Hybrid pyrimidine alkynyls inhibit the clinically resistance related Bcr-AblT315I mutant

Article abstract of DOI:10.1016/j.bmcl.2015.07.006

abstract A series of pyrimidine alkynyl derivatives were designed and synthesized as new Bcr-Abl inhibitors by hybriding the structural moieties from GNF-7, ponatinib and nilotinib. One of the most potent compounds 4e strongly suppresses Bcr-Abl^WT and Bcr-Abl^T315I kinase with IC₅₀ values of 5.0 and 9.0 nM, and inhibits the proliferation of K562 and murine Ba/F3 cells ectopically expressing Bcr-Abl^T315I cells with IC₅₀ values of 2 and 50 nM, respectively. It also displays good pharmacokinetics properties with an oral bioavailability of 35.3% and T_1/2 value of 48.7 h, and demonstrates significantly suppression on tumor growth in xenografted mice of K562 and Ba/F3 cells expressing Bcr-Abl^T315I. These inhibitors may serve as lead compounds for further developing new anticancer drugs overcoming the clinically acquired resistance against current Bcr-Abl inhibitors.

Full text of DOI:10.1016/j.bmcl.2015.07.006

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3458–3463
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hybrid pyrimidine alkynyls inhibit the clinically resistance related
Bcr-AblT315I mutant
Xiaoyun Lu a, , , Zhang Zhang b,a, , Xiaomei Ren a, Xiaofeng Pan a, Deping Wang a, Xiaoxi Zhuang a,
⇑
Jingfeng Luo a, Rongmin Yu b, Ke Ding a,
⇑
a Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, No. 190, Kaiyuan Avenue,
Science Park, Guangzhou 510530, China
b Biotechnological Institute of Chinese Materia Medica and Department of Pharmacology, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrimidine alkynyl derivatives were designed and synthesized as new Bcr-Abl inhibitors by
hybriding the structural moieties from GNF-7, ponatinib and nilotinib. One of the most potent com-
pounds 4e strongly suppresses Bcr-AblWT and Bcr-AblT315I kinase with IC50 values of 5.0 and 9.0 nM,
and inhibits the proliferation of K562 and murine Ba/F3 cells ectopically expressing Bcr-AblT315I cells with
IC50 values of 2 and 50 nM, respectively. It also displays good pharmacokinetics properties with an oral
bioavailability of 35.3% and T1/2 value of 48.7 h, and demonstrates significantly suppression on tumor
growth in xenografted mice of K562 and Ba/F3 cells expressing Bcr-AblT315I. These inhibitors may serve
as lead compounds for further developing new anticancer drugs overcoming the clinically acquired
resistance against current Bcr-Abl inhibitors.
Received 3 June 2015
Revised 1 July 2015
Accepted 4 July 2015
Available online 9 July 2015
Keywords:
Pyrimidine alkynyl
Bcr-AblT315I
Hybride
Ó 2015 Elsevier Ltd. All rights reserved.
Chronic myelogenous leukemia (CML)
Clinical resistance
Chronic myeloid leukemia (CML) is a hematological malignancy
representing about 20% of adult leukemia and characterized by the
occurrence of the Philadelphia (Ph) chromosome. The first genera-
tion Bcr-Abl inhibitor imatinib has shown significant clinical ben-
efit and become the first-line treatment of CML.1,2 However,
many patients eventually develop acquired resistance to imatinib.
The 2-year incidence of resistance reaches 80% in the blastic phase,
40–50% in the accelerated phase, and 8–10% in the chronic phase.3
Point mutation in the kinase domain of Bcr-Abl is the primary
mechanism to imatinib resistance, and about 100 point mutations
have been identified to date.4–6 Nilotinib (1),7,8 dasatinib9,10 and
bosutinib11 have been approved for the treatment of adults in all
phases of CML with resistance to the first Bcr-Abl inhibitor drug,
whereas bafetinib12 has also been developed in phase II clinical tri-
als. However, these second-generation inhibitors are not capable of
inhibiting all of the resistant mutants, especially the most notable
Bcr-AblT315I gatekeeper mutation accounting for 15–20% in all clin-
ical acquired resistance.13 Thus, T315I mutation induced resistance
remains a serious medical problem.
the kinase domain of Abl protein. The bulky isoleucine side chain
also makes a steric clash to prevent inhibitor binding into the
hydrophobic pocket.14,15 Several third generation inhibitors are
capable of inhibiting the Bcr-AblT315I mutant have been identi-
fied.16,17 Examples include the type I inhibitors PPY-A,18 SGX-
393,19,20 c-Src/Abl dual inhibitors TG100598 and TG101223,21,22
and aurora inhibitors MK-0457,23,24 PHA-739358,25,26 and
AT9283,27,28 as well as non-ATP competitive or allosteric inhibitors
ON01238029 and DCC2036.30 However, these molecules have to be
formulated for intravenous administration, and clinical develop-
ment for MK-0457 has been discontinued due to cardiac
toxicity.24,31
Recently, the ‘third-generation’ type II Bcr-Abl inhibitors
GZD824,32 GNF-7 (2),33 AP24534 (3)34,35 and 5-(arenethynyl)he-
tero-monocyclic derivatives36 were reported to strongly inhibit
Bcr-AblT315I and other mutants. AP24534 (ponatinib, IclusigÒ) has
been approved for the treatment of resistant or intolerant CML
and Ph+ ALL patients against imatinib, especially those harboring
Bcr-Abl (T315I) mutation.35,37 However, US FDA soon temporarily
suspended the marketing of this drug due to the increasing num-
bers of blood clots observed in ponatinib-treated patients.37 The
drug was later reauthorized for sale after a revised indication state-
ment and a boxed warning were made by the manufacture. As
acquired resistance increasingly observed in the clinic, the newly
Structurally, the T315I mutation diminished a key hydrogen
bond interaction between the inhibitors and Thr315 residue in
⇑
Corresponding authors. Tel.: +86 20 32015349; fax: +86 20 32015299.
E-mail addresses: lu_xiaoyun@gibh.ac.cn (X. Lu), ding_ke@gibh.ac.cn (K. Ding).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2015.07.006
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

X. Lu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3458–3463
3459
N
N
O
H
N
N
N
N
H
CF3
N
Hydrophobic tail
N
1
. Nilotinib
N
N
R
O
Hinge binding region
CF3
N
O
Hybriding
N
N
N
H
N
H
CF3
HN
N
N
O
4
N
2. GNF-7
N
Small size linker
O
N
N
N
N
N
H
CF3
3. AP24534(ponatinib)
Figure 1. Design of new Bcr-Abl inhibitors by hybriding the chemical structures of nilotinib, GNF-7 and AP24534.
discovered Bcr-Abl inhibitors are required to not only suppress
T315I mutant but also display reduced side effects.
The synthesis of compounds 4a–4l is outlined in Scheme 1. The
Sonogashira coupling was used as the key reaction in the synthetic
route.38 Briefly, the iodo-intermediate 7 was afforded by amide
Structural feature analysis reveals that almost all of the type-II
Bcr-Abl inhibitors contain three crucial chemical elements: a
kinase hinge binding region, a hydrophobic group binding to the
back pocket of protein and a suitable linker between these two
moieties. Based on the pharmacophore investigation, a series of
N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-3-
(2-(pyrimidin-5-yl)ethynyl) benzamides (4a–4l) were designed
and synthesized as new potential Bcr-Abl inhibitors by hybriding
the key elements from GNF-7, ponatinib and nilotinib (Fig. 1).
In the new inhibitors, a substituted pyrimidine is used as a
hinge binding group, the 3-(4-methyl-1H-imidazol-1-yl)-5-
(trifluoromethyl)phenyl hydrophobic moiety is ‘borrowed’ from
the corresponding design in nilotinib. A small triple bond linker
is also introduced by following AP24534 to avoid the potential
steric clash with I315 in the resistant mutant.
condensation
of
3-(4-methyl-1H-imidazol-1-yl)-5-(trifluo-
romethyl)aniline (6) with freshly prepared 3-iodo-4-methylben-
zoyl chloride starting with 3-iodo-4-methylbenzoic acid (5).
Treatment of
7
under palladium catalysis afforded the
Sonogashira coupling products, which was further deprotected to
produce the alkyne 8. The desired products 4a–4l were obtained
by using another Sonogashira coupling of 8 with substituted 3-bro-
mopyrimidine (9).
Kinase inhibitory activities of the designed compounds 4a–4l
against Bcr-AblWT and Bcr-AblT315I were evaluated by using a well
established FRET-based Z’-Lyte assay.39 FDA-approved drugs ima-
tinib, nilotinib, dasatinib and ponatinib were used as positive con-
trols to validate the screening conditions. As shown in Table 1,
imatinib, nilotinib and dasatinib potently inhibit Bcr-AblWT, but
N
N
O
N
N
H2N
I
O
OH
+
I
a
b
N
H
CF3
CF3
6
5
7
N
N
N
N
N
R
N
N
R
Br
O
9
N
O
N
CF3
c
N
H
CF3
H
8
4a-4l
Scheme 1. Reagents and conditions: (a) i. SOCl2, reflux; ii. THF, Et3N, 0 °C–rt; 90%, (b) i. Pd(dppf)2Cl2, CuI, DIPEA, DMF, 80 °C; ii. K2CO3, MeOH, rt, 90%; (c) Pd(OAc)2, PCy3, CuI,
DIPEA, DMF, 80 °C, 60–80%.

3460
X. Lu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3458–3463
Table 1
Bioactivities of new hybrid Bcr-Abl inhibitors
N
N
N
R
N
O
N
H
CF3
Compd
R
Kinase inhibition (IC50
,
lM)
Cell inhibition (IC50, lM)
WT
Bcr-AblWT
Bcr-AblT315I
K562
0.001
Ba/F3
Ba/F3T315I
0.078
4a
4b
NH2-
0.008
0.007
0.002
0.003
H
N
0.010
0.030
0.125
0.005
0.006
0.019
0.187
0.009
0.007
0.006
0.027
0.002
0.085
0.113
1.158
0.050
H
N
4c
4d
4e
0.008
0.023
0.002
H
N
H
N
H
N
4f
0.018
0.019
0.013
0.017
1.077
HO
H
4g
4h
N
0.093
0.028
0.458
0.024
0.030
0.023
0.049
0.038
2.711
0.135
H
N
H
N
4i
0.238
0.161
0.111
0.208
2.396
N
4j
0.904
>1
>1
>1
>1
0.640
>0.925
0.355
0.683
>1
>10
4k
>10
N
N
4l
0.3
0.292
8.433
O
Imatinib
Nilotinib
Dasatinib
AP24534
GNF-7a
–
–
–
–
–
0.399
0.014
0.003
0.001
0.133
>1
>1
>1
0.0009
0.061
0.280
0.022
0.001
0.003
<0.005
–
–
–
–
–
>1
>1
>1
0.006
0.011
a
Reported activity data.33
0 0.8 4 20 100 nM
0 4 20 100 500 nM
pABL
pABL
ABL
ABL
CRKL
pCRKL
pSTAT5
CRKL
E316
pCRKL
pSTAT5
E286
D381
I315
STAT5
GAPDH
STAT5
GAPDH
1 2 3 4 5
1 2 3 4 5
M318
K562
Ba/F3 Bcr-Abl T315I
Figure 3. Compound 4e inhibits Bcr-Abl signaling in K562 and Ba/F3 stable cells
expressing Bcr-AblT315I. K562 is a human CML cell with positive Bcr-AblWT. Cells
were treated with 4e at the indicated concentrations for 4.0 h, and whole cell
lysates were then subjected to Western blot analyses. The results are from three
independent experiments.
Figure 2. The predicted binding mode of 4e with Bcr-AblT315I. Hydrogen bonds are
indicated by yellow hatched lines to key amino acids.
with low nanomolar IC50 values. Similar to the reported data,
GNF-7 also potently suppresses the enzymatic activity of T315I
mutation with IC50 values of 61 nM.33
Under the experimental conditions, the first hybrid compound
4a with a free amino group at R position inhibits the kinase
they are all inactive against Bcr-AblT315I. However, ponatinib sup-
presses the enzymatic activity of wild-type and the T315I mutant

X. Lu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3458–3463
3461
Figure 4. (A) Effect of 4e on cell cycling and apoptosis in K562 cells. (B) Effects of 4e on cell cycling and apoptosis in Ba/F3-T315I cells. K562 cells and Ba/F3-T315I cells were
both exposed to 4e at the indicated concentrations for 24 h, then cells were fixed and analyzed by flow cytometry. Data represent the mean 95% CI of three independent
experiments.
Table 2
The pharmacokinetic profiles of 4b and 4e in rats
Compd
Pharmacokinetics parameters
g/L ⁄ h) Cmax g/L)
Route
Dose (mg/kg)
AUC0–1
(l
(
l
Tmax (h)
T1/2 (h)
F (%)
4e
4b
Oral
Iv
Oral
Iv
25
10
25
10
251684
285187
48574
35809
6205
49750
3090
4.5
0.033
2
48.7
71.7
7.8
35.3
27.1
10975
0.033
7.5
activities of both Bcr-AblWT and Bcr-AblT315I with IC50 values of
0.008 and 0.007 M, respectively, which is almost comparable to
Antiproliferative activities of the new hybrids are also examined
against Bcr-Abl positive K562, and murine Ba/F3 cells ectopically
expressing Bcr-AblWT and Bcr-AblT315I (termed as Ba/F3 WT and
Ba/F3 T315I cells). The results were summarized in Table 1.
Consistent with their kinase inhibitory activities, most of the com-
pounds show promising anti-proliferative activities. For instance,
the most promising compound 4e strongly inhibits the prolifera-
tion of K562, Ba/F3 WT and Ba/F3 T315I cells with IC50 values of
2, 2 and 50 nM, respectively, which is similar to that of GNF-7.
A molecular docking study was also performed to get insight
investigation on the binding mode of compound 4e with Bcr-
AblT315I (PDB code: 3IK3) by using Glide module (Glide, version
5.7 Schrödinger, LLC, New York) with standard precision scoring
function. Compound 4e binds to the ATP binding site of Bcr-
AblT315I with a DFG-out conformation (Fig. 2), which is similar to
that of ponatinib.35,37 The N-cyclopropyl amino pyrimidinyl motif
of 4e forms two essential hydrogen bonds with the backbone of
Met318 in the hinge region of Bcr-AblT315I. This observation high-
lights the great contribution of a hydrogen-donating NH moiety
to the kinase inhibitory activity, which has been confirmed by
varying the substituent in R-position (i.e., compounds 4j, 4k and
l
that of ponatinib and about 10-times more potent that GNF-7
(Table 1). Further structural optimization studies reveal that the
substituent at R-position significantly affect the kinase inhibitory
activities against Bcr-AblWT and Bcr-AblT315I. Although the methyl
amino analogue 4b displays identical potency to that of 4a, the
ethyl (4c), iso-propyl (4d), 2-hydroxyethyl (4f), cyclo-hexanyl
(4g), tert-butyl (4h) and 4-phenyl (4i) substituted compounds
are significantly less potent than 4a. The IC50 values are 0.30 and
0.19, 0.125 and 0.187, 0.018 and 0.019, 0.093 and 0.458, 0.028
and 0.024, 0.238 and 0.161 l
M, respectively, against Bcr-AblWT
and Bcr-AblT315I. N,N-disubstituted moieties such as piperidinyl
(4j), pyrrolidin-1-yl (4k) and morpholino (4l) groups are all detri-
mental to the inhibitory activities against both wild-type Bcr-Abl
and the resistant mutant. The resulting compounds almost totally
abolish their inhibitory effect against Bcr-AblT315I kinase (IC50
>1.0 lM). Finally, we are pleased to find the cyclopropyl substi-
tuted compound 4e is as potent as to compound 4a but is with
improved solubility, which inhibits Bcr-AblWT and Bcr-AblT315I
with IC50 values of 5.0 and 9.0 nM, respectively.

3462
X. Lu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3458–3463
bioavailability of 35.3% and a half-life of 48.7 h. It is also notewor-
thy that the Cmax of compound 4e reaches 6205 g/L (about 12 M)
l
l
after a 25 mg/kg oral administration, which is about 1333 folds of
its IC50 value against Bcr-Abl T315I, indicating the potential in vivo
efficacy in animal models.
Giving its promising kinase activity and PK properties, com-
pound 4e is further evaluated in subcutaneous K562 xenograft
model of human CML. The animals were orally dosed with 4e at
3.3 and 10 mg/kg/day. As shown in Figures 5 and 4e dose-depen-
dently inhibits the growth of the K562 tumor xenograft when
administered once daily for 16 consecutive days via oral gavage
and almost completely eradicates the tumor at doses of 10 mg/kg/
day after 8 days of treatment. Notably, after the cessation of treat-
ment (16 days of dosing), there was no sign of tumor recurrence in
the following 7 days. The compound 4e is well tolerated with no
mortality or significant body loss (<5% relative to vehicle-matched
controls) during the treatments.
Figure 5. Compound 4e potently suppresses tumor growth in K562 xenograft
model of human CML. Mice bearing K562 xenografts were dosed orally once a day
with 4e at 3.3 and 10 mg/kg dosages for 16 consecutive days. The data are
representative of two independent experiments.
Compound 4e also demonstrates promising antitumor effect in
an allograft model of Ba/F3 T315I cells. The animals were adminis-
tered 4e at doses of 10 and 30 mg/kg via oral gavarge once daily for
16 days. As shown in Figure 6, compound 4e dose-dependently
inhibits tumor growth in the allograft models of Ba/F3 T315I cells.
Although compound 4e does not show obvious inhibition of tumor
growth at dose of 10 mg/kg/day, it induces almost complete tumor
regression at a dose of 30 mg/kg/day after a 16 days consecutive
treatment.
In summary, a series of pyrimidine alkynyl derivatives are
designed and synthesized as new Bcr-AblT315I inhibitors by hybrid-
ing the structural moieties from GNF-7, ponatinib and nilotinib.
The resulting compounds strongly suppressed the activities of
Bcr-AblWT and Bcr-AblT315I kinases and potently inhibited the pro-
liferation of K562 and Ba/F3 T315I cells. The most promising com-
pound 4e displays comparable potency with that of ponatinib and
GNF-7 in cancer cell antiproliferative assay. Compound 4e also
dose-dependently induces the G0/G1phase arrest and apoptosis
of K562 and Ba/F3 T315I cells. It also displays good pharmacokinet-
ics properties and potently suppresses tumor growth in xeno-
grafted mice bearing K562 and Ba/F3 cells expressing Bcr-AblWT
and Bcr-AblT315I in vivo assay. These inhibitors might serve as lead
compounds for further developing new anticancer drugs.
Figure 6. Compound 4e potently suppressed tumor growth in allografted models of
Ba/F3 cells expressing Bcr-AblT315I. Mice bearing allografted Ba/F3 Bcr-AblT315I cells
were dosed orally once a day with 4e at 10 mg/kg and 30 mg/kg dosages for 16
consecutive days. The data are representative of two independent experiments.
4l). The amide forms three hydrogen bonds with Glu286 and
Asp381, and the trifluoromethylphenyl group binds deeply into a
hydrophobic back pocket. The alkynyl linker made favorable van
der Waals interactions with the gatekeeper Ile315 avoiding steric
clash.
Acknowledgements
We thank National High Technology Research and Development
(863) for Young Scientists program (No. 2015AA020906), the Key
Program of Chinese Academy of Sciences (No. KJZD-EW-L02), the
Pearl River S&T Nova Program of Guangzhou (201506010086)
Taking 4e as an example, the kinase inhibition of the new inhi-
bitors is further validated by investigating their suppressing effect
on the activation of Bcr-Abl and its downstream signals in cells
harboring different status of Bcr-Abl kinase. As shown in Figures
3 and 4e dose-dependently inhibits the phosphorylation of
Bcr-Abl in both K562 and Ba/F3 T315I cells. The phosphorylation
level of the downstream signal proteins such as CRKL and STAT5
were also obviously decreased, while the total protein levels of
Bcr-Abl, CRKL, STAT5 and GAPDH remained unchanged. The data
further support strong target inhibition of the newly designed
compounds. Not surprisingly, the three FDA-approved drugs
(imatinib, nilotinib and dasatinib) demonstrated no obvious
suppression of Bcr-AblT315I activation.32
Technological
Innovation
Program
of
Foshan
City
(2013HK100212) and Youth Innovation Promotion Association of
Chinese Academy of Sciences.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.07.
006.
Further flow cytometric analysis revealed that compound 4e
also significantly induced the G0/G1 phase arrest and apoptosis
in a dosage dependent manner in both K562 (Fig. 4A) and Ba/F3-
T315I cells (Fig. 4B), respectively, which is a consequent response
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