for (C20H22ClN3Oꢀ0.77H2O): C, 64.97; H, 6.42; N, 11.36. Found: C,
64.88; H, 6.90; N, 10.87.
Compound 19b (0.18 g) was synthesized from 18a (0.30 g,
1.0 mmol), butyryl chloride (0.16 mL, 1.5 mmol) according to
the procedure used to synthesize 19a as a white solid in 48.5%
yield. Mp: 175–178 °C. IR (cmꢃ1): 3416, 3267, 2921, 1620,
1466. 1H NMR (300 MHz, DMSO-d6): d 0.87 (t, 3H, –CH2CH2CH3),
1.58 (m, 2H, –CH2CH2CH3), 2.39 (m, 5H, –CH2CH2CH3, Ar-CH3),
2.78 (m, 2H, CH2), 3.32 (m, 1H, CH2), 3.96 (m, 1H, CH2), 6.78
(s, 1H, CH-Ar), 6.79–7.60 (m, 8H, Ar-H), 10.76 (s, 1H, –NH),
10.96 (s, 1H, NH). EI-MS (m/z): 371 (M+). Anal. Calcd for
(C24H25N3O): C, 77.60; H, 6.78; N, 11.31. Found: C, 77.24; H,
6.77; N, 11.32.
5.1.43. 6-Chloro-1-(3-hydroxyphenyl)-2-(3-aminobutyl)-1,2,3,4-
tetrahydro-b-carboline (17f)
Compound 17f (0.21 g) was synthesized from 16f (0.4 g,
0.97 mmol) according to the procedure used to synthesize 16d as
a white solid in 57.5% yield. Mp: 180–182 °C. IR (cmꢃ1): 3288,
2936, 1600, 1452, 1286. 1H NMR (300 MHz, DMSO-d6): d 1.19
(m, 4H, –NCH2CH2CH2CH2N–), 1.44 (m, 2H, NH2), 2.55 (m, 6H, –
NCH2CH2CH2CH2NH2, CH2), 3.63 (m, 2H, CH2), 4.55 (s, 1H, CH-Ar),
6.66–7.44 (m, 8H, Ar-H, –OH), 10.48 (s, 1H, NH). EI-MS (m/z):
369 (M+). Anal. Calcd for (C21H24ClN3Oꢀ0.5H2O): C, 66.57; H, 6.65;
N, 11.09. Found: C, 66.62; H, 6.87; N, 11.48.
5.1.48. 6-Methyl-1-(indol-3-yl)-2-isobutyryl-1,2,3,4-tetrahydro-
b-carboline (19c)
Compound 19c (0.24 g) was synthesized from 18a (0.30 g,
1.0 mmol), isobutyryl chloride (0.16 mL, 1.5 mmol) according to
the procedure used to synthesize 19a as a white solid in 64.7%
yield. Mp: 186–189 °C. IR (cmꢃ1): 3414, 3236, 1611, 1469, 745.
1H NMR (300 MHz, DMSO-d6): d 1.04 (m, 6H, –CH(CH3)2), 2.39 (s,
3H, Ar-CH3), 2.80 (s, 2H, CH2), 2.96 (m, 1H, –CH(CH3)2), 3.42 (m,
1H, CH2), 4.03 (m, 1H, CH2), 6.77 (s, 1H, CH-Ar), 6.78–7.55 (m,
8H, Ar-H), 10.78 (s, 1H, –NH), 10.95 (s, 1H, NH). EI-MS (m/z): 371
(M+). Anal. Calcd for (C24H25N3O): C, 77.60; H, 6.78; N, 11.31.
Found: C, 77.19; H, 6.80; N, 11.15.
5.1.44. 6-Methyl-1-(indol-3-yl)-1,2,3,4-tetrahydro-b-carboline
(18a)
A mixture of 5-methyltryptamine (2.21 g, 10 mmol), 1H-in-
dole-3-carbaldehyde (1.45 g, 10.00 mmol) in toluene (40 mL)
was stirred at 130 °C for 3 h. Then evaporate most of the toluene
and the resulting residue was filtered under vacuum to obtain a
light yellow solid. To a solution of the solid in CHCl3 (20 mL),
CF3COOH (8 mL) in CHCl3 (10 mL) was added by dropwise. After
stirring at room temperature for 24 h, the mixture was poured
into water (100 mL). The pH of the aqueous layer was adjusted
to 9 by adding ammonia water. Afterward, the mixture was ex-
tracted with ethyl acetate (3ꢂ 100 mL) and dried over Na2SO4.
Removal of the solvent in vacuo afforded the crude product which
was purified by column chromatography on silica gel by eluting
with a mixture of dichloromethane/methanol (20:1) to give the
desired product 18a as an amber solid (1.47 g, 48.7%). Mp: 164–
166 °C.
5.1.49. 6-Chloro-1-(indol-3-yl)-2-acetyl-1,2,3,4-tetrahydro-b-
carboline (19d)
Compound 19d (0.19 g) was synthesized from 18b (0.32 g,
1.0 mmol), acetyl chloride (0.11 mL, 1.5 mmol) according to the
procedure used to synthesize 19a as an amber solid in 52.8% yield.
Mp: 203–205 °C. IR (cmꢃ1): 3421, 2676, 1625, 1475, 1036. 1H NMR
(300 MHz, DMSO-d6): d 2.13 (s, 3H, –COCH3), 2.82 (m, 1H, CH2),
3.37 (m, 1H, CH2), 3.91 (m, 1H, CH2), 3.49 (m, 1H, CH2), 6.80 (s,
1H, CH-Ar), 6.95–7.60 (m, 8H, Ar-H), 10.99 (s, 1H, –NH), 11.15 (s,
1H, NH). EI-MS (m/z): 363 (M+). Anal. Calcd for (C21H18ClN3O): C,
69.32; H, 4.99; N, 11.55. Found: C, 69.28; H, 5.18; N, 11.61.
5.1.50. 6-Chloro-1-(indol-3-yl)-2-butyryl-1,2,3,4-tetrahydro-b-
carboline (19e)
Compound 19e (0.26 g) was synthesized from 18b (0.32 g,
1.0 mmol), butyryl chloride (0.16 mL, 1.5 mmol) according to the
procedure used to synthesize 19a as a white solid in 67.0% yield.
Mp: 248–249 °C. IR (cmꢃ1): 3426, 3245, 1620, 1445, 1208. 1H
NMR (300 MHz, DMSO-d6): d 0.89 (m, 3H, –CH2CH2CH3), 1.58 (m,
2H, –CH2CH2CH3), 2.39 (m, 2H, –CH2CH2CH3), 2.82 (m, 2H, CH2),
3.32 (m, 1H, CH2), 3.96 (m, 1H, CH2), 6.80 (s, 1H, CH-Ar), 6.95–
7.60 (m, 8H, Ar-H), 10.99 (s, 1H, –NH), 11.16 (s, 1H, NH). EI-MS (
m/z): 391 (M+). Anal. Calcd for (C23H22ClN3O): C, 70.49; H, 5.66;
N, 10.72. Found: C, 70.24; H, 5.69; N, 10.61.
5.1.45. 6-Chloro-1-(indol-3-yl)-1,2,3,4-tetrahydro-b-carboline
(18b)
Compound 18b (1.35 g) was synthesized from 5-chlorotrypta-
mine (1.8 g, 9.25 mmol) and 1H-indole-3-carbaldehyde (1.34 g,
9.25 mmol) according to the procedure used to synthesize 18a as
a white solid in 52.5% yield. Mp: 135–137 °C.
5.1.46. 6-Methyl-1-(indol-3-yl)-2-acetyl-1,2,3,4-tetrahydro-b-
carboline (19a)
To a solution of 18a (0.30 g, 1.0 mmol), TEA (0.2 mL) in THF
(20 mL) was added acetyl chloride (0.11 mL, 1.5 mmol) over a per-
iod of 20 min. The mixture was stirred at room temperature for 2 h,
and then was purified by column chromatography on silica gel by
eluting with a mixture of petroleum ether/ethyl acetate (3:1) to
yield title compound 19a as a white solid (0.26 g, 77.0%). Mp:
171–173 °C. IR (cmꢃ1): 3409, 3260, 1622, 1442, 753. 1H NMR
(300 MHz, DMSO-d6): d 2.13 (s, 3H, –COCH3), 2.38 (s, 3H, Ar-CH3),
2.75 (m, 1H, CH2), 2.84 (m, 1H, CH2), 3.37 (m, 1H, CH2), 3.90 (m,
1H, CH2), 6.77 (s, 1H, CH-Ar), 6.78–8.30 (m, 8H, Ar-H), 10.75 (s,
1H, –NH), 10.96 (s, 1H, NH). EI-MS (m/z): 343 (M+). Anal. Calcd
5.1.51. 6-Chloro-1-(indol-3-yl)-2-isobutyryl-1,2,3,4-tetrahydro-
b-carboline (19f)
Compound 19f (0.23 g) was synthesized from 18b (0.32 g,
1.0 mmol), isobutyryl chloride (0.16 mL, 1.5 mmol) according to
the procedure used to synthesize 19a as a white solid in 60.1%
yield. Mp: 205 °C dec. IR (cmꢃ1): 3306, 2976, 1587, 1438, 1228.
1H NMR (300 MHz, DMSO-d6): d 1.04 (m, 6H, –CH(CH3)2), 2.82 (d,
2H, CH2, J = 5.52), 2.96 (m, 1H, –CH(CH3)2), 3.34 (m, 1H, CH2),
4.04 (m, 1H, CH2), 6.80 (s, 1H, CH-Ar), 6.94–7.55 (m, 8H, Ar-H),
10.99 (s, 1H, –NH), 11.18 (s, 1H, NH). EI-MS (m/z): 391 (M+). Anal.

F. Liu et al. / Bioorg. Med. Chem. 18 (2010) 4167–4177
4177
Calcd for (C23H22ClN3O): C, 70.49; H, 5.66; N, 10.72. Found: C,
70.22; H, 5.70; N, 10.65.
lyzed using Microsoft Excel to obtain the IC50 of the test
compounds.
5.1.52. 6-Chloro-1-(indol-3-yl)-2-(b-aminopropionyl)-1,2,3,4-
tetrahydro-b-carbolinehydrochloride (19g)
5.2.3. SRB proliferation assays
Sulforhodamine B, trichloroacetic acid, trizma base, and acetic
acid were purchased from Sigma Chemical Co. (St. Louis, USA).
Lung cancer A549 was cultured in RPMI 1640 medium, supple-
mented with 10% fetal bovine serum at 37 °C in humidified air un-
der 5% CO2. A pilot-screening operation was initiated in which the
panel lines were inoculated onto a series of standard 96-well plates
on day 0, in the majority of cases at 20,000 cells/well, and then pre-
incubated in the absence of drug for 24 h. Test agents were then
added in 10-fold dilutions starting from the highest soluble con-
centration, and incubated for a further 48 h. Following this, the
cells were fixed in situ, washed, and dried. SRB was added, fol-
lowed by further washing and drying of the stained, adherent cell
mass. The inhibition of cell proliferation was assessed by measur-
ing changes in total optical density after a culture of each cell line
that was subjected to 48 h of drug treatment. The results were ob-
tained in one independent experiment run in triplicate.
Compound 19g was synthesized according to the procedure
used to synthesize 10a and then followed by 11a as a white solid
in 55.6% overall yield. Mp: 222–224 °C. IR (cmꢃ1): 3410, 1651,
1600, 1452, 1289. 1H NMR (300 MHz, DMSO-d6): d 2.95 (m, 4H, –
COCH2CH2NH2), 3.67 (m, 3H, CH2, CH2), 4.10 (m, 1H, CH2), 4.25
(s, 1H, CH-Ar), 6.20 (m, 2H, NH2), 6.67–8.54 (m, 8H, Ar-H), 10.65
(s, 1H, NH), 11.26 (s, 1H, NH). EI-MS (m/z): 406 (M+). Anal. Calcd
for (C23H24Cl2N4Oꢀ0.5H2O): C, 61.07; H, 5.57; N, 12.38. Found: C,
61.26; H, 5.83; N, 12.32.
5.2. Bioactivity test
5.2.1. Preparation of Eg5
Coding regions were PCR amplified from a template (obtained
in our lab) containing full-length human Eg5. The primers used
were, forward 50-TATAGG GCG AAT TCC GCC ATG GCG TCG CAG
CCA-30 and reverse 50-ACG GGC TGC AGC AAG CTC GAG TTT
TAAACG TTC TAT-30. The region encoding residues 2-386 was sub-
cloned into pET28a (NOVAGEN). Protein expression in Escherichia
coli cells was induced with 0.5 mM IPTG. Cells were harvested after
20 h of growth at 20 °C and then disrupted by sonication. The sol-
uble lysate was clarified by centrifugation and applied to a SP-Se-
pharose column (Amersham Pharmacia Biotech) in a buffer A
(20 mM Na–PIPES, pH 6.3; 20 mM NaCl; 1 mM MgCl2; 1 mM Na–
EGTA). Protein was eluted with a linear gradient of 20–1000 mM
NaCl. Eg5 was identified by SDS–PAGE, and then applied to
Mono-Q columns (Amersham Pharmacia Biotech) in a buffer B
(20 mM Tris–HCl, pH 8.8; 1 mM MgCl2; 1 mM Na–EGTA). A gradi-
ent from 0 to 1000 mM NaCl was used to elute Eg5) Fractions were
analyzed by SDS–PAGE. The most concentrated fraction was dia-
lyzed against ATPase buffer (20 mM Na–PIPES, pH 7.5; 1 mM
MgCl2; 1 mM Na–EGTA) and then aliquoted, frozen in liquid nitro-
gen, stored at ꢃ80 °C.
Acknowledgements
This work was supported by the Natural Science Foundation of
China (Nos. 30772624 and 30801437), Specialized Research Fund
for the Doctoral Program of Higher Education P.R.C. (Nos.
20060316006 and 20090096110003) and ‘The Six Top Talents’ of
Jiangsu Province (No. 06-C-23), and the National Major Science
and Technology Project of China (Innovation and Development of
New Drugs) (2008ZX09401-001 and 2009ZX09501-003) for finan-
cial supports.
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