Structure guided design of potent indole-based ATX inhibitors bearing hydrazone moiety with tumor suppression effects

Article abstract of DOI:10.1016/j.ejmech.2020.112456

ATX was capable of catalyzing the hydrolysis of LPC to the lipid mediator LPA which attracted considerable attention on the development of potent ATX inhibitors. Herein, driven by the HTS product indole-based lead 1, a hybridization strategy was utilized to construct the trifluoroacetyl hydrazone moiety through assembling the phenyl thiazole fragment to the indole skeleton of lead 1. After a systematic structure guided optimization, by cycling the phenyl thiazole to the compacted benzothiazole or decreasing the lipophilicity, two promising ATX inhibitors (9j and 25a) were identified with IC₅₀ values of 2.1 nM and 19.0 nM, respectively. All compounds were tested a panel of cancer cell lines and a preliminary affinity on breast cancer cell lines (SI > 16.5) were observed which shed a light on their potential application of breast cancer relevant cases. Through a dedicated docking study, the intramolecular pseudo-ring within the trifluoroacetylhydrazone moiety played a significant role in constraining the binding poses of 9j and 25a. Finally, a binding free energy calculation was conducted to examine the contribution of different interactions in binding affinity.

Full text of DOI:10.1016/j.ejmech.2020.112456

Journal Pre-proof
Structure guided design of potent indole-based ATX inhibitors bearing hydrazone
moiety with tumor suppression effects
Hongrui Lei, Changtao Li, Yu Yang, Fang Jia, Ming Guo, Minglin Zhu, Nan Jiang, Xin
Zhai
PII:
S0223-5234(20)30427-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112456
EJMECH 112456
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 February 2020
Revised Date: 29 April 2020
Accepted Date: 10 May 2020
Please cite this article as: H. Lei, C. Li, Y. Yang, F. Jia, M. Guo, M. Zhu, N. Jiang, X. Zhai,
Structure guided design of potent indole-based ATX inhibitors bearing hydrazone moiety with tumor
suppression effects, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112456.
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Graphical Abstract

Structure guided design of potent indole-based ATX inhibitors bearing hydrazone moiety
with tumor suppression effects
Hongrui Lei, Changtao Li, Yu Yang, Fang Jia, Ming Guo, Minglin Zhu, Nan Jiang, Xin Zhai1*
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China.
Abstract: ATX was capable of catalyzing the hydrolysis of LPC to the lipid mediator LPA which attracted
considerable attention on the development of potent ATX inhibitors. Herein, driven by the HTS product indole-based
lead 1, a hybridization strategy was utilized to construct the trifluoroacetyl hydrazone moiety through assembling the
phenyl thiazole fragment to the indole skeleton of lead 1. After a systematic structure guided optimization, by cycling
the phenyl thiazole to the compacted benzothiazole or decreasing the lipophilicity, two promising ATX inhibitors (9j
and 25a) were identified with IC50 values of 2.1 nM and 19.0 nM, respectively. All compounds were tested a panel of
cancer cell lines and a preliminary affinity on breast cancer cell lines (SI >16.5) were observed which shed a light on
their potential application of breast cancer relevant cases. Through a dedicated docking study, the intramolecular
pseudo-ring within the trifluoroacetylhydrazone moiety played a significant role in constraining the binding poses of
9j and 25a. Finally, a binding free energy calculation was conducted to examine the contribution of different
interactions in binding affinity.
Keywords: structure-based optimization, ATX inhibitors, trifluoroacetyl hydrazone, antiproliferation, binding mode
analysis
1. Introduction
Autotaxin (ATX), originally discovered as an autocrine motility stimulating factor, is a member of nucleotide
pyrophosphatase/phosphodiesterase family which can catalyze the transformation from lysophosphatidyl choline (LPC)
to lysophosphatidic acid (LPA) [1, 2]. The hydrolysis product LPA is a bioactive lipid widespread in many body fluids
and is involved in various physiological and pathological processes through corresponding G-protein coupled
receptors (GPCRs) [3,4]. Downregulation of the ATX-LPA signaling axis has been linked to various diseases, such as
inflammation [5], fibrosis [6,7], pruritus [8] and cancers [9,10].
The active domain of ATX has three binding sites: a Zn2+ containing catalytic site, a hydrophobic pocket and a
hydrophobic tunnel [11]. The uncovered small molecule ATX inhibitors were typically classified into four types
according to their X-ray binding mode with ATX enzyme [12]. The represent compounds in each type were mapped in
Figure 1. Generally, type I ~ type III inhibitors featured an acidic head, furthermore, type I inhibitor PF-8380 binds
directly with one of zinc ions [13,14]. However, three types of ATX inhibitors suffered a poor druggability with no
molecule entering clinical trials. To date, the sole ATX inhibitor entity in clinical trial was the type IV compound
GLPG-1690 developed by Galapagos [15-18].
Corresponding author.
E-mail address: zhaixin_syphu@126.com (X. Zhai).

Fig. 1. ATX structure, function and ATX inhibitors with different binding modes
Our previous screening efforts led to the identification of a small indole-3-carboxylic lead 1 with an ATX inhibitory
activity of 740 nM. In order to discover non-acidic entities as potent ATX inhibitors with specific binding mode, a
combination strategy between GLPG-1690 and lead 1 was conducted. The phenyl thiazole moiety of GLPG-1690 was
fixed on the indole scaffold through a trifluoroethylidene hydrazine hybrid which furnished 29 new compounds
(8aa~8ka, 10a and 10j).
Fig. 2. Design strategy of target compounds. (A) Structure of target compounds from emergence of GLPG-1690 and lead 1; (B) Binding mode

of GLPG-1690 in ATX active site; (C) Overlap of 8ka with GLPG-1690 in ATX binding site. (D) Further optimization of compounds through
cyclization and/or decreasing lipophilicity.
After a primary SARs study with FS-3 based ATX enzyme assay, further modification efforts were made through
cyclization and/or decreasing lipophilicity. In this sense, two novel series of compounds (9a~9j, 18da, 20aa, 20fa, 25a
and 25b) were designed and synthesized. 9a~9j were obtained by cyclizing the phenyl thiazole moiety to a fused
benzothiazole fragment. In parallel, in view of the relatively high lipophilicity of compounds 8aa~10j, we altered the
5-thiazole indole moiety to the more hydrophilic 5-piperazine or 5-piperidine indole fragment which gave rise to 18da,
20aa, 20fa, 25a, 25b. To the end, 9j and 25a turned out to be the most potent ATX inhibitors with IC50 values of 2.1
nM and 19.0 nM, respectively. Furthermore, all compounds were tested antiproliferative effects on four cancer cell
lines which showed a selectivity profile on breast cancer cell lines over other cells.
2. Results and discussion
2.1 Chemistry
All compounds were synthesized as displayed in Scheme 1, 2 and 3. The starting material indole-5-carbonitrile (1)
was utilized to undergo a typical N-methylation or N-ethylation reaction in satisfying yields. Subsequently, the -CN
group of 2 was treated with NaHS under the catalysis of MgCl2 to give the thioamide derivative 3 in 72.0% yield [19].
Next, the thiazole ring was furnished through a cyclization reaction between 3 and 1, 3-dichloroacetone in 78.9% yield
[19]. Subsequently, the trifluoroacetylation reaction was conducted onto the position-3 of the indole skeleton to result
in 5 in 78.1% yield [20]. Then, the N-alkylation between 5 and various amines were conducted in a K2CO3/MeCN
system to bring 6a~6k in 80.1~83.2% yields. Finally, compounds 8, 9 and 10 series were obtained through a
condensation reaction between 6 and different 2-hydrazine thiazole derivatives with catalysis of p-TsOH in moderate
yields [21].
Scheme 1. Reagents and conditions:(i) iodoethane, NaH, DMF, 25 ; (ii) NaHS, MgCl2·6H2O, DMF, 25 , 3 h; (iii) 1,3-dichloropropan-2-one,
toluene, reflux, 2.5 h; (iv) (CF3CO)2O, DMF, 0 to 25 ; (v) amine, K2CO3, MeCN, r.t., 2 h;(vi) hydrazine, p-TsOH, toluene, reflux, 3h;

As shown in Scheme 2 and 3, compounds 18da, 20aa, 20fa, 25a and 25b were synthesized in similar manners.
Firstly, 2,4,5-trifluoronitrobenzene (11) was involved in C-alkylating and decarboxylation reactions in sequence with
total 50.5% yield [22]. Taking the decarboxylation product 13 as a synthon, N-alkylating was implemented with
piperidine-4-carboxylic acid or piperazine to give 14 or 21 in satisfying results, respectively. The
piperidine-4-carboxylic acid derivative 14 was utilized to furnish the amide products 15 with a HATU protocol. 15
was then subjected to a reductive cyclization in the presence of Na2S2O4 in THF/H2O mixed solvents in 83.1~86.0%
yields [22]. With the indole skeleton 16 in hand, a formylation or trifluoroacetylation reaction were conducted with
treatment of POCl3 or (CF3CO)2O to give corresponding 17or 19 in acceptable yields. 17 and 19 were reacted with
2-hydrazine thiazole derivative driven by p-TsOH to result in 18da, 20aa, and 20fa in moderate yields.
Scheme 2. Reagents and conditions: (i) methyl 3-oxobutanoate, t-BuOK, THF, r.t., 2 h; (ii) concentrated sulfuric acid, HOAc, 50~70 , 2 h;
(iii)piperidine-4-carboxylic acid, K2CO3, MeCN, r.f., 2 h; (iv) amine, HATU, DIPEA, DMF, r.t. ,1 h; (v) Na2S2O4, THF, H2O, 70 , 2h; (vi)
(CF3CO)2O, DMF, r.t., 1.5 h; (vii) 4-(4-fluorophenyl)-2-hydrazinylthiazole-5-carbonitrile, p-TsOH, toluene, r.f., 3 h; (viii) POCl3, DMF, 0 to r.t.,
1.5 h; (ix) 4-(4-fluorophenyl)-2-hydrazinylthiazole-5-carbonitrile, HOAc, r.f., 3 h.
The piperazine derivative 21 was subjected to
a
typical N-alkylating reaction with 2-chloro-N,
N-dimethylethan-1-amine hydrochloride to give 22, which further underwent a reduction cyclization with a Na2S2O4
protocol to bring the indole 23 in 69.1% yield. 23 was subsequently equipped with trifluoroacetyl by treatment with
(CF3CO)2O in DMF gave rise to 24 in 60.0 % yield. Finally, 24 was reacted with hydrazine derivatives to give birth to
25a and 25b in reasonable yields.
Scheme 3. Reagents and conditions: (i) piperazine, TEA, MeCN, r.f., 2h; (ii) 2-chloro-N, N-dimethylethan-1-amine hydrochloride, NaHCO3,

i-PrOH, r.f. , 5 h; (iii) Na2S2O4, THF/H2O, 70 , 2 h; (iv) (CF3CO)2O, DMF, r.t., 1.5 h; (v) hydrazine, p-TsOH, toluene, r.f., 3 h;
The hydrazine derivatives 7a and 7b were synthesized as depicted in Scheme 4. The ester group in 26 was treated
with NaH followed by condensation with MeCN to furnish the active methylene derivative 27 [15]. 27 was subjected
to a cyclization reaction with thiourea to bring the 2-aminethiazole species 28 [15]. 28 was experiencing a sequential
Sandmeyer and hydrazinolysis reaction to obtain R4 substituted 7a. In the meantime, 7b was originated from a
hydrazinolysis reaction between 2-SH-benzothiazole 29 and hydrazine hydrate in quantitative yield.
Scheme 4. Reagents and conditions: (i) NaH, MeCN, Tol, r.f., 2 h; (ii) thiourea, I2, EtOH, r.t., 2 h; (iii) tert-butyl nitrite, CuCl2, MeCN, r.t., 1 h; (iv)
H2N-NH2·H2O, EtOH, r.f., 1.5 h.
2.2 Biological activity and discussion
2.2.1 In vitro ATX enzymatic assays and SARs study
2.2.1.1 The mergence strategy for compounds 8aa~8ka
Taken the originally discovered lead 1 and the phenyl thiazole moiety in GLPG-1690 as the template, combination
strategy was utilized to construct the trifluoroacetyl hydrazone linker. The dimethyl amine group in lead 1 was
replaced with various secondary cyclic amines for a better topology and hydrophobic tunnel occupancy. In the phenyl
thiazole fragment, different R4 (-F, -OCH3, -Cl) were explored to search the effect in binding with hydrophobic pocket.
The substituent in indole position-1 was fixed to ethyl. In this sense, compounds 8aa~8ka were thoroughly prepared
and advanced to FS-3 based ATX enzymatic assays. The LLE values of the chemical entities were calculated
according to the IC50 values and the cLogP values obtained. It should be stated that a positive (+) LLE value was
highly preferred for a promising entity [23-25].
Generally, compounds 8aa~8ka exerted ATX inhibition effect in IC50 values around 1 μM. The R4 substituent
exerted hardly influence in ATX inhibition. Taking the amine portion for a view, compounds containing morpholine,
piperidine, pyrroline, 4-methyl piperidine and N-methyl piperazine all displayed unsatisfying results with IC50 values
over 1 μM. Alternatively, the -OH containing amines as well as proline were observed encouraging activities, such as
8ca (0.61 μM), 8hb (0.87 μM), 8ib(0.91 μM), 8jb (0.56 μM) and 8ka (0.78 μM). However, there was still a big gap
between the promising results with the positive control GLPG-1690 (0.0037 μM) and PF-8380 (0.0043 μM). It should
be also noted that only 8ca (LLE 0.37) and 8cb (LLE 0.30) achieved positive LLE values. Consequently, further
optimizations were put on the agenda.
Table 1. FS-3 based ATX enzymatic assay of target compounds (8aa~8ka, 10a and 10j)

Compd.
8aa
8ab
10a
8ba
8bc
8ca
8cb
8da
8db
8dc
8ea
8eb
8ec
R1
R4
-F
cLogP
6.63
6.50
6.85
7.84
7.84
4.84
4.71
7.49
7.36
8.01
6.81
6.81
7.34
6.63
7.05
7.70
8.15
LLE
aATX IC50 (μM, FS-3)
2.37±0.19
2.06±0.15
2.97±0.13
3.43±0.47
6.07±0.51
0.61±0.07
0.98±0.11
1.31±0.21
3.05±0.63
2.45±0.57
3.02±0.37
1.85±0.19
1.21±0.11
1.54±0.27
0.93±0.13
2.13±0.25
>10
-2.00
-1.81
-2.32
-3.38
-3.62
0.37
-OCH3
-F
-F
-Cl
-F
-OCH3
-F
0.30
-2.61
-2.84
-3.40
-2.29
-2.08
-2.42
-1.82
-2.02
-3.03
-4.15
-OCH3
-Cl
-F
-OCH3
-Cl
8fa
-F
8fb
-OCH3
-Cl
8fc
8gb
-OCH3

8gc
8ha
8hb
8hc
8ia
-Cl
-F
8.15
6.77
6.74
7.40
6.16
6.61
6.68
6.87
6.74
7.40
6.57
6.13
4.21±0.91
5.27±0.51
0.87±0.17
1.41±0.11
1.25±0.17
0.91±0.11
2.46±0.31
2.05±0.25
0.56±0.11
1.43±0.17
1.72±0.20
0.78±0.11
-4.54
-2.49
-1.68
-OCH3
-Cl
-2.55
-1.26
-F
8ib
8ic
-OCH3
-Cl
-1.57
-2.07
-2.18
-1.49
-2.56
-1.81
-1.02
8ja
8jb
8jc
-F
-OCH3
-Cl
10j
8ka
-F
-F
GLPG-1690
PF-8380
-
-
-
-
-
-
3.84
3.80
3.59
3.57
0.0037±0.0012
0.0043±0.0011
adata were the results in at least two independent assays.
2.2.1.2 Examine the effect of position-1 substituent in indole backbone (10a and 10j)
First of all, the effect of the position-1 substituent of indole skeleton was taken into consideration. We altered the
originally ethyl to methyl which brought out compounds 10a and 10j. Unexpectedly, there were no significant
discrimination in enzymatic activities of these two compounds (2.97 μM for 10a and 1.72 μM for 10j) with their
corresponding entities 8aa (2.37 μM) and 8ja (2.05 μM). In this sense, we kept the original ethyl group in this site for
further modification.
2.2.1.3 The cyclization of phenyl thiazole to benzothiazole for compounds 9a~9j
Taking 8ka as a template, the binding mode of the primary 29 compounds were analyzed by docking with ATX. As
depicted in Fig. 2C, compared with GLPG-1690, 8ka was detected a slightly bigger molecular volume. The extended
volume also drove the molecule itself a little bit away from the exact binding site. To resolve the pending problem, the
phenyl thiazole pharmacophore was shrunk by a cyclization method to an alternative benzothiazole. To this end,
compounds 9a~9j were prepared accordingly and the ATX enzymatic results were listed in Table 2.
Table 2. FS-3 based ATX enzymatic assay of target compounds (9a~9j)

Compd.
9a
cLogP
6.24
7.46
7.10
6.42
7.89
6.37
6.03
LLE
-0.54
-1.49
-1.20
-0.48
-2.42
-0.23
1.65
aATX IC50 (μM, FS-3)
0.201±0.011
9b
0.107±0.013
9d
0.126±0.021
9e
0.116±0.017
9g
0.337±0.024
9h
HO
0.072±0.015
N
9j
0.0021±0.001
adata were the results inat least two independent assays.
Surprisingly, 9a, 9b, 9d, 9e and 9g were all detected sub-micromolar IC50 values (0.107~0.337 μM). Better yet,
consistent with the primary portions of compounds, molecules bearing -OH-containing amines displayed much better
inhibitory effects. The prolinol derivative 9h was observed an improved activity with an IC50 value of 0.072 μM. Best
of all, 9j bearing a piperidine-4-OH moiety exerted an amazing 0.0021 μM IC50 value. Given the highlighting result,
elaborate efforts were made to the construction of binding model of 9j with ATX. Except for the essential H-bonding
and π-π interactions, an intra-molecular H-bond was detected which accounted for the formation of a pseudo-ring
within the trifluoroacetyl hydrazone moiety. The intramolecular pseudo-ring may provide the extra good ATX activity
of 9j among this chemotype species (Fig. 5). Furthermore, the LLE value of 9j (1.65) turned out to be an encouraging
perspective.
2.2.1.4 Decreasing the lipophilicity for compounds 18da, 20aa, 20fa, 25a and 25b
Although the 5-thiazole indole group played a significant role in the formation of essential H-bonding and π-π
interactions with residues Trp255 and Phe275, it did result in the high lipophilicity of the total entity. Consequently,
much more hydrophilic pharmacophores piperazine and piperidine were embedded to the position-5 of the indole
skeleton which furnished compounds 18da, 20aa, 20fa, 25a and 25b. Among the piperidine derivatives bearing
amines without a -OH, 18da (0.057 μM) with a -CF3 group may verified a positive effect in the inhibitory profile over
20aa (0.104 μM) and 20fa (0.159 μM) since the amines involved (morpholine, pyrroline and N-ethyl piperazine)
generally brought little differences in potency (8aa 2.37μM, 8da 1.31 μM and 8fa 1.54 μM). Surprisingly, the
piperazine containing compound 25a exerted 0.019 μM ATX inhibitory activity. The subsequent docking study
showed that 25a adopted a similar binding mode with 9j.
Table 3. FS-3 based ATX enzymatic assay of target compounds (18da, 20aa, 20fa, 25a and 25b)

R4
F
N
N
O
HN
N
HN
N
CN
N
F3C
S
CN
R5
S
R3
N
N
N
F
R2
N
F
N
N
H
H
25a, 25b
18da, 20aa, 20fa
aATX IC50
Compd.
R5
R4
cLogP
LLE
(μM, FS-3)
18da
20aa
20fa
-CF3
-H
-
-
7.25
6.39
6.94
-1.01
-0.41
-1.14
0.057±0.021
0.104±0.032
0.159±0.017
-H
-
25a
25b
-
-
-
-
-F
6.66
6.53
0.06
0.019±0.005
0.167±0.021
-OCH3
-0.75
adata were the results in at least two independent assays.
2.2.2 Common properties of target compounds and their in vitro cytotoxicity effects
Accumulating evidence suggested that downregulation of the ATX-LPA signaling played a key role in many cancer
types especially breast cancer [26-31]. Herein, we focused our attention on the effects of the target compounds on
breast cancer cell lines.
2.2.2.1 Common properties of target compounds
Before the cell-based biological evaluation, the druggability of the compounds was assessed by the prediction of
several key parameters, tPSA (topological polar surface area) and the representative drug-likeness score other than the
former mentioned cLogP (calculated LogP) and LLE value. Generally, except for 8cb, all compounds were estimated
tPSA values lower than 100, which turned out to be a preferred factor. Given that almost all compounds exerted >5
cLogP values (8ca 4.84, 8cb 4.71), we alternatively put our attention on the evaluation of the drug-likeness score
through an empirical model [32]. Taking PF-8380 (scored 0.72) and GLPG-1690 (scored 1.82) as the reference, the
prediction details of the optimal 9j and 25a were present in Fig. 3. Theoretically, the drug-likeness score was expected
the bigger the better. Herein, we believed that a score value bigger than 0.72 (PF-8380) was considered acceptable.
Overall, more than a half compounds were detected > 0.72 drug-likeness score which further elucidated their
druggability.
Fig. 3. Representative drug-likeness model sore results of PF-8380, GLPG-1690, 9j and 25a. (The green curve indicates that a compound is
possibly not a drug, while the blue curve suggests the compound may possess drug-likeness properties.)
2.2.2.2 In vitro cytotoxicity effects of target compounds

The ATX-LPA signaling axis has shown great involvement in breast cancer which verified the potential application
of ATX inhibitors in breast cancer cases [33]. To this end, the antiproliferative activity of target compounds were
evaluated against two breast cancer cell lines, MCF-7 and MDA-MB-231. In the meantime, A549 (EGFR-positive
human lung cancer cell line) and H2228 (ALK-positive human lung cancer cell line) were chosen to examine potential
off-target effects. The cytotoxicity results were described as half-maximal inhibitory concentration (IC50) values and
present in Table 4.
It could be noted that most compounds exhibited moderate to excellent cytotoxic activities in two breast cancer cell
lines (MCF-7 and MDA-MB-231) with IC50 values below 5 µM. As expected, most compounds exerted poor
antiproliferative activities in A549 and H2228 cell (most of which >15 μM), which preliminarily suggested their
affinity for breast cancer cell lines. The positive control in enzymatic assays PF-8380 and GLPG-1690 were observed
not obvious antiproliferative effects in all tested samples.
Table 4. Antiproliferative effects of target compounds
MCF-7
MDA-MB-231
A549
H2228
Compd.
PSA
Druglikeness
(aIC50, μM)
(aIC50, μM)
(aIC50, μM)
(aIC50, μM)
8aa
8ab
10a
8ba
8ca
8cb
8da
8db
8dc
8ea
8eb
8ec
8fa
8fb
8fc
72.09
79.64
72.93
64.19
92.82
100.36
64.55
72.10
64.55
67.66
75.21
67.66
67.72
75.27
67.72
71.73
89.02
81.48
80.11
87.65
80.11
79.91
87.45
79.91
80.74
0.52
0.65
0.58
0.79
0.61
0.63
0.71
0.81
0.81
0.81
0.92
0.92
0.62
0.74
0.73
1.05
0.56
0.56
0.18
0.27
0.29
0.97
1.10
1.07
0.92
2.31±0.17
ND
4.97±0.51
5.12±0.29
2.72±0.41
5.46±1.20
1.27±0.21
1.23±0.18
2.37±0.41
5.17±0.61
1.56±0.23
ND
>15
>15
>15
ND
3.25±0.37
1.57±0.21
0.65±0.11
1.51±0.27
2.85±0.43
ND
9.41±1.26
>15
>15
5.71±0.85
>15
>15
>15
ND
>15
6.31
>15
13.72±1.27
>15
5.03±0.27
2.10±0.31
2.79±0.37
ND
ND
11.72±2.11
4.93±0.46
ND
ND
8.21±1.26
1.56±0.31
2.69±0.43
ND
2.60
>15
4.33±1.01
4.67±1.05
5.73±0.93
2.23±0.11
4.26±0.53
ND
>15
ND
>15
ND
3.21±0.46
ND
13.80±1.28
9.81±1.07
10.93±1.43
ND
>15
8gb
8hb
8hc
8ia
ND
ND
ND
1.21±0.22
ND
>15
1.79±0.23
>15
6.92±1.03
>15
ND
8ib
8ic
2.43±0.35
3.41±0.50
ND
>15
>15
ND
>15
8ja
8jb
8jc
5.13±0.47
1.37±0.27
ND
10.27±0.93
ND
ND
3.98±0.32
2.33±0.29
4.51±0.52
>15
>15
ND
10j
4.83±0.61
9.21±2.01
9.65±1.28

8ka
9a
84.63
54.25
46.35
46.71
49.82
46.35
63.64
62.07
78.59
86.13
81.76
69.23
76.77
77.96
73.50
0.98
0.44
0.72
0.63
0.72
0.82
0.40
0.85
0.53
0.22
0.82
1.00
1.01
1.82
0.72
3.25±0.35
2.73±0.26
3.12±0.37
1.79±0.35
2.78±0.41
ND
ND
7.31±0.81
9.37±1.13
>15
>15
ND
7.30±1.31
ND
9b
7.93±1.07
6.31±0.57
8.51±1.41
>15
9d
ND
8.01±0.97
7.43±1.26
ND
9e
1.45±0.43
2.08±0.27
3.49±0.61
0.31±0.07
ND
9g
9h
1.92±0.23
0.43±0.09
0.75±0.16
1.67±0.21
ND
>15
ND
9j
10.98±1.21
9.07±1.31
ND
7.18±0.97
>15
18da
20aa
20fa
25a
3.67±0.41
1.14±0.17
0.79±0.14
1.23±0.31
6.27±1.14
4.17±0.51
>15
>15
ND
1.01±0.11
2.91±0.17
8.32±0.87
2.31±0.27
13.40±2.18
>15
>15
25b
11.53±1.47
>15
GLPG-1690
PF-8380
>15
8.21±1.23
11.27±2.11
adata were the results in at least two independent assays; ND: not determined;
Among the compounds that exerted lower than 5 µM antiproliferative effects towards MCF-7 or MDA-MB-231 cell
lines, several compounds (8ca, 8cb, 8hc, 8jb, 9e, 9j, 18da, 20fa, 25a and 25b) were observed encouraging IC50 values
that was below 1.5 µM. These results suggested that the hydrophilic property of the amine portion affected the
cell-based assay viabilities (proline, prolinol, 4-OH piperidine and piperazine derivatives were much more hydrophilic
than other amines involved) to a great degree. Consistent with the enzymatic assay, the most potent ATX inhibitors
25a and 9j were detected the most powerful antiproliferative activities. 25a showed impressive antiproliferative
effects towards MCF-7 (1.01 µM) and MDA-MB-231 (0.79 µM) cell lines. Better yet, to our great satisfaction, 9j
presented both sub-micromolar IC50 values on MCF-7 (0.43 µM) and MDA-MB-231 cell lines (0.31µM). These
results did elucidate the promising application of 9j in the regulation of breast cancer relevant cases.
Given the versatile role of ATX in tumorigenesis, development and metastasis, there is great interest in the
exploration of combination of ATX inhibitors with other antitumor agents to bring synergistic effects, which is the
following aim of us in the near future.
2.4 Molecular docking and binding mode studies
2.4.1 Molecular docking of 9j & 25a with ATX and binding mode studies
To better verify the binding mode, molecular docking models of 9j and 25a were performed with ATX based on
the cocrystal structure of GLPG-1690 and ATX (PDB 4MHP) [15].

Fig. 5. The binding models of 9j with ATX. (A) Predicted binding conformation for 9j (magenta sticks) in the binding site cavity of ATX (PDB
4MHP). (B) Predicted binding conformation of 9j in the binding site of ATX and overlapping with GLPG-1690 (yellow sticks). (C) 2D diagram of
the interactions between 9j and ATX. (Hydrogen bonds are indicated by green dashed lines and π-π interactions are shown with red dashed lines).
As shown in Figure 5A and 5C, the S-atom of the 5-thiazole indole moiety in 9j made hydrogen bonds (3.4 Å) with
residue Trp255. Moreover, two essential π-π interactions were observed between Trp255/thiazole ring and
Phe275/indole skeleton. Apparently, 9j presented a highly consistent binding manner with the reference GLPG-1690
(Fig. 5B). Most importantly, the intramolecular pseudo-ring formed within the trifluoroacetyl hydrazone moiety by a
1.6 Å H-bond may account for the typical feature of this chemotype ATX inhibitors and well uncovered their binding
mode.
Fig. 6. Binding models of 25a with ATX. (A) Predicted binding pose for 25a (magenta sticks) in the binding site cavity of ATX (PDB 4MHP).
(B) Predicted binding conformation of 25a in the binding site of ATX and overlapping with GLPG-1690 (yellow sticks). (C) 2D diagram of the
interactions between 25a and ATX. (H-bonds are indicated by green dashed lines and π-π interactions are shown with red dashed lines).
The concrete binding model of 25a with ATX was presented in Fig. 6A and 6C. The nitrile group and the S-atom of
the CN-thiazole moiety formed two principle H-bonds with Trp276 which greatly enhanced the binding efficiency of
25a with the hydrophobic pocket of ATX. The indole core was involved in two fundamental π-π interactions with
Phe250 and Phe275. Besides, a Pi-sigma interaction was observed between Trp255 and piperazine. Although this
Pi-sigma interaction was weaker than the π-π interaction of thiazole ring in 9j with Trp255, it did present an ideal
binding pose and highly improve the hydrophilicity. Similarly, 25a was detected an intramolecular pseudo-ring within
the trifluoroacetyl hydrazone moiety. Although the pseudo-ring in 25a turned out to be exo-type (endo-type in 9j), they
both played a significant role in the constraining of the binding conformations of compounds.
2.4.2 Comparison of binding free energies of compounds 9j, 8ka, and 25a with ATX
Various researches have suggested that binding free energy that calculated through MM/GBSA method was proved
a good correlation with biological activity [34], thus the binding affinity of the promising compounds (9j & 25a)
towards ATX were evaluated by the calculated binding free energy. The less potent 8ka was utilized as a reference.

The results were depicted in Table 5 and Fig. 7.
Table 5. Complexes binding free energy calculated (kcal/mol) by MM-GBSA algorithm
Complex
9j-ATX
ΔG_Bind
-89.396
-67.433
-81.631
ΔG_Bind_Coulomb
-27.168
ΔG_Bind_Hbond
-3.591
ΔG_Bind_Lipo
-53.436
ΔG_Bind_Packing
-5.035
ΔG_Bind_vdW
-66.219
8ka-ATX
25a-ATX
-21.985
-1.061
-24.358
-4.432
-53.802
-24.198
-3.085
-43.559
-4.843
-61.629
The MM-GBSA method calculates the accurate binding free energies includingΔG_Bind (the binging free energy),
ΔG_Bind_Coulomb, ΔG_Bind_Hbond, ΔG_Bind_Lipo, ΔG_Bind_Packing and ΔG_Bind_vdW. The frames extracted
from the final 1 ns (totally 100 ns) trajectory were selected for the analysis of the energy contribution of the three
complexes (9j-ATX, 8ka-ATX and 25a-ATX).
Fig. 7. (A) The spectrum of intermolecular interaction between protein-ligand complexes determined from MM/GBSA calculation. (B)
Superimposition of GLPG-1690 (yellow lines), 8ka (cyan lines), 9j (pink lines), 25a (magenta lines) within the active site of ATX.
Generally, it was observed that the tendency of the predicted ΔG_Bind values of 9j-ATX (–89.396 kcal/mol),
25a-ATX (–81.631 kcal/mol), and 8ka-ATX (–67.433 kcal/mol) are consistent with the experimentally determined
enzymatic activities. It was also noticed that in all three complexes, ΔG_Bind_Coulomb, ΔG_Bind_Lipo and
ΔG_Bind_vdW contribute dominantly to the binding free energy of ligands with ATX. It was hypothesized that the
well occupancy of the hydrophobic pocket of ATX binding site may play the key role for the identification of efficient
allosteric ATX inhibitors.
3. Conclusion
With the aim to develop novel allosteric specific ATX inhibitors, a hybrid strategy was implemented by
incorporating the phenyl thiazole moiety onto the HTS product indole-based 1, which furnished 29 new compounds
(8aa~8ka, 10a and 10j). After a FS-3 based ATX inhibitory assay, further optimization was carried out according to
the preliminary SARs conclusion. On one hand, the phenyl thiazole group was shrunk to the more tolerable
benzothiazole which bring compounds 9a~9j, among which 9j was identified to be most powerful with an amazing 2.1
nM ATX inhibitory profile. On the other hand, the 5-thiazole moiety on indole fragment was altered to piperazine (25a
and 25b) and piperidine (18da, 20aa and 20fa) motif to improve the hydrophilicity. Although the representative 25a
exhibited the IC50 value of 19.0 nM which was inferior to 9j in enzymatic assay, it was hypothesized that the
introduction of piperazine could highly decrease the lipophilicity of the total entity. All compounds were advanced to a
panel of cancer cell lines and a preferred affinity towards breast cancer cell lines was presented. The most promising
9j was detected optimal activities on MCF-7 (0.43 µM) and MDA-MB-231(0.31 µM) cell lines. Through a dedicated

docking study, except the essential H-bond and π-π interactions, it was apparently shown that the intramolecular
pseudo-ring within the trifluoroacetyl hydrazone moiety played a significant role in constraining the binding poses of
9j and 25a. Taken together, 9j and 25a deserved further study which may eventually confirm their potential
applications.
4. Experimental section
4.1 Chemistry
The melting points were obtained through a Büchi Melting Point B-540 facility (BüchiLabortechnik, Flawil,
Switzerland) which were uncorrected. Mass spectra (MS) were conducted in ESI mode on Agilent 1100 LC-MS
1
(Agilent, Palo Alto, CA, USA) and a mixture of H2O/MeCN (4:6) was utilized as the eluting system. The H and 13C
NMR spectra were performed by Bruker ARX-400 spectrometers (Bruker Bioscience, Billerica, MA, USA) using
TMS as internal standard. Column chromatography was run on 200-300 mesh silica gel from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). Unless otherwise stated, all materials were Commercially available and were
used without further purification.
4.1.1 1-ethyl-1H-indole-5-carbonitrile (2)
60% NaH (100.2 g, 5.0 mol) was suspended in DMF (800 mL) at ambient temperature. 1H-indole-5-carbonitrile
(284.0 g, 2.0 mol) was added when the temperature reached to 40 . After the gas escaped, the solution was cooled to
25 . Then iodoethane (468.0 g, 3.0 mol) was added and the reaction mixture was stirred at room temperature for 2 h.
When completed, the mixture was diluted with water (500 mL). After stirring for 0.5 h, the mixture was filtered to
obtain the solid 2 in 88.1% yield.
4.1.2 1-ethyl-1H-indole-5-carbothioamide (3)
To a solvent of DMF (600 mL) was added NaSH (85.0 g, 3.5 mol), MgCl2·6H2O (406.0 g, 2 mol) and 2 (170 g, 0.7
mol) under stirring. The reaction mixture was stirred at 25 for 3 h until completion. After cooling to room
temperature, water was added, filtered. The residue was transferred to 1 M HCl solution (600 mL) and stirred for 20
min and filtered. The resulting solid was washed with plenty of water to afford the title compound 3 as a yellow solid
1
in 72.1% yield. H NMR (600 MHz, DMSO) δ 9.58 (s, 1H), 9.33 (s, 1H), 8.25 (d, J = 1.6 Hz, 1H), 7.84 (dd, J = 8.7,
1.8 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.47 (d, J = 3.5 Hz, 1H), 6.56 – 6.54 (m, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.35 (t, J
= 7.2 Hz, 3H).
4.1.3 4-(chloromethyl)-2-(1-ethyl-1H-indol-5-yl) thiazole (4)
At room temperature, the 1,3-dichloropropan-2-one (63 g, 0.5 mol) and 3 (102 g, 0.5 mol) was dissolved in toluene
(600 mL), stirring at reflux for 2.5 h. When completed, the solvent was removed in vacuo and the residue was
triturated with water. The resulting precipitate was filtered, washed with water to give a yellow solid after drying in
78.9% yield. 1H NMR (600 MHz, DMSO) δ 8.17 (d, J = 1.5 Hz, 1H), 7.74 (dd, J = 8.6, 1.7 Hz, 1H), 7.69 (s, 1H), 7.59
(d, J = 8.6 Hz, 1H), 7.49 (d, J = 3.1 Hz, 1H), 6.58–6.57 (m, 1H), 4.87 (s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.38 (t, J = 7.2
Hz, 3H).
4.1.4 1-(5-(4-(chloromethyl)thiazol-2-yl)-1-ethyl-1H-indol-3-yl)-2,2,2-trifluoroethan-1-one (5)
At room temperature, 4 (108 g, 0.4 mol) was dissolved in DMF (800 mL). 2,2,2-trifluoroacetic anhydride (0.6 mol)
was added dropwise at 0 . After addition, the reaction mixture was stirred at ambient temperature for 1.5 h. When the
reaction is completed as indicated by the TLC result, the mixture was poured into water (2500 mL) and stirred for 30
min. The solution was filtered to give 5 as a solid 113.5 g in 78.1% yield. 1H NMR (600 MHz, DMSO) δ 8.80 (d, J =

1.4 Hz, 1H), 8.70 (d, J = 1.6 Hz, 1H), 7.98 (dd, J = 8.6, 1.8 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.82 (s, 1H), 4.93 (s,
2H), 4.45 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 168.60, 153.30, 141.29, 137.88,
129.17, 127.27, 123.28, 119.66, 119.54, 118.63, 115.73, 113.08, 108.81, 42.62, 41.44, 15.61.
4.1.5 General procedure for preparation of intermediate 6 (6a-6k)
Amine (0.2 mol) and K2CO3 (0.25 mol) were added to a stirred solution of 5 (0.1 mol) in MeCN (200 mL) at room
temperature. After a 2 h stirring, the reaction mixture was filtered and the filtrate was evaporated. The residue was
taken up in water, and extracted with EA twice. Then the combined organic phases were washed with brine, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo to give off 6 in 80.1% to 83.5% yield.
4.1.6 General procedure for preparation of target compounds (8aa~8ka)
To a solution of 6 (0.01 mol) in toluene (30 mL), hydrazine (0.01 mol) and p-TsOH (0.02 mol) were added at
ambient temperature. After addition, the resulting mixture was heated to reflux and stirred for 3 h. After cooling to
room temperature, the solvent was removed under reduced pressure. The residue was dissolved in DCM (100 mL),
washed with an aqueous saturated NaHCO3 solution. The organic layers were then dried over Na2SO4, filtered and
concentrated in vacuo. The crude products were further purified by chromatography on silica gel to afford the desired
compound.
4.1.6.1
(Z)-2-(2-(1-(1-ethyl-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazinyl)-4-(4-fl
uorophenyl)thiazole-5-carbonitrile (8aa) Yellow solid; Yield: 21.7%; HPLC(%): 95.1%; M. p.: 213.8-214.2 ; MS(ESI)
m/z: 640.24 [M+H], 638.20 [M-H]; 1H NMR (400 MHz, DMSO) δ 12.18 (s, 1H), 8.07 (s, 1H), 7.98 (s, 1H), 7.95 (dd,
J = 8.8, 5.5 Hz, 2H), 7.86 – 7.83 (m, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 7.34 (t, J = 8.9 Hz, 2H), 4.35 (q, J =
7.1 Hz, 2H), 3.75 (s, 1H), 3.58 (s, 4H), 2.58 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).
4.1.6.2
(Z)-2-(2-(1-(1-ethyl-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazinyl)-4-(4-
methoxyphenyl)thiazole-5-carbonitrile (8ab) Yellow solid; Yield: 27.1%; HPLC(%): 92.7%; M. p.: 167.0-168.7 ;
MS(ESI)m/z: 652.20 [M+H], 650.27 [M-H]; 1H NMR (400 MHz, DMSO) δ 8.12 (s, 1H), 8.05 (s, 1H), 7.88 (d, J = 8.8
Hz, 2H), 7.82 (d, J = 8.7 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.40 (s, 1H), 7.03 (d, J = 8.9 Hz, 2H), 4.34 (q, J = 7.0 Hz,
2H), 3.80 (s, 4H), 3.64 (s, 2H), 3.59 – 3.53 (m, 4H), 2.48 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 168.51, 161.41, 160.72, 159.98, 153.94, 147.86, 147.30, 145.95, 136.83, 132.68, 129.29, 126.38, 126.21,
124.58, 123.91, 120.78, 119.36, 117.31, 116.74, 114.49, 114.31, 111.58, 106.31, 66.50 (2C), 58.23, 55.74, 53.51(2C),
41.49, 15.84.
4.1.6.3(Z)-2-(2-(1-(1-ethyl-5-(4-(piperidin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazin
yl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8ba) Yellow solid; Yield: 28.1%; HPLC(%): 91.9%; M. p.:
120.0-121.3 ; ESI-MS(m/z): 638.32[M+H]; 1H NMR (600 MHz, CDCl3) δ 8.04 (s, 1H), 7.94 (s, 1H), 7.89 (d, J = 8.6
Hz, 1H), 7.85 (dd, J = 8.4, 5.4 Hz, 2H), 7.46 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.00 (t, J = 8.5 Hz, 2H), 4.23 (q, J = 7.3
Hz, 2H), 3.83 (s, 2H), 2.64 (s, 4H), 1.65 (s, 4H), 1.52 (t, J = 7.3 Hz, 3H), 1.38 (d, J = 21.7 Hz, 2H).
4.1.6.4
(Z)-4-(4-chlorophenyl)-2-(2-(1-(1-ethyl-5-(4-(piperidin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylide
ne)hydrazinyl)thiazole-5-carbonitrile (8bc) Yellow solid; Yield: 24.9%; HPLC(%): 93.3%; M. p.: 162.8-163.4
;
MS(ESI)m/z: 654.27 [M+H], 652.24 [M-H]; LHR-59: 1H NMR (400 MHz, DMSO) δ 8.20 (s, 1H), 8.11 (s, 1H), 7.93

(s, 1H), 7.91 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.70 (s, 2H), 7.52 (d, J = 8.1 Hz, 2H), 4.34 (q, J = 7.0 Hz, 2H), 4.23 (s,
2H), 3.03 (m, 4H), 1.64 (s, 4H), 1.44 (s, 2H), 1.41 (m, J = 6.8 Hz, 3H).
4.1.6.5
(Z)-((2-(3-(1-(2-(5-cyano-4-(4-fluorophenyl)thiazol-2-yl)hydrazono)-2,2,2-trifluoroethyl)-1-ethyl-1H-indol-5-yl)thiaz
ol-4-yl)methyl)-L-proline (8ca) Yellow solid; Yield: 26.3%; HPLC(%): 92.1%; M. p.: 183.0-184.2 ; MS(ESI) m/z:
1
668.46 [M+H], 690.41 [M+Na], 666.24 [M-H]; H NMR (400 MHz, DMSO) δ 12.15 (s, 2H), 8.07 (s, 1H), 7.98 (s,
1H), 7.95 (dd, J = 8.7, 5.6 Hz, 2H), 7.87 (d, J = 8.7 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.59 (s, 1H), 7.35 (t, J = 8.8 Hz,
2H), 4.36 (q, J = 7.0 Hz, 2H), 4.24 (d, J = 13.9 Hz, 1H), 4.10 (d, J = 13.9 Hz, 1H), 3.77 – 3.69 (m, 1H), 3.28 (d, J =
10.3 Hz, 1H), 2.87 (dd, J = 17.5, 8.6 Hz, 1H), 2.09 (td, J = 17.1, 8.4 Hz, 1H), 1.94 – 1.81 (m, 1H), 1.72 (ddd, J = 28.8,
14.2, 8.1 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 172.56, 168.86, 164.53, 162.07, 160.28,
151.43, 137.19, 133.11, 129.58, 126.34, 123.22, 121.10, 120.49, 119.55, 118.97, 118.74, 116.50, 115.69, 113.05,
111.98, 102.39, 65.92, 53.61, 52.80, 41.65, 29.11, 23.39, 15.79.
4.1.6.6
(Z)-((2-(3-(1-(2-(5-cyano-4-(4-methoxyphenyl)thiazol-2-yl)hydrazono)-2,2,2-trifluoroethyl)-1-ethyl-1H-indol-5-yl)thi
azol-4-yl)methyl)-L-proline (8cb) Yellow solid; Yield: 28.1%; HPLC(%): 95.2%; M. p.: 129.7-130.5 ; MS(ESI)m/z:
680.27 [M+H], 678.27 [M-H]; 1H NMR (400 MHz, DMSO) δ 12.07 (s, 2H), 8.05 (s, 1H), 7.95 (s, 1H), 7.88 (s, 2H),
7.86 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.57 (s, 1H), 7.05 (d, J = 8.8 Hz, 2H), 4.36 (q, J = 7.1 Hz, 2H), 4.20 (d, J = 14.0
Hz, 1H), 4.06 (d, J = 13.9 Hz,1H), 3.80 (s, 3H), 3.69 – 3.61 (m, 1H), 3.23 (s, 1H), 2.82 (dd, J = 17.2, 8.4 Hz, 1H), 2.05
(td, J = 16.8, 8.3 Hz, 1H), 1.90 – 1.79 (m, 1H), 1.70 (ddd, J = 29.1, 14.6, 8.4 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H). 13C
NMR (101 MHz, DMSO) δ 172.81, 168.72, 161.10, 161.02, 151.98, 137.21, 133.11, 129.43(2C), 126.42, 126.35,
125.39, 123.32, 123.15, 121.11, 120.43, 119.56, 118.60, 115.91, 114.72(2C), 113.05, 112.00, 108.81, 102.19, 65.89,
55.81, 53.46, 52.82, 41.65, 29.17, 23.47, 15.78.
4.1.6.7
(Z)-2-(2-(1-(1-ethyl-5-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazinyl)-4-(
4-fluorophenyl)thiazole-5-carbonitrile (8da) Yellow solid; Yield: 24.3%; HPLC(%): 96.0%; M. p.: 125.0-126.9 ;
MS(ESI)m/z: 624.31 [M+H], 622.27 [M-H]; 1H NMR (400 MHz, DMSO) δ 10.88(s, 1H), 8.17 (s, 1H), 8.08 (s, 1H),
7.95 (dd, J = 8.8, 5.5 Hz, 2H), 7.83 (d, J = 8.6 Hz, 1H), 7.78 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.30 (t, J = 8.8 Hz, 2H),
4.44 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.27(s, 4H), 1.87 (s, 4H), 1.43 (t, J = 7.2 Hz, 3H). 13C NMR (151 MHz, DMSO)
δ 170.04, 163.79, 162.15, 160.57, 147.88, 136.99, 132.44, 130.52, 129.97, 129.91, 126.55, 125.38, 121.36, 120.99,
120.82, 119.96, 117.32, 116.15, 116.00, 115.93, 115.79, 111.57, 104.72, 53.59(2C), 52.60, 41.44, 23.12(2C), 15.81.
4.1.6.8
(Z)-2-(2-(1-(1-ethyl-5-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazinyl)-4-(
4-methoxyphenyl)thiazole-5-carbonitrile (8db) Yellow solid; Yield: 24.9%; HPLC(%): 96.1%; M. p.: 120-121.5
;
MS(ESI)m/z: 636.24 [M+H], 634.27 [M-H]; 1H NMR (400 MHz, DMSO) δ 10.61 (s, 1H), 8.19 (s, 1H), 8.07 (s, 1H),
7.87 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 8.1 Hz,1H), 7.70 (s, 2H), 7.00 (d, J = 8.7 Hz, 2H), 4.35 (s, 2H), 4.32 (d, J = 7.1
Hz, 2H), 3.79 (s, 3H), 3.18 (s, 4H), 1.84 (s, 4H), 1.43 (t, J = 7.1 Hz, 3H).
4.1.6.9
(Z)-4-(4-chlorophenyl)-2-(2-(1-(1-ethyl-5-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylid
ene)hydrazinyl)thiazole-5-carbonitrile (8dc) Yellow solid; Yield: 25.4%; HPLC(%): 94.3%; M. p.: 143.2-144.6 ;

MS(ESI)m/z: 640.18 [M+H], 638.20 [M-H]; 1H NMR (400 MHz, DMSO) δ 10.25 (s, 1H), 8.25 (s, 1H), 8.10 (s, 1H),
7.94 (s, 1H), 7.91 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.71 (s, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.53 (s, 1H), 7.50 (s, 1H),
4.37 (s, 2H), 4.35 – 4.29 (m, 2H), 3.20 (s, 4H), 1.85 (s, 4H), 1.42 (t, J = 7.2 Hz, 3H).
4.1.6.10
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydra
zinyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8ea) Yellow solid; Yield: 27.3%; HPLC(%): 93.7%; M. p.:
128.3-128.9 ; MS(ESI)m/z: 653.27 [M+H], 651.20 [M-H]; 1H NMR (400 MHz, DMSO) δ 8.15 (s, 1H), 8.13 (s, 1H),
7.98 (d, J = 6.6 Hz, 1H), 7.97 – 7.94 (m, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.43 (s, 1H), 7.37 –
7.30 (m, 1H), 7.29 (d, J = 8.7 Hz, 1H), 4.33 (d, J = 7.1 Hz, 2H), 3.74 (s, 2H), 3.00 (s, 4H), 2.89 (s, 2H), 2.74 (s, 2H),
2.61 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H).
4.1.6.11
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydra
zinyl)-4-(4-methoxyphenyl)thiazole-5-carbonitrile (8eb) Yellow solid; Yield: 28.4%; HPLC(%): 95.5%; M. p.:
137.3-138.6 ; MS(ESI)m/z: 665.36 [M+H]; 1H NMR (400 MHz, DMSO) δ 8.12 (s, 2H), 7.89 (d, J = 8.9 Hz, 2H),
7.79 (dd, J = 8.6, 1.5 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.01 (d, J = 8.9 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H),
3.79 (s, 3H), 3.72 (s, 2H), 2.89 (m, 4H), 2.67 (m, 4H), 2.53 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H).
4.1.6.12
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-ethylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazi
nyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8ec) Yellow solid; Yield: 26.5%; HPLC(%): 94.3%; M. p.:
125.9-126.7 ; MS(ESI)m/z: 669.23 [M+H], 667.20 [M-H]; 1H NMR (400 MHz, DMSO) δ 8.17 (s, 1H), 8.15 (s, 1H),
7.94 (d, J = 8.7 Hz, 2H), 7.79 (dd, J = 8.6, 1.6 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.42 (s, 1H),
4.33 (q, J = 7.1 Hz, 2H), 3.73 (s, 2H), 2.95 (m, 4H), 2.67 (m, 4H), 2.57 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H).
4.1.6.13
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-ethylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazi
nyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8fa) Yellow solid; Yield: 24.9%; HPLC(%): 92.7%; M. p.:
135.7-136.6 ; MS(ESI)m/z: 667.47 [M+H];1H NMR (400 MHz, DMSO) δ 8.19 (s, 1H), 8.15 (s, 1H), 7.99 – 7.94 (m,
2H), 7.79 (dd, J = 8.6, 1.6 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.29 (t, J = 8.9 Hz, 2H), 4.33 (q, J = 7.1 Hz,
2H), 3.74 (s, 2H), 2.89 (m, 4H), 2.84 (m, 2H), 2.67 (s, 4H), 1.42 (t, J = 7.2 Hz, 3H), 1.11 (t, J = 7.1 Hz, 3H). 13C NMR
(151 MHz, DMSO) δ 169.03, 163.64, 162.01, 160.86, 153.51, 136.61, 132.24, 131.31, 129.92, 129.87, 126.57, 125.73,
124.31, 121.98, 120.50, 120.44, 118.35, 116.91, 115.98, 115.92, 115.83, 111.21, 106.24, 57.05, 51.45(2C), 50.56(2C),
46.15, 41.37, 15.91, 9.13.
4.1.6.14
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-ethylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazi
nyl)-4-(4-methoxyphenyl)thiazole-5-carbonitrile (8fb) Yellow solid; Yield: 26.7%; HPLC(%): 97.2%; M. p.:
80.1-81.9 ; MS(ESI)m/z: 679.31 [M+H]; 1H NMR (400 MHz, DMSO) δ 8.15 (s, 1H), 8.13 (s, 1H), 7.91-7.87 (m, 2H),
7.79 (dd, J = 8.6, 1.6 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.00 (d, J = 8.9 Hz, 2H), 4.33 (dd, J = 14.2, 7.0
Hz, 2H), 3.79 (s, 2H), 3.73 (s, 2H), 2.74 (m, 10H), 1.42 (t, J = 7.2 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H).
4.1.6.15
(Z)-4-(4-chlorophenyl)-2-(2-(1-(1-ethyl-5-(4-((4-ethylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluor

oethylidene)hydrazinyl)thiazole-5-carbonitrile (8fc) Yellow solid; Yield: 28.1%; HPLC(%): 91.3%; M. p.:
143.2-144.6 ; MS(ESI)m/z: 683.24 [M+H]; 1H NMR (400 MHz, DMSO) δ 8.17 (s, 1H), 8.14 (s, 1H), 7.94 (d, J = 8.6
Hz, 2H), 7.79 (dd, J = 8.6, 1.5 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.43 (s, 1H), 4.33 (q, J = 7.1
Hz, 2H), 3.76 (s, 2H), 2.80 (m, 10H), 1.42 (t, J = 7.2 Hz, 3H), 1.15 – 1.08 (t, 3H). 13C NMR (101 MHz, DMSO) δ
169.01, 160.48, 153.29, 140.49, 136.66, 134.17, 133.35, 132.29, 129.91, 129.08(2C), 128.99(2C), 126.56, 125.77,
121.87, 120.55, 120.27, 117.88, 117.08, 111.25, 105.85, 56.83, 51.30(2C), 50.17(2C), 41.38, 27.30, 15.87, 10.01.
4.1.6.16
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-methylpiperidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydra
zinyl)-4-(4-methoxyphenyl)thiazole-5-carbonitrile (8gb) Yellow solid; Yield: 28.4%; HPLC(%): 95.3%; M. p.:
112.8-113.7 ; MS(ESI)m/z: 664.18 [M+H], 662.23 [M-H]; 1H NMR (400 MHz, DMSO) δ 10.51 (s, 1H), 8.14 (s, 1H),
8.07 (s, 1H), 7.89 (s, 1H), 7.87 (s, 1H), 7.80 (dd, J = 8.6, 1.2 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.62 (s, 1H), 7.01 (s,
1H), 6.99 (s, 1H), 4.34 (q, J = 7.1 Hz, 2H), 4.12 (s, 2H), 3.79 (s, 3H), 3.22 (d, J = 11.2 Hz, 2H), 2.65 (s, 2H), 1.63 (d, J
= 12.9 Hz, 2H), 1.43 (t, J = 7.2 Hz, 3H), 1.27 (d, J = 11.7 Hz, 1H), 1.23 (s, 2H), 0.85 (d, J = 6.4 Hz, 3H). 13C NMR
(101 MHz, DMSO) δ 169.65, 161.47, 160.57, 149.29, 136.91, 132.23, 129.21, 127.97, 126.77, 126.64, 125.55, 124.25,
121.51, 120.79, 119.99, 117.78, 114.39, 111.47, 105.24, 105.17, 103.74, 55.72, 52.68, 41.43, 31.96, 28.98, 27.29,
26.80, 21.51, 15.85.
4.1.6.17
(Z)-4-(4-chlorophenyl)-2-(2-(1-(1-ethyl-5-(4-((4-methylpiperidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-triflu
oroethylidene)hydrazinyl)thiazole-5-carbonitrile (8gc) Yellow solid; Yield: 29.6%; HPLC(%): 95.7%; M. p.:
110.2-111.8 ; MS(ESI) m/z: 668.23 [M+H], 666.27 [M-H]; 1H NMR (400 MHz, DMSO) δ 8.20 (s, 1H), 8.09 (s, 1H),
7.93 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.6 Hz, 1H), 7.72 – 7.67 (m, 2H), 7.52 (d, J = 8.5 Hz, 2H), 4.41 – 4.28 (q, 2H),
4.21 (s, 2H), 3.29 (d, J = 10.6 Hz, 2H), 2.76 (s, 2H), 1.66 (d, J = 12.8 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H), 1.34-1.24 (m,
2H), 0.88 (d, J = 7.3 Hz, 1H), 0.86 (d, J = 6.1 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 169.94, 160.41, 136.85, 134.23,
133.99, 133.23, 132.18, 130.04, 129.36(2C), 129.09(2C), 129.00, 126.66, 125.33, 124.47, 121.77, 121.33, 120.71,
120.46, 117.73, 111.38, 105.77, 52.47(2C), 41.40, 31.54(2C), 28.70, 21.39, 15.87, 9.06.
4.1.6.18
(S,Z)-2-(2-(1-(1-ethyl-5-(4-((2-(hydroxymethyl)pyrrolidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethy
lidene)hydrazinyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8ha) Yellow solid; Yield: 25.7%; HPLC(%): 95.5%; M.
p.: 128.4-129.3 ; MS(ESI) m/z: 654.20 [M+H], 652.23 [M-H]; 1H NMR (400 MHz, DMSO) δ 10.05 (s, 1H), 8.19 (s,
1H), 8.11 (s, 1H), 7.95 (s, 2H), 7.81 (s, 1H), 7.71 (s, 1H), 7.70 – 7.66 (m, 1H), 7.29 (s, 2H), 5.22 (s, 1H), 4.49 (s, 1H),
4.34 (s, 1H), 4.33 (s, 2H), 3.58 (s, 2H), 3.08 (s, 2H), 1.96 (s, 1H), 1.74 (s, 2H), 1.67 (s, 2H), 1.42 (s, 3H).
4.1.6.19
(S,Z)-2-(2-(1-(1-ethyl-5-(4-((2-(hydroxymethyl)pyrrolidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroeth
ylidene)hydrazinyl)-4-(4-methoxyphenyl)thiazole-5-carbonitrile (8hb) Yellow solid; Yield: 24.7%; M. p.:
125.7-126.4 ; MS(ESI)m/z: 666.24 [M+H], 664.23 [M-H]; 1H NMR (400 MHz, DMSO) δ 9.94 (s, 1H), 8.14 (s, 1H),
8.09 (s, 1H), 7.89 (s, 1H), 7.87 (s, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.02 (s, 1H), 7.00 (s, 1H),
5.32 (s, 1H), 4.47 (s, 1H), 4.34 (dd, J = 14.3, 7.1 Hz, 2H), 4.30 – 4.09 (m, 1H), 3.79 (s, 3H), 3.58 (s, 2H), 3.08 (s, 1H),
1.97 (s, 1H), 1.75 (d, J = 7.6 Hz, 2H), 1.67 (s, 1H), 1.43 (t, J = 7.1 Hz, 3H), 1.23 (s, 2H).
4.1.6.20

(S,Z)-4-(4-chlorophenyl)-2-(2-(1-(1-ethyl-5-(4-((2-(hydroxymethyl)pyrrolidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl
)-2,2,2-trifluoroethylidene)hydrazinyl)thiazole-5-carbonitrile (8hc) Yellow solid; Yield: 31.1%; HPLC(%): 92.9%; M.
p.: 80.1-81.8 ; MS(ESI) m/z: 670.23 [M+H], 668.20[M-H]; 1H NMR (400 MHz, DMSO) δ 8.22 (s, 1H), 8.14 (s, 1H),
7.94 (s, 1H), 7.92 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.53 (s, 1H), 7.51 (s, 1H), 5.23 (s, 1H),
4.46 (s, 1H), 4.33 (dd, J = 14.1, 7.0 Hz, 2H), 3.63 (s, 1H), 3.58 (s, 2H), 1.94 (m, 1H), 1.76 (m, 2H), 1.66 (m, 1H), 1.42
(t, J = 7.2 Hz, 3H), 1.23 (s, 2H).
4.1.6.21
(Z)-2-(2-(1-(1-ethyl-5-(4-((3-hydroxyazetidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydra
zinyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8ia) Yellow solid; Yield: 28.7%; HPLC(%): 93.9%; M. p.:
174.7-175.6 ; MS(ESI)m/z: 626.47 [M+H], 624.27[M-H]; 1H NMR (400 MHz, DMSO) δ 8.23 (s, 1H), 8.19 (s, 1H),
7.97 (s, 2H), 7.79 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.53 (s, 1H), 7.28 (t, J = 8.5 Hz, 2H), 5.74 (s,1H), 4.33
(d, J = 6.7 Hz, 2H), 4.12 (s, 2H), 3.96 (s, 2H), 3.86 (s, 1H), 3.44 (s, 2H), 1.42 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 169.74, 164.01, 161.56, 160.83, 136.68, 132.34, 131.33, 129.90, 129.82, 126.50, 125.39, 123.32, 122.06,
120.49, 119.50, 118.33, 117.70, 115.95, 115.74, 113.04, 111.21, 108.82, 106.32, 63.96, 60.53, 46.20, 41.35, 27.29,
15.87.
4.1.6.22
(Z)-2-(2-(1-(1-ethyl-5-(4-((3-hydroxyazetidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydr
azinyl)-4-(4-methoxyphenyl)thiazole-5-carbonitrile (8ib) Yellow solid; Yield: 28.5%; HPLC(%): 94.7%; M. p.:
157.1-158.4 ; MS(ESI) m/z: 638.43 [M+H], 636.27[M-H]; 1H NMR (400 MHz, DMSO) δ 8.20 (s, 1H), 7.98 (s, 1H),
7.89 (s, 2H), 7.79 (s, 1H), 7.67 (d, J = 7.2 Hz, 1H), 7.48 (s, 1H), 7.00 (s, 2H), 5.68 (s, 1H), 4.32 (s, 2H), 4.03 (s, 1H),
3.89 (s, 1H), 3.80 (s, 2H), 3.77 (s, 2H), 1.43 (s, 3H). 13C NMR (101 MHz, DMSO) δ 169.53, 161.73, 160.36, 151.70,
136.66, 132.35, 129.17, 128.53, 127.34, 126.47, 125.96, 125.50, 124.73, 123.31, 122.02, 120.51, 120.36, 119.49,
118.58, 117.18, 114.28, 111.23, 106.14, 64.03, 60.70, 55.68, 46.15, 41.36, 15.87.
4.1.6.23
(Z)-4-(4-chlorophenyl)-2-(2-(1-(1-ethyl-5-(4-((3-hydroxyazetidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-triflu
oroethylidene)hydrazinyl)thiazole-5-carbonitrile (8ic) Yellow solid; Yield: 27.8%; HPLC(%): 96.3%; M. p.:
169.7-170.7 ; MS(ESI)m/z: 642.41 [M+H], 640.49[M-H]; 1H NMR (400 MHz, DMSO) δ 8.23 (s, 1H), 8.18 (s, 1H),
7.94 (d, J = 7.8 Hz, 2H), 7.80 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.59 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 5.85
(s, 1H), 4.33 (d, J = 6.8 Hz, 2H), 4.22 (s, 1H), 4.05 (s, 2H), 3.63 (s, 3H), 3.58 (s, 2H), 1.42 (t, J = 6.7 Hz, 3H). 13C
NMR (101 MHz, DMSO) δ 172.44, 169.97, 160.51, 149.90, 136.73, 134.08, 133.94, 133.47, 132.39, 132.07, 129.36,
129.04, 128.50, 126.51, 125.96, 125.31, 124.69, 121.99, 121.21, 120.53, 118.48, 118.07, 111.28, 106.19, 63.82, 60.30,
46.20, 41.37, 27.30, 15.88.
4.1.6.24
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydr
azinyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8ja) Yellow solid; Yield:28.1%; HPLC(%): 95.0%; M. p.:
1
142.7-143.5 ; MS(ESI)m/z: 654.27 [M+H], 652.31[M-H]; H NMR (400 MHz, DMSO) δ 12.63 (s, 1H), 10.65 (s,
1H), 8.06 (s, 1H), 7.98 – 7.94 (m, 2H), 7.94 (d, J = 4.6 Hz, 2H), 7.91 (s, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.37 (t, J = 8.8
Hz, 2H), 4.98 (s, 1H), 4.40 (s, 2H), 4.39 – 4.33 (m, 2H), 3.35 (s, 2H), 3.23 (s, 2H), 3.01 (s, 1H), 1.92 (d, J = 11.4 Hz,
2H), 1.70 (d, J = 11.8 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 171.28, 169.45, 164.70,

162.22, 146.51, 137.48, 133.46, 130.35, 130.27, 129.13, 128.66, 126.36, 126.23, 122.93, 122.79, 121.36, 120.07,
118.26, 116.67, 116.45, 114.92, 112.29, 101.30, 55.37, 54.20, 50.53, 47.12, 41.75, 31.63, 29.69, 15.75.
4.1.6.25
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydr
azinyl)-4-(4-methoxyphenyl)thiazole-5-carbonitrile (8jb) Yellow solid; Yield: 25.7%; HPLC(%): 94.6%; M. p.:
1
137.9-138.4 ; MS(ESI)m/z: 666.24 [M+H] , 664.20[M-H]; H NMR (400 MHz, DMSO) δ 8.21 (s, 2H), 7.90 (d, J =
8.7 Hz, 2H), 7.79 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.40 (s, 1H), 7.00 (d, J = 8.8 Hz, 2H), 4.61 (s, 1H),
4.33 (q, J = 7.0 Hz, 2H), 3.80 (s, 3H), 3.74 (s, 2H), 3.49 (s, 1H), 3.00 (q, J = 7.1 Hz, 4H), 2.87 (s, 2H), 2.32 (s, 2H),
1.74 (d, J = 9.8 Hz, 2H), 1.45 (d, J = 7.9 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 169.12,
169.00, 161.81, 160.28, 136.53, 132.26, 129.14, 127.57, 126.47, 125.68, 124.90, 122.19, 120.69, 120.54, 120.41,
118.87, 116.84, 114.23, 111.11, 107.29, 106.50, 55.68, 51.02, 46.23, 41.32, 34.21, 15.87.
4.1.6.26
(Z)-4-(4-chlorophenyl)-2-(2-(1-(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifl
uoroethylidene)hydrazinyl)thiazole-5-carbonitrile (8jc) Yellow solid; Yield: 26.8%; HPLC(%): 95.6%; M. p.:
141.0-142.8 ; MS(ESI) m/z: 670.27 [M+H], 668.20[M-H];1H NMR (400 MHz, DMSO) δ 8.20 (s, 1H), 8.15 (s, 1H),
7.93 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.61 (s, 1H), 7.52 (d, J = 8.6 Hz, 2H), 4.81
(s, 1H), 4.33 (q, J = 7.2 Hz, 2H), 4.09 (s, 2H), 3.63 (s, 1H), 3.09 (d, J = 7.3 Hz, 2H), 2.70 (d, J = 24.5 Hz, 2H), 1.80 (s,
2H), 1.56 (s, 2H), 1.42 (t, J = 7.2 Hz, 3H).
4.1.6.27
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethyli
dene)hydrazinyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (8ka) Yellow solid; Yield: 26.4%; HPLC(%): 92.3%; M. p.:
102.4-103.5 ; MS(ESI) m/z: 683.27 [M+H], 681.31[M-H]; 1H NMR (400 MHz, DMSO) δ 9.44 (s, 1H), 8.18 (s, 1H),
8.17 (s, 1H), 7.97 (dd, J = 8.2, 5.7 Hz, 2H), 7.79 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.40 (s, 1H), 7.28 (t, J =
8.7 Hz, 2H), 4.86 (s, 1H), 4.33 (dd, J = 14.2, 7.0 Hz, 2H), 3.72 (s, 2H), 3.59 (s, 2H), 2.86 (s, 4H), 2.72 (m, 6H), 1.42 (t,
J = 7.2 Hz, 3H).
4.1.6.28
(Z)-4-(4-chlorophenyl)-2-(2-(2,2,2-trifluoro-1-(1-methyl-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indol-3-yl)ethylide
ne)hydrazinyl)thiazole-5-carbonitrile (10a) Yellow solid; Yield: 28.9%; HPLC(%): 91.5%; M. p.: 127.3-128.5 ;
MS(ESI)m/z: 626.18 [M+H]; 1H NMR (400 MHz, DMSO) δ 8.09 (s, 1H), 8.08 (s, 1H), 7.98 – 7.94 (m, 2H), 7.84 (dd,
J = 8.6, 1.5 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.42 (s, 1H), 7.32 (t, J = 8.9 Hz, 2H), 3.92 (s, 3H), 3.68 (s, 2H), 3.59 –
3.55 (m, 4H), 2.51 (m, 4H). 13C NMR (151 MHz, DMSO) δ 168.58, 163.91, 162.26, 160.53, 153.69, 153.59, 153.50,
137.85, 134.39, 130.31, 130.08, 130.02, 126.26, 126.15, 123.41, 120.88, 119.16, 117.05, 116.80, 116.23, 116.09,
111.60, 103.64, 66.37, 53.43(2C), 46.23(2C), 40.54.
4.1.6.29
(Z)-4-(4-fluorophenyl)-2-(2-(2,2,2-trifluoro-1-(5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1-methyl-1H-indol
-3-yl)ethylidene)hydrazinyl)thiazole-5-carbonitrile (10j) Yellow solid; Yield: 29.4%; HPLC(%): 90.3%; M. p.:
94.7-95.3 ; MS(ESI)m/z: 640.49 [M+H], 638.23[M-H]; 1H NMR (400 MHz, DMSO) δ 9.07 (d, J = 1.3 Hz, 0.35H),
8.25 (s, 0.66H), 8.19 (s, 0.65H), 8.06 (dd, J = 8.8, 5.6 Hz, 0.75H), 7.98 (dd, J = 8.5, 5.3 Hz, 1.29H), 7.96 (s, 0.32H),
7.82 (dd, J = 8.6, 1.5 Hz, 0.70H), 7.68 (d, J = 2.5 Hz, 0.31H), 7.62 (d, J = 8.5 Hz, 0.94H), 7.52 (s, 0.86H), 7.36–7.31

(m, 0.71H), 7.28 (d, J = 8.9 Hz, 1.46H), 4.74 (s, 1H), 3.91 (s, 3H), 3.87 (s, 1H), 3.58 (s, 2H), 3.03 – 2.94 (m, 4H), 1.80
(m, 2H), 1.52 (m, 2H).
4.1.7 General procedure for preparation of the target compounds 9 (9a, 9b, 9d, 9e, 9g, 9h, 9j)
To a solution of 6 (0.01 mol) in toluene (30 mL), 2-hydrazinylbenzo[d]thiazole (0.01 mol) and p-TsOH (0.02 mol)
were added at ambient temperature. After addition, the resulting mixture was heated to reflux and stirred for 3 h. After
cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved in DCM
(100 mL), washed with an aqueous saturated NaHCO3 solution. The organic layers were then dried over Na2SO4,
filtered and concentrated in vacuo. Crude products were further purified by chromatography on silica gel to afford the
desired compound.
4.1.7.1
(Z)-4-((2-(3-(1-(2-(benzo[d]thiazol-2-yl)hydrazono)-2,2,2-trifluoroethyl)-1-ethyl-1H-indol-5-yl)thiazol-4-yl)methyl)m
orpholine (9a). Yellow solid; Yield:19.1%; HPLC(%): 93.1%; M.p.: 120.0-122.1 ; MS(ESI) m/z: 571.18 [M+H],
593.23 [M+Na], 569.20 [M-H]; 1H NMR (400 MHz, DMSO) δ 12.15 (s, 1H), 8.03 (s, 2H), 7.86 – 7.81 (m, 1H), 7.72
(t, J = 9.1 Hz, 2H), 7.36 (s, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 4.34 (q, J =
7.1 Hz, 2H), 3.58 (s, 3H), 3.55 – 3.49 (m, 4H), 2.42 (s, 4H), 1.44 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ
170.87, 168.33, 154.25, 143.20, 136.81, 132.51, 126.92, 126.79, 126.40, 126.28, 123.57, 122.79, 122.66, 120.84,
120.55, 119.79, 116.54, 114.44, 111.62, 104.18, 66.58 (2C), 58.29, 53.53 (2C), 41.54, 15.81.
4.1.7.2
(Z)-2-(2-(1-(1-ethyl-5-(4-(piperidin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazinyl)benz
o[d]thiazole (9b) Yellow solid; Yield: 25.2%; HPLC(%): 95.1%; M. p.: 123.6-124.3 ; MS(ESI) m/z: 569.20 [M+H],
567.23 [M-H];1H NMR (400 MHz, DMSO) δ 12.00 (s, 1H), 8.03 (s, 1H), 7.82 (dd, J = 8.6, 1.5 Hz, 1H), 7.72 (d, J =
4.1 Hz, 1H), 7.70 (d, J = 5.1 Hz, 1H), 7.34 (s, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.6
Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 3.59 (s, 1H), 2.43 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H), 1.30 (s, 2H).
4.1.7.3
(Z)-2-(2-(1-(1-ethyl-5-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazinyl)ben
zo[d]thiazole (9d) Light yellow solid; Yield: 24.7%; HPLC(%): 92.1%; M.p.: 126.7-127.2 ; MS(ESI) m/z: 555.24
[M+H], 553.27 [M-H]; 1H NMR (400 MHz, DMSO) δ 8.04 (s, 2H), 7.82 (dd, J = 8.6, 1.6 Hz, 1H), 7.72 (d, J = 3.5 Hz,
1H), 7.70 (d, J = 4.8 Hz, 1H), 7.35 (s, 1H), 7.27 (dd, J = 11.1, 4.0 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.14–7.08 (m,
1H), 4.34 (q, J = 7.1 Hz, 2H), 3.73 (s, 2H), 2.55 (m, 4H), 1.64 (m, 4H), 1.43 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 170.68, 167.70, 154.62, 136.23, 131.92, 126.27, 126.22, 125.73, 123.09, 122.15, 121.94, 120.44, 120.37,
120.24, 119.24, 117.69, 115.48, 114.00, 111.04, 103.76, 54.72, 53.31(2C), 40.96, 23.00(2C), 15.27.
4.1.7.4
(Z)-2-(2-(1-(1-ethyl-5-(4-(piperazin-1-ylmethyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydrazinyl)benz
o[d]thiazole (9e) Light yellow solid; Yield: 28.4%; HPLC(%): 96.4%; M.p.: 124.1-125.2 ; MS(ESI) m/z: 584.23
[M+H], 582.20 [M-H]; 1H NMR (400 MHz, DMSO) δ 8.04 (s, 1H), 8.03 (s, 1H), 7.83 (dd, J = 8.6, 1.5 Hz, 1H), 7.72
(dd, J = 10.3, 5.2 Hz, 2H), 7.39 (s, 1H), 7.29 (t, J = 7.6 Hz,1H), 7.21 (d, J = 7.8 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 4.34
(q, J = 7.1 Hz, 2H), 3.64 (s, 2H), 2.74 (s, 4H), 2.61 (s, 4H), 2.41 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). 13C NMR (151 MHz,
DMSO) δ 170.83, 168.45, 161.85, 153.91, 153.79, 136.79, 132.54, 126.90, 126.73, 126.30, 123.07, 122.77, 122.63,
121.26, 120.82, 119.67, 116.85, 114.49, 111.62, 104.12, 57.19, 53.78(2C), 50.96(2C), 44.09, 41.50, 15.78.

4.1.7.5
(Z)-2-(2-(1-(1-ethyl-5-(4-((4-methylpiperidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethylidene)hydra
zinyl)benzo[d]thiazole (9g) Yellow solid; Yield: 26.5%; HPLC(%): 91.0%; M.p.: 99.9-100.6 ; MS(ESI) m/z: 583.24
[M+H], 581.23 [M-H]; 1H NMR (400 MHz, DMSO) δ 12.06 (s, 1H), 8.03 (s, 2H), 7.82 (d, J = 8.6 Hz, 1H), 7.71 (t, J
= 6.6 Hz, 2H), 7.33 (s, 1H), 7.27 (t, J = 7.4 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 4.34 (q, J = 6.9
Hz, 2H), 3.59 (s, 2H), 2.86 (d, J = 11.1 Hz, 2H), 2.00 (t, J = 10.9 Hz, 2H), 1.49 (d, J = 12.6 Hz, 2H), 1.43 (t, J = 7.2
Hz, 3H), 1.10 (d, J = 10.3 Hz, 2H), 1.10 (m, J = 10.3 Hz, 1H), 0.81 (d, J = 6.4 Hz, 3H).
4.1.7.6
(S,Z)-(1-((2-(3-(1-(2-(benzo[d]thiazol-2-yl)hydrazono)-2,2,2-trifluoroethyl)-1-ethyl-1H-indol-5-yl)thiazol-4-yl)methyl
)pyrrolidin-2-yl)methanol (9h) Yellow solid; Yield: 27.3%; HPLC(%): 92.0%; M.p.: 80.0-81.2 ; MS(ESI) m/z:
585.44 [M+H], 583.49 [M-H]; 1H NMR (400 MHz, DMSO) δ 8.07 (s, 1H), 8.05 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.73
(s, 1H), 7.71 (s, 1H), 7.50 (s, 1H), 7.27 (t, J = 7.2 Hz, 2H), 7.21 (s, 1H), 7.14 (t, J = 7.4 Hz, 2H), 5.32 (s, 1H), 4.66 (s,
1H), 4.35 (d, J = 6.8 Hz, 2H), 4.24 (s, 1H), 3.92 (s, 1H), 3.60 – 3.43 (m, 2H), 1.83 (s, 2H), 1.61 (s, 2H), 1.55 (m, 1H),
1.44 (t, 3H), 1.18 (s, 2H).
4.1.7.7
(Z)-1-((2-(3-(1-(2-(benzo[d]thiazol-2-yl)hydrazono)-2,2,2-trifluoroethyl)-1-ethyl-1H-indol-5-yl)thiazol-4-yl)methyl)pi
peridin-4-ol (9j) Yellow solid; Yield: 26.8%; HPLC(%): 96.7%; M. p.: 79.4-81.8 ; MS(ESI) m/z: 585.23 [M+H],
583.20 [M-H]; 1H NMR (400 MHz, DMSO) δ 12.13 (s, 1H), 8.05 (s, 1H), 8.02 (s, 1H), 7.83 (dd, J = 8.7, 1.4 Hz, 1H),
7.71 (t, J = 6.8 Hz, 2H), 7.33 (s, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 4.54 (s,
1H), 4.34 (q, J = 7.1 Hz, 2H), 3.58 (s, 2H), 3.42 (m, 1H), 2.76 (d, J = 11.3 Hz, 2H), 2.13 (t, J = 10.0 Hz, 2H), 1.68 (d,
J = 9.9 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H), 1.41–1.33 (m, 2H). 13C NMR (101 MHz, DMSO) δ 171.01, 168.17, 154.83,
136.72, 132.51, 126.83, 126.69, 126.36, 123.56, 122.79, 122.69, 122.52, 121.42, 120.84, 120.74, 119.74, 116.20,
114.44, 111.55, 104.15, 66.49, 57.95, 51.21(2C), 41.47, 34.65(2C), 15.76.
4.1.8 Methyl 2-(4,5-difluoro-2-nitrophenyl)-3-oxobutanoate (12)
At ambient temperature, methyl 3-oxobutanoate (25 g, 0.21 mol) and t-BuOK (24 g, 0.21 mol) were dissolved in
THF (120 mL). After stirring for 1 h, 11 (17.7 g, 0.1 mol) was added and the reaction stirred at room temperature for 2
h. After completion, a 2 M solution of HCl was added until the pH value of the solution was equal to 2. It was then
extracted twice with EA, followed by collection of organic layers. The organic portion was concentrated under
vacuum to afford the expected compound 12 as a brown oil in 93.0% yield that was used in the next step without
further purification.
4.1.9 1-(4,5-difluoro-2-nitrophenyl) propan-2-one (13)
To a stirring solution of HOAc (50 mL), concentrated sulfuric acid (5.5 g, 0.15 mol) was added dropwise slowly.
After addition, 12 (5.4 g, 0.05 mol) was added slowly and the mixture was heated to 50 . When the CO2 was escaped,
the reaction was allowed to warmed up to 70 and stirred at this temperature for 2h. After cooling down to room
temperature, the solution was diluted with water (300 mL) and extracted with DCM (100 mL×2). The combined
organic layers were washed with a 20% solution of NaOH and saturated aqueous NaCl solution successively, then
concentrated under reduced pressure to give a brown oil. The crude mixture was triturated in MeOH and stirred at 0
for 15min. A brown solid 13 was obtained by filtration in 54.3% yield.
4.1.10 1-(2-fluoro-4-nitro-5-(2-oxopropyl)phenyl)piperidine-4-carboxylic acid (14)

To a stirring solution of 13 (4.3 g, 0.02 mol) in MeCN, K2CO3(8 g, 0.06 mol) and piperidine-4-carboxylic acid (5.2
g, 0.04 mol) were added successively at ambient temperature. The reaction mixture was refluxed for 2 h. After cooling
to room temperature, the solvent was evaporated in vacuo. The crude product was diluted with water and DCM, and
layers are separated. The aqueous phase was acidified to pH 2 with 2 M HCl (aq.), then filtered and dried to afford the
desired compound 14 as a yellow solid in 82.1% yield.
4.1.11 General procedure for preparation of intermediate (15)
A mixture of 14 (4.5 g, 14 mmol), DIPEA (6 g, 56 mmol) and the substitute amines (15.4 mmol) were dissolved in
DMF (50 mL) at 25 , followed by the addition of HATU (5.8 g, 15.4 mmol). The mixture was stirred in the same
temperature for 2 h until completion. In most cases, the mixture was diluted with water and extracted with EA three
times. The organic layers were combined and washed with LiCl solution. Solvent was evaporated in vacuo to give the
compound 15. In other cases, the final product could precipitate from the solvent and a filtration is conducted to obtain
the desired product in good yield.
4.1.12 General procedure for preparation of intermediate (16)
To a mixture of THF (15 mL) and H2O (30 mL), 15(0.02 mol) was added at interior temperature. Add Na2S2O4(5 eq.)
in batches slowly with stirring. The resulting suspension was heated at 70 for 2 h. The mixture was cooled and
evaporated in vacuo. The mixture was diluted with water, extracted with EA for three times and the combined organic
phases was washed with brine, dried over Na2SO4, filtered, and concentrated to a yellow solid in 83.1% to 86.0%
yield.
4.1.13 General procedure for preparation of intermediate (17)
At room temperature, 16(0.01 mol) was dissolved in DMF (30 mL). 2,2,2-trifluoroacetic anhydride (2 eq.) was
added dropwise at 0 . After addition, the reaction mixture was stirred at ambient temperature for 1.5 h. When the
reaction is completed as indicated by the TLC result, the mixture was poured into water (100 mL) and extracted with
DCM three times. The organic layers were combined and washed with LiCl solution. It was then concentrated in
vacuo to give 17 as a solid in 58.0% to 60.1% yields.
4.1.14 General procedure for preparation of target compound (18da, 20aa, 20fa)
To a solution of 17(0.01mol) in toluene (30 mL), 4-(4-fluorophenyl)-2-hydrazinylthiazole-5-carbonitrile (0.01mol)
and p-TsOH (0.02mol) were added at ambient temperature. After addition, the resulting mixture was heated to reflux
and stirred for 3 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue
was dissolved in DCM (100 mL), washed with an aqueous saturated NaHCO3 solution. The organic layers were then
dried over Na2SO4, filtered, and concentrated in vacuo. The crude products were further purified by chromatography
on silica gel to afford the desired compound.
4.1.14.1
(Z)-4-(4-fluorophenyl)-2-(2-(2,2,2-trifluoro-1-(6-fluoro-2-methyl-5-(4-(morpholine-4-carbonyl)piperidin-1-yl)-1H-ind
ol-3-yl)ethylidene)hydrazinyl)thiazole-5-carbonitrile (20aa) Yellow solid; Yield: 21.3%; HPLC(%): 92.5%; M.p.:
168.3-169.2 ; MS(ESI)m/z: 531.46 [M+H]; 1H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 11.79 (s, 1H), 8.64 (s, 1H),
8.03 (dd, J = 8.7, 5.5 Hz, 2H), 7.39 (t, J = 8.8 Hz, 2H), 7.10 (d, J = 8.5 Hz, 1H), 6.90 (t, J = 8.2 Hz, 1H), 3.58 (s, 2H),
3.56 (m, 4H), 3.48 (m, 2H), 3.26 (d, J = 11.3 Hz, 2H), 2.78 (t, J = 10.2 Hz, 2H), 2.73 (m, 1H), 2.64 (s, 3H), 1.85–1.69
(m, 4H). 13C NMR (101 MHz, DMSO) δ 173.33, 170.37, 164.59, 162.77, 162.12, 160.74, 150.07, 147.65, 143.27,
139.46, 130.24, 130.15, 129.78, 116.54,116.30, 115.93, 115.17, 107.66, 104.68, 85.25, 66.88, 66.70, 56.50, 51.97(2C),

37.33, 29.39(2C), 19.02, 15.13.
4.1.14.2
(Z)-4-(4-fluorophenyl)-2-(2-(2,2,2-trifluoro-1-(6-fluoro-2-methyl-5-(4-(pyrrolidine-1-carbonyl)piperidin-1-yl)-1H-ind
ol-3-yl)ethylidene)hydrazinyl)thiazole-5-carbonitrile (18da) Yellow solid; Yield: 23.5%; HPLC(%): 96.3%; M. p.:
136.8.3-137.4 ; MS(ESI) m/z: 531.46 [M+H]; 1H NMR (400 MHz, DMSO) δ 12.56 (s, 1H), 11.83 (s, 1H), 7.96 (dd,
J = 8.8, 5.4 Hz, 2H), 7.38 (t, J = 8.9 Hz, 2H), 7.14 (d, J = 8.6 Hz, 1H), 6.94 (t, J = 8.1 Hz, 1H), 3.21 (m, 2H), 3.06 (m,
4H), 2.25 (s, 3H), 1.86 (dd, J = 13.1, 6.6 Hz, 2H), 1.77 (dd, J = 13.5, 6.6 Hz, 2H), 1.72 (m, 4H), 1.20 (t, J = 7.3 Hz,
3H). 13C NMR (101 MHz, DMSO) δ 172.95, 170.91, 164.70, 162.80, 162.24, 160.47, 148.99, 146.56, 139.27, 134.96,
132.89, 130.38, 130.29, 129.30, 117.09, 116.64, 116.42, 115.19, 114.93, 107.48, 94.72, 52.12(2C), 46.16, 28.92(2C),
27.32, 26.20, 24.28, 19.04, 12.67.
4.1.14.3
(Z)-2-(2-(1-(5-(4-(4-ethylpiperazine-1-carbonyl)piperidin-1-yl)-6-fluoro-2-methyl-1H-indol-3-yl)-2,2,2-trifluoroethyli
dene)hydrazinyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (20fa) Yellow solid; Yield: 21.7%; HPLC(%): 91.8%; M.
p.: 174.3-175.8 ; MS (ESI)m/z: 531.46 [M+H]; 1H NMR (400 MHz, DMSO) δ 12.68 (s, 1H), 11.76 (s, 1H), 8.61 (s,
1H), 8.00 (dd, J = 8.8, 5.5 Hz, 2H), 7.36 (t, J = 8.8 Hz, 2H), 7.06 (d, J = 8.5 Hz, 1H), 6.87 (t, J = 8.2 Hz, 1H), 3.47 (m,
4H), 3.22 (m, 2H), 2.79–2.71 (m, 2H), 2.70 (s, 1H), 2.60 (s, 3H), 2.34 (m, 6H), 1.81–1.62 (m, 4H), 0.98 (t, J = 7.6 Hz,
3H). 13C NMR (101 MHz, DMSO) δ 173.01, 170.37, 164.58, 162.76, 162.10, 160.74, 147.61, 143.29, 139.44, 130.23,
130.14, 129.76, 116.67, 116.53, 116.32, 115.91, 115.17, 107.65, 104.65, 85.21, 56.47, 51.94, 51.91(2C), 37.42, 36.22,
29.44(2C), 18.99, 12.16.
4.1.15 1-(4-fluoro-2-nitro-5-(piperazin-1-yl)phenyl)propan-2-one (21)
To a stirring solution of 13(4.2 g, 0.05 mol) in MeCN, TEA (6 g, 0.15 mol) and piperazine (3.5 g, 0.1 mol) were
added successively at ambient temperature. The reaction mixture was refluxed for 2 h. After cooling to room
temperature, the solvent was evaporated in vacuo. The crude product was diluted with water, filtered and dried to
afford the desired compound 21 as a yellow solid in 87.5%yield.
4.1.16 1-(5-(4-(2-(dimethylamino)ethyl)piperazin-1-yl)-4-fluoro-2-nitrophenyl)propan-2-one (22)
To a mixture of 2-chloro-N, N-dimethylethan-1-amine hydrochloride (2.1 g, 0.015 mol) and NaHCO3(2.5 g, 0.03
mol) dissolved in i-PrOH (40 mL) was added compound 21(2.8 g, 0.01 mol) at 25 . The reaction mixture was stirred
at reflux for 5h. After cooling to room temperature, filtered and the filtrate was evaporated under vacuum. The residue
was suspended in water (100 mL) and extracted with EA (50 mL×2). Then the organic phases were collected and
concentrated in vacuo to get the compound 22 as a yellow solid in 81.1% yield.
4.1.17 2-(4-(6-fluoro-2-methyl-1H-indol-5-yl)piperazin-1-yl)-N,N-dimethylethan-1-amine (23)
To a mixture of THF (5 mL) and H2O (30 mL), 15(4 mmol) was added at interior temperature. Add Na2S2O4(5 eq.)
in batches slowly with stirring. The resulting suspension was stirred at reflux for 2 h. The mixture was cooled and
evaporated in vacuo. The residue was brought up to water, extracted with n-butyl alcohol (50 mL×2) and the combined
organic phases was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to a brown solid in
69.0% yield.
4.1.18 1-(5-(4-(2-(dimethylamino)ethyl)piperazin-1-yl)-6-fluoro-2-methyl-1H-indol-3-yl)-2,2,2-trifluoroethan-1-one
(24)
At room temperature, 23 (0.01 mol) was dissolved in DMF (30 mL) and 2,2,2-trifluoroacetic anhydride (2 eq.) was

added dropwise at 0 . After addition, the reaction mixture was stirred at ambient temperature for 1.5 h. When the
reaction is completed as indicated by the TLC result, the mixture was poured into water (100 mL) and extracted with
DCM three times. The organic layers were combined and washed with LiCl solution. It was then concentrated in
vacuo to give 24 as a solid in 60.2% yield.
4.1.19 General procedure for preparation of target compound (25a, 25b)
To a solution of 24 (0.001 mol) in toluene (30 mL), hydrazine (0.001 mol) and p-TsOH (0.002 mol) were added at
ambient temperature. After addition, the resulting mixture was heated to reflux and stirred for 3 h. After cooling to
room temperature, the solvent was removed under reduced pressure. The residue was dissolved in DCM (100 mL),
washed with an aqueous saturated NaHCO3 solution. The organic layers were then dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo. The crude products were further purified by chromatography on silica gel to afford
the desired compound.
4.1.20.1
(Z)-2-(2-(1-(5-(4-(2-(dimethylamino)ethyl)piperazin-1-yl)-6-fluoro-2-methyl-1H-indol-3-yl)-2,2,2-trifluoroethylidene)
hydrazinyl)-4-(4-fluorophenyl)thiazole-5-carbonitrile (25a) Yellow solid; Yield: 23.5%; HPLC(%): 93.1%; M. p.:
164.0-165.8 ; MS(ESI)m/z: 617.43 [M+H], 615.31 [M+H]; 1H NMR (400 MHz, DMSO) δ 11.79 (s, 1H), 7.96 (dd, J
= 8.3, 5.6 Hz, 2H), 7.35 (t, J = 8.7 Hz, 2H), 7.13 (d, J = 8.5 Hz, 1H), 6.90 (t, J = 8.1 Hz, 1H), 3.16 (d, J = 5.4 Hz, 2H),
2.97 (s,4H), 2.74 (s, 6H), 2.71 (s, 2H), 2.62 (s, 4H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSO) δ 164.37, 161.91,
160.36, 148.86, 146.42, 138.64, 134.79, 134.69, 131.83, 131.76, 130.08, 130.00, 129.65, 117.36, 117.19, 116.33,
116.11, 115.69, 114.32, 107.23, 55.27, 53.13(2C), 52.33, 51.44(2C), 45.64, 43.00(2C).
4.1.20.2
(Z)-2-(2-(1-(5-(4-(2-(dimethylamino)ethyl)piperazin-1-yl)-6-fluoro-2-methyl-1H-indol-3-yl)-2,2,2-trifluoroethylidene)
hydrazinyl)-4-(4-methoxyphenyl)thiazole-5-carbonitrile (25b) Yellow solid; Yield: 24.7%; HPLC(%): 93.6%; M. p.:
202.5-203.6 ; MS(ESI)m/z: 629.27 [M+H], 627.43 [M+H]; 1H NMR (400 MHz, DMSO) δ 11.90 (s, 1H), 7.89 (d, J
= 8.8 Hz, 2H), 7.15 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.92 (t, J = 8.3 Hz, 1H), 3.81 (s, 3H), 3.19 (t, J = 6.0
Hz, 2H), 2.99 (s, 4H), 2.75 (s, 6H), 2.64 (s, 4H), 2.51 (s, 2H), 2.26 (s, 3H). 13C NMR (101 MHz, DMSO) δ 166.78,
154.44, 152.01, 144.83, 140.58, 137.63, 135.04(2C), 130.90, 128.54, 125.82, 122.88, 122.70, 121.10, 120.37(2C),
120.19, 113.07, 100.58, 92.20, 61.42, 58.79(3C), 57.94, 57.08(2C), 51.33, 48.67(2C).
4.2 Biological section
4.2.1 In vitro FS-3 based ATX enzyme assay
Starting from 10 μM highest concentration, 10 μL of a diluted series of compounds, 1/10 dilution, were added to the
wells. Human ATX protein (Echelon Biosciences, Inc. Salt Lake City,UT) was used at a final concentration of 0.4/0.64
μg/mL. The enzyme was diluted in 50 mM Tris-HCl at pH 8.0, 5 mM KCl, 250 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,
and 0.1% fatty acid free BSA to a total volume of 20 μL. The enzyme complex was added to compounds followed by
incubating for 45 min at room temperature with shaking. The reaction was started from the addition of 20 μL of 1 μM
FS-3(Echelon Biosciences, Inc. Salt Lake City,UT) diluted in the buffer mentioned above. The rate of increase in
fluorescence was measured at 37 °C every minute for 30 min using a Multi-Mode Microplate Reader (Thermo
Varioskan Flash, excitation 485 nm, emission 528 nm). Data was analyzed using Statistical Product and Service
Solutions (SPSS).
4.2.2 MTT assay

Cells were introduced into 96-well plates at a density of approximate 5×104/well. 24 h later, triplicate wells were
treated with media and various concentrations of compounds. After 72 h, the drug containing medium was replaced by
100 µL fresh medium with 5 mg/mL MTT solution. After 4 h of incubation, the medium with MTT was removed, and
100 µL of DMSO was added to each well. The plates were gently agitated until the purple formazan crystals were
dissolved, and the OD490 was determined using a microplate reader (MK3, Thermo, Germany). The data were
calculated and plotted as the percent viability compared to the control. The IC50 was defined as the concentration that
reduced the absorbance of the untreated wells by 50% of the vehicle in the MTT assay.
4.3 Molecular modeling study
4.3.1 Molecular docking
The molecular docking was performed with Schrodinger 2016. The protein files (PDB 5MHP and 5L0K) were
obtained from Protein Data Bank (http://www.rcsb.org/pdb/). The complex was prepared through several steps, such as
delete waters beyond 0 Å and analyze the work space. The complex was then split into ligands, water, other.
Subsequently, the protein model was created a binding grid within 15 Å radius around the reference ligand
(GLPG-1690). GLPG-1690, 8ka, 9j and 25a were drawn with ChemBioDraw 3D and thoroughly minimized by
CHARMm force field. Finally, possible conformations were searched in the binding site using the Glide protocol in
default settings (XP precision). The binding results were inspected with Discovery Studio 2016 and the 2D binding
mode figure was generated from it. The 3D figures were obtained from Pymol (The Pymol Molecular Graphics
System, Version 1.4.1. Schrodinger, LLC).
4.3.2 Binding free energy analysis
Taking the binding results of 8ka, 9j and 25a in a maestro format as the template, the complexes was analysis in
Schrodinger 2016 under ‘task’ view with MM-GBSA method. The superimposition of GLPG-1690, 8ka, 9j and 25a
were implemented for a better visual effect to analyze the binding free energy contributions. Generally, the ΔG_Bind
(binging free energy), ΔG_Bind_Coulomb, ΔG_Bind_Hbond, ΔG_Bind_Lipo, ΔG_Bind_Packing and ΔG_Bind_vdW
results was selected as the main factor in a excel file and the column diagram was finally mapped for a direct
inspection.
Acknowledgements
This work was supported by National Natural Science Foundation of China (No. 81872751), Development Project
of Ministry of Education Innovation Team (No. IRT1073) and Technology Innovation Guide Fund of Shenyang
Pharmaceutical University (No. DFJJ2018201).
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