Design, synthesis, and biological evaluation of novel water-soluble triptolide derivatives: Antineoplastic activity against imatinib-resistant CML cells bearing T315I mutant Bcr-Abl

Article abstract of DOI:10.1016/j.bmc.2010.01.052

Imatinib (STI571) is the frontline targeted-therapeutic agent for patients with chronic myelogenous leukemia (CML). However, resistance to imatinib due to point mutations in Bcr-Abl kinase domain is an emerging problem. We recently reported that triptolide (compound 1) could effectively kill CML cells including those harboring T315I mutant Bcr-Abl. In the present study, we designed a series of C-14 triptolide derivatives with C-14-hydroxyl substituted by different amine esters (3-18): 3-6 and 13 (by aliphatic chain amine esters); 7-9, 11, 12 and 15-18 (by alicyclic amine esters with different size), and 10 and 14 (by aralkylamine esters).The compounds were examined for their antineoplastic activity against CML cells (including KBM5-T315I cells) in terms of proliferation inhibition, apoptosis and signal transduction. Nude mouse xenograft model was also used to evaluate the in vivo activity. Compounds 2-9, 11-14, 17 and 18 exhibited a potent inhibitory activity against KBM5 and KBM5-T315I cells. This series of derivatives down-regulated Bcr-Abl mRNA. Compounds 4, 5, 8 and 9 were further examined for their impact on signaling and apoptosis with immunoblotting. Compound 5 was chosen for evaluation in a nude mouse xenograft model. The stereo-hindrance of C-14 group appeared to be responsible for the antitumor effect. The computational small molecule-protein docking analysis illustrated the possible interaction between compound 9 and RNA polymerase II. Our results suggest that this series of derivatives may be promising agents to overcome imatinib-resistance caused by the Bcr-Abl-T315I mutation.

Full text of DOI:10.1016/j.bmc.2010.01.052

Bioorganic & Medicinal Chemistry 18 (2010) 1806–1815
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel water-soluble
triptolide derivatives: Antineoplastic activity against imatinib-resistant
CML cells bearing T315I mutant Bcr-Abl
Fang Xu a,b, , Xianping Shi b, , Shichang Li a, Jieshun Cui a, Zhongzheng Lu b, Yanli Jin b, Yongcheng Lin a,
b,
Jiyan Pang a, , Jingxuan Pan
*
*
a School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275, People’s Republic of China
b Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510089, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Imatinib (STI571) is the frontline targeted-therapeutic agent for patients with chronic myelogenous leu-
kemia (CML). However, resistance to imatinib due to point mutations in Bcr-Abl kinase domain is an
emerging problem. We recently reported that triptolide (compound 1) could effectively kill CML cells
including those harboring T315I mutant Bcr-Abl. In the present study, we designed a series of C-14 tri-
ptolide derivatives with C-14-hydroxyl substituted by different amine esters (3–18): 3–6 and 13 (by ali-
phatic chain amine esters); 7–9, 11, 12 and 15–18 (by alicyclic amine esters with different size), and 10
and 14 (by aralkylamine esters).The compounds were examined for their antineoplastic activity against
CML cells (including KBM5-T315I cells) in terms of proliferation inhibition, apoptosis and signal trans-
duction. Nude mouse xenograft model was also used to evaluate the in vivo activity. Compounds 2–9,
11–14, 17 and 18 exhibited a potent inhibitory activity against KBM5 and KBM5-T315I cells. This series
of derivatives down-regulated Bcr-Abl mRNA. Compounds 4, 5, 8 and 9 were further examined for their
impact on signaling and apoptosis with immunoblotting. Compound 5 was chosen for evaluation in a
nude mouse xenograft model. The stereo-hindrance of C-14 group appeared to be responsible for the
antitumor effect. The computational small molecule-protein docking analysis illustrated the possible
interaction between compound 9 and RNA polymerase II. Our results suggest that this series of deriva-
tives may be promising agents to overcome imatinib-resistance caused by the Bcr-Abl-T315I mutation.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 13 December 2009
Revised 19 January 2010
Accepted 20 January 2010
Available online 25 January 2010
Keywords:
Triptolide
Derivatives
Synthesis
RNA polymerase
Inhibitor
Modeling
Tyrosine kinase inhibitors
Imatinib-resistance
CML
1. Introduction
subsequent signal transduction.6 Of newly diagnosed patients with
chronic-phase CML, 82% showed complete cytogenetic response on
The cytoplasmic tyrosine kinase Bcr-Abl, the product of a fusion
gene formed by reciprocal chromosomal translocation
treatment with imatinib over a median follow-up of 54 months.7
However, there are primary refractory disease and acquired
resistance to imatinib, particularly in patients with advanced-
phase disease.5 Although several mechanisms have been proposed
to explain acquired resistance to imatinib, mutation in the kinase
domain of Bcr-Abl is believed to be the predominant mecha-
nism.8–12 Approximately 50 point mutations have been identified
to be associated with clinical resistance to imatinib.13 To overcome
acquired resistance to imatinib, new tyrosine kinase inhibitors
such as nilotinib, dasatinib and INNO-406 have been developed
and are effective against all but the T315I mutation.14–16 The prog-
nosis for patients whose disease does not respond to imatinib, nil-
otinib and dasatinib is poor. Hence, novel strategies or compounds
to overcome this challenging resistance are required, and, as dem-
onstrated by recent reports,17,18 lowering the expression of Bcr-Abl
is a promising strategy.
a
t(9,22)(q34;q11), is found in approximately 95% patients with
chronic myelogenous leukemia (CML) and 30% of adult patients
with acute lymphoblastic leukemia.1–3 The aberrant tyrosine ki-
nase activity of this chimeric protein is responsible for malignant
transformation by activating multiple signal transduction path-
ways.4 Therefore, Bcr-Abl has been an important target for leuke-
mia therapeutics.5 Imatinib mesylate (STI571, Gleevec, Norvartis)
was the first small molecule inhibitor approved by the US Food
and Drug Administration. Imatinib effectively inhibits tyrosine ki-
nase activity by occupying the ATP-binding pocket of Bcr-Abl and
stabilizes the kinase in an inactive conformation, thus abrogating
* Corresponding authors. Tel./fax: +86 20 87332788 (J. Pan).
E-mail addresses: panjx2@mail.sysu.edu.cn (J. Pan), or cespjy@mail.sysu.edu.cn
(J. Pang).
Tripterygium wilfordii Hook. f., one of the traditional Chinese
medicine, has been used for centuries to treat inflammation and
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.052

F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
1807
autoimmune diseases.19–21 Among the many small molecules ex-
tracted and purified from this shrub-like vine, triptolide (com-
pound 1) (Fig. 1) is the key biologically active component that
mediates immunosuppression and anti-inflammation.22 Besides,
triptolide possesses antitumor activities against a broad range of
human cancer cells.23–25 Recently, we and others have demon-
strated that triptolide is an inhibitor of RNA polymerase I and II,
causing global transcription inhibition.26–29 Global transcriptional
or translational inhibition would result in different degrees of de-
creases in protein levels among different proteins due to different
degradation rates. We and others have demonstrated that tripto-
lide at low nanomolar concentrations can effectively kill imati-
nib-resistant CML cells bearing T315I Bcr-Abl, hypereosinophilic
molecular interaction between the derivatives of 1 and the target,
RNA polymerase II.
In this study, we designed a series of novel triptolide derivatives
by substituting C-14-hydroxyl with different amine esters (3–18).
The antitumor activity of these analogues was evaluated in imati-
nib-sensitive KBM5 cells and imatinib-resistant KBM5-T315I CML
cells in vitro and in a nude mouse model. Computational docking
analysis of these derivatives to RNA polymerase II was also con-
ducted. Our results demonstrated that this series of novel C-14
substituted derivatives of triptolide are promising antitumor drug
candidates to overcome the imatinib-resistance.
2. Chemistry
leukemia cells bearing T674I PDGFRa, and mast leukemia cells
bearing D816V KIT by inhibiting transcription of Bcr-Abl, PDGFR
a
Cationic amine group can facilitate the contact between bio-
macromolecule and small molecules by electrostatic interactions.
In most instances, a limited numbers of electrostatic interactions
contribute to the binding energy of association. Hydrogen bonds
and salt-bridges are believed to confer specificity to interactions
due to their dependence on the precise location of participating
atoms.34,35 We hypothesized that the characteristic hydrogen-
bonded C-14 in 1 substituted with amine groups may potently in-
hibit the growth of CML cells with Bcr-Abl-T315I mutation. We de-
signed a series of C-14 triptolide derivatives with C-14-hydroxyl
substituted by different amine esters (3–18): 3–6 and 13 (by ali-
phatic chain amine esters); 7–9, 11, 12 and 15–18 (by alicyclic
amine esters with different size), and 10 and 14 (by aralkylamine
esters) (Fig. 1). 14 was the combination of functional moieties of
1 and imatinib.
and KIT, respectively.26,27,29,30 Importantly, triptolide inhibits the
growth of xenografted Bcr-Abl-T315I CML cells in nude mice,30
and primary cells from CML patient with clinical resistance to
imatinib.29,30 These findings suggest that triptolide may be a prom-
ising agent in leukemia cells harboring mutant tyrosine kinase;
however, its clinical development has been challenged by severe
toxicity and water-insolubility.
Structure–activity relationship analysis in previous reports has
demonstrated that the characteristic hydrogen-bonded C14 in tri-
ptolide is a key functional group for its antitumor effect.23,31 At-
tempts to improve the solubility of
1 were documented
previously.31,32 For example, 14-succinyl triptolide sodium salt
(PG490-88) is a prodrug that is converted to triptolide in serum
and it retains all the toxicity of 1.32 Substituting C14-hydroxyl
group of 1 with fluoride can keep the antitumor activity while low-
ering the side effects.33 Nevertheless, little is known about the
The synthetic approach used for the preparation of compounds
2–18 is outlined in Scheme 1. Chloroacetyl of 1 at 0 °C, gave 2 in
Figure 1. Structure of triptolide (compound 1) and its derivatives (2–18).

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F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
Scheme 1. Synthetic route for compounds 2–18.
74.2% yield. Ammonolysis of 2 with different amines synthesized
3–18 in 55–90% yields. Compounds 2–18 were fully characterized
by HRMS and NMR (1H and 13C). They afforded HRMS spectra with
the m/z values corresponding to [M]+. Their NMR spectra were in
full agreement with the given structures. The purity was judged
from mass spectrometry, 1H NMR, and being single spots on TLC
developed by different eluting solvents.
T315I cells were incubated for 72 h with increasing concentrations
of various derivatives of triptolide. Cell viability of both cell types
was examined by the MTS assay, and IC50 values were listed in
Table 1. Representative dose–response curve were shown in Figure
2. Compounds 10, 15 and 16 exhibited weak potency, while all the
rest analogues including 14 exhibited potent inhibitory effect
against both KBM5 and KBM5-T315I cells. We discovered that
the bigger the substituted group was, the weaker the antitumor ef-
fect was (compared with 7 and 16), and that different heteroatom
might give different effects (compared 4, 5 and 6; 10 and 14). To
confirm the effect of triptolide derivatives on imatinib-resistant
CML cells, we used an additional pair of murine myeloid cells
32D stably transfected with either wild-type or T315I Bcr-Abl.
The data confirmed that imatinib-resistant 32D-T315I cells were
also sensitive to the derivatives (4, 5, 8 and 9). The IC50 values were
listed in Table 2. Taken together, the results support that the ste-
reo-hindrance of C-14 group is important for the antitumor effect
of these triptolide derivatives.
3. Results and discussion
3.1. Triptolide derivatives inhibits growth of imatinib-sensitive
and -resistant CML cells
T315I in Bcr-Abl is a notorious point mutation eliciting resis-
tance to imatinib. We recently found a KBM5 CML cell line harbor-
ing wild-type Bcr-Abl and a variant line harboring T315I Bcr-Abl
(KBM5-T315I) with different sensitivities to imatinib; IC50 values
were 0.28 lM and 5.4 l
M, respectively.30,36 KBM5 and KBM5-

F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
1809
Table 1
was assayed by MTS. The IC50 values in MEF and NHFB cells ranged
from 8800 to 11,500 nM) (Table 2), implying that there is a thera-
peutic window to use triptolide derivatives to treat CML.
IC50 values of derivatives of compound 1 based on 72-h incubation
Compound
R
IC50 (nM)
KBM5-T315I
KBM5
3.2. Derivatives of triptolide inhibit RNA polymerase activity
and induce apoptosis
1
2
10.3
27.2
8.3
52.8
Cl
We next examined whether the derivatives of 1, like their
parental compound triptolide, exerted their effect on the expres-
sion of Bcr-Abl which is the critical driving force for the transfor-
mation of CML cells. Based on IC50 values, compounds 4, 5, 8, or
9 were selected in such experiments. After 18-h treatments with
compounds 4, 5, 8, or 9, respectively, KBM5 versus KBM5-T315I,
32D-Bcr-Abl versus 32D-T315I cells were analyzed by immuno-
blotting or RT-PCR. The results showed that the protein levels of
Bcr-Abl were decreased by the four compounds in a dose-depen-
dent manner (Fig. 3A). Concomitantly, the mRNA levels of Bcr-
Abl were remarkably diminished (Fig. 3B). These results together
indicated that derivatives of 1 inhibit Bcr-Abl at the transcriptional
level leading to decreased protein levels.
Because triptolide is an inhibitor of RNA polymerase II (pol II),
we examined whether these analogues possessed similar activities.
As shown in Figure 3A, the phosphorylation of pol II was dimin-
ished in a dose-dependent manner in both KBM5 and KBM5-
T315I cells by these analogues. Similar findings were also obtained
in HeLa cells (cervical carcinoma), HCT116 (colon cancer), and
Du145 (prostate carcinoma) (data not shown), indicating a general
inhibitory activity of these compounds in a variety of cancer cells
of different tissue origins. In aggregate, treatment with the deriva-
tives of triptolide leads to inhibition of RNA polymerase resulting
in downregulation of Bcr-Abl mRNA level and Bcr-Abl protein level
regardless of mutation status the Bcr-Abl gene.
N
N
3
4
46.7
32.2
24.6
45.4
N
5
6
52.5
20.7
O
N
327.1
160.8
N
N
H
7
8
51.3
19.2
29.1
11.7
NH2
OH
N
H
9
18.2
19.7
NH
10
5020
734
Cl
O
H
3.3. Compound 5 inhibited growth of the xenografted KBM5-
T315I cells in nude mice
N
11
12
18.6
774
14.2
950
OMe
NH2
H
N
We examined the in vivo effect of compound 5 on imatinib-
resistant CML cells using the nude mouse xenograft model. Four
nu/nu BALB/c mice were injected with KBM5-T315I cells. Fourteen
days after inoculation of tumor cells, when the size of tumor
reached approximately 50 mm3, the mice were randomized to re-
ceive treatment with normal saline (placebo) or compound 5 dis-
solved in normal saline, 0.5 mg/kg/d, intraperitoneally (ip), for
two weeks (four animals each group). We found that this dosage
was well tolerated by pilot dose-finding experiments (data not
shown). The growth curves (the estimated tumor size calculated
from the tumor dimension versus time) are shown in Figure 4.
The data revealed that compound 5 potently inhibited the growth
of KBM5-T315I tumors. The body weight of the mice remained sta-
ble, with no significant differences between treated and control
mice (data not shown). Motor activity and feeding behavior of
the mice were all normal. Whole blood cell counts did not reveal
any significant myelosuppression in the treated mice (data not
shown), nor were aspartate and alanine aminotransferase activities
increased in the treatment group. No mice died. Overall, surveil-
lance of morbidity and mortality did not reveal any significant tox-
icity of the compound 5 at the dosage used.
N
13
14
496
297
548
N
H
NH
N
N
214.4
N
OH
N
15
16
2800
5420
1550
1040
OH
NH CH3
11
N
17
18
78
86
348
308
3.4. Molecular docking
NH
Triptolide does not directly inhibit the activity of tyrosine ki-
nase; rather, it inhibits the activity of RNA polymerase II (pol
II).26–28 We therefore performed the molecular docking to eluci-
date, at an atomic level, the potential interactions that govern
the recognition and the binding of triptolide derivatives (represen-
tative compounds 4, 5, 8 and 9) to pol II. To date, only the
The effect of the derivatives (4, 5, 8 and 9) on normal cells was
also determined. Normal fibroblasts [mouse embryonic fibroblasts
(MEF) and normal human fibroblasts (NHFB)] were treated with
increasing concentrations of the derivatives for 72 h, cell viability

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F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
Figure 2. Derivatives of 1 inhibit growth of chronic myelogenous leukemia (CML) cells expressing wild-type Bcr-Abl and T315I-mutant Bcr-Abl at nanomolar concentrations.
Cell viability (percent relative to control) was determined by MTS assay. Dose–response curves are shown. Error bars indicate standard errors (SE).
Table 2
series of 14-hydroxyl substituted by amine esters. Cationic amine
group can facilitate the contact of bio-macromolecule with small
molecules by electrostatic interaction. We synthesized a new ser-
IC50 values of triptolide derivatives in normal cells and murine CML cells after 72-h
exposure
Compound
IC50 (nM)
32D-Bcr-Abl
ies of C-14 triptolide derivatives with C-14-hydroxyl substituted
by different amine esters (3–18).We found that the bigger the
substituted group was, the weaker the antitumor effect was (com-
pared with 7 and 16), and that different heteroatom might give dif-
ferent effect (compared 4, 5 and 6; 10 and 14). Taken together, the
results support that the stereo-hindrance of C-14 group is impor-
tant for the antitumor effect of these triptolide derivatives.
We have found a group of novel water-soluble derivatives of tri-
ptolide (3–9, 11–14, 17 and 18) with anticancer activity in vivo and
in vitro.
MEF
9200
11,000
11,500
11,100
10,400
NHFB
32D-T315I
1
4
5
8
9
8800
11,400
10,400
11,400
10,000
32
73
70
50
61
34
80
80
44
74
structures of subunits Rpb6, Rpb8 and the Rpb4/Rpb7 dimer have
been determined by NMR and X-ray crystallography in human
pol II.37 For the fission yeast Schizosaccharomyces pombe showed
considerable sequence homology to the subunits of the human
pol II (>53%), we chose the largest subunit of Schizosaccharomyces
pombe pol II (pdb ID: 3h0g) as the receptor to conduct the docking.
The docking result showed that compound 9 had the highest score
in the modeling which was in accord with the pharmacological re-
sults. The structure of the ligand (9)-pol II complex was shown in
Figure 5.
5. Experimental section
5.1. Chemistry
Melting points (mp) were determined on X-4 micromelting
point apparatus. The NMR spectra were performed on Varian Inova
300NB NMR spectrometer using TMS as an internal standard. The
NMR experiments include 1H NMR, 13C NMR. HREIMS were mea-
sured on a Thermo MAT95XP high-resolution mass spectrometer
and ABI 4800 plus MALDI-TOF/TOF. EIMS were measured on a
Thermo DSQ EI-mass spectrometer. Si gel (200–300 mesh, Qingdao
Haiyang Chemical Co. Ltd) was used for column chromatography
(CC). Silica gel precoated aluminum cards with fluorescent indica-
tor visualizable at 254 nm (Merck) were used for thin layer chro-
matography (TLC).
4. Conclusions
The prognosis of CML resistant to imatinib is poor. Point muta-
tions in Bcr-Abl, which prevent imatinib binding, are responsible
for most of the cases of acquired clinical resistance to imatinib.
In the present study, a series of novel derivatives of 1 were synthe-
sized. We evaluated their antitumor activity with CML cells
(including KBM5-T315I cells) in terms of proliferation inhibition,
apoptosis, and signal transduction. Nude mouse xenograft model
was also demonstrated the in vivo antitumor activity of 9. In con-
trast to previous studies that aimed at improving water solubility
and exploiting the structure–activity relationship of 1 via C-14
modification, we focused on increasing its effect against imati-
nib-resistant CML cells bearing T315I Bcr-Abl via synthesis of the
5.1.1. 14-Chloracetyl triptolide ester (2)
Chloroacetyl chloride (0.039 mL, 0.490 mmol) was added into a
mixture of triptolide (1) (50 mg, 0.139 mmol), 4-dimethylamino-
pyridine (85 mg, 0.696 mmol) in dichloromethane (1.75 mL) at
0 °C. The reaction mixture was stirred at 25 °C for 24 h and washed
with HCl (5%, 10 mL ꢀ 3) and then water (10 mL ꢀ 3) successively,
and then dried with anhydrous magnesium sulfate and

F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
1811
Figure 3. Derivatives of 1 decrease Bcr-Abl mRNA and activity of RNA polymerase II. KBM5 versus KBM5-T315I, and 32D-Bcr-Abl versus 32D-T315I cells were exposed to the
derivatives of 1 at the indicated concentrations for 18 h. (A) Cell lysates were analyzed by Western blotting with the specific antibodies against the proteins as indicated,
immunoblots of phospho-c-Abl, Bcr-Abl, phospho-RNA polymerase II, PARP and actin are shown. (B) Semi-quantitative analysis of RT-PCR in CML cells after 18 h of treatment
with the derivatives. Ethidium bromide-stained RT-PCR products of Bcr-Abl and GAPDH mRNAs from KBM5 and KBM5-T315I cells treated with control medium or derivatives
(50 nM, 100 nM) for 18 h are shown. C denoted control.
concentrated under reduced pressure. The resulting residue was
purified by chromatography on silica gel (30 g) eluting with
CH2Cl2/CH3OH (100:0.5), then (100:1) to give 2 (45 mg, 74.2%) as
a colorless solid. Mp 122–124 °C. 1H NMR (CDCl3, 300 MHz) d
5.30 (s, 1H), 5.10 (s, 1H), 4.67 (s, 2H), 4.19 (d, J = 6.6 Hz, 2H), 3.84
(d, J = 3 Hz, 1H), 3.56 (d, J = 3 Hz, 1H), 3.49 (d, J = 5.7 Hz, 1H), 2.69
(m, 1H), 2.18 (m, 2H), 1.91 (m, 2H), 1.57 (m, 1H), 1.22 (m, 1H),
1.06 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H). 13C
NMR (CDCl3, 75 MHz) d 173.0, 166.9, 159.7, 125.5, 72.89, 69.9,
63.5, 63.0, 61.4, 59.5, 55.4, 55.0, 40.9, 40.3, 35.7, 29.9, 28.1, 23.4,
17.5, 17.1, 16.7, 13.8. HREIMS m/z 436.1282 (calcd for C22H25O7Cl1:
436.1283).
5.1.2. General procedure for the synthesis of compounds 3–18
Example: triptolidyl 2-(pyrrolidin-1-yl) acetate (3). To a solu-
tion of 14-chloracetyl triptolide ester (2) (10 mg, 0.0229 mmol) in
CH3CN(1 mL), was added at 0 °C pyrrolidine (4.07 mg,
0.0573 mmol) in THF (0.5 mL), The reaction mixture was stirred
at 25 °C for 24 h and concentrated under reduced pressure. The
resulting residue was purified by chromatography on silica gel
(15 g) eluting with CH2Cl2/MeOH (100:1), then (100:2) to give 3
(45 mg, 74.2%). Yield: 78% of a colorless solid. Mp 152–154 °C. 1H
NMR (CDCl3, 300 MHz) d 5.13 (s, 1H), 4.68 (s, 2H), 4.19 (d,
J = 7.2 Hz, 2H), 3.89 (d, J = 3.3 Hz, 1H), 3.55 (d, J = 3.3 Hz, 1H),
3.48 (d, J = 5.4 Hz, 1H), 2.75 (m, 1H), 2.35 (m, 2H), 2.35 (m, 2H),

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F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
75 MHz) d 173.0, 169.5, 159.7, 125.5, 71.2, 69.9, 66.9, 66.9, 63.6,
63.2, 61.3 , 59.7, 59.6, 55.5, 55.1, 53.1, 53.1, 40.4, 35.8, 29.9, 28.3,
23.6, 17.6, 17.2, 16.8, 13.8. HREIMS m/z 487.2200 (calcd for
C26H33O8N1: 487.2201).
5.1.5. Triptolidyl 2-(1-methylpiperazine-1-yl) acetate (6)
Yield: 75% of a colorless oil; 1H NMR (CDCl3, 300 MHz) d 5.11 (s,
1H), 4.67 (s, 2H), 4.21 (m, 1H), 3.83 (d, J = 3.3 Hz, 1H), 3.54 (d,
J = 3.0 Hz, 1H), 3.47 (d, J = 5.4 Hz, 1H), 3.35 (d, J = 16.8 Hz,
29.4 Hz, 2H),2.70 (m, 2H), 2.37 (s, 1H), 2.37 (m, 2H), 2.17 (m, 2H)
, 2.15 (m, 2H), 1.90 (m, 2H), 1.56 (m, 1H), 1.45 (m, 2H), 1.27 (m,
1H), 1.26 (s, 3H), 1.05 (s, 3H), 0.96 (d, J = 6.9 Hz, 3H), 0.84 (d,
J = 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d 173.2, 168.9, 159.7,
125.5 , 71.2, 69.9, 63.5, 63.2, 61.3, 59.6, 55.4, 55.0, 54.6, 54.6,
52.3, 52.3, 45.6, 40.4, 40.3, 35.7, 29.9, 29.7, 23.5, 17.3, 17.1, 16.8,
13.8. HREIMS m/z 500.2515 (calcd for C27H36O7N2: 500.2517).
Figure 4. Compound 5 abrogates the growth of tumors transplanted in nude mice.
The growth curves of subcutaneous xenografts of imatinib-resistant KBM5-T315I
cells are shown. The estimated tumor sizes are plotted against the number of days
since compound 5 treatment began (day 14 after inoculation). Placebo: normal
saline. The error bars represent standard errors.
5.1.6. Triptolidyl 2-(butan-1-amine-1-yl) acetate (7)
Yield: 95% of a colorless solid. Mp 114–116 °C.1H NMR (CDCl3,
300 MHz) d 5.30 (s, 1H), 5.10 (s, 1H), 4.67 (s, 2H), 4.25 (q,
J = 15 Hz, 22.2 Hz, 2H), 3.84 (d, J = 3.3 Hz, 1H), 3.72 (d, J = 3.3 Hz,
1H), 3.56 (d, J = 3 Hz, 1H), 3.49 (q, J = 6.0 Hz, 12.0 Hz, 1H), 2.87
(m, 2H), 2.69 (m, 1H), 2.35 (m, 1H), 2.30 (m, 1H), 2.17 (m, 2H),
1.91 (m, 2H), 1.59 (d, J = 4.8 Hz, 1H), 1.59 (d, J = 4.5 Hz, 1H), 1.57
(m, 1H), 1.26 (m, 2H), 1.26 (s, 2H), 1.06 (s, 3H), 0.97 (d, J = 6.9 Hz,
3H), 0.86 (d, J = 6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d 173.1,
166.9, 159.7, 125.5, 72.87, 69.9, 63.5, 63.2, 63.2, 61.4, 61.2, 59.6,
59.5, 55.4, 55.0, 40.9, 40.3, 35.7, 29.9, 29.7, 28.0, 23.4, 17.5, 17.1,
16.7, 13.8. HREIMS m/z 473.2399 (calcd for C26H35O7N1, 473.2408).
2,20 (m, 2H), 1.93 (m, 2H), 1.90 (m, 2H), 1.90 (m, 2H),1.85 (m, 1H),
1.58 (m, 1H), 1.26 (s, 1H), 1.06 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.86
(d, J = 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d 173.0, 162.2, 159.9,
125.4, 71.0, 70.0, 63.5, 63.3, 61.2, 59.6, 55.4, 55.0, 53.6, 53.6, 40.4,
40.3, 35.7, 29.9, 28.0, 23.5, 23.5, 23.4, 17.6, 17.1, 16.7, 13.8. HREIMS
m/z 471.2253 (calcd for C26H33O7N1: 471.2252).
5.1.3. Triptolidyl 2-(1-methylpiperidine-1-yl) acetate (4)
Yield: 83% of a colorless solid. Mp 122–124 °C. 1H NMR (CDCl3,
300 MHz) d 5.12 (s, 1H), 4.67 (s, 2H), 3.83 (d, J = 3.0 Hz, 1H), 3.53 (d,
J = 2.7 Hz, 1H), 3.47 (d, J = 5.7 Hz, 1H), 3.33 (d, J = 6.3 Hz, 2H), 2.65
(m, 1H), 2.57 (m, 2H), 2.57 (m, 2H), 2.17 (m, 2H), 1.95 (s, 1H), 1.90
(m, 2H), 1.85 (s, 1H), 1.64 (m, 2H), 1.64 (m, 2H), 1.45 (m, 2H), 1.26
(s, 1H), 1.00 (s, 3H), 1.06 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.85 (d,
J = 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d 172.9, 162.6, 159.8,
125.5, 70.9, 69.9, 63.6, 63.3, 61.2, 59.6, 55.4, 55.1, 53.9, 53.9,
40.4, 40.3, 35.7, 29.9, 28.2, 25.9, 25.9, 23.9, 23.4, 17.6, 17.1, 16.7,
13.8. HREIMS m/z 485.2409 (calcd for C27H35O7N1: 485.2408).
5.1.7. Triptolidyl 2-(amine-1-yl) acetate (8)
Yield: 90% of a colorless solid. Mp 223–225 °C .1H NMR (CDCl3,
300 MHz) d 4.68 (s, 2H), 3.91 (d, J = 3.3 Hz, 2H), 3.52 (d, J = 2.4 Hz,
1H), 3.42 (d, J = 10.5 Hz, 1H), 3.38 (d, J = 5.4 Hz, 1H), 2.74 (d,
J = 10.8 Hz, 1H), 2.32 (m, 1H), 2.23 (m, 1H), 2.16 (t, J = 5.7 Hz,
1H), 1.99 (d, J = 13.2 Hz, 1H), 1.61 (s, 2H), 1.56 (m, 2H), 1.23 (m,
2H), 1.13 (s, 3H), 1.02 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H).
13C NMR (CDCl3) d 173.0, 173.0, 160.0, 125.4, 73.4, 69.9, 66.2,
65.7, 60.8, 60.1, 56.8, 54.6, 40.5, 40.5, 35.8, 29.9, 28.3, 23.8, 17.8,
17.2, 16.9, 13.8, HREIMS m/z 417.1809 (calcd for C22H27O7N1:
417.1782).
5.1.4. Triptolidyl 2-(morpholine-1-yl) acetate (5)
Yield: 75% of a colorless solid. Mp 98–99 °C. 1H NMR (CDCl3,
300 MHz) d 5.13 (s, 1H), 4.67 (s, 2H), 3.83 (d, J = 3.0 Hz, 1H), 3.76
(t, J = 4.5 Hz, 2H), 3.76 (t, J = 4.5 Hz, 2H), 3.54 (d, J = 3.0 Hz, 1H),
3.48 (dd, J = 5.7 Hz, 1H), 3.35 (q, J = 16.5 Hz, 28.2 Hz, 2H), 2.68
(m, 1H), 2.64 (m, 2H), 2.64 (m, 2H), 2.17 (m, 2H), 1.90 (m, 2H),
1.56 (m, 1H), 1.45 (m, 2H), 1.26 (s, 1H), 1.21 (m, 1H), 1.01 (s,
3H), 0.98 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H). 13C NMR (CDCl3,
5.1.8. Triptolidyl 2-(aminomethanol-1-yl) acetate (9)
Yield: 85% of a colorless solid. Mp 131–133 °C. 1H NMR (CDCl3,
300 MHz) d 4.68 (s, 2H), 3.90 (s, 1H), 3.86 (m, 2H), 3.52 (m, 2H),
3.56 (d, J = 3 Hz, 1H), 3.42 (m, 1H), 3.38 (d, J = 5.4 Hz, 1H), 2.70
Figure 5. Molecular docking analysis of RNA polymerase II to derivative 9. (A) Docking pocket of protein with compound 9 (orange color). Opaque type surface was created of
the active site. (B) Details interaction between compound 9 to protein with some residues labeled.

F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
1813
(m, 2H), 2.36 (m, 1H), 2.30 (m, 1H), 2.23 (m, 1H), 2.16 (m, 2H), 1.97
(s, 1H), 1.90 (m, 1H), 1.57 (dd, J = 5.1, 12.3 Hz, 1H), 1.26 (s, 1H), 1.20
(m, 1H), 1.13 (s, 3H), 1.02 (d, J = 6.9 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H).
13C NMR (CDCl3, 75 MHz) d 173.7, 170.2, 159.7, 125.5, 73.5, 69.9,
66.2, 65.7, 60.7, 60.1, 59.7, 56.7, 55.6, 54.5, 40.5, 40.2, 35.8, 29.9,
28.3, 23.7, 17.8, 17.1, 16.9, 13.7. MS: 462.1913 (calcd for
C24H31O8N1)
136.5, 134.6, 125.2, 107.6. HREIMS m/z 172.0740 (calcd for
C9H8N4, 172.0743).
5.1.14. Triptolidyl 2-(4-(pyridin-3-yl)pyrimidin-2-amine-1-yl)
acetate (14)
Yield: 70% of a colorless solid. Mp 167–169 °C. 1H NMR (CDCl3,
300 MHz) d 9.23 (s, 1H), 8.73 (d, J = 3.3 Hz, 1H), 8.43 (d, J = 5.1 Hz,
1H), 8.30 (d, J = 7.8 Hz, 1H), 7.40 (m, 1H), 7.06 (d, J = 4.2 Hz, 1H),
5.18 (s, 1H), 4.68 (s, 2H), 3.90 (d, J = 3 Hz, 1H), 3.52 (d, J = 2.1 Hz,
1H), 3.42 (m, 2H), 3.38 (d, J = 5.1 Hz, 1H), 2.69 (m, 1H), 2.28 (m,
1H), 2.18 (m, 2H), 1.91 (m, 2H), 1.70 (s, 1H), 1.57 (m, 1H), 1.22
(m, 1H), 1.13 (s, 3H), 1.02 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.6 Hz,
3H). 13C NMR (CDCl3, 75 MHz) d 173.0, 166.7, 163.2, 162.8, 159.1,
151.2, 149.4, 148.2 134.3, 132.7, 125.5, 123.5, 107.6, 73.5, 69.9,
66.2, 60.8, 60.1, 56.8, 54.6, 55.0, 40.5, 40.1, 35.8, 29.9 , 28.3, 23.8,
17.8, 17.2, 17.0, 13.7. MS m/z 573.2217 (calcd for C31H32O7N4).
5.1.9. Triptolidyl 2-(4-chloroaniline-1-yl) acetate (10)
Yield: 55% of a yellow solid. Mp 95–97 °C. 1H NMR (CDCl3,
300 MHz) d 7.13 (d, J = 9 Hz, 1H), 7.13 (d, J = 9 Hz, 1H), 6.56 (d,
J = 9 Hz, 1H), 6.56 (d, J = 9 Hz, 1H), 5.11 (s, 1H), 4.70 (s, 2H), 4.19
(q, J = 15.0, 21.6 Hz, 2H), 3.84 (d, J = 3 Hz, 1H), 3.56 (d, J = 3 Hz,
1H), 3.49 (d, J = 5.7 Hz, 1H), 2.69 (m, 1H), 2.35 (m, 1H), 2.16 (m,
2H), 1.92 (m, 2H), 1.57 (m, 1H), 1.26 (m, 2H), 1.07 (s, 3H), 0.98
(d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H). 13C NMR (CDCl3,
75 MHz) d 172.9, 166.9, 159.6, 145.4, 129.0, 129.0, 125.6, 114.2,
109.7, 109.7, 73.0, 69.9, 63.5, 63.1, 61.4, 59.5, 55.4, 55.3, 40.8,
40.4, 35.7, 29.9, 28.1, 23.5, 17.5, 17.2, 16.8, 13.9. HREIMS m/z
527.1716 (calcd for C28H30O7N1Cl1: 527.1705).
5.1.15. Triptolidyl 2-(2,20-azanediyldiethanol-1-yl) acetate (15)
Yield: 70% of a colorless oil. 1H NMR (CDCl3, 300 MHz) d 4.69 (s,
2H), 4.55 (s, 1H), 4.19 (d, J = 3.9 Hz, 2H), 3.90 (s, 1H), 3.90 (s, 1H),
3.83 (m, 2H), 3.83 (m, 2H), 3.56 (m, 1H), 3.50 (s, 2H), 3.49 (m,
1H), 2.27 (m, 2H), 2.27 (m, 2H), 2.69 (m, 1H), 2.18 (m, 2H), 1.91
(m, 2H), 1.57 (m, 1H), 1.22 (m, 1H), 1.06 (s, 3H), 0.88 (d,
J = 6.9 Hz, 3H), 0.81 (d, J = 6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d
173.3, 166.7, 160.2, 125.5, 73.44, 68.0, 63.8, 63.0, 62.2, 62.2, 61.1,
60.6, 60.6, 59.2, 55.7, 55.6, 47.8, 40.3, 35.3, 29.9, 28.1, 23.7, 17.7,
17.1, 16.5, 13.7. MS m/z 506.1972 (calcd for C26H35O9N1).
5.1.10. Triptolidyl 2-(methyl 2-aminopropanoate-1-yl) acetate
(11)
Yield: 75% of a colorless solid. Mp 110–112 °C. 1H NMR (CDCl3,
300 MHz) d 5.30 (s, 1H), 5.11 (s, 1H), 4.68 (s, 2H), 4.19 (q, J = 15.0,
21.9 Hz, 2H), 3.85 (d, J = 3 Hz, 1H),3.74 (s, 1H), 3.57 (d, J = 3 Hz, 1H),
3.50 (d, J = 5.7 Hz, 1H), 3.47 (s, 3H), 2.69 (m, 1H), 2.17 (m, 2H), 1.92
(m, 2H), 1.92 (s, 1H), 1.8 (s, 1H),1.57 (dd, J = 4.2, 13.2 Hz, 1H), 1.26
(s, 3H), 1.06 (s, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H).
13C NMR (CDCl3, 75 MHz) d 175.4, 173.9, 169.7, 157.5, 125.6, 72.9,
69.9, 63.5, 63.0, 61.4, 60.0, 59.5, 55.4, 55.0, 40.9, 40.3, 38.8, 35.7,
29.9, 29.6, 28.1, 23.4, 17.5, 17.1, 16.7, 13.8. HREIMS m/z 503.2151
(calcd for C26H33O9N1, 503.2150).
5.1.16. Triptolidyl 2-(dodecan-1-amine-1-yl) acetate (16)
Yield: 80% of a colorless oil. 1H NMR (CDCl3, 300 MHz) d 5.12 (s,
1H), 4.67 (s, 2H), 4.31 (t, J = 6.6 Hz, 1H), 4.09 (d, J = 6.6 Hz, 1H), 3.83
(d, J = 3 Hz, 1H), 3.57 (s, 1H), 3.54 (d, J = 3 Hz, 1H), 3.49 (d,
J = 6.0 Hz, 1H), 2.66 (m, 1H), 2.64 (m, 1H) 2.16 (m, 2H), 1.91 (m,
2H), 1.77 (m, 2H),1.57 (m, 1H), 1.52 (m, 2H), 1.26 (s, 2H), 1.26 (s,
2H), 1.26 (s, 2H), 1.26 (s, 2H), 1.26 (s, 2H), 1.26 (s, 2H), 1.26 (s,
2H), 1.26 (s, 2H), 1.22 (m, 1H), 1.06 (s, 3H), 1.01 (s, 3H), 0.97 (d,
J = 6.9 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d
173.5, 166.1, 160.0, 125.8, 72.9, 71.9, 66.2, 61.8, 61.7, 58.6, 56.2,
53.2, 48.1 , 40.6, 40.2, 35.9, 30.2, 30.0, 30.0, 30.0, 30.0, 30.0, 30.0,
30.0, 30.0, 29.4, 28.8, 23.8, 23.0, 17.5, 15.9, 15.5, 14.5, 14.4. HREIMS
m/z 585.3660 (calcd for C34H51O7N1, 585.3660).
5.1.11. Triptolidyl 2-(ethane-1,2-diamine-1-yl) acetate (12)
Yield: 80% of a colorless solid. Mp 130–132 °C. 1H NMR (CDCl3,
300 MHz) d 5.13 (s, 2H), 4.68 (s, 2H), 3.84 (d, J = 3.3 Hz, 1H), 3.75 (s,
1H), 3.55 (d, J = 3.0 Hz, 1H), 3.50 (s, 1H), 3.49 (s, 1H), 3.46 (d,
J = 7.2 Hz, 2H), 3.41 (d, J = 5.4 Hz, 2H), 2.93 (m, 1H), 2.93 (m, 1H),
2.70 (m, 2H), 2.16 (m, 2H), 1.91 (m, 2H), 1.61 (s, 1H), 1.27 (m,
1H), 1.05 (s, 3H), 0.99 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H).
13C NMR (CDCl3, 75 MHz) d 173.3, 169.4, 159.7, 125.9, 71.6, 69.9,
63.6, 63.1, 61.5, 59.7, 55.6, 55.0, 48.3, 41.6, 40.3, 37.2, 35.7, 29.9,
28.3, 23.5, 17.6, 17.2, 16.8, 13.8. HREIMS m/z 460.2217 (calcd for
C24H32O7N2, 460.2204).
5.1.17. Triptolidyl 2-(diethylamine-1-yl) acetate (17)
Yield: 70% of a colorless solid. Mp 50–52 °C. 1H NMR (CDCl3,
300 MHz) d 5.11 (s, 1H), 4.67 (s, 2H), 3.83 (d, J = 3 Hz, 1H), 3.54
(d, J = 3 Hz, 1H), 3.49 (t, J = 5.7 Hz, 2H), 2.75 (m, 2H), 2.75 (m,
2H), 2.69 (m, 1H), 2.32 (m, 2H), 2.18 (m, 2H), 1.91 (m, 2H), 1.57
(m, 1H), 1.22 (m, 1H), 1.12 (t, J = 7.2 Hz, 3H), 1.12 (t, J = 7.2 Hz,
3H), 1.07 (s, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H).
13C NMR (CDCl3, 75 MHz) d 173.0, 170.5, 159.8, 125.6, 73.4, 69.9,
63.6, 63.3, 61.2, 59.7, 55.4, 55.1, 53.7, 53.7, 47.6, 40.5, 35.8, 29.9,
28. 2, 23.6 17.6, 17.2, 16.8, 13.8, 12.5, 12.5. HREIMS m/z
473.2407 (calcd for C26H35O7N1: 473.2408).
5.1.12. Triptolidyl 2-(piperazine-1-yl) acetate (13)
Yield: 75% of a colorless solid. Mp 230–232 °C. 1H NMR (CDCl3,
300 MHz) d 5.10 (s, 1H), 4.67 (s, 2H), 3.84 (d, J = 3.3 Hz, 1H), 3.55 (d,
J = 2.7 Hz, 1H), 3.50 (d, J = 4.8 Hz, 2H), 3.43 (s, 1H), 3.38 (s, 1H), 3.29
(m, 2H), 3.29 (m, 2H), 2.96 (m, 2H), 2.96 (m, 2H), 2.70 (m, 1H), 2.15
(m, 2H), 1.91 (m, 2H), 1.58 (dd, J = 4.8, 12.6 Hz, 1H), 1.27 (s, 1H),
1.22 (m, 1H), 1.03 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.86 (d,
J = 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d 172.9, 168.9, 159.6,
125.6, 71.7, 69.9, 63.5, 63.1, 61.5, 59.8, 58.7, 55.6, 55.1, 49.4,
49.4, 43.6, 43.6, 40.4, 35.7, 30.0, 28.4, 23.5, 17.6, 17.2, 16.8, 13.9.
HREIMS m/z 486.2364 (calcd for C26H34O7N2: 486.2361).
5.1.18. Triptolidyl 2-(methanamine-1-yl) acetate (18)
Yield: 85% of a colorless solid. Mp 109–111 °C. 1H NMR (CDCl3,
300 MHz) d 5.13 (s, 1H), 4.68 (s, 2H), 4.31 (t, J = 6.6 Hz, 2H), 3.90 (s,
1H), 3.84 (d, J = 3.0 Hz, 1H), 3.76 (m, 3H), 3.56 (d, J = 3 Hz, 1H), 3.37
(d, J = 5.7 Hz, 1H), 2.77 (m, 1H), 2.69 (s, 1H), 2.16 (m, 2H), 1.94 (m,
2H), 1.60 (m, 1H), 1.27 (m, 1H), 1.06 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H),
0.86 (d, J = 6.9 Hz, 3H). 13C NMR (CDCl3, 75 MHz) d 173.8, 167.7,
158.0, 125.6, 84.1, 71.8, 69.9, 64.3, 63.9, 61.4, 59.6, 57.6, 55.4,
47.6, 39.9, 35.7, 29.8, 27.7, 23.4, 17.2, 17.2, 15.3, 14.2, HREIMS m/
z 431.1939 (calcd for C23H29O7N1: 431.1939).
5.1.13. 4-(Pyridin-3-yl)pyrimidin-2-amine
4-(Pyridin-3-yl)pyrimidin-2-amine was synthesized follow the
literature. Yield 90% of three steps of a pale yellow slice. Mp
172 °C (sublimation), 1H NMR (MeOD, 300 MHz) d 9.20 (s, 1H),
8.62 (s, 1H), 8.49 (s, 1H), 8.32 (s, 1H), 7.55 (s, 1H), 7.17 (s, 1H).
13C NMR (MeOD, 75 MHz) d 163.9, 160.0, 151.6, 149.2, 148.8,

1814
F. Xu et al. / Bioorg. Med. Chem. 18 (2010) 1806–1815
5.2. Molecular modeling
5.3.4. Semi-quantitative RT-PCR
Total RNA was extracted from KBM5 and KBM5-T315I cells
treated with or without triptolide using Trizol reagent (Invitrogen).
After quantification by spectrophotometry, the first-strand cDNA
All molecular studies were performed on a HP xw6600 worksta-
tion with Intel Xeon E5430 2.66 GHz processor running Red Hat LI-
NUX enterprise 5 and SYBYL8.1. The crystal structure was downloaded
from the PDB (http://www.rcsb.org) (PDB ID code: 3H0G). Firstly,
we deleted all chains except chain A of the protein and chain A
was used in the study. Secondly, hydrogen atoms were added to
the protein. Ligand structures were built with SYBYL8.1 [SYBYL8.1 (Tri-
pos, Inc., St. Louis, MO)] and minimized with the Powell method
and MMFF94 force field until a rmsd gradient of 0.005 kcal/
(mol Å) was reached. Docking calculations were performed using
surflex-dock of SYBYL8.1 and the sfxc mode was chosen with default
values. The protocol was produced based on the automatic mode
and C-score calculations were performed.
was synthesized from 1
lL of total RNA with the use of the RNA
PCR Kit (AMV) Ver.3.0 (TaKaRa, Dalian, China) and random prim-
ers. Bcr-Abl gene amplification involved the following primers38
:
forward 50-TTCAGAAGCTTCTCCCTGACAT-30 (exon 13 of Bcr gene),
reverse 50-CTTCGTCTGAGATACTGGATTCCT-30 (exon 9 of Abl gene),
with an expected PCR product of 1614 bp. The PCR products were
electrophoresed on 1% agarose gel.
5.3.5. Nude mouse xenograft model
nu/nu BALB/c mice were bred at the animal facility of Sun Yat-
sen University. The mice were housed in barrier facilities with a
12-h light dark cycle, with food and water available ad libitum.
3 ꢀ 107 of KBM5-T315I cells were inoculated subcutaneously on
the flanks of 6–8-week-old male nude mice. Tumors were mea-
sured every other day with use of calipers. Tumor volumes were
calculated by the following formula: a2 ꢀ b ꢀ 0.4, where a is the
smallest diameter and b is the diameter perpendicular to a. Com-
pound 5 was dissolved in normal saline. Mice in the control group
received the same volume of normal saline. The body weight, feed-
ing behavior and motor activity of each animal were monitored as
indicators of general health. At the end of the experiments, blood
samples were collected for assay of lactate dehydrogenase, and
aspartate and alanine aminotransferase activity, and complete
blood count. The animals were then euthanized, and tumor xeno-
grafts were immediately removed, weighed, stored and fixed. All
animal studies were conducted with the approval of the Sun Yat-
sen University Institutional Animal Care and Use Committee.
5.3. Bioactivity assay
5.3.1. Cell lines
KBM5 and KBM5-T315I cells were cultured as described pre-
viously.30,36 In brief, KBM5 cells expressing 210 kDa wild-type
Bcr-Abl were cultured in Iscove’s modified Dulbecco’s medium
(Invitrogen, Guangzhou, China) supplemented with 10% heat-
inactivated fetal calf serum (FCS). KBM5-T315I cells were rou-
tinely maintained in the same medium but with 1 lM imatinib,
which was removed before experiments with a wash-out period
of 2–3 days. KBM5 cells expressing the 210 kDa wild-type Bcr-
Abl was derived from a female CML patient. The KBM5-T315I
subline was originally established by exposure to increasing con-
centrations of imatinib. KBM5-T315I cells, harboring a threonine-
to-isoleucine substitution at position 315 of Abl, are resistant to
imatinib, and they only have a marginal increase Bcr-Abl gene
copies and expression.38–40 The 32D myeloid cells stably
expressing either 210 kDa wild-type Bcr-Abl (32D-Bcr-Abl) or
T315I Bcr-Abl (32D-T315I) were established and maintained in
RPMI 1640 with 10% fetal bovine serum as described previ-
ously.41 MEF (normal murine embryonic fibroblast) and NHFB
(normal human fibroblast) cells were cultured in DMEM supple-
mented with 10% fetal calf serum.41
Acknowledgements
This study was supported by grants from the Major Research
Plan of the National Natural Science Fund of China (Grant
90713036 to J. Pan), the National High Technology Research and
Development Program of China (863 Program Grant
2008AA02Z420 to J. Pan), the National Basic Research Program of
China (973 Program Grant 2009CB825506 to J. Pan), and China
Postdoctoral Science Foundation (Grant 20080440794 to F.X.).
5.3.2. Cell viability assay
Cell viability was evaluated by 3-(4,5-dimethyl-2-yl)-5-(3-carb-
oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) as-
say (CellTiter 96 Aqueous One Solution reagent, Promega,
Shanghai, China) as described previously.42 The optical density
was read on a 96 well plate reader at a single wavelength of
490 nm. The drug concentration resulting in a 50% inhibition of cell
proliferation (IC50) was determined.
References and notes
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Engl. J. Med. 1999, 341, 164.
2. Ren, R. Nat. Rev. Cancer 2005, 5, 172.
3. Melo, J. V.; Barnes, D. J. Nat. Rev. Cancer 2007, 7, 441.
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