Discovery of 2-arylamino-4-(1-methyl-3-isopropylsulfonyl-4-pyrazol-amino)pyrimidines as potent anaplastic lymphoma kinase (ALK) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.06.021

Abstract A new series of 2,4-diamino pyrimidine derivatives with a sulfone-substituted pyrazole right side-chain were discovered as potent anaplastic lymphoma kinase inhibitors. Structure-activity relationship of the left side-chain on phenyl substitutions were explored which delivered many potent ALK inhibitors. Among them, 29a showed favorable pharmacokinetic profiles in rats and dogs together with significant antitumor efficacy in EML4-ALK fusion xenograft model. Graphical abstract

Full text of DOI:10.1016/j.bmcl.2015.06.021

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-arylamino-4-(1-methyl-3-isopropylsulfonyl-4-
pyrazol-amino)pyrimidines as potent anaplastic lymphoma kinase
(ALK) inhibitors
b
b
b
b
b
Peilong Zhang a,c, Jiaqiang Dong b, , Boyu Zhong , Deyi Zhang , Can Jin , Xuejing Meng , Desheng Sun ,
Xiangyuan Xu b, Yong Zhou b, Zhi Liang b, Minghua Ji b, Hailong Li b, Tao Xu b, Guowei Song b, Ling Zhang b,
Gang Chen b, Hongbin Yuan b, Joe Shih b, Ruihao Zhang b, Guojun Hou b, Ying Jin b, Qiong Yang b
⇑
a Department of Applied Chemistry, Beijing Institute of Technology, Zhongguancun South Street, Beijing 100081, China
b Crown Bioscience Inc. (Taicang), 6 Beijing West Road, Taicang Economic Development Area, Taicang 215400, China
c Beijing Pearl Biotech Ltd, No. 203, Section 2, Wangjing Lize Zhongyuan, Chaoyang District, Beijing 100102, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 2,4-diamino pyrimidine derivatives with a sulfone-substituted pyrazole right side-chain
were discovered as potent anaplastic lymphoma kinase inhibitors. Structure–activity relationship of the
left side-chain on phenyl substitutions were explored which delivered many potent ALK inhibitors.
Among them, 29a showed favorable pharmacokinetic profiles in rats and dogs together with significant
antitumor efficacy in EML4-ALK fusion xenograft model.
Received 15 April 2015
Revised 27 May 2015
Accepted 5 June 2015
Available online xxxx
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
ALK
Kinase inhibitor
NSCLC
PK properties
Antitumor efficacy
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase
that belongs to the insulin receptor superfamily, which contains
other members such as IGF-1 receptor (IGF-1R), insulin related
receptor (IR) and leukocyte tyrosine kinase (LTK). The kinase was
firstly identified in 1994 as a part of the nucleophosmin (NPM)–
ALK fusion protein found in around 75% of ALK-positive anaplastic
large cell lymphoma (ALCL) cases.1 In adult mice, ALK expression is
low and primarily expressed in brain tissue and plays an important
role in the development and function of the central nervous sys-
tem.2 Although the physiological function of ALK in the normal
body is unclear, ALK fusion proteins have been found in many
human cancers.3
(KRAS) mutations.6 Thus the development of selective ALK inhibi-
tors would be a promising treatment option for ALK-driven
cancers.
As the first ALK inhibitor approved by FDA in 2011, crizotinib (1,
PF2341066) was initially designed and developed as a c-Met
inhibitor which also showed good antitumor efficacy in ALK-de-
pendent xenograft models.7 After the initial dramatic responses
to crizotinib, however, the patients eventually developed drug
resistance within 12 months after treatment with crizotinib. The
proposed mechanisms of acquired resistance include secondary
(point) mutations (especially for gatekeeper mutation L1196M),
ALK gene amplification, and up-regulation of ‘ALK-independent’
cell-signaling bypass.8 Now several second generation ALK inhibi-
tors have been or are being developed at different clinical stages,9
including the second ALK inhibitor ceritinib (2, LDK-378) approved
by FDA in April, 2014.
The 2,4-diarylamino pyrimidine analogs (DAAPs) were exten-
sively explored in this area, resulting in the discovery of several
potent and selective ALK inhibitors (Fig. 1) such as LDK-378
(2),10 NVP-TAE-684 (3),11 AP-26113 (4),12 CEP-37440,13 etc (among
them, the development of 3 was discontinued.10). These inhibitors
share the common 2,4-diarylamino unit.
Most recently, ALK has become a hot drug target4 in cancer
therapy along with the identification of the oncogenic fusion gene
echinoderm micro-tubule-associated protein-like 4 (EML4)—ALK
in around 5% non-small cell lung cancer (NSCLC) patients.5
Notably, EML4-ALK translocation is almost mutually exclusive
with the other oncogenes such as epidermal growth factor receptor
(EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
⇑
Corresponding author. Tel.: +86 512 3301 1318; fax: +86 512 5387 9801.
E-mail address: dongjiaqiang@crownbio.com (J. Dong).
http://dx.doi.org/10.1016/j.bmcl.2015.06.021
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang, P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.021

2
P. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
NH2
Cl
N
O
N
Cl
HN
N
NH
O
O
S
Cl
F
O
N
N
N
H
NH
1
, crizotinib (PF2341066)
2
, ceritinib (LDK378)
Cl
N
Cl
N
Cl
N
HN
N
N
NH
HN
N
N
NH
O
HN
N
N
NH
O
O
S
O
O
P
O
N
H
O
N
N
N
N
N
OH
4
3
, AP26113
5
, NVP-TAE684
Figure 1. Known ALK inhibitors.
Recently, by applying a bioisosteric strategy, we discovered a
substituted anilines 11a–11i. The intermediates 11j–11l contain-
ing trifluoromethoxy, trifluoromethyl and isopropyl groups were
obtained from 16 through bromination using NBS, followed by
Suzuki coupling and subsequent hydrogenation. Anilines 16 were
in turn prepared from starting materials 14, 15 and 18 respectively.
The Negishi coupling between in situ formed organozinc reagent
21 and bromide 9 provided the product 22 with a total 25% yield.
The last hydrogenation on 22 afforded aniline 11m.
Scheme 3 summarized the synthesis of the right hand piece and
its coupling with the left hand piece to provide the final products.
Starting from 3-nitropyrazole 23, methylation followed by nitra-
tion produced dinitro compound 24. Subsequent nucleophilic sub-
stitution by isopropyl thiol, oxidation with m-CPBA and reduction
of the nitro compound 25 afforded amino pyrazole 26, which
reacted with trichloropyrimidine to provide key intermediate 27.
Buchwald coupling between 27 and amines 11 followed by depro-
tection afforded desired molecules 28, which were further func-
tionalized to deliver the final products 29.
series of potent ALK inhibitors with a unique amino pyrazole
side-chain replacing 4-aryl on DAAPs scaffold, exemplified by the
molecule 6 and 7 (Fig. 2) which showed good in vitro potency
against ALK in enzyme and cellular assays.14
In the left hand of the structures, there is a structural feature,
4-amino aniline. However, it is generally considered to be a
structural alert for creating potential safety liability through the
formation of the reactive p-diiminoquinone species A (Scheme 1).
This metabolic liability has been investigated by Novartis10 and
Cephalon15 during their studies of ALK inhibitors.
To avoid this problem, we decided to optimize our leading
compound by moving out N on morpholine (Fig. 3) to deliver a
new ALK inhibitor with good anti-tumor efficacy and improved
PK properties.
General synthetic routes of new ALK inhibitors are shown in
Schemes 2 and 3. The left side-chain anilines were synthesized in
several steps. As shown in Scheme 2, SNAr substitution of fluoride
8 with different alcohols in DMF under basic condition afforded 9
in good yield. Intermediate 13 containing fluorine was prepared
from phenol 12 through an etherification followed by the nitration
using HNO3. With 9 and 13 in hand, Suzuki coupling followed by
catalytic hydrogenation using PtO2 or Pd/C provided key alkoxy
For those compounds containing different R1 substitution, we
firstly evaluated their potency against ALK in enzyme16 and cellu-
lar17 assays. As shown in Table 1, the simple alkoxy substitution
was tolerated and compounds 28b and 29b containing
cyclopropyloxy displayed the best activities. Slightly bulky substi-
tutions in compounds such as 29c, 29d and 29e diminished the
potencies in H2228 cell line. By the comparison between 28a,
28b and 29a, 29b, N-methylation contributed to improve the activ-
ities, especially in cellular assays. In addition, the targets 29f and
29g with carbon attached to the phenyl ring directly were 3 to 5
folds less potent, demonstrating the importance of oxygen linkage.
Based on the structure of ALK and known literatures,10,18 we
believe that the relatively bulky R1 substitutions in Table 1 should
provide good ALK selectivities and these will be studied in the
future.
Cl
N
Cl
N
HN
N
N
NH
O
O
S
O
HN
N
NH
N
O
S
O
N
N
O
N
N
O
With the best cyclopropyloxy group set on R1, the effect of R2
substitution was then explored. R2 is at the para-position of R1
and might have some potential metabolic liabilities. As shown in
Table 2, several substitutions were tested against ALK. Molecules
29h and 29b with H and Me respectively showed the most potent
profiles but 29h displayed much higher in vivo clearance in rat
N
N
6
7
Figure 2. Our early ALK inhibitors.
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P. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Cl
Cl
N
Cl
N
N
R1
R1
R1
HN
N
N
N
H
N
N
N
N
H
HN
N
N
N
H
R2
Oxidation
R2
Nu
R2
Nu
R3
R4
R3
R4
A
R3
R4
B
Scheme 1. Metabolic liability of 4-amino aniline through reactive adduct diiminoquinone.
than 29b (79.8 vs 22.7 mL/min/kg), possibly due to the existence of
the metabolically labile para-H. The substitutions with electro-
withdrawing groups such as F, Cl and CN did not improve the
potency.
Piperidine substitution in molecule points to the solvent area
once bound to the ALK enzyme and SAR on this region is summa-
rized on Table 3. Compared with piperidine, molecules 28l, 29l,
28m and 29m containing the 4-member and 5-member hetero-
cyclic substitutions showed slightly less potency than 28a and
28b. The simple alkylated piperidines such as 29n, 29o and 29p
showed very good ALK inhibition while compound 29q with
Me
Currently, remaining
the core scaffold.
Cl
N
N
N
HN
N
H
N
H
O
Substitution to
S
O
O
improve potency
and selectivity.
Me
Me
Me
N
O
Substitution to
further reduce
metabolism.
To mitigate the
metabolite liability.
Figure 3. Our SAR strategy.
NO2
NH2
NO2
NO2
O
O
O
F
R1
R1
R1
a
R2
Cl, Br
9
R2
R2
Cl, Br
8
R2
c
b
( )n = 0 or 1
( )n = 0 or 1
N
N
Boc
10
NO2
Boc
HO
11a-11i
O
d, e
11a: R1= iPr, R2=Me, n=1
11b: R1=Cyclopropyl, R2=Me, n=1
11c: R1=Cyclopropylmethyl, R2=Me, n=1
11d: R1=1,3-difluoropropan-2-yl, R2=Me, n=1
11e: R1=Cyclopropyl, R2=H, n=1
11f: R1=Cyclopropyl, R2=F, n=1
11g: R1=Cyclopropyl, R2=Cl, n=1
11h: R1=Cyclopropyl, R2=CN, n=1
11i: R1=Cyclopropyl, R2=Me, n=0
F
F
Br
12
Br
13
NO2
F3CO
NH2
R1'
f, g
NH2
Br
NH2
R1'
14
NO2
b, c
R1'
h
f
Me
F3C
Br
Br
17
16
N
Boc
11j-11l
15
j
NO2
NO2
11j: R1'=OCF3
11k: R1'=CF3
11l: R1'= iPr
i
19
18
NH2
NO2
O
O
I
ZnI
N
k
l
m
N
Boc
Boc
N
Boc
11m
N
Boc
22
20
21
Scheme 2. Reagents and conditions: (a) R1-OH, tBuONa, DMF, rt, 85–95%; (b) boronic acid pinacol ester, Pd(PPh3)2Cl2, Na2CO3, dioxane/H2O, 100 °C, 80–90%; (c) PtO2 or Pd/C,
H2, MeOH, rt, 70–85%; (d) cyclopropyl bromide, Cs2CO3, KI, NMP, 150 °C, 43%; (e) HNO3, Ac2O, ꢀ5 °C, 72%; (f) Fe, NH4Cl, 80 °C, 98%; (g) methyl boronic acid, Pd(dppf)Cl2,
K2CO3, dioxane, 80 °C, 72%; (h) NBS, DMF, rt, 99%; (i) prop-1-en-2-boronic acid pinacol ester, Pd(dppf)Cl2, NaHCO3, dioxane/H2O, 95 °C, 85%; (j) Pd/C, H2, MeOH, rt, 79%; (k) Zn,
TMSCl, BrCH2CH2Br, DMF, 65 °C; (l) 9, Pd(dppf)Cl2, CuI, DMF, 80 °C, 25% for two steps; (m) Pd/C, H2, MeOH, rt, 55%.
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P. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
N
N
N
N
NO2
NO2
NO2
d, e
f
N
N
c
a, b
H2N
O2N
N
HN
O
S O
S
23
24
25
26
Cl
Cl
N
S
N
N
N
N
N
Cl
HN
N
N
H
HN
N
N
H
N
N
g, h
i or j
O
O
R1
R1
S
N
O
O
Cl
N
N
H
O
S
R2
R2
O
27
( ) ( )n = 0 or 1
N
( ) ( )n = 0 or 1
N
n = 0 or 1
n = 0 or 1
H
28
R
29
Scheme 3. Reagents and conditions: (a) CH3I, tBuONa, DMF, 0 °C, 92%; (b) HNO3, H2SO4, 100 °C, 79%; (c) isopropyl thiol, K2CO3, CH3CN, rt, 96%; (d) m-CPBA, DCM, rt, 98%; (e)
Pd/C, H2, MeOH, rt, 77%; (f) 2,4,5-trichloropyrimidine, Et3N, EtOH, 70 °C, 70%; (g) 11, Pd(OAc)2, Xantphos, Cs2CO3, THF, sealed tube,130 °C, 20–60%; (h) TFA, DCM, rt, 70–90%;
(i) for methylation, HCHO, NaBH(OAc)3, MeOH, AcOH, THF, rt, 50–70%; for 29p, 3-oxetanone, NaBH3CN, ZnCl2, MeOH, 48 °C, 95%; for other alkylation, RX, K2CO3, CH3CN, 80 °C,
50–85%; (j) for acylation, acyl chloride or isocyanate, Et3N, DCM, 0 °C–rt, 40–80%.
The pharmacokinetic19 properties of 29a were evaluated in rats
and dogs and the compound showed favorable clearance, accept-
Table 1
SAR of ortho-substitution of aniline
able half life and good bio-availabilities in both species (Table 4).
Cl
N
N
N
Then this compound was evaluated on a well-established NCI-
H2228 (EML4-ALK NSCLC) xenograft model in mice. As shown in
Figure 4, compound 29a showed dose-dependent antitumor effi-
cacy20 when the animals were treated orally once daily for two
weeks. There was almost no anti-tumor inhibition at 3 mg/kg,
however, a significant tumor regression was observed at the dose
of 30 mg/kg without obvious body weight loss. Notably, almost
complete tumor regression at 30 mg/kg was observed after the
dosing was stopped for several days.
HN
N
N
H
O
R1
S
O
N
R
In summary, we disclosed the discovery of a series of new 2,4-
diarylaminopyrimides (DAAPs) with a novel 3-sulfonyl-4-amino
pyrazole side-chain as potent ALK inhibitors. We performed exten-
sive SAR exploration on the left hand side of the scaffold and deliv-
ered many active compounds displaying nanomolar inhibitory
activity against ALK enzymes in wild-type and mutant assays.
Compound R1
R
ALK
WT
IC50
(nM)
ALK
(L1196M)
IC50 (nM)
Karpas-299 H2228
cell
cell
IC50 (nM)
IC50
(nM)
28a
29a
H
8
4
20
8
17
10
225
96
i
PrO
∗
Me
28b
29b
O
H
Me
2
2
4
4
9
6
122
52
∗
28c
29c
H
10
17
48
43
74
262
226
Table 2
O
O
Me 15
SAR of R2 substitution
∗
∗
F
Cl
29d
Me 10
23
62
403
N
S
N
F
N
F3CO
HN
N
N
H
29e
29f
Me
6
10
49
20
74
374
∗
O
O
O
F3C
Me 22
Me 12
1078
∗
R2
i
Pr
29g
28
—
660
∗
N
(dimethylamino)ethyl did not improve the potency. Molecules 29r,
29s and 29t containing electro-withdrawing groups also main-
tained the ALK potency but with much lower in vivo
exposure which might be due to stability issue (data not shown).
Lastly, the 3-substituted piperidine 29u demonstrated very good
cell activity. However, one enantiomer of 29u did not show
promising in vivo efficacy and the other one displayed unfavorable
hERG tolerability.
Compound
R2
ALK WT
IC50 (nM)
ALK (L1196M)
IC50 (nM)
Karpas-299
cell
IC50 (nM)
H2228
cell
IC50 (nM)
29h
29b
29i
29j
29k
H
Me
F
Cl
CN
3
2
3
4
8
5
4
4
10
13
7
6
17
15
100
36
52
125
103
165
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P. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 3
SAR of R3
Cl
N
S
N
N
HN
N
N
H
O
R1
O
R3
Compound
R1
R3
ALK WT
IC50 (nM)
ALK (L1196M)
IC50 (nM)
Karpas-299 cell
IC50 (nM)
H2228 cell
IC50 (nM)
i
PrO
∗
∗
∗
NH
28l
29l
4
2
13
5
60
17
288
270
∗
∗
∗
i
PrO
N
Me
i
PrO
28m
4
7
5
4
28
11
364
131
NH
N
i
PrO
∗
∗
Me
29m
O
∗
∗
N
R
29n
29o
R = iPr
2
4
5
7
9
58
43
OMe
14
∗
O
29p
29q
10
2
10
5
19
15
44
∗
NMe2
199
∗
O
Me
Me
29r
26
29
—
149
∗
SO2Me
29s
29t
9
4
11
6
18
20
59
82
∗
O
∗
∗
NHEt
O
∗
29u
3
7
3
32
N
Me
Table 4
1000
800
600
400
200
0
Vehicle (0.5% MC/0.5% Tween 80) QD x 2 weeks p.o.
29a 3 mg/kg QD
Pharmacokinetic properties of 29a in Sprague-Dawley rats and beagle dogs
PK parametersa
Ratb
Dogc
29a 10 mg/kg QD
i.v.
29a 30 mg/kg QD
Dose (mg/kg)
t1/2 (h)
AUC0-1 (ng h/mL)
Vd (L/kg)
1
1
5.1
988
7.5
16.9
4.9
637
11
CL (mL/min/kg)
26.2
p.o.
Dose (mg/kg)
Cmax(ng/mL)
tmax(h)
AUC0-1 (ng h/mL)
F (%)
5
199
6.7
3122
63.2
3
85.8
2.0
952
49.2
16
18
20
22
24
26
28
30
32
34
36
38
Days after Tumor Inoculation
a
b
c
Values are means data from three animals.
Vehicle: DMSO (10%) + 20% HP-b-CD (90%).
Vehicle: 20% HP-b-CD.
Figure 4. Antitumor efficacy of 29a in a 14-day mouse H2228 xenograft model.
Acknowledgments
These compounds also showed very good cell anti-proliferation
activity. Compound 29a showed good pharmacokinetic properties
in rats and dogs. Meanwhile, 29a also demonstrated good dose-de-
pendent anti-tumor efficacy in xenograft mice model. Currently,
our efforts toward the identification of a drug candidate are ongo-
ing and the result will be reported in the future.
We thank Dr. Shaosong Chu for his pioneer work; Ms. Kefeng
Gong for her help in chemical analysis; Dr. QiyongMou for his con-
tribution on side-chain synthesis; Dr. Chengcheng Wang for pro-
viding ALK enzymes; Dr. Hepeng Shi for his support and useful
discussion.
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6
P. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
13. CEP-37440 might have very similar structure to compound 5 based on the
Supplementary data
information from Ref. 9.
14. Unpublished results.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.
021.
15. Mesaros, E. F.; Thieu, T. V.; Wells, G. J.; Zificsak, C. A.; Wagner, J. C.; Breslin, H. J.;
Tripathy, R.; Diebold, J. L.; McHugh, R. J.; Wohler, A. T.; Quail, M. R.; Wan, W. H.;
Lu, L. H.; Huang, Z. Q.; Albom, M. S.; Angeles, T. S.; Wells-Knecht, K. J.; Aimone,
L. D.; Cheng, M. G.; Ator, M. A.; Ott, G. R.; Dorsey, B. D. J. Med. Chem. 2012, 55,
115.
16. Enzyme inhibition assays: the ALK WT activity was determined in a 384-well
HTRF format using HTRFÒ KinEASE™-TK Assay Kit (Cat# 62TK0PEC, Cisbio) and
recombinant human ALK enzyme (Cat# ALK-20110607,Crownbio). The final
assay conditions were 50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM
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40 nM SEB reagent and 10 ng ALK WT enzyme, TK Substrate-biotin and SEB
reagent were provided by Cisbio kit. The assay reaction time was 30 min after
which the antibody detection mix was added. The L1196M mutant ALK
enzyme assay was done in
Crownbio (Cat# ALKm-20110923) and 5
a
similar format, mutant enzyme was from
M ATP was used in the assay buffer.
l
IC50 was calculated by Xlift software using four-parameter logistic
concentration–response curve.
17. Cell anti-proliferation assay: cells were seeded in 96-well tissue culture plate,
and incubated at 37 °C, 5% CO2 for 2 hours, then cells were exposed to various
concentrations of compounds and further cultured for 96 h. Cell proliferation
was determined using CellTiter-Glo assay kit (Promega, G7572). IC50 was
calculated by Xlift software using four-parameter logistic concentration–
response curve.
18. Kinoshita, K.; Kobayashi, T.; Asoh, K.; Furuichi, N.; Ito, T.; Kawada, H.; Hara, S.;
Ohwada, J.; Hattori, K.; Hong, W. S.; Park, M. J.; Takanashi, K.; Tsukaguchi, T.;
Sakamoto, H.; Tsukuda, T.; Oikawa, N. J. Med. Chem. 2011, 54, 6286.
19. PK studies: 29a (mesylate salt) was formulated as a solution in DMSO (10%)
and 20% HP-b-CD (90%) and it was administrated to male Sprague-Dawley rats
(n = 3 per group) by intravenous (i.v., 1 mg/kg) or oral (p.o., 5 mg/kg). After
dosing, blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 24 h for i.v.
dosing and at 0.25, 0.5, 1, 2, 4, 6, 8, 24 h for p.o. dosing. The samples were
centrifuged and the collected plasma was analyzed by LC–MS/MS. The PK
parameters were calculated by Winnonlin 6.3 software Non-Compartmental
model. 29a (mesylate salt) was formulated as a solution in 20% HP-b-CD and it
was administrated to male beagle dogs (n = 3 per group) by intravenous (i.v.,
1 mg/kg) or oral (p.o., 3 mg/kg). Sample collection and analysis followed the
same method as above.
20. In vivo efficacy: Balb/c nude mice inoculated with H2228 were randomized
into four groups (8 mice per group) with an average tumor volume of around
200 mm3. Then the mice were dosed by vehicle and 29a (mesylate salt,
formulated in 0.5% MC and 0.5% Tween 80, oral, QD) at dose levels of 3, 10 and
30 mg/kg. Mice were monitored for the behavior and body weight loss, and
tumors were measured twice weekly in two dimensions using a caliper, and
the volume is expressed in mm3 using the formula: V = 0.5 a ꢁ b2 where a and b
are the long and short diameters of the tumor, respectively. Tumor growth was
calculated by T/C% = (T ꢀ T0)/(C ꢀ C0) ꢁ 100, where T and T0 are the mean
tumor volumes on a specific experimental day and on the first day of treatment
respectively for the treatment groups, where C and C0 are the data for the
control group. Meanwhile, tumor growth inhibition (TGI) was calculated by
TGI = (1 ꢀ T/C) ꢁ 100%.
Please cite this article in press as: Zhang, P.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.021