Discovery of T-1101 tosylate as a first-in-class clinical candidate for Hec1/Nek2 inhibition in cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2020.112118

Highly expressed in cancer 1 (Hec1) plays an essential role in mitosis and is correlated with cancer formation, progression, and survival. Phosphorylation of Hec1 by Nek2 kinase is essential for its mitotic function, thus any disruption of Hec1/Nek2 prote

Full text of DOI:10.1016/j.ejmech.2020.112118

Journal Pre-proof
Discovery of T-1101 tosylate as a first-in-class clinical candidate for Hec1/Nek2
inhibition in cancer therapy
Shih-Hsien Chuang, Ying-Shuan E. Lee, Lynn Y.L. Huang, Chi-Kuan Chen, Chun-
Liang Lai, Yu-Hsiang Lin, Ju-Ying Yang, Sheng-Chuan Yang, Lien-Hsiang Chang,
Ching-Hui Chen, Chia-Wei Liu, Her-Sheng Lin, Yi-Ru Lee, Kuan Pin Huang, Kuo Chu
Fu, Hsueh-Min Jen, Jun-Yu Lai, Pei-Shiou Jian, Yu-Chuan Wang, Wen-Yun Hsueh,
Pei-Yi Tsai, Wan-Hua Hong, Chia-Chi Chang, Diana ZC. Wu, Jinn Wu, Meng-Hsin
Chen, Kuo-Ming Yu, Ching Yuh Chern, Jia-Ming Chang, Johnson Y.N. Lau, Jiann-Jyh
Huang
PII:
S0223-5234(20)30085-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112118
EJMECH 112118
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 October 2019
Revised Date: 18 January 2020
Accepted Date: 3 February 2020
Please cite this article as: S.-H. Chuang, Y.-S.E. Lee, L.Y.L. Huang, C.-K. Chen, C.-L. Lai, Y.-H. Lin, J.-
Y. Yang, S.-C. Yang, L.-H. Chang, C.-H. Chen, C.-W. Liu, H.-S. Lin, Y.-R. Lee, K.P. Huang, K.C. Fu, H.-
M. Jen, J.-Y. Lai, P.-S. Jian, Y.-C. Wang, W.-Y. Hsueh, P.-Y. Tsai, W.-H. Hong, C.-C. Chang, D.Z. Wu,
J. Wu, M.-H. Chen, K.-M. Yu, C.Y. Chern, J.-M. Chang, J.Y.N. Lau, J.-J. Huang, Discovery of T-1101
tosylate as a first-in-class clinical candidate for Hec1/Nek2 inhibition in cancer therapy, European
Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112118.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

©
2020 Published by Elsevier Masson SAS.

Graphical abstract
Discovery of T-1101 tosylate as a first-in-class clinical candidate for
Hec1/Nek2 inhibition in cancer therapy
Shih-Hsien Chuang, Ying-Shuan E. Lee, Lynn Y. L. Huang, Chi-Kuan Chen,
Chun-Liang Lai, Yu-Hsiang Lin, Ju-Ying Yang, Sheng-Chuan Yang, Lien-Hsiang
Chang, Ching-Hui Chen, Chia-Wei Liu, Her-Sheng Lin, Yi-Ru Lee, Kuan Pin Huang,
Kuo Chu Fu, Hsueh-Min Jen, Jun-Yu Lai, Pei-Shiou Jian, Yu-Chuan Wang, Wen-Yun
Hsueh, Pei-Yi Tsai, Wan-Hua Hong, Chia-Chi Chang, Diana ZC Wu, Jinn Wu,
Meng-Hsin Chen, Kuo-Ming Yu, Ching Yuh Chern, Jia-Ming Chang, Johnson Y. N.
Lau, and Jiann-Jyh Huang
Optimized from lead compound 7, T-1101 tosylate (9j tosylate) showed improved oral
pharmacokinetics and promising in vitro/vivo anticancer activity. T-1101 tosylate is a
first-in-class clinical candidate for Hec1/Nek2 inhibition currently in clinical trials for
cancer therapy.
-
1 -

Discovery of T-1101 tosylate as a first-in-class clinical candidate for
Hec1/Nek2 inhibition in cancer therapy
Dedicated to Dr. Henry Feng and Dr. Jeff Wang for making everything possible.
a,1
a,1
b
c
a
a
a
a
Shih-Hsien Chuang, Ying-Shuan E. Lee, Lynn Y. L. Huang, Chi-Kuan Chen, Chun-Liang Lai,
a
a
a
a
Yu-Hsiang Lin, Ju-Ying Yang, Sheng-Chuan Yang, Lien-Hsiang Chang, Ching-Hui Chen,
a
a
a
a
a
Chia-Wei Liu, Her-Sheng Lin, Yi-Ru Lee, Kuan Pin Huang, Kuo Chu Fu, Hsueh-Min Jen,
d
d
d
d
a
Jun-Yu Lai, Pei-Shiou Jian, Yu-Chuan Wang, Wen-Yun Hsueh, Pei-Yi Tsai, Wan-Hua Hong,
b
e
e
a
b
d
Chia-Chi Chang, Diana ZC Wu, Jinn Wu, Meng-Hsin Chen, Kuo-Ming Yu, Ching Yuh Chern,
a
b
,a,d
Jia-Ming Chang, Johnson Y. N. Lau and Jiann-Jyh Huang*
a
Development Center for Biotechnology, National Biotechnology Research Park, Taipei City 11571,
Taiwan
b
Taivex Therapeutics Corporation, 2nd Floor, Dongxing Rd., Songshan Dist., Taipei City 10511,
Taiwan
c
Genomics Research Center, Academia Sinica, Taipei City, Taiwan
d
Department of Applied Chemistry, National Chiayi University, No. 300, Syuefu Rd., Chiayi City
60004, Taiwan
e
Xenobiotic Laboratories, Inc., Plainsboro, New Jersey, U.S.A.
*
Corresponding author. Department of Applied Chemistry, National Chiayi University, No. 300,
Syuefu Rd., Chiayi City 60004, Taiwan. Email address: lukehuang@mail.ncyu.edu.tw (J.-J. Huang).
1
Authors have contributed equally to this work.
1

ABSTRACT
Highly expressed in cancer 1 (Hec1) plays an essential role in mitosis and is correlated with
cancer formation, progression, and survival. Phosphorylation of Hec1 by Nek2 kinase is essential for
its mitotic function, thus any disruption of Hec1/Nek2 protein–protein interaction has potential for
cancer therapy. We have developed T-1101 tosylate (9j tosylate, 9j formerly known as TAI-95),
optimized from 4-aryl-N-pyridinylcarbonyl-2-aminothiazole of scaffold 9 by introducing various
C-4’ substituents to enhance potency and water solubility, as a first-in-class oral clinical candidate
for Hec1 inhibition with potential for cancer therapy. T-1101 has good oral absorption, along with
potent in vitro antiproliferative activity (IC : 14.8–21.5 nM). It can achieve high concentrations in
5
0
Huh-7 and MDA-MB-231 tumor tissues, and showed promise in antitumor activity in mice bearing
human tumor xenografts of liver cancer (Huh-7), as well as of breast cancer (BT474, MDA-MB-231,
and MCF7) with oral administration. Oral co-administration of T-1101 halved the dose of sorafenib
(25 mg/kg to 12.5 mg/kg) required to exhibit comparable in vivo activity towards Huh-7 xenografts.
Cellular events resulting from Hec1/Nek2 inhibition with T-1101 treatment include Nek2
degradation, chromosomal misalignment, and apoptotic cell death. A combination of T-1101 with
either of doxorubicin, paclitaxel, and topotecan in select cancer cells also resulted in synergistic
effects. Inactivity of T-1101 on non-cancerous cells, a panel of kinases, and hERG demonstrates
cancer specificity, target specificity, and cardiac safety, respectively. Subsequent salt screening
showed that T-1101 tosylate has good oral AUC (62.5 µM·h), bioavailability (F = 77.4%), and
thermal stability. T-1101 tosylate is currently in phase I clinical trials as an orally administered drug
for cancer therapy.
Keywords: thiazole; 2-aminothiazole; Hec1; Nek2; anticancer
Abbreviations: ADC, antibody–drug conjugate; AUC, area under the curve; CDE, CDOCKER
energy; Cenp-E, centromere-associated protein E; CIE, CDOCKER interaction energy; ClogP,
calculated logarithm of the partition coefficient; CMC, carboxymethyl cellulose; DMSO,
dimethylsulfoxide; dppf, 1,1'-bis(diphenylphosphino)ferrocene; DSC, differential scanning
2

calorimetry; Eg5, egg protein 5; F%, oral bioavailability; GI , the concentration for 50% of
5
0
maximal inhibition of cell proliferation; GLP, good laboratory practice; Hec1, highly expressed in
cancer 1; hERG, human ether-a-go-go-related gene; IC , the half maximal inhibitory concentration;
5
0
IV, intravenous; KSP, kinesin spindle protein; Mcl-1, myeloid cell leukemia 1; Mps1, monopolar
spindle 1; MTA, microtubule-targeting agent; Ndc80, kinetochore protein Ndc80 homolog; Nek2,
NIMA (never in mitosis gene a)-related kinase 2; NMP, N-methyl-2-pyrrolidone; Nuf2, kinetochore
protein Nuf2, PARP, poly(ADP-ribose) polymerase; PBS, phosphate buffered saline; PDB, protein
data bank; PO, per os; Spc24, kinetochore protein Spc24; Spc25, kinetochore protein Spc25,
TBABr , tetrabutylammonium tribromide; T/C, treatment-tumor-volume/control-tumor-volume;
3
TGA, thermogravimetric analysis; THF, tetrahydrofuran; XIAP, X-linked inhibitor of apoptosis
protein; XRPD, X-ray powder diffraction; acac, acetylaceto
3

1
. Introduction
Suppression of cell proliferation by targeting mitosis is one of the most successful anti-cancer
therapeutic strategies [1]. Anticancer drugs paclitaxel [2], docetaxel [3], carbazitaxel [4], epothilones
5], Vinca alkaloids [6], and even the payloads MMAE [7] and DM1 [8] used in the marketed
[
antibody–drug conjugates (ADC) are mitotic inhibitors. These drugs exert their anticancer effects by
interfering with the dynamic instability of microtubules [9] in dividing cells, then triggering the
spindle checkpoint [10] to arrest the cell cycle and induce apoptosis. Thus they are referred to as
microtubule-targeting agents (MTA) [11]. However, microtubules are also the major cytoskeletal
components of neurons. Mechanism-based neurotoxicities [12] (including peripheral neuropathy
[
13]) are inevitably encountered in patients treated with MTA. The peripheral neuropathy can be
dose-limiting [14] and treatment must be discontinued. Currently, no medication has proved
effective for the prevention or management of peripheral neuropathy. Mitotic targets other than
microtubules are thus emerging. Polo-like kinase 1, Aurora kinases, Mps1, Cenp-E, and KSP/Eg5
are examples [1] and some of their inhibitors have shown less neurotoxicity in clinical trials [15].
Highly expressed in cancer 1 (Hec1) plays an essential role in mitosis and is a key component
of the kinetochore that regulates the spindle checkpoint [16−18]. With Nuf2, Spc24, and Spc25,
Hec1 forms a dumbbell-shaped heterotetramer called the Ndc80 complex, in which Hec1 and Nuf2
dimerize as a sub-complex facing the microtubule-binding interface [19−21]. Hec1 has
microtubule-binding activity at its N-terminal region and is responsible for normal kinetochore–
microtubule attachment [19,22,23]. Except for its mitotic functions, Hec1 has oncogenic properties.
Overexpression of Hec1 leads to mitotic checkpoint hyperactivation and induces tumor formation in
transgenic mice [24]. The Hec1 overexpression is observed in breast [25], pancreatic [26], liver
[
27,28], colon [29], and prostate [30] cancer tissues and cells [31], and is significantly associated
with poor clinical outcomes for cancer patients [25,26,32]. RNA interference of Hec1 causes mitotic
catastrophe in cancer cells [27,33], suppresses tumor growth in vivo [33], and enhances the
sensitivity of human ovarian cancer cells to paclitaxel [34]. Modification of the N-terminus of Hec1
also inhibits the proliferation of cancer cells both in vitro and in vivo [35]. A screen of 6,000
4

druggable genes in a panel of epithelial ovarian cancer cells identifies Hec1 as one of the four genes
required for cancer cell survival [36]. For its critical roles in mitosis and cancer, Hec1 may serve as a
promising drug target for cancer therapy.
As phosphorylation of Hec1 by Nek2 kinase is essential for its mitotic function [37–39],
disruption of Hec1/Nek2 protein–protein interaction provides an alternative route to RNA
interference for Hec1 inactivation. Discovered by Wu and Lee et al. [40],
4-aryl-N-(4-arylcarbonyl)-2-aminothiazole 1 (INH1, Fig. 1a) is the first reported small-molecule
Hec1/Nek2 inhibitor identified from the genetic screening of a commercial compound library. INH1
inhibits the proliferation of a panel of breast cancer cells with IC values of 10.5–20.5 µM and
5
0
shows cellular responses attributed to Hec1 inhibition. In a subsequent medicinal chemistry study,
Qiu and Lee et al. discovered that aminothiazoles 2 and 3 show improved in vitro potency (IC :
5
0
1.1–3.4 µM, Fig. 1a) by focusing on the C-6’ position and the arylcarbonyl group of 1 [41]. In a
recent report [42], Hu and Lee et al. demonstrated new analogs of 1–3 with enhanced potency,
among them aminothiazole 4 (INH154) with a C-4’ piperidinyl group which showed the best in vitro
potency (IC : 0.2 µM for HeLa, 0.12 µM for MDA-MB-468). It is also active in vivo. Using 1–3
5
0
and their analogs as the leads, He et al. reported aminothiazole 5 (Lyt-2010) with a urea moiety
possessing low mircromolar IC values (Fig. 1a ) [43]. Zhu et al. reported aminothiazole 6 (TH-39)
5
0
with a (E)-4-(tert-butyl)styryl moiety having an IC value of 0.79 µM for K562 cells (Fig. 1a) [44].
50
Ferrara, Brancale, and Degrassi et al. identified SM-15 with a 1,3-disubstituted indole scaffold as the
first microtubule–Hec1 inhibitor [45].
Inspired by these pioneering studies [40−42], we used compounds 1–3 as the leads to discover
new Hec1/Nek2 inhibitors with improved potencies and pharmacokinetic properties (Fig. 1b) [46].
Our work derives 4-aryl-N-(4-pyridylcarbonyl)-2-aminothiazoles
7
and
8
bearing
a
4
3
-methyoxyphenoxy group at the C-4’ position that show low nanomolar in vitro potencies (IC :
5
0
2.0–48.6 nM for 7, 16.3–42.8 nM for 8). Compounds 7 and 8 have high IV AUC and are active in
vivo for mice bearing MDA-MB-231 xenografts. Cellular responses to 7 and 8 resulting from
Hec1/Nek2 inhibition [46,47] are consistent with those reported for 1–3. Both compounds showed
5

great selectivity in counter-screens: they are inactive toward normal cells, kinases, and hERG.
a. Reported Hec1/Nek2 inhibitors
4'
2'
O
4'
N
4
N
S
X
O
6
'
NH
N
N
R
NH
S
1
2
3
. R = H, X = CH (INH1), IC50 = 10.5 20.5 M
. R = Me, X = CH, IC50 = 1.7 2.5 M
4 (INH154)
. R = H, X = N, IC50 = 1.1 3.4 M
IC : 0.2 M (HeLa), 0.12 M (MB468)
50
O
N
S
NH
O
N
NH
CF3
NH
t-Bu
S
Cl
5
(Lyt-2010)
6 (TH-39)
IC : 5.6 M (MB468), 7.8 M (MB231)
IC : 0.79 M (K562)
50
50
b. Previous work
O
4'
X
O
4
N
S
N
MeO
NH
7. X = H, IC : 32.0 48.6 nM, F%: 12.4%, CLogP: 5.71
50
8
. X = F, IC : 16.3 42.8 nM, F%: 4.6%, CLogP: 5.35
50
c. This work
Y
X
4'
O
4
''
R
4
N
S
N
O
Z
NH
scaffold 9
X = O, CH , NH, S; Y, Z = CH, N
2
R = Me, MeO(CH2)n (n = 1 or 2), Me N(CH )
2
2 2
Fig. 1. Structures of Hec1/Nek2 inhibitors. (a) Reported Hec1/Nek2 inhibitors 1–6 and their in vitro
antiproliferative activities. (b) Improved Hec1/Nek2 inhibitors 7 and 8 reported in the previous study.
(c) The structure of scaffold 9 reported in this work.
Although compounds 7 and 8 have good in vitro potency, once per day consecutive intravenous
administration is required to show the corresponding in vivo potency [46]. When orally administered,
compounds 7 and 8 need a very high dose (150 mg/kg), along with a more frequent twice per day
schedule. The low oral bioavailabilities of 7 (12.4%) and 8 (4.6%) arising from their low water
solubility, predicted from their high ClogP values (5.71 for 7, 5.35 for 8) [48], are responsible for
6

their unsatisfactory oral in vivo potency. The even lower oral bioavailability of 8 results from the
reduced basicity of the pyridine nitrogen by the ortho fluoride (Fig. 1b) that makes it less soluble in
gastric acid. To pursue an orally administered clinical candidate for Hec1/Nek2 inhibition, 7 (without
fluoride) is the better lead [46].
Accordingly, we expanded the C-4’ substituent in lead 7 and redesigned it into scaffold 9 whose
oral AUC and bioavailability could be further enhanced (Fig. 1c). The C-4’ substituent in scaffold 9
consists of a one-atom linker O, CH , NH, or S (X) connecting to an aryl/heteroaryl group bearing a
2
C-4’’ alkoxy group (RO–). The heteroaryl groups are six-membered pyridinyl and pyrazinyl groups
(Y and/or Z = N) which increase the water solubility of 9, thereby enhancing the oral bioavailability.
The alkoxy group at the C-4’’ position of 9 was presumed to modulate the pharmacokinetic
properties but was also found to promote the in vitro potency. Our study eventually led to compound
9j having a C-4’ 5-(2-methoxyethoxy)pyrazin-2-ylthio group that possessed high oral AUC and low
nanomolar in vitro potency. Compound 9j, formerly known as TAI-95, had been reported in our
previously publications [49,50] which demonstrate its pharmacology on breast and liver cancers.
In this paper, we report the medicinal chemistry efforts that led to the discovery of 9j through
intermediate compounds 9a–i and 9k. Pharmaceutical and more pharmacological properties of 9j
were investigated in this study which confirmed that 9j is suitable for further development. After salt
screening and preclinical development, the original code number TAI-95 (for research) of 9j was
changed to T-1101 (for a clinical candidate). T-1101 tosylate is the first Hec1/Nek2 inhibitor
approved by the US FDA as an investigational new drug. As of this writing, it is in phase I clinical
trials to study its maximum tolerated dose, pharmacokinetics, and responses in patients with
advanced refractory solid tumors.
2. Results and discussion
2.1 Chemistry
Scheme 1 presents the synthesis of 4-aryl-N-(4-pyridylcarbonyl)-2-aminothiazoles 9b, 9c, 9e,
and 9i–k of scaffold 9 bearing either one of a C-4’ arylmethyl, arylamino, aryloxy, or pyrazinylthio
7

substituent (cf. Fig. 1c, X = CH , NH, O, and S in 9). 1-(4-Iodo-2,6-dimethylphenyl)ethanone (10)
2
served as the starting material to react with TBABr in CH CN to give the α-brominated product 11
3
3
in 98% yield. Compound 11 was condensed with thiourea in refluxing EtOH to form the
-aminothiazole moiety, producing compound 12 in 83% yield. Acylation of 12 by isonicotinoyl
chloride hydrochloride in CH Cl using Et N as the base gave
-aryl-N-(4-pyridylcarbonyl)-2-aminothiazole 13 bearing a C-4’ iodo group in 66% yield. C–C
coupling of 13 with (4-methoxybenzyl)zinc(II) bromide using Pd(PPh ) as the catalyst in THF [51]
2
2
2
3
4
3
4
gave compound 9b bearing a C-4’ 4-methoxybenzyl group in 73% yield. C–N coupling of 13 with
4
9
4
9
-methoxyaniline using PdCl (dppf)/dppf/t-BuOK catalytic system [52] in THF formed compound
2
c having a C-4’ 4-methoxyanilino group in 28% yield. C–O coupling of 13 with
-(3-methoxypropoxy)phenol (using a CuI and Fe(acac) co-catalyzed system) [53] gave compound
3
e bearing a C-4’ 4-(3-methoxypropoxy)phenoxy group in 19% yield. C–S coupling of 13 with C-5
substituted pyrazine-2-thiols catalyzed by CuI in the presence of K CO in DMF gave the desired
2
3
9i–k having a C-4’ pyrazinethio group with C-4’’ methoxy, 2-methoxyethoxy, and
2-(dimethylamino)ethoxy groups in 11%, 59%, and 15% yields, respectively.
Scheme 1. Synthesis of 9b, 9c, 9e, and 9i–k. Reagents and conditions: (i) TBABr , CH CN, rt, 98%.
3
3
(
ii) thiourea, EtOH, reflux, 83%. (iii) isonicotinoyl chloride hydrochloride, Et N, CH Cl , 66%. (iv)
3 2 2
for 9b, (4-methoxybenzyl)zinc(II) bromide, Pd(PPh ) , THF, reflux, 73%. (v) for 9c,
3
4
4
-methoxyaniline, PdCl (dppf), dppf, t-BuOK, THF, 100 °C, 28%. (vi) for 9e,
2
4
-(3-methoxypropoxy)phenol, CuI, Fe(acac) , K CO , DMSO, 160 °C, 19%. (vii) for 9i–k, CuI,
3
2
3
8

K CO , DMF, 100 °C; 9i using 5-methoxypyrazine-2-thiol, 11%; 9j using
2
3
5
1
-(2-methoxyethoxy)pyrazine-2-thiol, 59%; 9k using 5-[2-(dimethylamino)ethoxy]pyrazine-2-thiol,
5%.
Scheme 2 presents the synthesis of compounds 9d and 9f–h of scaffold 9 bearing a C-4’ aryloxy,
pyridinyloxy, or pyrazinyloxy substituent. Compound 13 was reacted with KOH in co-solvents of
PEG-400 and H O using CuI as the catalyst [54] to give compound 14 having a C-4’ hydroxyl group
2
in 43% yield. O-Arylation of 14 with aryl bromide, pyridinyl bromide, or pyrazinyl bromides using
CuI and Fe(acac) as the catalysts [53] gave the desired 9d and 9f–h in 11–54% yields.
3
Scheme 2. Synthesis of 9d and 9f–h. Reagents and conditions: (i) CuI, KOH, PEG-400, H O, 120
2
°
C,
43%.
(ii)
CuI,
Fe(acac)3,
K CO ,
DMSO,
160
°C;
9d
using
2
3
1-bromo-4-(2-methoxyethoxy)benzene, 48%; 9f using 5-bromo-2-(2-methoxyethoxy)pyridine, 54%;
9g using 2-bromo-5-methoxypyrazine, 11%; 9h using 2-bromo-5-(2-methoxyethoxy)pyrazine, 42%.
For the preparation of compound 9a of scaffold 9 bearing a C-4’ (4-methoxyphenyl)thio
substituent (Scheme 3), direct C–S coupling of 13 with 4-methoxybenzenethiol (cf. Scheme 1) was
not feasible as the thiol group cleaved the amide functionality. Compound 9a was alternatively
prepared by using 10 as the starting material to couple with 4-methoxybenzenethiol using CuI as the
catalyst and K CO as the base in N-methyl-2-pyrrolidone (NMP). The reaction gave sulfide 15 in
2
3
6
2% yield. Bromination of 15 using TBABr gave 16 in 85% yield, which was subsequently reacted
3
with thiourea to form 2-aminothiazole 17 in 64% yield. Amidation of 17 with isonicotinoyl chloride
hydrochloride gave the targeted 9a in 89% yield.
9

Scheme 3. Synthesis of 9a. Reagents and conditions: (i) 4-methoxybenzenethiol, CuI, K CO , NMP,
2
3
1
50 °C, 62%. (ii) TBABr , CH CN, rt, 85%. (iii) thiourea, EtOH, reflux, 64%. (iv) isonicotinoyl
3 3
chloride hydrochloride, Et N, CH Cl , 89%.
3
2
2
2.2 In vitro antiproliferative activity and oral pharmacokinetics of 9a–k of scaffold 9
Cancer cells HeLa (cervical), K562 (leukemia), MDA-MB-468 (breast), and MDA-MD-231
(breast) were used to evaluate the in vitro antiproliferative activity of 9a–k of scaffold 9 bearing
different C-4’ substituents. The chemical structure, in vitro potency, oral AUC, oral bioavailability,
and ClogP value of the compounds are presented in Table 1. The data for lead 7 [46] are also
included for comparison. We first studied the effect of the one-atom linker (X, Table 1 and Fig. 1c)
on the in vitro potency of 9 by replacing the C-4’ oxygen in lead 7 with either a sulfur, methylene, or
amino linker. Compound 9a, using a sulfur atom as the linker, exhibited comparable potency (IC :
5
0
2
2.0–45.0 nM) to that of lead 7 using an oxygen atom (IC : 32.0–48.6 nM). Compounds 9b and 9c,
50
using a methylene or an amino linker, showed reduced potency (IC : 33.0–72.0 nM for 9b, 62.0–
5
0
129 nM for 9c). As a result, oxygen and sulfur atoms were used exclusively as the one-atom linkers
in the following optimizations.
We then investigated the effect of the C-4’’ alkoxy substituent of scaffold 9 (RO–, Table 1 and
Fig. 1c). Replacement of the C-4’’ methoxy group in 7 with a 2-methoxyethoxy group generated
compound 9d having greatly enhanced in vitro potency (IC : 9.1–12.8 nM). Compound 9d was the
5
0
most potent analog of scaffold 9 and ~2–5 times more potent than lead 7 (IC : 32.0–48.6 nM).
5
0
Elongation of the C-4’’ alkoxy in 9d by methylene provided compound 9e with twice-reduced
potency (IC : 14.1–22.3 nM) to that of 9d. Compound 9e was still more potent than lead 7. The use
5
0
of a 2-methoxyethoxy side chain also increased the IV and oral AUCs in rats. At the same dose, the
1
0

IV AUC increased from 34.3 to 63.4 µM·h and the oral AUC increased from 42.4 to 533.8 µM·h for
to 9d. The oral bioavailability (F%) increased from 12.4% (7) to 84.2% (9d). The increased IV
7
AUC implied that the C-4’’ 2-methoxyethoxy side chain contributed to the better intrinsic
physicochemical properties of 9d in vivo.
11

Table 1. In vitro antiproliferative activity, oral pharmacokinetics, and ClogP values of compounds 7 and 9a–k
a
antiproliferative IC50 (nM)
oral pharmacokinetic parameter
compound
ArX–
d
ClogP
b
oral dose
c
oral AUC
(µM·h)
HeLa
K562 MB468 MB231
species
formulation
F%
(mg/kg)
4
8.6
34.9
± 2.7
37.0
± 1.0
32.0
± 1.0
42.4
(34.3)
7
rat
20
A
e
12.4 5.18
±
1.9
2
5.1
22.0
± 7.4
45.0
± 9.0
34.0
± 9.0
9
a
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5.47
5.15
4.68
±
4.1
72.0
33.0
± 4.4
66.0
± 15.0
49.0
± 2.0
9b
±
12.6
MeO
6
2.0
129
9
c
–
–
–
±
10.1 ± 15.2
5
33.8
rat
20
10
B
C
e
84.2
–
9
5.4
.1
9.9
± 4.8
11.9
± 0.4
12.8
± 2.1
(63.4)
9
d
5.07
5.46
±
mouse
–
4.29
–
1
±
6.6
4.5
20.6
± 6.9
22.3
± 2.1
14.1
± 3.2
9
e
–
–
–
1
2

1
1.0
10.1
± 9.8
10.8
± 2.3
11.2
± 1.1
9
f
mouse
rat
10
20
10
10
C
B
C
C
20.1
–
4.78
±
4.6
25.6
24.4
± 12.7
24.9
± 10.2
33.7
± 0.3
591.9
(96.7)
9g
e
61.2 3.76
±
9.3
15.1
12.0
± 7.6
20.5
± 4.9
13.4
± 0.9
9h
mouse
mouse
43.7
57.0
–
–
3.65
4.25
±
7.3
15.5
14.7
± 6.6
17.0
± 3.1
16.5
± 4.3
9
i
±
2.5
2
1.1
18.0
± 0.4
21.5
± 0.2
14.8
± 1.2
9
j
mouse
mouse
10
10
C
C
137
–
–
4.14
4.35
±
6.1
32.8
59.2
± 2.2
53.9
± 8.7
94.5
± 11.5
9
k
13.9
±
4.7
a
b
c
The IC values are expressed as the mean ± SD (n ≥ 2). Rat: Sprague–Dawley rat; mouse: CD-1 mouse; IV dose for rat: 2.0 mg/kg. Formulation:
5
0
d
A, 1% CMC in saline; B, 5% DMSO, 10% Cremophor® EL, and 85% saline; C, 10% DMSO, 25% PEG200, and 65% saline. The AUC values
were averaged from the concentrations of the compound in three rats or mice; variation was generally <20%. IV AUC.
e
1
3

In our previous study, we observed that the oral bioavailability of 7 (and its analogs) increases with
lower values of ClogP (increasing water solubility). The ClogP values of 7, 9a, 9b, 9d, and 9e all exceed
5
.0 (5.07–5.47), except for 9c (ClogP 4.68) with reduced in vitro potency (Table 1). To lower ClogP, we
replaced the phenyl group in 7, 9a, and 9d (ArX in Table 1) by a pyridinyl or pyrazinyl group which
carries either one or two nitrogen atoms. For compound 9f, the pyridinyl analog of 9d, the replacement
lowers ClogP from 5.07 to 4.78. As anticipated, the oral AUC in mice increased from 4.29 µM·h to 20.1
µM·h. A five times increase (approximately) in oral AUC was observed when one carbon in the phenyl
group was replaced by one nitrogen. The replacement also retained the in vitro potency: 9f shows
similar IC (10.1–11.2 nM) to that of 9d (9.1–12.8 nM).
5
0
After observing increased oral AUC by reducing the ClogP using a nitrogen atom, we further used
a pyrazinyl group carrying two nitrogen atoms to replace the phenyl group in 7. The replacement
provided 9g with increased in vitro potency (IC50: 24.4–33.7 nM) compared to that of 7 (IC50: 32.0–48.6
nM). The IV AUC of 9g (96.7 µM·h) became ~3 times that of 7 (34.3 µM·h). It also showed high oral
AUC (591.9 µM·h) and higher oral bioavailability (61.2%) in rats. Combined with the increased AUC
we observed from 9d to 9f, these results indicated that the increased oral AUCs for the analogs of
scaffold 9 correlated with the number of nitrogen atoms in the heteroaryloxy group (ArX, X = O, Table
1
2
) along with the preservation of in vitro potency. We then replaced the C-4’’ methoxy group in 9g by a
-methoxyethoxy group that increased the oral AUC from 7 to 9d. Compound 9h was subsequently
generated with preserved in vitro potency (IC : 12.0–20.5 nM) and doubled oral AUC (43.7 µM·h)
5
0
compared to that of 9f (20.1 µM·h). These results also demonstrated that the oral AUC values of 9d and
f–h increased with the lower ClogP values. This is possibly correlated with the presence of additional
9
nitrogen atoms which increase the water solubility. The presence of 2-methoxyethoxy side chain also
enhances the physicochemical properties of 9d and 9f–h.
In the final stage of our optimization, we evaluated compounds 9i–k of scaffold 9 which possess a
sulfur atom linking the pyrazinyl group to the C-4’ position (ArX, X = S, Table 1) with an alkoxy
substituent at the C-4’’ position. Structurally similar to 7, 9a, and 9g, compound 9i bearing a C-4’’
1
4

methoxy group showed slightly enhanced in vitro potency (IC : 14.7–17.0 nM). The oral AUC of 9i
5
0
(43.7 µM·h) in mice was higher than those of 9d, 9f, and 9h, and was estimated to be higher than that of
9
g despite its ClogP value (4.25) being higher than those of 9g (3.76) and 9h (3.65). The result
suggested that the sulfur linker provided better pharmacokinetic properties than the oxygen linker to
scaffold 9. More observations came from 9j, which possesses a C-4’’ 2-methoxyethoxy group, showing
~3 times higher AUC (137 µM·h) than that of its oxygen analog 9h (43.7 µM·h), though its ClogP (4.14)
is higher than that of 9h (3.65). The use of a 2-methoxyethoxy group also correlated with higher AUC
from 9i to 9j (~2.4-fold) as 7 to 9d. The lower ClogP of 9j (4.14), compared to 9i (4.25), may account
for the AUC improvement. Replacement of the C-4’’ methoxyethoxy in 9j with
-(dimethylamino)ethoxy group generated compound 9k with a higher ClogP (4.35), leading to reduced
oral AUC (13.9 µM·h). Compound 9k also showed reduced in vitro potency (IC : 32.8–94.5 nM)
a
2
5
0
compared to 9j. As compound 9j, lately designated as T-1101, displayed the best oral AUC and excellent
in vitro antiproliferative activity, more extensive pharmacological and in vivo assessments were
conducted.
2
.3 Mechanism of action, pharmacology, and spectrum of activity of 9j/T-1101
To characterize the Hec1 inhibitory activity of 9j, a coimmunoprecipitation assay was performed
on DMSO- (vehicle) and 9j-treated K562 cells for the immunoprecipitation of Nek2 (Fig. 2a). A
reduced amount of coimmunoprecipitated Hec1 was observed in cells treated with 100 nM of 9j in
comparison with cells treated with DMSO. The results demonstrated that the disruption of Hec1/Nek2
protein–protein interaction by 9j took place in the cell. Other observations indicated that the level of
Nek2 was lowered in a time-dependent manner in K562 cells treated with 9j (Fig. 2b), which is
consistent with the literature [40–42] that address cellular responses to Hec1/Nek2 inhibition. The
detached Nek2 from the Hec1/Nek2 complex resulting from 9j could be degraded through the
proteasomal pathway, as reported by Fry et al. [55,56] Nek2 is also found to highly correlate with drug
resistance, rapid relapse, and poor clinical outcome in cancer patients [57]. These two cellular
1
5

phenotypes are also observed in MDA-MB-231 [49] and Huh-7 [50] cells.
1
6

Fig. 2. Comparative disruption of Hec1/Nek2 interaction by compound 9j in the cell. (a) Coimmunoprecipitation analysis of K562 cells treated
with DMSO or 100 nM of 9j for 6.0 h. (b) Time-dependent Western blotting of Hec1 and Nek2 in K562 cells treated with DMSO or 1.0 µM of 9j.
(c) Immunofluorescent staining of DNA and α-tubulin in MDA-MB-468 cells treated with DMSO or 1.0 µM of 9j for 24 or 48 h. The percentages
of mitotic abnormalities (including chromosomal misalignment and formation of multipolar spindles) in mitotic cells were 23%, 45%, and 64% for
DMSO (24 and 48 h), 9j (24 h), and 9j (48 h), respectively. (d) Cell cycle profile of HeLa cells treated with DMSO or 1.0 µM of 9j for 24 h.
1
7

As Hec1 is essential for mitosis, intervening in its functions leads to mitotic abnormalities [40–
42]. In MDA-MB-468 cells, compound 9j caused mitotic abnormalities including chromosomal
misalignment and formation of multipolar spindles (Fig. 2c). The percentages of mitotic
abnormalities increased from 45% at 24 h to 64% at 48 h when cells were treated with 9j at a
concentration of 1.0 µM. The mitotic catastrophe led to the apoptosis of cancer cells (Fig. 2d), as the
sub-G population of HeLa cells increased to 20.44% (when treated with 1.0 µM of 9j for 24 h)
1
compared to cells treated with DMSO (3.64%). Furthermore, the amount of apoptotic marker
proteins cleaved caspase-3 and PARP increased and the amount of antiapoptotic proteins Mcl-1 and
XIAP decreased in HeLa cells treated with 1.0 µM of 9j for 24 h (Supplementary Fig. S1). Amounts
of cell-cycle related proteins cyclin A1, cyclin B1, and cyclin D1 were also reduced (Supplementary
Fig. S2).
For the spectrum of activity, compound 9j is potent against the proliferation of a panel of breast
and liver cancer cells with low nanomolar to submicromolar in vitro GI values [49,50]. It also
5
0
inhibited the growth of U937 (leukemia), COLO 205 (colon cancer), and U2OS (osteosarcoma)
cancer cells with GI values of 12.4, 29.1, and 83.2 nM, respectively (this study). Furthermore, 9j is
5
0
active against multi-drug resistant cancer cells including MES-SA/Dx5 (uterine sarcoma) and
NCI/ADR-RES (ovarian carcinoma) that are resistant to paclitaxel, and down-regulates the
expression of P-glycoprotein in HeLa and MDA-MB-231 cells. Compound 9j shows a synergistic
effect with paclitaxel, doxorubicin, and topotecan in several liver cancer cells [50], demonstrating its
potential to combine with conventional chemotherapy drugs for cancer therapy. Furthermore, 9j was
inactive toward normal cells, kinases, and hERG, supporting its safety which is encouraging for
further evaluation.
2.4 Concentrations of 9j in tumor tissues and its in vivo antitumor activities
SCID mice bearing human Huh-7 and MDA-MB-231 xenografts were intravenously
administered with 9j at a dose of 10 mg/kg. Then, concentrations of 9j in plasma and tumor tissues
were recorded over time. In Huh-7 tumor tissues, the concentrations of 9j were 3.38, 2.66, 2.07,
0.937, 0.507, and 0.0599 µM at 0.50, 1.0, 4.0, 8.0, 12, and 24 h, respectively (Fig. 3). All these
1
8

concentrations were greater than the GI value of 9j for Huh-7 (34.98 nM), even at 24 h (59.9 nM).
5
0
The tumor-to-plasma concentration ratios gradually increased from 20.7% at 30 min, reaching a
maximum value of 60.5% at 12 h, then slowly decreased to 41.1% at 24 h. The high ratios (20.7–
60.5%) indicated the significant distribution of 9j from plasma to Huh-7 tissues. In MDA-MB-231
tissues, the concentration of 9j in the tumors was 2.95 µM (IC for MDA-MB-231: 14.8 nM) and
5
0
the ratio was 24.1% at 2.0 h. The high concentrations of 9j in tumor tissues relative to its low GI50
and IC values suggest that 9j would show promising in vivo antitumor activity in a xenograft
5
0
model.
Fig. 3. Concentrations of 9j in plasma and Huh-7 tumor tissues. ▲, 9j in plasma; ●, 9j in tumor
tissues; ■, tumor-to-plasma concentration ratios of 9j. The concentrations of 9j in tumor tissues
were 3.38, 2.66, 2.07, 0.937, 0.507, and 0.0599 µM at 0.50, 1.0, 4.0, 8.0, 12, and 24 h, respectively.
The tumor-to-plasma concentration ratios of 9j at different time points were 20.7% (30 min), 26.1%
(1.0 h), 33.2% (4.0 h), 35.0% (8.0 h), 60.5% (12 h), and 41.1% (24 h).
The in vivo antitumor activities of 9j as monotherapy are summarized in Table 2 [49,50]. When
given orally at 25 or 50 mg/kg dose twice per day, 9j shows significant in vivo activity in mice
bearing various human cancer xenografts, including live cancer cells Huh-7 and breast cancer cells
1
9

BT-474, MCF-7, and MDA-MB-231. The T/C values were 18–52% at day 28. Compared to leads 7
and 8 having respective T/C values of 50% and 57% at a dose of 150 mg/kg for MDA-MB-231
(Table 2) [46], 9j induces greater growth inhibition with T/C value of 40% at the lower dose of 25
mg/kg. The enhanced in vivo activity of 9j may possibly be credited to its better in vitro potency,
higher oral AUC (Table 1), and/or good tumor tissue distribution (Fig. 3).
Table 2. A concise summary of the in vivo antitumor activities of compounds 9j, 7, and 8
a
compound
xenograft
Huh-7
dose (mg/kg)
T/C (%)
18
9j
9j
9j
9j
50
50
BT-474
19
MCF-7
50
52
MDA-MB-231
MDA-MB-231
MDA-MB-231
25
40
7
150
150
50
8
57
a
Compounds 9j, 7, and 8 were administered orally twice per day to mice bearing the indicated
human cancer xenograft at the indicated dose for 28 consecutive days. The T/C values were
calculated from the tumor sizes on day 28.
In our previous study [50], we demonstrated the significant in vitro potency of 9j against a
panel of liver cancer cells (GI : 15.4–70.8 nM) and its additive/synergistic effect with sorafenib,
5
0
currently an approved drug for the treatment of advanced inoperable hepatocellular carcinoma (HCC)
58,59]. To further evaluate the potential of 9j with sorafenib in vivo (combination therapy of HCC),
[
mice bearing hepatic Huh-7 xenografts were administered PO with sorafenib (25 mg/kg, once per
day), 9j (2.5 mg/kg, twice per day) with sorafenib (12.5 mg/kg, once per day), or 9j (2.5 mg/kg,
twice per day) for 28 consecutive days. The average tumor sizes of the treatment groups were all
reduced compared to the control group, possessing T/C values of 25%, 26%, and 61% for sorafenib
alone, 9j combined with sorafenib, and 9j alone, respectively (Fig. 4). The results indicated that
co-administration of 9j with sorafenib showed synergistic effect in vivo, halving the dose of
sorafenib to show comparable in vivo antitumor activity (T/C: 26%) to sorafenib monotherapy (T/C:
2
0

25%). This indicated that compound 9j and sorafenib co-administered (PO) has the potential as a
combination regimen for HCC treatment.
Fig. 4. In vivo antitumor activity of 9j combined with sorafenib toward Huh-7 xenografts. ×, vehicle;
♦
, sorafenib alone (25 mg/kg); ●, 9j (2.5 mg/kg) with sorafenib (12.5 mg/kg); ▲, 9j alone (2.5
mg/kg). The T/C values for sorafenib alone, 9j with sorafenib, and 9j alone at day 28 were 25%,
26%, and 61%, respectively.
2.5 Salt Screening
The oral AUC of 9j presented in Table 1 was measured using formulations containing organic
solvents (DMSO and PEG200). Without the solvents to enhance solubility, the AUC dropped to 43.5
µM·h with 50.6% oral bioavailability when 9j was suspended in 1% CMC (Table 3). Therefore, 9j
was reacted with various acids to form the corresponding salts to enhance the oral absorption and
stability [60,61]. The most basic group in 9j was the pyridinyl that has a calculated pK of 2.2
a
(conjugate acid) [62]. Pharmaceutically acceptable strong acids including hydrochloric acid,
methanesulfonic acid, and toluenesulfonic acid with pK values less than zero were used. The oral
a
pharmacokinetic parameters of the salts were measured and the results are shown in Table 3. Among
the salts, 9j tosylate had the best oral AUC (62.5 µM·h) compared to its hydrochloride (50.9 µM·h)
2
1

and mesylate (47.5 µM·h) salts at a dose of 10 mg/kg in mice, despite its oral bioavailability (77.4%)
being slightly lower to that of the hydrochloride (84.0%). The AUC values of all the salts were
greater than 1,000 times the IC values of 9j. As 9j tosylate showed the best oral AUC, its oral
5
0
pharmacokinetics were also evaluated on beagle dogs. At a dose of 10 mg/kg, it had an oral AUC of
.61 µM·h with 24.9% bioavailability. The higher pH gastric fluid in beagle dogs [63] may reduce
8
the solubility of 9j tosylate, thereby causing its lower bioavailability. Cross-species pharmacokinetic
data were used to predict the oral bioavailability of 9j tosylate in humans who are considered to have
an intermediate stomach pH which is between that of mice and dogs.
Table 3. Salt screening of 9j
a
oral pharmacokinetic parameter
salt of 9j
species
b
c
Tmax (h)
0.50
1.0
Cmax (µM) AUC (µM·h)
T (h)
F%
1/2
free base
CD-1 mouse
6.30
8.23
8.44
8.13
0.98
43.5
50.9
47.5
62.5
8.61
2.57
2.81
3.38
2.81
50.6
84.0
58.5
77.4
24.9
hydrochloride CD-1 mouse
mesylate
tosylate
tosylate
CD-1 mouse
CD-1 mouse
beagle dog
1.5
1.0
1.0
4.53
a
b
Dose: 10 mg/kg (equal to 9j free base) formulated with 1% CMC in saline. The AUC values were
averaged from the concentrations of the compound in three mice or beagle dogs; variation was
c
generally <20%. Absolute bioavailability.
Compound 9j and its salts (listed in Table 3) were further analyzed by differential scanning
calorimetry (DSC). Their thermograms are displayed in Fig. 5. Except for 9j hydrochloride showing
several exothermic and endothermic transitions at 100–180 °C (decomposition), 9j mesylate, 9j free
base, and 9j tosylate show single explicit endothermic peaks at 159.4, 172.0, and 218.7 °C,
respectively. Further analysis of the salts was performed using X-ray powder diffraction (XRPD) and
2
2

the results indicated that 9j free base and all its salts were crystalline compounds (data not shown).
The endothermic peak of 9j tosylate at 218.7 °C was at its melting point as the value was close to the
melting point (213–214 °C) measured by melting point apparatus. A loss of weight when heating 9j
tosylate was not observed from thermogravimetric analysis (TGA) before its melting point,
confirming its stability for formulation development. Compound 9j tosylate had the highest oral
AUC, the highest melting point, and good thermal stability compared to its salt analogs. Thus, it was
selected as a candidate for preclinical development.
Fig. 5. DSC thermograms of 9j and its salts: (a) free base, (b) hydrochloride, (c) mesylate, and (d)
tosylate.
2.6 Interaction of 9j with Hec1 from docking simulations
The interaction of 9j with Hec1 was expected to resemble that of its structurally similar
precedents 1 and 4 which are found to occupy the surface formed by the residues Leu394 to Ile408
in the coiled-coil region (Arg281 to Leu535) of Hec1 [42]. The reported homology model for 1 and
4
was thus re-built and docked with 9j using BIOVIA Discovery Studio 2017. Compound 4 was also
docked to verify the model and to compare with 9j. The results shown in Fig. 6a and 6b indicate that
2
3

the same 4-aryl-N-pyridinylcarbonyl-2-aminothiazole scaffold belonging to 4 and 9j have different
binding poses in the region formed by residues Leu394, Glu397, Glu398, Lys400, and Tyr401.
However, the piperidinyl group in 4 and the 2-(methoxy)ethoxy group in 9j occupied a similar
hydrophobic region around Gly404. The interactions of 4 and 9j with Hec1 can be analyzed in detail
by use of the two-dimension diagram depicted in Fig. 6c, which shows that 4 used its
2,6-dimethylphenyl group to interact with Tyr401, while 9j used its pyrazinyl group to interact with
Tyr401. Glu397 was found to interact with the thiazolyl group of 4, but the residue was not
important for 9j. Compound 4 used its pyridinyl group to interact with Leu394, while 9j used its
thiazolyl group for Leu394. Additional interactions were found for 9j, in which the two methyl
groups interacted with Leu394 and Tyr401. The binding of 9j to Hec1 was stronger than that of 4
(Fig. 6d), as the calculated binding energies –CDE and –CIE for 9j (11.8 and 35.1 Kcal/mol) were
higher than those of 4 (11.7 and 25.7 Kcal/mol). The stronger binding of 9j may account for its
better in vitro antiproliferative activity (IC : 21.5 nM) compared to that of 4 (IC : 120 nM) for
5
0
50
MDA-MB-486 inhibition.
2
4

Fig. 6. Docking simulation of 4 and 9j in the coiled-coil region (Arg281 to Leu535) of Hec1. (a)
Binding pose of 4. (b) Binding pose of 9j. (c) Two-dimensional diagram of ligand–protein
interactions for 4 and 9j with Hec1. The structures of 4 and 9j are aligned with the same residues
they interacted with. (d) Calculated binding energy and antiproliferative activities of 4 and 9j. CDE
and CIE stand for CDOCKER energy and CDOCKER interaction energy, respectively.
2.7 Brief description of the safety pharmacology of 9j
Central nervous system effects of 9j were evaluated on rats using the Irwin test [64]. Behaviors
including locomotor activity, restlessness, lethargy, tremors, convulsions, touch response, etc. did not
change when the rats were given 9j as an oral single dose of up to 60 mg/kg. For potential
cardiopulmonary effects of 9j, the study was carried out on beagle dogs. An oral single dose of 40
mg/kg did not show any remarkable cardiopulmonary findings. Furthermore, a 28-day GLP
compliance, repeated-dose toxicology study of 9j was performed on rats. No unexpected findings at
high oral dose (60 mg/kg) were found in clinical pathology, serum biochemistry, and organ change
examinations other than those expected from chemotherapeutic agents. The related symptoms caused
by 9j in rats were reversible when the administration was discontinued. These studies demonstrated
a positive safety profile for 9j and determined that it was suitable to investigate in clinical trials.
2
5

3. Conclusions
After preclinical development, 9j tosylate was designated as T-1101 tosylate and approved by
the US FDA as an investigational new drug at the end of 2016. As of this writing, it was in phase I
clinical trials (ClinicalTrials.gov identifiers NCT03195764 and NCT03349073) as an oral drug to
evaluate its safety and pharmacokinetics on patients with advanced refractory solid tumors and their
treatments. T-1101 tosylate is the first-in-class Hec1/Nek2 inhibitor clinical candidate as no such
inhibitor has been used or investigated in clinical trials as of this writing. Further progress of its
clinical data will be reported in due course.
4. Experimental section
4.1 Chemistry
Reagents and starting materials were used as purchased. Purification by column
chromatography was conducted using Merck Reagents Silica Gel 60 (particle sizes of 0.063–0.200
mm, 70–230 mesh ASTM). Proton and carbon-13 NMR spectra were recorded on an Agilent
4
00-MR (400 MHz) spectrometer using CDCl or DMSO-d as solvents. Multiplicities are
3 6
abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling constant
Hz). Carbon-13 chemical shifts are referenced to the center peak of CDCl (δ 77.0 ppm) or
(
3
DMSO-d (δ 39.5 ppm). The following abbreviations are used for NMR assignment: Ar for arene, Py
6
for pyridine, Pyz for pyrazine, and Th for thiazole. EI-MS were recorded on an Agilent 5973 mass
spectrometer (70 eV) coupled with an Agilent 6890 Series GC System. ESI-MS spectra were
recorded on a Thermo Finnigan TSQ Quantum mass spectrometer. High-resolution mass spectra
were obtained on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Compound
purities (for biological evaluation) were greater than 95%, determined by reversed-phase HPLC.
None of the compounds for biological evaluation contains the substructures having pan-assay
activities (PAINS) inspected by the program at http://zinc15.docking.org/patterns/home/.
4.1.1. 2-Bromo-1-(4-iodo-2,6-dimethylphenyl)ethanone (11)
A solution of 1-(4-iodo-2,6-dimethylphenyl)ethanone (10, 1.028 g, 3.750 mmol, 1.0 equiv) in
CH CN (30 mL) was added with TBABr (3.225 g, 6.688 mmol, 1.8 equiv). The reaction mixture
3
3
2
6

was stirred at room temperature for 12 h. The solution was concentrated under reduced pressure,
added with water, and extracted with EtOAc. The organic layer was washed with brine, dried over
MgSO , and concentrated under reduced pressure to give 11 (1.298 g, 3.677 mmol) as orange solids
4
1
13
in 98% yield: H NMR (DMSO-d ): δ 7.51 (s, 2 H, ArH), 4.74 (s, 2 H, CH ), 2.13 (s, 6 H, CH ); C
6
2
3
NMR (DMSO-d ): δ 200.4 (C=O), 138.8, 136.5, 136.0, 96.7, 38.5 (C–Br), 18.7; EI-MS m/z (relative
6
+
intensity) 354 (8), 352 (M , 9), 260 (14), 259 (100), 231 (25), 117 (6), 104 (14), 103 (11), 78 (5), 77
(7); CAS # N.A.
4.1.2. 4-(4-Iodo-2,6-dimethylphenyl)thiazol-2-amine (12)
A reaction mixture containing 11 (4.16 g, 11.8 mmol, 1.0 equiv) and thiourea (1.17 g, 15.4
mmol, 1.3 equiv) in 95% EtOH (45 mL) was heated under reflux for 6.0 h. The solution was
concentrated under reduced pressure, added with water, and adjusted to pH 8.0–9.0 using saturated
Na CO . The resulting precipitate was collected and vacuum dried to give 12 (3.23 g, 9.78 mmol) as
2
3
1
pink solids in 83% yield: H NMR (DMSO-d ): δ 9.18 (brs, 2 H, NH ), 7.55 (s, 2 H, ArH), 6.84 (s, 1
6
2
13
H, ThH), 2.10 (s, 6 H, CH ); C NMR (DMSO-d ): δ 170.0 (Th-C2), 140.6, 136.3, 136.0 (Th-C4),
3
6
+
1
1
4
29.4, 106.6, 97.4, 19.8 (CH ); EI-MS m/z (relative intensity) 330.0 (M , 29), 324 (10), 271 (18),
3
36 (4), 160 (6), 144 (15), 115 (7), 106 (16), 91 (100), 78 (14), 51 (6); CAS # N.A.
.1.3. N-[5-(4-Iodo-2,6-dimethylphenyl)thiazol-2-yl]isonicotinamide (13)
A solution of 12 (1.420 g, 4.301 mmol, 1.0 equiv) and isonicotinoyl chloride hydrochloride
(
1.148 g, 6.449 mmol, 1.5 equiv) in CH Cl (30 mL) was added with Et N (1.81 mL, d = 0.726 g/mL,
2 2 3
1.31 g, 12.9 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature for 12 h. The
solution was concentrated under reduced pressure and added with hot water. The resulting
precipitate was collected and vacuum dried to give 13 (1.236 g, 2.840 mmol) as white solids in 66%
1
yield: H NMR (CDCl ): δ 8.72 (d, J = 4.5 Hz, 2 H, PyH), 7.50 (d, J = 4.5 Hz, 2 H, PyH), 7.06 (s, 2
3
1
3
H, ArH), 6.78 (s, 1 H, ThH), 4.75 (brs, 1 H, NH), 1.81 (s, 6 H, CH ); C NMR (CDCl ): δ 163.7
3
3
(C=O), 159.5 (Th-C2), 150.2 (Py-C2), 147.0, 138.8, 138.4, 136.2, 133.1, 121.0 (Py-C3), 112.4, 95.0,
+
1
9.8 (CH ); EI-MS m/z (relative intensity) 435 (M , 100), 402 (3), 329 (39), 313 (32), 287 (9), 270
3
(21), 255 (10), 202 (8), 186 (25), 144 (6), 128 (8), 123 (12), 106 (65), 78 (61), 51 (18); CAS #
2
7