Design, synthesis and evaluation of tetrahydroisoquinolines as new kinesin spindle protein inhibitors

Article abstract of DOI:10.1248/cpb.57.567

In this study, a series of tetrahydroisoquinolines have been synthesized and identified as novel kinesine spindle protein (KSP) inhibitors based on the pharmacophore we have mapped and the crystal structure of monastrol bound to the target protein. The KS

Full text of DOI:10.1248/cpb.57.567

June 2009
Chem. Pharm. Bull. 57(6) 567—571 (2009)
567
Design, Synthesis and Evaluation of Tetrahydroisoquinolines as New
Kinesin Spindle Protein Inhibitors
Cheng JIANG,a,b Qidong YOU, ,a,b Fei LIU,a,b Wutong WU,c Qinglong GUO,a Jiwang CHERN,d
*
d
Lei YANG,c and Mengling CHEN
a Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University; b Department of
Medicinal Chemistry, China Pharmaceutical University; c School of Life Science and Technology, China Pharmaceutical
University; 24 Tongjiaxiang, Nanjing 210009, P. R. China: and d School of Pharmacy, College of Medicine, National
Taiwan University; No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan.
Received December 15, 2008; accepted February 25, 2009; published online March 16, 2009
In this study, a series of tetrahydroisoquinolines have been synthesized and identified as novel kinesine
spindle protein (KSP) inhibitors based on the pharmacophore we have mapped and the crystal structure of
monastrol bound to the target protein. The KSP inhibitory activities of all the designed compounds were tested
using cloned Human KSP protein. All thirteen compounds were more potent than the control, monastrol, in
Human KSP protein adenosine triphosphatase (ATPase) assays. Three compounds (1b, 1g, 1h) exhibited over 100
times higher potency than monastrol. Cytotoxic results in vitro by MTT method indicated that nine of these com-
pounds (1a, 1b, 1c, 1d, 1e, 1g, 1h, 1j, 1k) were more active than monastrol. In particular, compounds 1b and 1g,
each of which contains a hydrophilic group on the side chain at the 2-position, exhibited excellent cell-killing ac-
tivities against HepG2 cells.
Key words kinesin spindle protein inhibitor; tetrahydroisoquinoline; pharmacophore; anticancer
Kinesin spindle protein (KSP), also known as Hs Eg5, is a tains four parts of the pharmacophore, although the hy-
member of the kinesin superfamily of molecular motors that drophobic group of part 2 is not obvious. R-Mon-97 (IC50ꢀ
utilize the energy generated from the hydrolysis of ATP to 150 nM),27) in which there is a hydrophobic group of part 2
transport vesicles, organelles, and microtubules.1) It is also but without the side chain of part 3, is sixty times more po-
required for centrosome separation during prophase or tent in inhibiting KSP activity than monastrol. This phenom-
prometaphase.2) Inhibition of KSP can prevent the formation enon suggests that the hydrophobic group of part 2 is essen-
of normal bipolar spindle which leads to mitotic arrest with a tial for inhibiting KSP activity. S-Mon-97 (IC50ꢀ650 nM),27)
characteristic monoastral phenotype, followed by apoptosis
in the transformed cells without disturbing the micro-
tubules.3,4) Therefore, KSP inhibition represents a novel and
specific mechanism for targeting the mitotic spindle without
any neuropathy-associated, mechanism-based side effects
which are quite common for taxanes and other microtubule-
targeting natural products. In recent years, several classes of
KSP inhibitors have been reported as novel anti-cancer
agents.5—23)
Fig. 1. The Four Parts of an Active KSP Inhibitor in Our Model (Part 1:
Aromatic Ring; Part 2: Hydrophobic Group; Part 3: Side Chain; Part 4:
Flexible Heterocyclic Ring)
Based on the KSP inhibitors reported in the literature, we
have identified the pharmacophore and performed binding
mode analysis for KSP inhibitors.24,25) In our previous work,
we identified the pharmacophore of KSP inhibitors which
consists of four chemical features (one hydrogen-bond ac-
ceptor, one hydrogen-bond donor, one aromatic ring, and one
hydrophobic group).24) After mapping the pharmacophore
with several classes of KSP inhibitors, we found that most
KSP inhibitors contained four parts: an aromatic ring (part
1), a hydrophobic group (part 2), a side chain (part 3) and a
flexible heterocyclic ring (part 4) which connected the other
three components (Fig. 1). Moreover, the hydrogen-bond ac-
ceptor or hydrogen-bond donor at the side chain can improve
KSP inhibitory activity to a certain extent. In 2004, the crys-
tal structure of monastrol bound to KSP, in complex with
ADP, was reported by Yan et al.26) This binding mode of KSP
inhibitors has been studied extensively, offering us a good
opportunity to discover novel KSP inhibitors as anti-cancer
agents.
Molecular Design As shown in Fig. 2, monastrol con- Fig. 2. Design of New KSP Inhibitors (1) Based on the Pharmacophore
∗ To whom correspondence should be addressed. e-mail: youqidong@gmail.com
© 2009 Pharmaceutical Society of Japan

568
Vol. 57, No. 6
Fig. 3. Tetrahydroisoquinoline Is Selected as the Core of New KSP Inhibitors
which does not have a hydrophobic group of part 2 but pos-
sesses an aryl group in the side chain of part 3, is also six-
teen times more potent than monastrol. This suggests that the
introduction of aryl group to part 3 may increase the KSP in-
hibitory activity.
Based on this pharmacophore, we identified and analyzed
some representative KSP inhibitors. A novel series of KSP
inhibitors were then designed (1 in Fig. 2). Part 1 of the
newly designed KSP inhibitors is the same as that of monas-
trol, S-Mon-97, and R-Mon-97, which is generally consid-
ered to be essential for potent KSP inhibitory activity. In sev-
eral patents,28,29) a side chain was attached to the N atom of a
six-ring core which may be used to replace part 4. Our team
have reported a series of tetrahydro-b-carbolines as KSP in-
hibitors recently,30,31) which are different from those reported
by Sunder-Plassmann et al.32) It could be found that the
Fig. 4. The Binding Site of Monastrol with KSP
tetrahydro-b-carboline core is shared by part 2 and part 4 of indole ring of Trp127 were close to the side chain of monas-
our pharmacophore. Benzene ring is a typical hydrophobic trol, which may explain why an aryl group in part 3 can in-
group, thus a phenyl group could be introduced to fit part 2 crease the binding affinity to KSP. From the crystal structure,
of our pharmacophore. After changing the indole group of it is clear that the side chain of part 3 is exposed to the sol-
tetrahydro-b-carboline core into phenyl group, a tetrahy- vent, as was reported in previous literature.10,12) Thus, a hy-
droisoquinoline core could be gained (as shown in Fig. 3). drophilic group such as an amino group at this location may
Although, recently, a series of 4-phenyl tetrahydroisoquino- also increase the KSP inhibitory activity. Taken into consid-
lines were reported as potent KSP inhibitors,14) in which eration all the information, a series of molecules were de-
there is an aryl group connected to the C4 atom of the re- signed which are listed in Table 1.
ported tetrahydroisoquinolines, in our newly designed com-
Chemistry The synthesis of the tetrahydroisoquinoline-
pounds, a 3ꢁ-hydroxyphenyl group was attached to C1 posi- based compounds is presented in Chart 1. Condensation of
tion of tetrahydroisoquinolines. In the structure of these 1- piperoethylamine (2) with 3-hydroxybenzaldehyde (3), fol-
(3ꢁ-hydroxyphenyl)-tetrahydroisoquinoline compounds, the lowed by cyclization in the presence of HCl, gave the inter-
tetrahydroisoquinoline ring is shared by part 2 and part 4 of mediate 5. In the last step, acylation with different acids led
KSP inhibitor 1. To facilitate the synthesis, a methylenedioxy to thirteen desired compounds which were confirmed by IR,
substitution was introduced to the tetrahydroisoquinoline 1H-NMR, and elemental analysis.
core. Combining these fragments, an aromatic ring (part 1), a
KSP ATPase Assay The motor and linker domain of
hydrophobic group (part 2), a side chain (part 3) and a flexi- human KSP protein was cloned, expressed in Escherichia
ble heterocyclic ring (part 4), as shown in Fig. 2, are all pres- coli BL21 (DE3) cells and then purified. The effects of tem-
ent in the newly designed compounds.
perature, pH, metal ions and dimethyl sulfoxide (DMSO) on
In order to optimize the design of KSP inhibitor 1, the ATPase activity were investigated to validate the assay. KSP
binding model of compound 1 with KSP was analyzed and inhibitory activity was assessed by measuring the release
compared with the crystal structure of monastrol bound to of inorganic phosphate from ATP hydrolysis through ab-
KSP. As shown in Fig. 4, the benzene ring of Tyr211 and the sorbance detection of a malachite green–phosphate com-

June 2009
569
Table 1. Bioactivity of the Synthesized KSP Inhibitors
chain may contribute to cytotoxicity against cells. Overall,
the result from the cytotoxicity assay has good correlation
with the KSP inhibitory activities.
KSP IC50
Cytotoxicitya)
Compounds
R
(mM)
(mM)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
–CH2CH2CH3
–CH2CH2NH2
–CH3
PhCH2–
–OCH2CH3
–OCH3
4-OH–Ph–
4-CH3–Ph–
Naphth–CH2–
4-OCH3–Ph–
4-Cl–Ph–
3-Cl–Ph–
4-NO2–PhCH2–
—
1.09
0.02
0.51
1.02
0.17
4.66
0.04
0.06
0.23
0.95
0.35
3.65
7.14
7.45
12.65
8.34
Conclusion
In summary, we report the design, synthesis, and evalua-
tion of a series of tetrahydroisoquinolines derivatives (1a—
m) as novel KSP inhibitors based on the crystal structure of
monastrol bound to KSP protein, as well as pharmacophore
analysis based on the available literature. Generally, all thir-
teen synthesized compounds were more potent than monas-
trol in human KSP protein ATPase assays. Three KSP in-
hibitors (1b, 1g, 1h) had over 100 times higher potency than
monastrol. In vitro cytotoxicity assay using the MTT method
indicated that nine compounds (1a, 1b, 1c, 1d, 1e, 1g, 1h, 1j,
1k) were more cytotoxic than monastrol. Compounds 1b and
1g, both containing a hydrophilic group at the end of the side
chain at 2-position, exhibited excellent cell-killing activities
against HepG2. This discovery may yield novel lead com-
pounds for further modification/optimization in the develop-
ment of potent KSP inhibitors as anti-cancer agents.
15.44
13.01
10.33
21.41
6.08
10.97
25.33
19.22
14.21
29.92
36.38
20.45
Monastrol
a) Presented as IC50 values against HepG2 cells.
Experimental
Chemistry All chemicals were obtained commercially and used without
further purification. Melting points were determined on Mel-Temp II appara-
tus. Infrared spectra were acquired on Nicolet Impact 410 spectrophotometer
1
using a KBr film. The absorption band is given in cmꢂ1. H-NMR spectra
were recorded on a Bruker ACF-300 spectrometer (300 MHz). Chemical
shifts are presented in ppm relative to tetramethylsilane. Mass spectra were
obtained on a Mariner Mass Spectrum, a GC-2010 mass spectrometer, or a
MAT-212 mass spectrometer. Elemental analyses were determined on a
Carlo Erba 1106 elementary analysis apparatus.
3-[(2-(3,4-Dioxolo)phenylethylimine)methyl]phenol (4) Piperoethyl-
amine 2 (10.00 g, 0.06 mol) was added into a solution of 3-hydroxybenz-
aldehyde 3 (7.40 g, 0.06 mol) in ethanol (60 ml) at room temperature. The
mixture was stirred for 10 min and cooled. The precipitate (4) was collected
by filtration as an off-white powder (14.80 g, yield 90.8%): mp 153—
155 °C. IR (cmꢂ1): 3474, 3050, 2906, 2867, 1649, 1595, 1502, 1454, 1273,
1247, 1038, 923, 775, 686. 1H-NMR (CDCl3, ppm) d: 2.83 (t, Jꢀ7.2 Hz,
2H, –CH2CH2N), 3.74 (t, Jꢀ7.2 Hz, 2H, –CH2CH2N), 5.95 (s, 2H,
–OCH2O–), 6.68—7.25 (m, 7H, Ar-H), 8.17 (s, 1H, NꢀCH–), 9.54 (s, 1H,
–OH). EI-MS: 269 [M].
Reaction conditions: (a) EtOH, room temperature; (b) 24% HCl, 60 °C; (c) RCOOH,
PyBOP, TEA, dry THF.
Chart 1. General Route for the Synthesis of Tetrahydroisoquinolines
plex.33) The IC50 values of the target compounds against KSP
were determined by measuring the activities of microtubule-
activated ATPase using monastrol as the control.
As shown in Table 1, all thirteen compounds were more
potent than monastrol in inhibiting KSP activity. Compound
1b, 1g and 1h, with the side chains being aminopropionyl,
4ꢁ-hydroxybenzoyl and 4ꢁ-methylbenzoyl respectively, were
1-(3ꢀ-Hydroxyphenyl)-6,7-dioxolo-1,2,3,4-tetrahydroisoquinoline (5)
The mixture of 4 (10.00 g, 0.037 mol) and HCl (24%, 50 ml) was heated to
60 °C under N2. Eight hours later, the mixture was cooled and filtered. The
over 100 times more potent than monastrol. This finding solid was washed with acetone to yield the title compound as an off-white
powder (5.40 g, yield 47.6%): mp 213—215 °C. IR (cmꢂ1): 3423, 3224,
proved that tetrahydroisoquinolines with a 3ꢁ-hydroxyphenyl
group attached to C1 are potent KSP inhibitors. It was also
found that an aryl group attached to N2, as well as a hy-
2934, 2769, 1589, 1503, 1486, 1243, 1039, 938, 787, 700. 1H-NMR
(DMSO-d6, ppm) d: 2.88—2.99 (m, 2H, –CH2CH2N), 3.13—3.20 (m, 2H,
–CH2CH2N), 5.31 (s, 1H, NH), 5.93 (s, 2H, –OCH2O–), 6.19 (s, 1H, CH),
drophilic group at the end of the side chain attached to N2,
could increase the KSP inhibitory activity of the compound.
Taken together, these results confirm the hypothesis we pro-
posed during the pharmacophore design.
6.68—7.19 (m, 6H, Ar-H), 9.55 (s, 1H, –OH). EI-MS: 269 [M].
General Procedure for the Synthesis of the Target Compounds 1a,
1c—m A mixture of the proper organic acid (0.98 mmol), PyBOP (0.51 g,
0.98 mmol), TEA (0.3 ml) and dry THF (15 ml) was stirred at room tempera-
ture for 10 min. Compound 5 (0.30 g, 0.98 mmol) was then added. The mix-
ture was stirred for 3 h and filtered. The filtrate was purified by column chro-
matography (petroleum ether/ethyl acetateꢀ2/1).
Cytotoxic Activity in Vitro All the tetrahydroisoquino-
lines were evaluated for cytotoxicity to human liver cancer
HepG2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay. As shown in
Table 1, nine compounds (1a, 1b, 1c, 1d, 1e, 1g, 1h, 1j, 1k)
exhibited more potent growth inhibition of HepG2 than
monastrol. In particular, compounds 1b and 1g, with the side
chains being aminopropionyl and 4ꢁ-hydroxybenzoyl respec-
tively, exhibited excellent cell-killing activities against
HepG2 cells. Interestingly, there is a hydrophilic group at the
end of the side chain of part 3 in each of the two structures,
1-(3ꢀ-Hydroxyphenyl)-2-butyryl-6,7-dioxolo-1,2,3,4-tetrahydroiso-
quinoline (1a) White powder, yield 57.1%; mp 159—160 °C. IR (cmꢂ1):
1
3192, 2957, 2873, 1593, 1485, 1275, 1231, 1038, 925, 774, 706. H-NMR
(DMSO-d6, ppm) d: 0.87 (t, Jꢀ7.37 Hz, 3H, –CH2CH2CH3), 1.53 (m, 2H,
–CH2CH2CH3), 2.33 (m, 2H, –CH2CH2N), 2.6 (m, 2H, –CH2CH2CH3),
2.61—2.81 (m, 2H, –CH2CH2N), 5.95 (s, 2H, –OCH2O–), 6.68 (s, 1H, CH),
6.49—6.60, 6.76—7.09 (m, 6H, Ar-H), 9.24 (s, 1H, –OH). EI-MS: 339 [M].
Anal. Calcd for C20H21NO4: C, 70.78; H, 6.24; N, 4.13. Found: C, 70.78; H,
6.24; N, 4.00.
1-(3ꢀ-Hydroxyphenyl)-2-acetyl-6,7-dioxolo-1,2,3,4-tetrahydroisoquino-
line (1c) White powder, yield 55.7%; mp 198—200 °C. IR (cmꢂ1): 3265,
which suggested that the terminal –OH or –NH2 of the side 2903, 1618, 1584, 1484, 1448, 1235, 1037, 921, 883, 775. 1H-NMR

570
Vol. 57, No. 6
(DMSO-d6, ppm) d: 2.09 (s, 3H, –COCH3), 2.64—2.85 (m, 2H,
–CH2CH2N), 3.31—3.70 (m, 2H, –CH2CH2N), 5.98 (s, 2H, –OCH2O–), 6.71
(s, 1H, CH), 6.52—6.63, 6.77—7.12 (m, 6H, Ar-H), 9.28 (s, 1H, –OH). EI-
MS: 311 [M]. Anal. Calcd for C18H17NO4: C, 69.44; H, 5.50; N, 4.50.
Found: C, 69.48; H, 5.52; N, 4.54.
1-(3ꢀ-Hydroxyphenyl)-2-phenylacetyl-6,7-dioxolo-1,2,3,4-tetrahy-
droisoquinoline (1d) White powder, yield 21.1%; mp 184—185 °C. IR
(cmꢂ1): 3098, 2895, 1590, 1481, 1275, 1229, 1038, 921, 742, 692. 1H-NMR
(DMSO-d6, ppm) d: 2.60 (s, 2H, –CH2CO), ca. 2.6 (m, 2H, –CH2CH2N),
3.81 (m, 2H, –CH2CH2N), 5.97 (s, 2H, –OCH2O–), 6.68 (s, 1H, CH), 6.53—
6.62, 6.75—7.30 (m, 11H, Ar-H), 9.29 (s, 1H, –OH). EI-MS: 387 [M]. Anal.
Calcd for C24H21NO4: C, 74.40; H, 5.46; N, 3.62. Found: C, 74.40; H, 5.47;
N, 3.54.
1-(3ꢀ-Hydroxyphenyl)-2-ethoxycarboxyl-6,7-dioxolo-1,2,3,4-tetrahy-
droisoquinoline (1e) White powder, yield 44.8%; mp 180—181 °C. IR
(cmꢂ1): 3255, 2973, 2877, 1660, 1596, 1487, 1438, 1249, 1111, 928, 775.
1H-NMR (DMSO-d6, ppm) d: 1.26 (m, 3H, –CH2CH3), 2.61—2.89 (m, 2H,
–CH2CH2N), 3.16—3.22 (m, 2H, –CH2CH2N), 4.18 (m, 2H, –CH2CH3),
5.92 (s, 2H, –OCH2O–), 6.63 (s, 1H, CH), 6.49, 6.71—7.16 (m, 6H, Ar-H).
EI-MS: 341 [M]. Anal. Calcd for C19H19NO5: C, 66.85; H, 5.61; N, 4.10.
Found: C, 66.73; H, 5.70; N, 4.03.
1-(3ꢀ-Hydroxyphenyl)-2-methoxycarboxyl-6,7-dioxolo-1,2,3,4-tetrahy-
droisoquinoline (1f) White powder, yield 49.8%; mp 244—246 °C. IR
(cmꢂ1): 3263, 2904, 1658, 1594, 1487, 1448, 1246, 1217, 1039, 942, 927,
776, 700. 1H-NMR (CDCl3, ppm) d: 2.62—2.80 (m, 2H, –CH2CH2N),
3.20—3.50 (m, 2H, –CH2CH2N), 3.76 (s, 3H, –CH3), 5.91 (s, 2H,
–OCH2O–), 6.63 (s, 1H, CH), 6.50, 6.71—7.29 (m, 6H, Ar-H). EI-MS: 327
[M]. Anal. Calcd for C18H17NO5: C, 66.05; H, 5.23; N, 4.28. Found: C,
65.92; H, 5.36; N, 4.16.
1-(3ꢀ-Hydroxyphenyl)-2-(4ꢀ-hydroxyphenylacetyl)-6,7-dioxolo-1,2,3,4-
tetrahydroisoquinoline (1g) White powder, yield 55.0%; mp 140 °C. IR
(cmꢂ1): 3308, 2954, 1734, 1562, 1484, 1233, 1037, 939, 778. 1H-NMR
(DMSO-d6, ppm) d: 2.63—2.89 (m, 2H, –CH2CH2N), 3.21—3.60 (m, 2H,
–CH2CH2N), 5.98 (s, 2H, –OCH2O–), 6.97 (s, 1H, CH), 6.63—6.65, 6.79—
7.23 (m, 10H, Ar-H), 9.34 (s, 1H, –OH), 9.81 (s, 1H, –OH). EI-MS: 389
[M]. Anal. Calcd for C23H19NO5.C2H5OH: C, 68.96; H, 5.75; N, 3.19.
Found: C, 68.68; H, 5.88; N, 2.90.
1-(3ꢀ-Hydroxyphenyl)-2-(4ꢀ-methylbenzoyl)-6,7-dioxolo-1,2,3,4-tet-
rahydroisoquinoline (1h) White powder, yield 31.6%; mp 186—187 °C.
IR (cmꢂ1): 3414, 3170, 2886, 1594, 1440, 1235, 1033, 932, 829, 781, 706.
1H-NMR (DMSO-d6, ppm) d: 2.34 (s, 3H, –CH3), 2.60—2.85 (m, 2H,
–CH2CH2N), 3.20—3.48 (m, 2H, –CH2CH2N), 5.99 (s, 2H, –OCH2O–), 6.79
(s, 1H, CH), 6.45—6.71, 7.12—7.26 (m, 10H, Ar-H), 9.38 (s, 1H, –OH). EI-
MS: 387 [M]. Anal. Calcd for C24H21NO4: C, 74.40; H, 5.46; N, 3.62.
Found: C, 74.47; H, 5.53; N, 3.51.
7.90 (m, 10H, Ar-H), 9.37 (s, 1H, –OH). EI-MS: 407 [M]. Anal. Calcd for
C23H18ClNO4·0.5H2O: C, 66.26; H, 4.56; N, 3.36. Found: C, 66.38; H, 4.70;
N, 3.26.
1-(3 -Hydroxyphenyl)-2-(4 -nitrophenylacetyl)-6,7-dioxolo-1,2,3,4-
tetrahydroisoquinoline (1m) Pale yellow powder, yield 8.7%; mp 153—
155 °C. IR (cmꢂ1): 3415, 3079, 2931, 1618, 1519, 1483, 1345, 1236, 1037,
922, 858, 773, 698. 1H-NMR (DMSO-d6, ppm) d: 2.66—2.80 (m, 2H,
–CH2CH2N), 2.67 (s, 2H, –CH2CO), 3.34—3.50 (m, 2H, –CH2CH2N), 5.93
(s, 2H, –OCH2O–), 6.77 (s, 1H, CH), 6.48—6.73, 6.90—8.14 (m, 10H, Ar-
H). EI-MS: 432 [M]. Anal. (C24H20N2O6) C, H, N. Calcd: 66.66, 4.66, 6.48;
Found: 66.44, 4.86, 6.41.
ꢀ
ꢀ
1-(3 -Hydroxyphenyl)-2-(Boc- -alaninyl)-6,7-dioxolo-1,2,3,4-tetrahy-
ꢀ
b
droisoquinoline (1b ) Following the synthetic procedure of compound
(1a), the title compound was obtained from compound 5 (0.50 g, 1.64
mmol), (0.31 g, 1.64 mmol), PyBOP (0.85 g, 1.64 mmol) as a colorless oil
(0.62 g, yield 86.1%). IR (cmꢂ1): 3342, 2974, 1686, 1617, 1484, 1453, 1366,
1237, 1167, 1038, 936, 864, 778, 702. 1H-NMR (DMSO-d6, ppm) d: 1.41 (s,
9H, CH3ꢃ3), 2.52 (m, 2H, –CH2CH2CO), 3.45 (m, 4H, –CH2CH2N and
–CH2CH2NHBoc), 5.93 (s, 2H, –OCH2O–), 6.73 (s, 1H, CH), 6.54—6.62,
6.76—7.26 (m, 6H, Ar-H). EI-MS: 440 [M].
ꢀ
1-(3 -Hydroxyphenyl)-2-( -aminopropionyl)-6,7-dioxolo-1,2,3,4-
tetrahydroisoquinoline (1b) Compound 1bꢁ (0.62 g, 1.40 mmol) was dis-
solved in ethyl acetate (25 ml), and the solution was saturated by HCl gas.
The mixture was stirred at room temperature for 3.5 h and evaporated to dry-
ness. Dry ether (10 ml) was added to the residue and the precipitate was col-
lected by filtration. The title compound was dried in vacuum to give 1b as a
pale yellow powder (0.20 g, yield 37.7%): mp 125 °C (dec.). IR (cmꢂ1):
3435, 3223, 2970, 1614, 1484, 1453, 1236, 1037, 922, 774. 1H-NMR
(DMSO-d6, ppm) d: 2.67—2.71 (m, 2H, –NCH2CH2CO), 2.77—2.86 (m,
2H, –CH2CO), 3.01—3.06 (m, 2H, –CH2CH2NH2), 3.32—3.45 (m, 2H,
–CH2CH2N), 5.98 (s, 2H, –OCH2O–), 6.74 (s, 1H, CH), 6.53—6.64, 6.79—
7.09 (m, 6H, Ar-H), 7.75 (br, 2H, –NH2), 9.34 (s, 1H, –OH). EI-MS: 340
[M]. Anal. Calcd for C19H20N2O4·HCl·1.5H2O: C, 56.51; H, 5.70; N, 6.94.
Found: C, 56.29; H, 5.96; N, 6.98.
ꢀ
b
Preparation of KSP The coding regions were PCR amplified from a
template (obtained in our laboratory) containing full-length human KSP. The
primers used were: forward 5ꢁ-TAT AGG GCG AAT TCC GCC ATG GCG
TCG CAG CCA-3ꢁ and reverse 5ꢁ-ACG GGC TGC AGC AAG CTC GAG
TTT TAA ACG TTC TAT-3ꢁ. The region encoding residues 2—386 was
sub-cloned into pET28a (NOVAGEN).34) Protein expression in E. coli cells
was induced with 0.5 mM IPTG. Cells were harvested after 20 h of growth at
20 °C and then lysed by sonication. The soluble lysate was clarified by cen-
trifugation and applied to a SP-Sepharose 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. KSP 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 gradient from 0—
1000 mM NaCl was used to elute KSP.35) Fractions were analyzed by SDS-
PAGE. The most concentrated fraction was dialyzed against ATPase buffer
(20 mM Na-PIPES, pH 7.5; 1 mM MgCl2; 1 mM Na-EGTA) and then
aliquoted, frozen in liquid nitrogen, and stored at ꢂ80 °C for future use.
ATPase Activity Assay All experiments were carried out at room tem-
perature. The reagents were added to the wells of a 96-well clear plate and
the final reaction of the assay contained 20 mM PIPES, pH 7.5, 5.0 mM
MgCl2, 1 mM EGTA, 10 mM paclitaxel, 0.6 mM tubulin (MT), 0.5 mM ATP,
2% DMSO containing inhibitors (DMSO had no effect on the ATPase activ-
ity as shown later) in a reaction volume of 100 ml. Reactions were started by
addition of ATP and the plates were incubated at 37 °C for 30 min. Mala-
chite-green based reagents were then added to detect the release of inorganic
phosphate.36) The plates were incubated for an additional 5 min at room tem-
perature, and 10 ml of 34% sodium citrate was added. The absorbance at
610 nm was determined using Multiskan Spectrum Microplate Spectropho-
tometer (Thermo Electron Corporation). The controls without KSP or MTs
were also measured to determine the background, which was subtracted
from all measured values. The controls with MTs but without KSP give the
nucleotide hydrolysis by MTs and should be subtracted from corresponding
values with KSP and the same concentration of MTs. The data were ana-
lyzed using Microsoft Excel to obtain the IC50 of the test compounds. IC50
values are reported as the averages of at least three independent determina-
tions; standard deviations are within ꢄ25—50% of IC50 values.
1-(3ꢀ-Hydroxyphenyl)-2-(b-naphthalin acetyl)-6,7-dioxolo-1,2,3,4- tet-
rahydroisoquinoline (1i) White powder, yield 14.0%; mp 224—226 °C.
1
IR (cmꢂ1): 3234, 2890, 1591, 1502, 1452, 1238, 1042, 925, 790, 703. H-
NMR (DMSO-d6, ppm) d: 2.64—2.85 (m, 2H, –CH2CH2N), 3.29—3.45 (m,
2H, –CH2CH2N), 4.25 (s, 2H, –CH2CO), 5.97 (s, 2H, –OCH2O–), 6.79 (s,
1H, CH), 6.57—6.71, 7.08—7.94 (m, 13H, Ar-H), 9.30 (s, 1H, –OH). EI-
MS: 437 [M]. Anal. Calcd for C28H23NO4: C, 76.87; H, 5.30; N, 3.20.
Found: C, 76.58; H, 5.46; N, 3.07.
1-(3ꢀ-Hydroxyphenyl)-2-(4ꢀ-methoxybenzoyl)-6,7-dioxolo-1,2,3,4-
tetrahydroisoquinoline (1j) White powder, yield 25.3%; mp 192 °C. IR
(cmꢂ1): 3203, 2887, 1589, 1447, 1252, 1176, 1038, 941, 836, 772, 744. 1H-
NMR (DMSO-d6, ppm) d: 2.63—2.86 (m, 2H, –CH2CH2N), 3.22—3.60 (m,
2H, –CH2CH2N), 5.99 (s, 2H, –OCH2O–), 6.79 (s, 1H, CH), 6.63—6.70,
6.98—7.97 (m, 10H, Ar-H), 9.34 (s, 1H, –OH). EI-MS: 403 [M]. Anal.
Calcd for C24H21NO5·H2O: C, 68.40; H, 5.46; N, 3.32. Found: C, 68.32; H,
5.62; N, 2.99.
1-(3ꢀ-Hydroxyphenyl)-2-(4ꢀ-chlorobenzoyl)-6,7-dioxolo-1,2,3,4-tet-
rahydroisoquinoline (1k) White powder, yield 25.0%; mp 191—193 °C.
IR (cmꢂ1): 3182, 1586, 1484, 1445, 1241, 1039, 941, 845, 771, 700. 1H-
NMR (DMSO-d6, ppm) d: 2.64—2.90 (m, 2H, –CH2CH2N), 3.39—3.50 (m,
2H, –CH2CH2N), 6.00 (s, 2H, –OCH2O–), 6.74 (s, 1H, CH), 6.66, 6.81—
7.54 (m, 10H, Ar-H), 9.37 (s, 1H, –OH). EI-MS: 407 [M]. Anal. Calcd for
C23H18ClNO4·0.5H2O: C, 66.26; H, 4.56; N, 3.36. Found: C, 65.99; H, 4.69;
N, 3.23.
1-(3ꢀ-Hydroxyphenyl)-2-(3ꢀ-chlorobenzoyl)-6,7-dioxolo-1,2,3,4-tet-
rahydroisoquinoline (1l) White powder, yield 37.5%; mp 91—93 °C. IR
(cmꢂ1): 3262, 2895, 1592, 1484, 1445, 1235, 1037, 940, 774, 697. 1H-NMR
(DMSO-d6, ppm) d: 2.63—2.86 (m, 2H, –CH2CH2N), 3.34—3.47 (m, 2H,
–CH2CH2N), 5.99 (s, 2H, –OCH2O–), 6.74 (s, 1H, CH), 6.65—6.67, 6.80—
In Vitro Cytotoxicity Assay (MTT Assay) The tested cells were intro-
duced into each well of a 96-well plate, with a density of 2500 cells/well.
The cells were then exposed to compounds of different concentrations (1.0,

June 2009
571
2.0, 4.0, 8.0, 16, 32 mM) (100 ml/well). Controls were performed in which
only culture media was added into wells containing cells. After 48 h incuba-
tion, 5 mg/ml MTT solution (20 ml/well) was added and cultured for 4 h, and
then the supernatant was discarded and DMSO was added in (100 ml/well),
respectively. The suspension was placed on micro-vibrator for 5 min and the
absorbance (A) was measured at 570 nm by the Universal Microplate Reader
(EL800, BIO-TEK INSTRUMENTS INC.). Triplicate experiments were
performed in a parallel manner for each concentration point and the results
were reported presented as mean. Cell inhibitory ratio was calculated by the
following formula:
15) Cox C. D., Breslin M. J., Whitman D. B., Coleman P. J., Garbaccio R.
M., Fraley M. E., Zrada M. W., Buser C. A., Walsh E. S., Hamilton K.,
Lobell R. B., Tao W., Abrams M. T., South V. J., Huber H. E., Kohlb
N. E., Hartmana G. D., Bioorg. Med. Chem. Lett., 17, 2697—2702
(2007).
16) Coleman P. J., Schreier J. D., Cox C. D., Fraley M. E., Garbaccio R.
M., Buser C. A., Walsh E. S., Hamilton K., Lobell R. B., Rickert K.,
Tao W., Diehl R., South V. J., Davide J. P., Kohlb N. E., Yan Y., Kuo L.,
Prueksaritanont T., Li C., Mahan E. A., Fernandez-Metzler C., Salata
J. J., Hartmana G. D., Bioorg. Med. Chem. Lett., 17, 5390—5395
(2007).
17) Garbaccio R. M., Tasber E. S., Neilson L. A., Coleman P. J., Fraley M.
E., Olson C., Bergman J., Torrent M., Buser C. A., Rickert K., Walsh
E. S., Hamilton K., Lobell R. B., Tao W., South V. J., Diehl R. E., Da-
vide J. P., Yan Y., Kuo L., Li C., Prueksaritanont T., Fernandez-Metzler
C., Mahan E. A., Slaughter D. E., Salata J. J., Kohl N. E., Huberd H.
E., Hartmana G. D., Bioorg. Med. Chem. Lett., 17, 5671—5676
(2007).
18) Roecker A. J., Coleman P. J., Mercer S. P., Schreier J. D., Buser C. A.,
Walsh E. S., Hamilton K., Lobell R. B., Tao W., Diehl R. E., South V.
J., Davide J. P., Kohl N. E., Yan Y., Kuo L., Li C., Fernandez-Metzler
C., Mahan E. A., Prueksaritanont T., Hartmana G. D., Bioorg. Med.
Chem. Lett., 17, 5677—5682 (2007).
inhibitory ratio (%)ꢀ[(AcontrolꢂAtreated)/Acontrol]ꢃ100%
The IC50 was taken as the concentration that caused 50% inhibition of cell
proliferation.
Acknowledgements This work was supported by a grant from “The Six
Top Talents” of Jiangsu Province (No. 06-C-023); Specialized Research
Fund for the Doctoral Program of Higher Education P.R.C. (No.
20060316006) and National Natural Science Foundation (30772624). This
work was also financially supported by Jiangsu Hengrui Medicine Co., Ltd.
References
1) Heald R., Cell, 102, 399—402 (2000).
2) Blangy A., Lane H. A., d’Hérin P., Harper M., Kress M., Nigg E. A.,
Cell, 83, 1159—1169 (1995).
3) Sharp D. J., Rogers G. C., Scholey J. M., Nature (London), 407, 41—
47 (2000).
4) Mayer T. U., Kapoor T. M., Haggarty S. J., King R. W., Schreiber S.
L., Mitchison T. J., Science, 286, 971—974 (1999).
5) Nakazawa J., Yajima J., Usui T., Ueki M., Takatsuki A., Imoto M.,
Toyoshima Y., Osada H., Chem. Biol., 10, 131—137 (2003).
6) DeBonis S., Skoufias D. A., Lebeau L., Lopez R., Robin G., Margolis
R. L., Wade R. H., Kozielski F., Mol. Cancer Ther., 3, 1079—1090
(2004).
7) Hotha S., Yarrow J. C., Yang J. G., Garrett S., Renduchintala K. V.,
Mayer T. U., Kapoor T. M., Angew. Chem., 115, 2481—2484 (2003).
8) Sakowicz R., Finer J. T., Beraud C., Crompton A., Lewis E., Fritsch
A., Lee Y., Mak J., Moody R., Turincio R., Chabala J. C., Gonzales P.,
Roth S., Weitman S., Wood K. W., Cancer Res., 64, 3276—3280
(2004).
19) Pinkerton A. B., Lee T. T., Hoffman T. Z., Wang Y., Kahraman M.,
Cook T. G., Severance D., Gahman T. C., Noble S. A., Shiaub A. K.,
Davisb R. L., Bioorg. Med. Chem. Lett., 17, 3562—3569 (2007).
20) Parrish C. A., Adams N. D., Auger K. R., Burgess J. L., Carson J. D.,
Chaudhari A. M., Copeland R. A., Diamond M. A., Donatelli C. A.,
Duffy K. J., Faucette L. F., Finer J. T., Huffman W. F., Hugger E. D.,
Jackson J. R., Knight S. D., Luo L., Moore M. L., Newlander K. A.,
Ridgers L. H., Sakowicz R., Shaw A. N., Sung C. M., Sutton D., Wood
K. W., Zhang S. Y., Zimmerman M. N., Dhanak D., J. Med. Chem., 50,
4939—4952 (2007).
21) DeBonis S., Skoufias D. A., Indorato R. L., Liger F., Marquet B., Lag-
gner C., Joseph B., Kozielski F., J. Med. Chem., 51, 1115—1125
(2008).
22) Cox C. D., Coleman P. J., Breslin M. J., Whitman D. B., Garbaccio R.
M., Fraley M. E., Buser C. A., Walsh E. S., Hamilton K., Schaber M.
D., Lobell R. B., Tao W., Davide J. P., Diehl R. E., Abrams M. T.,
South V. J., Huber H. E., Torrent M., Prueksaritanont T., Li C., Slaugh-
ter D. E., Mahan E., Fernandez-Metzler C., Yan Y., Kuo L. C., Kohl N.
E., Hartman G. D., J. Med. Chem., 51, 4239—4252 (2008).
23) Jiang C., You Q. D., Li Z. Y., Guo Q. L., Expert Opin. Ther. Patents,
16, 1517—1532 (2006).
9) Cox C. D., Breslin M. J., Mariano B. J., Coleman P. J., Buser, C. A.,
Walsh E. S., Hamilton K., Huber H. E., Kohl N. E., Torrent M., Yan
Y., Kuo L. C., Hartman G. D., Bioorg. Med. Chem. Lett., 15, 2041—
2045 (2005).
10) Fraley M. E., Garbaccio R. M., Arrington K. L., Hoffman W. F., Tas-
ber E. S., Coleman P. J., Buser C. A., Walsh E. S., Hamilton K., Fer-
nandes C., Schaber M. D., Lobell R. B., Tao W., South V. J., Yan Y.,
Kuo L. C., Prueksaritanont T., Shu C., Torrent M., Heimbrook D. C.,
Kohl N. E., Huber H. E., Hartman G. D., Bioorg. Med. Chem. Lett., 16,
1775—1779 (2006).
11) Garbaccio R. M., Fraley M. E., Tasber E. S., Olson C. M., Hoffman W.
F., Arrington K. L., Torrent M., Buser C. A., Walsh E. S., Hamilton
K., Schaber M. D., Fernandes C., Lobell R. B., Tao W., South V. J., Yan
Y., Kuo L. C., Prueksaritanont T., Slaughter D. E., Shu C., Heimbrook
D. C., Kohl N. E., Huber H. E., Hartman G. D., Bioorg. Med. Chem.
Lett., 16, 1780—1783 (2006).
12) Cox C. D., Torrent M., Breslin M. J., Mariano B. J., Whitman D. B.,
Coleman P. J., Buser C. A., Walsh E. S., Hamilton K., Schaber M. D.,
Lobell R. B., Tao W., South V. J., Kohl N. E., Yan Y., Kuo L. C., Pruek-
saritanont T., Slaughter D. E., Li C., Mahan E., Lu B., Hartman G. D.,
Bioorg. Med. Chem. Lett., 16, 3175—3179 (2006).
13) Kim K. S., Lu S., Cornelius L. A., Lombardo L. J., Borzilleri R. M.,
Schroeder G. M., Sheng C., Rovnyak G., Crews D., Schmidt R. J.,
Williams D. K., Bhide R. S., Traeger S. C., McDonnell P. A., Mueller
L., Sheriff S., Newitt J. A., Pudzianowski A. T., Yang Z., Wild R., Lee
F. Y., Batorsky R., Ryder J. S., Ortega-Nanos M., Shen H., Gottardis
M., Roussell D. L., Bioorg. Med. Chem. Lett., 16, 3937—3942 (2006).
14) Tarby C. M., Kaltenbach R. F., Huynh T., Pudzianowski A., Shen H.,
Ortega-Nanos M., Sheriff S., Newitt J. A., McDonnell P. A., Burford
N., Fairchild C. R., Vaccaro W., Chen Z., Borzilleri R. M., Naglich J.,
Lombardo L. J., Gottardis M., Trainor G. L., Roussell D. L., Bioorg.
Med. Chem. Lett., 16, 2095—2100 (2006).
24) Liu F., You Q. D., Chen Y. D., Bioorg. Med. Chem. Lett., 17, 722—726
(2007).
25) Jiang C., Chen Y. D., Wang X. J., You Q. D., J. Mol. Model., 13, 987—
992 (2007).
26) Yan Y., Sardana V., Xu B., Homnick C., Halczenko W., Buser C. A.,
Schaber M., Hartman G. D., Huber H. E., Kuo L. C., J. Mol. Biol.,
335, 547—554 (2004).
27) Cogan E. B., Birell G. B., Griffith O. H., Anal. Biochem., 271, 29—35
(1999).
28) Fraley M. E., Garbaccio R. M., Olson C. M., Tasber E. S., WO
2004058700 (2004).
29) Wang W. B., Ni Z. J., Barsanti P., Pecchi S., Xia Y., Brammeier N.,
Treutle M., Jazan E., Wayman K., Dibble D., Cheng J. K., WO
2005070930 (2005).
30) Ruan X. Q., You Q. D., Yang L., Wu W. T., Acta Pharm. Sinica, 43,
828—832 (2008).
31) Ruan X. Q., You Q. D., Yang L., Wu W. T., Acta Chim. Sinica, 66,
1731—1734 (2008).
32) Sunder-Plassmann N., Sarli V., Gartner M., Utz M., Seiler J., Huem-
mer S., Mayer T. U., Surreyb T., Giannisa A., Bioorg. Med. Chem., 13,
6094—6111 (2005).
33) Garcia-Saez I., DeBonis S., Lopez R., Trucco F., Rousseau B., Thuery
P., Kozielski F., J. Biol. Chem., 282, 9740—9747 (2007).
34) Blangy A., rnaud L., Nigg E. A., J. Biol. Chem., 272, 19418—19424
(1997).
35) Turner J., Anderson R., Guo J., Beraud C., Fletterick R., Sakowicz R.,
J. Biol. Chem., 276, 25496—25502 (2001).
36) Ohno T., Kodama T., J. Physiol., 441, 685—702 (1991).