Discovery of Novel Indole-Based Allosteric Highly Potent ATX Inhibitors with Great in Vivo Efficacy in a Mouse Lung Fibrosis Model

Article abstract of DOI:10.1021/acs.jmedchem.0c00506

Autotaxin (ATX) is the dominant catalytic enzyme accounting for the lipid mediator lysophosphatidic acid (LPA) through hydrolysis of lysophosphatidylcholine (LPC). There is great interest in developing nonacidic ATX inhibitors with a specific binding mode to serve as potential in vivo effective therapeutic tools. Herein, dating from a high-throughput screening (HTS) product Indole-1 (740 nM), a dedicated optimization campaign was implemented through derivatizing the-COOH group to versatile linkers that well-bridged the indole skeleton and the hydrophobic pocket binding groups. Ultimately, it was established that the coexistence of a carbamate linker and-OH-group-containing amines could generally furnish excellent indole-based ATX inhibitors with even below 1 nM in vitro activities. Two optimal entities were advanced to a bleomycin-induced mice pulmonary fibrosis model, which exerted promising efficacy in alleviating the damaged lung texture caused by bleomycin exposure. The novel carbamate-containing indole-based ATX inhibitors with a concrete binding mode may contribute to the identification of potential therapeutic agents to intervene in fibrotic diseases.

Full text of DOI:10.1021/acs.jmedchem.0c00506

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Article
Discovery of Novel Indole-based Allosteric Highly Potent ATX
Inhibitors with Great in vivo Efficacy in Mouse Lung Fibrosis Model
Hongrui Lei, Ming Guo, Xiaopeng Li, Fang Jia, Changtao Li,
Yu Yang, Meng Cao, Nan Jiang, Enlong Ma, and Xin Zhai
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00506 • Publication Date (Web): 01 Jun 2020
Downloaded from pubs.acs.org on June 1, 2020
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Discovery of Novel Indole-based Allosteric Highly Potent ATX Inhibitors with Great in vivo
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Efficacy in Mouse Lung Fibrosis Model
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Hongrui Lei , Ming Guo , Xiaopeng Li , Fang Jia , Changtao Li , Yu Yang , Meng Cao , Nan Jiang , Enlong Ma
b*, Xin Zhai a*
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aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China.
bDepartment of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.
Abstract
Autotaxin (ATX) is the dominant catalytic enzyme accounting for the lipid mediator lysophosphatidic acid (LPA)
through hydrolysis of lysophosphatidylcholine (LPC). There is great interest in developing non-acidic ATX inhibitors
with specific binding mode to serve as potential in vivo effective therapeutic tools. Herein, dating from an HTS
product Indole-1(740 nM), a dedicated optimization campaign was implemented through derivatizing the -COOH
group to versatile linkers that well bridged the indole skeleton and the hydrophobic pocket binding groups. Ultimately,
it was established that the coexistence of carbamate linker and a -OH group containing amines could generally furnish
excellent indole based ATX inhibitors with even below 1 nM in vitro activities. Two optimal entities were advanced
to a bleomycin induced mice pulmonary fibrosis model which exerted promising efficacy in alleviating the damaged
lung texture caused by bleomycin exposure. The novel carbamate-containing indole based ATX inhibitors with
concrete binding mode may contribute to the identification of potential therapeutic agents to intervene the fibrotic
diseases.
Introduction
Lysophosphatidic acid (LPA) is a bioactive serum-borne lipid mediator which exerts its physiological effect through
acting on specific G-protein coupled receptors (GPCRs) LPAR1-6.1 Accumulating evidence suggests that downregulation
of LPA signaling has been proven effective in various diseases, such as tumor metastasis,2,3 fibrosis,4-6 pruritis, multiple
7
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sclerosis, inflammation and so on. Typically, autotaxin (ATX) turns out to be the predominant enzyme accounting for
physiological source of extracellular LPA through a lysophosphatidylcholine (LPC) hydrolysis process (Figure 1).10,11
ATX is the sole member of ectonucleotide pyrophosphatase/phosphodiesterase family (ENPPs) that harbors lysoPLD/
12
ATX activity over lysophospholipids. Structurally, ATX is composed of two N-terminal somatomedin B (SMB)‐like
13
domains, a central catalytic phosphodiesterase (PDE) domain and an inactive nuclease-like domain. The catalytic bis-
14
Zinc site is connected by a T-junction to a hydrophobic pocket and a partial “hydrophobic tunnel” (Figure 2). The tunnel
referred to as the allosteric site binds steroid molecules, as well as the LPA product, which results in a modulation of ATX
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catalytic efficiency , while the hydrophobic pocket is designated the orthosteric site.
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Figure 1. Function of ATX and uncovered ATX inhibitors. FS-3 assay; LPC choline release assay; *Our screening lead.
Taken together, ATX is endowed with the dual roles as an LPA producer and carrier, transporting LPA to distal sites from
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where LPC can be taken. Initially, classical ATX inhibitors derived from lipid analogues shared great similarities with
sphingosine-1-phosphate (S1P) or LPA. With the massive research in depth, ATX inhibitors with distinct chemical
properties were designed which included lipid-based inhibitors,17 DNA aptamers,18 as well as small molecules.15,19-20
Furthermore, small molecules can be in turn classified in four distinct types (I, II, III and IV) depending on their mode of
binding to the ATX tripartite site.15
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Figure 2. Structure and active site of ATX. Type I inhibitor PF-8380 (magenta stick) and type IV inhibitor GLPG-1690 (yellow stick)
were superimposed in the active site (The H-bond was indicated in green dash line while the covalent interaction was shown in red line).
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1
22
Typically, PF-8380 (binds the hydrophobic pocket and the catalytic site, 1) and GLPG-1690 (binds the hydrophobic
pocket and the tunnel, 5) were known as the representative type I and type IV ATX inhibitors, which shared a great
uniformity in binding modes within the hydrophobic pocket. However, PF-8380, as well as the type II (binds the
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hydrophobic pocket, 2 ) and type III (binds the hydrophobic tunnel, 3 )entities featuring an acidic head suffered from
poor druggability with none of inhibitors entering clinical trials. Recently, a δ-norleucine based hydroxamic acid derivative
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GK442 (4) was tested in vivo effects in bleomycin (BLM)-induced mice lung fibrosis model with moderate protecting
effects. However, to date, the sole candidate in clinical trial was the type IV inhibitor GLPG-1690 (Ziritaxestat) developed
by Galapagos, which was proved efficacious to reduce extracellular matrix deposition in the pulmonary fibrosis model.22,
2
7-29
Typically,the type IV inhibitors could preclude the binding of bile acid and LPA derivatives to ATX by occupying both
the hydrophobic tunnel and the hydrophobic pocket, avoiding any interactions of substrates with the zinc atoms. Whereas
all types of ATX inhibitors block the formation of LPA, type IV inhibitors could also prevent LPA transport and delivery
to LPAreceptors throughATX channel occupancy. The advantages mentioned above of type IVATX inhibitors may provide
a rationale for their predominant prospect in ATX-LPA axis related diseases.
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Figure 3. Structure optimization process of indole-based ATX inhibitors.
Through a FS-3 based high throughput screening effort, an indole-3-carboxylic acid lead Indole-1 was identified (ATX
IC50: 740 nM). In pursuit of developing non-acidic type IV inhibitors, the carboxylic group was modified to achieve an
efficient occupancy of the hydrophobic pocket. Meanwhile, the tiny dimethylamine fragment was altered to various amines
to explore the possible interactions within the hydrophobic tunnel. Herein, optimization and structure-activity relationships
(SARs) of novel indole-based ATX inhibitors with high in vitro enzymatic activities and impressive in vivo effectiveness
in BLM-challenged mice pulmonary fibrosis model was described.
Results and Discussion
Starting from the HTS hit Indole-1, we conducted a systematic SARs-based optimization campaign in pursuit of
acquiring in vivo effective allosteric highly potent ATX inhibitors. As there was great interest to focus on non-acidic entities
as novel ATX inhibitors for specific binding mode and promising druggability, the -COOH group of Indole-1 was
derivatized in search of a highly tolerable linker that well bridged the indole skeleton and the hydrophobic pocket binding
moieties.
In vitro ATX inhibition assay: Structure-Activity Relationships and binding mode Studies. All synthesized
indole-based derivatives were tested for their in vitro inhibition effect on recombinant mouse ATX using the Amplex Red
PLD assay kit (Echelon Biosciences, Inc. Salt Lake City, UT). FS-3 was used as substrate, and the inhibition results are
presented in Tables 1~4. The molecular weight (MW), cLogP and PSA of the new synthetic compounds predicted using
30
ChemOffice Ultra or corresponding websites were included in the Tables. The ligand lipophilicity efficiency (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. 31-32 The parameters above are important properties
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of target compounds which may provide essential information for the evaluation of druggability.
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Incorporation of the Acyl hydrazone linker. Firstly, the acyl hydrazone was taken into consideration as a potential
linker connecting the indole skeleton and the hydrophobic pocket binding moieties. To this end, the carboxylic acid group
in Indole-1 was derivatized to the corresponding acyl hydrazones, and the dimethylamine group in Indole-1 was replaced
by various secondary amines with expanded volume for an efficient occupancy of the hydrophobic tunnel. An ethyl group
was introduced on position-1 of the indole core as an initial attempt to better fit the protein region. First of all, morpholine
containing compounds (20~23) were explored with the aldehydes/ketones fixed to 4-fluoro benzaldehyde (20), 4-
methyloxy benzaldehyde (21), piperonal (22) and 4-fluoroacetophenone (23). Generally, four compounds did not exert
ATX inhibitory activities superior to Indole-1 (740 nM). It seemed that electron-withdrawing groups on the
aldehyde/ketones in 20 (1370 nM) & 23 (1840 nM) presented a primary affinity over the electron-donating groups in 21
0
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(>10000 nM) and 22 (>10000 nM).
Table 1. FS-3 Based In Vitro Inhibitory Activities of Compounds 20~35 against ATX
IC50a(nM) ±SDb
Compd.
R
1
R
2
Ar
1
MW
cLogP
PSA
LLE
Indole-1
-
-
-
301.09
491.18
503.20
517.18
505.19
489.20
515.20
487.20
491.15
489.20
518.23
2.47
4.68
4.50
4.56
4.65
6.00
5.88
5.46
6.08
5.61
4.70
52.63
59.15
66.70
76.27
58.44
51.24
68.36
59.15
51.61
50.90
71.84
2.66
0.18
740±27
1370± 81
>10000
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2
0
1
H
H
<-0.50
<-0.56
0.09
22
23
H
>10000
-CH
H
3
1840 ± 170
3960 ± 213
1550 ± 170
1460 ± 145
1170 ± 127
2740 ± 154
2530 ± 230
2
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2
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4
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-1.60
-1.07
-0.62
-1.15
-1.05
-0.10
H
H
H
-CH
-CH
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H
H
530.21
503.22
529.21
518.23
534.20
532.24
477.09
588.24
4.61
54.01
51.24
68.36
54.78
54.78
54.07
73.50
77.96
0.51
<-2.26
<-2.14
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-1.03
-0.39
3.77
757 ± 45
>10000
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6.26
6.14
5.22
5.67
5.19
3.80
3.84
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H
1349 ± 98
2310 ± 131
1590 ± 95
2.7 ± 0.32
1.3 ± 0.11
3
3
4
5
H
-CH
-
3
PF-8380
-
-
-
-
GLPG-1690
-
4.05
adata were the results in at least two independent assays.
However, once morpholine was altered to piperidine, pyrrolidine and N-methyl piperazine, the tendency was vanished.
For example, the 4-F-phenyl containing 24 (3960 nM), 28 (2740 nM) and 29 (2530 nM) performed worse than the
corresponding alkyloxy phenyl derivatives 25 (1550 nM), 26 (1460 nM) and 30 (757 nM), especially, 30 performed similar
potency with Indole-1. In view of the atom volume, there was no clear trend between the F- and Cl-containing compounds,
such as 27 (2740 nM) vs 26 (1170 nM) and 33 (1349 nM) vs 34 (2310 nM). The 4-methyl piperidine derivatives 31 and 32
exerted IC50s over 10000 nM perhaps due greatly to the highly hydrophobic property of the 4-methylpiperidine ring. As
expected, the N-ethyl piperazine compounds (33~35) were detected similar ATX inhibition potency with the counterpart
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9~30 bearing N-methyl piperazine. In the meantime, the utilization of aldehyde or ketone may exert no obvious influence
in activity for that the 4-fluoro benzaldehyde products presented parallel enzymatic profile with 4-fluoroacetophenone (20
vs 23 or 33 vs 35). Taken together, the first trial may turn out to be a frustrating attempt with only micromolar activities
achieved. Hence, further modifications were put on agenda.
Exploring the indole N-alkyl substituent. The preliminarily involvement of the 1-indole ethyl resulted to be a temporary
expedient which may be a potential sally port for the following research. Generally, the former uncovered ATX inhibitors
1
5
bore no active hydrogens in the skeleton section. Hence, the primary ethyl group was replaced with a more versatile
methyl which led to the design and synthesis of 36~38 as well as 39~41. The morpholine and N-methyl piperazine with
relatively better potency above were selected as the reference amines.
Table 2. FS-3 Based In Vitro Inhibitory Activities of Compounds 36~41 against ATX
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Compd.
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Ar
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MW
cLogP
PSA
LLE
IC50a(nM) ±SDb
3
3
3
3
4
4
6
7
8
9
0
1
H
H
477.16
493.13
503.16
490.20
504.21
516.19
4.21
4.66
4.10
4.27
4.24
4.15
59.98
59.98
77.10
55.55
54.84
72.67
0.33
<-0.66
<-0.10
0.01
2870 ± 211
>10000
H
>10000
H
5240 ± 342
>10000
-CH
H
3
<-0.24
0.91
879 ± 77
adata were the results in at least two independent assays.
However, much to our disappointment, the 1-methyl derivatives (36, 39 and 41) exerted a little weaker potency relative
to the corresponding 20, 29 and 30. Even more frustrated, 37, 38 and 40 were detected loss of ATX inhibition effect. These
results suggested that the substituent on position-1 may not be a key factor for an efficient promotion of the activity.
Therefore, we retained the preliminarily tolerated ethyl group for the following identification.
Introduction of -OH containing amines. Given our hypothesis that the aliphatic amines stretched to the hydrophobic
tunnel or even exposed to the solvent region, the nature of the amine itself may account a lot for the binding affinity with
ATX. In this sense, we further introduced the -OH containing amines to the chloromethyl thiazole moiety in pursuit of
acquiring more efficient inhibitors. Subsequently, compounds 42~45, 46~48 and 49~52 were synthesized with the same
procedure as 20~41.
Table 3. FS-3 Based In Vitro Inhibitory Activities of Compounds 42~52 against ATX
IC50a(nM) ±SDb
Compd.
R
1
R
2
R
3
MW
cLogP
PSA
LLE
4
4
2
3
H
H
505.19
521.17
4.22
4.63
66.97
66.97
0.82
1.11
906±121.0
180±27.0
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4
4
4
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5
5
5
5
5
5
5
6
4
4
-CH
H
3
519.21
531.19
505.19
519.21
531.19
534.22
550.19
548.24
560.22
3.79
3.85
4.57
4.14
4.19
4.06
4.43
4.03
3.94
66.25
84.08
68.54
67.83
85.65
71.69
71.69
70.98
88.81
2.37
2.12
0.61
2.66
1.02
1.81
1.21
1.26
0.94
69.4±4.5
107.4±17.0
655±65.0
45
46
47
H
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-CH
H
15.7±1.1
3
4
4
5
5
5
8
9
0
1
2
610 ±47.0
134.9±13.8
231.1 ±25.3
507.9±31.5
1320± 127.0
H
H
-CH
H
3
adata were the results in at least two independent assays.
Surprisingly, except for 45 (IC50 =1320 nM), the -OH containing analogs 42~52 exerted sub-micromolar IC50 values or
even better. Among these, 44 and 47 were detected as the most potent ATX inhibitors with IC50 values of 69.4 nM and 15.7
nM, respectively. Given the obvious improvement in activities, we performed a docking study using the 3D template of 47
21
with the cocrystal structure of ATX (5MHP), and the computational results were presented in Figure 4.
Figure 4. (A) Binding mode of 47 with ATX; (B) Overlapping of 47 with GLPG-1690 within the active site of ATX.
As shown in Figure 4A, the indole skeleton made two essential π-π interactions with residues Phe250 and Phe275.
Meanwhile, another key π-π interaction was observed between the thiazole ring of 47 and Trp255. The prolinol moiety did
stretch into the solvent region through the hydrophobic tunnel. As expected, the acyl hydrazone linker typically bridged
the indole skeleton and the 4-fluorophenyl group and directed the 4-fluorophenyl group to the hydrophobic pocket formed
by Phe274-Phe275-Trp276 and other residues. In Figure 4B, 47 and GLPG-1690 were superimposed within the active site
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of ATX. It was apparently shown that 47 adopted a quite similar pose with the reference GLPG-1690. However, mainly
due to the rigid carbonyl group in the acyl hydrazone moiety, the 4-fluorophenylgroup was away from (perpendicular to)
the 4-fluorophenyl moiety in GLPG-1690 which may account for a poor occupancy of the hydrophobic pocket. This virtual
docking offered a potential rationale for the difference between 47 and GLPG-1690 (15.7 nM vs 1.3 nM).
The amide bond reversal leading to carbamate analogues with -OH containing amines. The apparent enhanced
potency of 47 encouraged us greatly for a new stage of modification. Given the rigidness property of the carbonyl group
on 3-position of indole backbone, it was hypothesized to conduct an amide bond reversal strategy to yield flexible linkers.
Ultimately, the versatile carbamate moiety was recommended as a surrogate for the former acyl hydrazone (Figure 5).
Various benzyl alcohols were introduced to implement a condensation reaction following the Curtius rearrangement
reaction of indole-3 carboxylic derivatives.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Amide bond reversal strategy to transform the acyl hydrazone group to the carbamate linker.
According to the SARs above, the well tolerated -OH containing amines, prolinol, 4-OH piperidine, N-hydroxyethyl
piperazine as well as diethanolamine were explored firstly. Among the 4-OH piperidine derivatives (53~59), 53~57 were
detected sub-micromolar IC50 values ranging from 117 nM to 970 nM. It was noteworthy that the unsubstituted phenyl
group 55 (970 nM) performed worse than others. 58 with a 1,3-benzodioxole moiety exerted even better activity (34.1 nM)
although an unfavorable drug-likeness score (0.92) was obtained. To our great surprise, the 3,4-difluoro derivative 59
displayed 1.01 nM activity which may suggest a considerable breakthrough. In view of the impressive potency of 59, we
performed a detailed virtual docking between 59 and ATX (Figure 6).
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 6. (A) Binding mode of 59 with ATX; (B) Overlapping of 59 with GLPG-1690 within the active site of ATX; (C) Overlapping of
9 with PF-8380 within the active site of ATX (The H-bonds/π-π interactions were indicated in green/red dash lines while the covalent
5
interaction was shown in red line).
As shown in Figure 6A, the thiazole ring and the indole skeleton of 59 produced similar π-π interactions as that in 47
with Trp255, Phe250 as well as Phe275. Importantly, in Figure 6B, although the carbamate moiety in 59 was away from
the 5-cyano thiazole fragment in GLPG-1690, it could be obviously found that the 3,4-difluorophenyl group in 59 were
almost overlapped totally with the 4-fluorophenyl counterpart. This may ideally account for the excellent enzymatic activity
of 59 in vitro.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As a continuation, the 4-OH piperidine was replaced with L-prolinol which furnished 60~65. Generally, 61 and 64 were
detected promising ATX IC50s around 35 nM which failed to exceed that of 59.
Subsequently, the extended -OH containing amine N-hydroxyethyl piperazine with an extra chain was explored after the
successfully synthesis of compounds 66 and 67. To our great surprise, 66 (0.43 nM) was detected sub-nanomolar activities
while 67 was 150 folds less potent (62.7 nM). This discrimination between the two compounds may result from the
hindrance of the 2-Cl substituent in the benzyl carbamate moiety of 67. To further verify the potential role of introducing
N-hydroxyethyl piperazine, a dedicated computational docking was implemented with 66 as a template and the results were
shown in Figure 7.
Figure 7. (A) Binding mode of 66 with ATX; (B) Overlapping of 66 with GLPG-1690 within the active site of ATX; (C) Overlapping of
6
6 with PF-8380 within the active site of ATX (The H-bonds/π-π interactions were indicated in green/red dash lines while the covalent
interaction was shown in red line).
Although the benzyl carbamate group in 66 was not thoroughly superimposed as that in 59 and the 4-fluorophenyl moiety
in GLPG-1690, it did adopt a highly similar binding pose with the phenyl thiazole part in GLPG-1690. An essential 1.5 Å
H-bond was detected between the carbamate NH- and Phe274 which greatly restrained the binding conformation of 66
within the hydrophobic pocket. It was noteworthy that the necessary H-bond was absent in 47 and 59. Most importantly,
the -OH group in 66 further approached the solvent region and formed a significant 2.9 Å H-bond with residue Gly256.
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Taken together, the presence of two necessary H-bonds well explained the exceeding of 66 (0.43 nM) over GLPG-1690
1.3 nM) in ATX inhibitory activity.
Table 4. FS-3 Based In Vitro Inhibitory Activities of Compounds 53~69 against ATX
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compd.
R
1
R
R
2
MW
cLogP
PSA
LLE
IC50a(nM) ±SD
5
5
5
5
5
3
4
5
6
7
3,5-di-Cl
4-Me
H
-CH
H
558.13
518.24
490.20
538.18
558.13
534.19
526.19
490.20
518.24
538.18
558.13
534.19
526.19
587.15
587.15
562.12
538.19
6.05
5.27
4.62
5.47
5.81
4.77
5.04
4.53
5.19
5.38
5.72
4.68
4.95
5.27
5.03
3.83
3.35
62.50
61.79
62.50
61.79
62.50
79.62
62.50
64.07
63.36
63.36
64.07
81.19
64.07
67.22
67.22
80.30
97.41
-0.84
0.66
0.39
0.21
0.09
1.70
2.96
1.38
1.24
0.32
0.18
1.78
0.58
3.10
1.17
2.84
3.16
613±78.5
117± 21.7
970 ±45.1
210 ± 17.1
127 ± 15.3
34.1 ± 2.1
1.01±0.2
3
H
2-Cl
-CH
H
3
2,3-di-Cl
58
59
H
3,4-di-F
H
H
6
6
6
6
6
6
0
1
2
3
4
5
H
121.8±13.1
36.9±5.3
4-Me
2-Cl
-CH
-CH
H
3
3
199.4±9.5
127.3 ± 8.7
34.7 ±4.5
297.2±31.7
0.43±0.05
62.7 ± 7.4
21.3±2.8
2,3-di-Cl
H
3,4-di-F
3,5-di-Cl
2,3-di-Cl
3,5-di-Cl
H
66
67
H
H
6
6
8
9
H
H
30.7±5.1
adata were the results in at least two independent assays.
After identifying the pretty powerful -OH containing carbamate derivatives, we then synthesized 68 and 69 to develop
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9
1
1
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1
1
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2
2
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the possible effect of the number of -OH group. The enzymatic results that the diethanolamine finally furnished moderate
ATX inhibitory activities (68 21.3 nM, 69 30.7 nM) suggested no regular pattern in the activity and the -OH count. In this
end, it could be preliminarily concluded that the combination of -OH containing amines, especially N-hydroxyethyl
piperazine, and the carbamate moiety generally provided the highly potent ATX allosteric inhibitors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Superimposing of 47, 59 and 66 with GLPG-1690 within the active site of ATX. For an intuitive vision, the binding
poses of the representative compounds 47, 59 and 66 were placed in the cocrystal structure of GLPG-1690 andATX (Figure
8
). Firstly, it could be easily seen that the benzyl carbamate moiety of 47 (cyan lines) was a little bit away from the exact
binding site of the hydrophobic pocket. Next, the amide reverse strategy from the acyl hydrazone did bring a highly flexible
linker carbamate. Although the carbamate fragment of 59 was twisted to some degree, its phenyl group surprisingly
achieved an utmost consistence with the benzene ring of GLPG-1690. Then, the -OH group in 66 stretched further
approaching the solvent region and formed a significant H-bond with Gly256. Finally, the principle H-bond between 66
and Phe274 may turn out to be the marked contribution to its extra good in vitro enzymatic activities.
Figure 8. The binding mode of 47 (cyan lines), 59 (orange lines) and 66 (pink lines) superimposed with GLPG-1690 (yellow lines) within
ATX active site. (The hydrogen bonds were indicated in green dash lines).
To conclude, after a dedicated SARs-guided optimization, we identified a series of highly potent ATX inhibitors (sub-
nanomolar activities) with specific binding modes through efficiently interactions with the hydrophobic pocket and the
allosteric partial hydrophobic tunnel. It was envisioned that the combination of carbamate linker with a -OH containing
amines could offer a robust ATX inhibitory potency. We hope it works to promote the efficiency in identifying novel ATX
inhibitors through this discovery.
Ex vivo ATX enzymatic assay and In vivo prophylactic effect of 59 and 66 on a Bleomycin-induced Mice
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Pulmonary Fibrosis Model. After verifying the in vitro enzymatic activities of target compounds (59 and 66), the next
stage was to evaluate their in vivo effects in inhibitingATX activity. The BLM-induced pulmonary fibrosis model in rodents
turn out to be the principle animal model to examine the compounds as potential therapies for IPF.33 It was well known
that the concentration and activity of ATX increase obviously which may account for the severity of fibrosis.34 The
C57Bl/6J mice were undergone oral treatment with GLPG-1690 (60mg/kg) or compounds 59 and 66 (20 or 60 mg/kg)
once a day for 31 days (day -3 to day 28). After three days (day +1), the mice in test groups (5 mice per group) were
subjected to a tracheal administration of BLM to build the IPF model (Figure 9A). Meanwhile, the model group was treated
with saline as surrogate of the test compounds whereas the control group was the untreated mice with normal lung
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
35
textures. Of note, death only occurred within the BLM model group (3 death in total). In the meantime, the body weight
changes of the mice were shown in Figure 9C and 9D. It was apparently that the test compounds (59, 66 and GLPG-1690)
could bring encouraging effects in protecting the BLM-treated mice with recovering of body weights. These results
preliminarily suggested a potential protective effect of the tested compounds on mice with injured lung tissues.
Ex vivo ATX enzymatic assay of 59 and 66 on a Bleomycin-induced Mice Pulmonary Fibrosis Model. The ex vivo
ATX lysoPLD activity was measured by the fluorescence excitation through the cleavage of the phospholipid bond in the
artificial substrate FS-3. A control sample was prepared to account for reference fluorescent absorption using 10 μL lung
homogenate from the control group followed by addition of FS-3 substrate and the standard buffer. The relative absorbance
values were directly utilized for an intuitive analysis of the ex vivo ATX inhibitory effects of compounds 59, 66 and GLPG-
1
9
690. The lower absorbance value indicates a high inhibition of the ex vivo ATX activity. The results were shown in Figure
B.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. (A) Schematic representation of experimental protocol; (B) The ex vivo enzymatic assay of GLPG-1690 (5), 59 and 66 (data
were drawn from the absorbance of the cleaved FS-3 by in vivo ATX; The tested lung homogenate samples were derived from the lung
tissues in the BLM model treated with Trypsin); (C-D) Body weight change upon BLM administration under the effect of GLPG-1690,
5
9 and 66. Values are presented as means (±SD); ***denotes statistical significance (p < 0.05, Mann-Whitney test).
It was of note that the model group presented a highATX activity that was two folds over that in the control group (~7.09
vs ~3.45). In the groups under treatment with ATX inhibitors, there was obvious decrease in ATX activity. A consistency
between the in vitro inhibition effect and ex vivo efficacy was observed for compounds 59 & 66 with GLPG-1690. 59
reduced the ex vivo ATX activity to a large degree (~3.85) and dose-dependent effect was present (~3.37 in 60 mg/kg).
Alternatively, 66 exerted a better ex vivo efficacy than GLPG-1690 (~4.03 in 60 mg/kg) in both 20 (~3.43) and 60 mg/kg
(~3.25), and a slightly dose-dependent effect was finally achieved. The promising ex vivo potency of 66 provided a rationale
for the potential utilization in the animal models.
In vivo Effectiveness of 59 and 66 on a Bleomycin-induced Mice Pulmonary Fibrosis Model. The promising
compounds 59 and 66 were tested for their efficacy in a 31-day model of pulmonary fibrosis induced by bleomycin. The
severity of the lung tissues was evaluated by the modified Ashcroft score (grade 0~8) which further support the direct
observation.36,37 The efficacy of inhibitors was evaluated based on histopathological changes in lung architecture through
38
H&E staining (Figure 10) and Masson staining (Figure 11) inspection. As shown in Figure 10A, bleomycin administration
resulted in thickening of the alveolar septa, alveolar inflammation, and formation of fibrotic masses (6.28). ATX inhibition
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by compounds 66, 59 and GLPG-1690 generally improved lung texture: contiguous fibrotic walls and fewer fibrotic lesions
were predominantly observed in microscopic field of H&E lung tissues. In the 59 group, the dose-dependent manner was
much more obvious that the 20 mg/kg group present a high level of red staining (4.66). While in the 60 mg/kg group of 59,
similar biological effect (3.20) was observed with that in GLPG-1690 group (2.84).
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
2
2
2
3
3
3
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3
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5
5
6
0
1
2
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0
1
2
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0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 10. In vivo inhibition of ATX function with inhibitors alleviated BLM-induced pulmonary fibrosis viewing through H&E staining.
(
A) Representative stained sections from control mice; (B-C) BLM-challenged mice that received 59 in 20 or 60 mg/kg dose; (D) BLM-
challenged mice that received GLPG-1690 in 60 mg/kg dose; (E) BLM-challenged mice that received vehicle as model group; (F-G)
BLM-challenged mice that received 66 in 20 or 60 mg/kg dose; (A1-G1: ×100 magnification, A2-G2: ×200 magnification); (H)
Quantitative histopathological analysis of fibrosis was performed by the modified Ashcroft score. Values are presented as means (±SD);
*
**denotes statistical significance (p < 0.05, Mann-Whitney test).
In the meantime, conform to the in vivo enzymatic assays, 66 displayed a better protection effect on the impaired
lung architecture than 59. In low doses (20 mg/kg), 66 could bring the BLM-exposed lung tissues to similar patterns
with the control group. Amazingly, in high doses (60 mg/kg), 66 (2.04) exceeded the effect of GLPG-1690 (2.84)
which suggested the potential application in fibrotic relevant diseases.
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1
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3
3
3
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1
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1
2
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0
1
2
3
4
5
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9
0
1
2
3
4
5
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8
9
0
Figure 11. In vivo inhibition of ATX function with inhibitors alleviated BLM-induced pulmonary fibrosis viewing through Masson
staining. (A) Representative stained sections from control mice; (B-C) BLM-challenged mice that received 59 in 20 or 60 mg/kg dose;
(D) BLM-challenged mice that received GLPG-1690 in 60 mg/kg dose; (E) BLM-challenged mice that received vehicle as model group;
(F-G) BLM-challenged mice that received 66 in 20 or 60 mg/kg dose; (A1-G1: ×100, A2-G2: ×200, magnification); (H) Quantitative
histopathological analysis of fibrosis was performed by the modified Ashcroft score. Values are presented as means (±SD); ***denotes
statistical significance (p < 0.05, Mann-Whitney test).
The Masson staining results of testing compounds were depicted in Figure 11. Generally, similar outcomes were
harvested with the H&E staining assays. It was noteworthy that a significant difference was observed between the high
doses of 66 (2.01) and GLPG-1690 (2.74). Although no superior results of 59 over GLPG-1690 were present in Masson
staining assay, the repairing effects of 66 on the damaged lung texture deserved more attention.
Synthesis of indole-based allosteric ATX inhibitors. First of all, the lead compound Indole-1 was synthesized
as depicted in Scheme 1. The commercially available indole-5-carbonitrile (6) was reacted with di-tert-butyl-di-
carbonate under 4-dimethylaminopyridine (DMAP) catalysis to obtain intermediate 7 in 94.3% yield. The cyano
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group of 7 was conveniently converted to thioamide in the presence of sodium bisulfide and magnesium chloride
hexahydrate to afford compound 8.39 Condensation of thioamide derivative (8) with 1,3-dichloropropan-2-one in
toluene gave rise to 9. Substitution of 9 with dimethylamine hydrochloride produced intermediate 10, which was de-
Boc by hydrochloric acid to resulted in 11. Intermediate 11 was acylated using 2,2,2-trifluoroacetic anhydride in the
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0
presence of N, N-dimethyl formamide at 25 °C to afford 12 in 76.3% yield. Finally, the trifluoroacetyl group in 12
was treated with 6 M NaOH at reflux to furnish Indole-1.41
Scheme 1. Synthesis of Indole-1a
aReagents and conditions: (a) Boc
one, toluene, reflux, 2.5 h; (d) dimethylamine·HCl, K
(CF CO) O, DMF, 55 ºC, 2 h; (g) 6 N NaOH (aq.), MeOH, 70 ºC, 2 h;
O, DMAP, CH
Cl
2
, 25ºC, 1 h; (b) NaHS, MgCl
2
2
2 2
·6H O, DMF, 25 ºC, 3 h; (c) 1,3-dichloropropan-2-
2
CO , MeCN, 25 ºC, 2 h; (e) 4 M HCl in MeOH, DCM, 25-50 ºC, 6 h; (f)
3
3
2
The synthetic efforts of acylhydrazone derivatives were outlined in Scheme 2. The starting material indole-5-carbonitrile
6
was utilized to undergo a typical N-alkylating (methylation or ethylation) reaction to obtain compound 13 in satisfying
yields. Subsequently, the -CN group of 13 was treated with NaHS under the catalysis of MgCl
2
·6H O to give the thioamide
2
39
derivative 14 in 72.0% yield. Next, a cyclization reaction took place between 14 and 1, 3-dichloroacetone to furnish the
thiazole species with a chloromethyl active site. Typically, a trifluoroacetylation reaction went ahead of the N-alkylation in
40
the presence of trifluoroacetic anhydride in DMF to result in 16 in 78.1% yield with the chloromethyl unchanged. Then,
the N-alkylation between 16 and various amines were conducted in a K CO /MeCN system to bring 17a~17k in 80.1~83.2%
yields. Subsequently, the trifluoroacetyl group in 17a~17k was hydrolyzed to corresponding -COOH derivatives 18a~18k
2
3
41
by treating with 6 M NaOH in reflux. 18a~18k subjected to a sequential esterification and hydrazinolysis reaction gave
rise to the acyl hydrazine species 19a~19k as the products. Finally, 19a~19k reacted with various aldehydes or ketones in
ethanol to furnish the title compounds 20~52 in reasonable yields.
Scheme 2. Synthesis of compounds 20-52a
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aReagents and conditions: (a) MeI or EtI, NaH, DMF, 25 ºC, 1 h; (b) NaHS, MgCl
2 4
reflux, 2.5 h; (d) (CF CO) O, DMF, 0 ºC to 25 ºC, 1.5 h; (e) amine, K CO , MeCN, 25 ºC, 2 h;(f) 6 N NaOH (aq.), 100 ºC, 2 h; (g) MeOH, H SO , 25
2
2
·6H O, DMF, 25 ºC, 3 h; (c) 1,3-dichloropropan-2-one, toluene,
3
2
2
3
2 2 2
ºC-reflux, 2 h; (h) H N-NH ·H O, EtOH, reflux, 2 h; (i) aldehyde or ketone, EtOH, reflux, 3 h.
Alternatively, the synthesis of the carbamate derivatives 53~69 was depicted in Scheme 3. 16 utilized as the starting
material underwent a N-alkylation with secondary amines to give 17i~17l, which was further transformed to the carboxylic
acid counterpart 18 series. Subsequently, the -OH group containing amines in 18i~18l were firstly undergoing a -OH
protecting step using (CH
3
CO) O as the acylating agent. The protected 3-indole carboxylic acid intermediates 18i-2~18l-
2
2 were then rearranged to the carbamate derivatives followed by an ester hydrolysis reaction to furnish the final compounds
53~59, 60~65, 66~67 and 68~69.42
Scheme 3. Synthesis of compounds 53-69a
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aReagents and conditions: (a) amines, K
CO
2
3
3 2 2 2
, MeCN, 25 ºC, 2 h; (b) 6 N NaOH (aq.), 100 ºC, 3 h; (c) (CH CO) O, CH Cl , 25 ºC, 2 h; (d) DPPA, TEA,
toluene, reflux, 2.5 h; (e) 1 N NaOH (aq.), dioxane, 25 ºC, 2 h.
Conclusion
In conclusion, we report the optimization campaign from an indole-3-carboxylic acid lead Indole-1 to the potent type
IV ATX inhibitors 59 (1.01 nM) and 66 (0.43 nM) with IC50 values even below 1 nM (GLPG-1690 1.3 nM). The SARs
study showed that carbamate linker ideally bridged the indole skeleton and hydrophobic pocket binding aromatic group
and brought an essential H-bond with residue Phe274 (the carbamate -NH in 66). Meanwhile, the -OH containing amines
well occupied the partial hydrophobic tunnel with the -OH in 66 made an extra H-bond with residue Gly256. In bleomycin
induced mice pulmonary fibrosis model, compound 66 exerted better ex vivo ATX inhibiting effects with lower
fluorescence absorbance (~3.25) than GLPG-1690 (~4.04) and a superior protection function on the injured lung tissues.
Taken together, the identification of the carbamate-bearing indole based ATX inhibitors with preferred in vitro and in vivo
potency may provide alternative therapy for fibrotic diseases.
Experimental
4
.1 Chemistry
The melting points were obtained through a Büchi Melting Point B-540 facility (Büchi Labor technik, Flawil,
Switzerland) which were uncorrected. Mass spectra (MS) were conducted in ESI mode on Agilent 1100 LC-MS (Agilent,
1
13
Palo Alto, CA, USA) and a mixture of H O/MeCN (4:6) was utilized as the eluting system. The H and C NMR spectra
2
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
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purification. The purity of all target compounds was determined by HPLC analysis with a variable wavelength detector
Agilent G1314F) that turned out to exceed 95% purity. The chromatography column was an Agilent column (ZORBAX
SB-C18, 5 μm, 4.6 mm×150 mm, Agilent Corporation). Flow rate: 0.5 mL/min. Wavelength: 254 nm. Column temperature:
5 °C. Mobile phase: MeOH: H O = 95:5. The optical active compounds with one or two stereocenters were material-
derived and unchanged in the transforming procedures, so the optical rotation degrees were not given here.
(
2
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.1.1 The synthetic procedures of the lead Indole-1
.1.1.1 Synthesis of tert-butyl 5-cyano-1H-indole-1-carboxylate (7)
Indole-5-carbonitrile (14.2 g, 0.1 mol) were introduced to a mixture of catalytic DMAP (0.25 g, 0.002 mol) in 80 mL
DCM and stirred for 10 min. Subsequently, di-tert-butyl di-carbonate (24.01 g, 0.11 mol) was added slowly and the system
was kept at ambient temperature for 1 h. Water (100 mL) was added to the mixture and the organic phase was separated,
concentrated to give white product 7 in 94.3% yield (22.82 g).
4
.1.1.2 Synthesis of tert-butyl 5-carbamothioyl-1H-indole-1-carboxylate (8)
To a solvent of DMF (100 mL) was added NaHS (5.6 g, 0.1 mol), MgCl ·6H O (20.3 g, 0.1 mol) and 7 (12.1 g, 0.05
2
2
mol) under stirring. The reaction mixture was stirred at 25℃ for 3 h until completion. Water (500 mL) was added and the
mixture was filtered. The residue was transferred to 0.5 M HCl solution (200 mL), stirred for 20 min and filtered. The
resulting solid was washed with plenty of water to afford 8 as a yellow solid in 77.3% yield (10.68 g).
4.1.1.3 Synthesis of tert-butyl 5-(4-(chloromethyl)thiazol-2-yl)-1H-indole-1-carboxylate (9)
The 1,3-dichloropropan-2-one (1.92 g, 0.015 mol) and 8 (4.14 g, 0.015 mol) was dissolved in toluene (40 mL), stirred
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 81.3% yield (4.25 g).
4
.1.1.4 Synthesis of tert-butyl 5-(4-((dimethylamino)methyl)thiazol-2-yl)-1H-indole-1-carboxylate (10)
Dimethylamine hydrochloride (0.2 mol) and K CO (0.25 mol) were added to a stirred solution of 9 (3.48 g, 0.01 mol)
2
3
in MeCN (35 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, stirred for 30 min, filtered to obtain yellow solid 10 in 75.3% yield (2.69 g).
4
.1.1.5 Synthesis of 1-(2-(1H-indol-5-yl)thiazol-4-yl)-N,N-dimethylmethanamine (11)
0 (2.51 g, 0.007 mol) was dissolved in DCM (10 mL) followed by slowly addition of 4 M HCl (g) in MeOH (20 mL).
1
The mixture was stirred at 50℃ for 6 h until the raw material was completely consumed. The solvent was removed in
vacuo followed by addition of water (100 mL). The aqueous portion was firstly extracted with DCM (40 mL) and the
organic layers were discarded. The pH of the aqueous phase was adjusted to 10 with 2 M NaOH and extracted with DCM
(60 mLⅹ2). The organic layers were combined, dried over Na
2
SO and concentrated in vacuo to bring 11 in 69.3% yield
4
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.1.1.6 Synthesis of 1-(5-(4-((dimethylamino)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethan-1-one (12)
At room temperature, 11 (1.03 g, 0.004 mol) was dissolved in DMF (20 mL). 2,2,2-trifluoroacetic anhydride (0.6 mol)
was added dropwise at 0℃. After addition, the reaction mixture was stirred at 55℃ for 2 h. When the reaction is completed
as indicated by the TLC result, the mixture was poured into water (80 mL) and stirred for 30 min. The solution was filtered
to give 12 as a brown solid in 76.3% yield (1.08 g).
0
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.1.1.7 Synthesis of 5-(4-((dimethylamino)methyl)thiazol-2-yl)-1H-indole-3-carboxylic acid (Indole-1)
Intermediate 12 (0.71g, 0.002 mol) were added to a mixed solvent of 10 mL 4 M aqueous NaOH and 3 mL MeOH which
was refluxed for 2 h. The reaction mixture was cooled to room temperature and the MeOH was purged followed by addition
of more water (20 mL). The aqueous solution was extracted with n-BuOH (20 mL), dried over Na SO , and the organic
2
4
layer was concentrated to give the crude product to be involved in column chromatography purification with an eluting
1
system of DCM: MeOH: HOAc=20:1:1 to furnish Indole-1 in 57.2% yield (0.35 g). H NMR (400 MHz, DMSO) δ 12.09
1
3
(s, 1H), 8.66 (s, 1H), 8.05 (s, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.38 (s, 1H), 4.64 (s, 2H). C NMR
(101 MHz, DMSO) δ 168.92, 159.18, 137.93, 133.75, 126.93, 126.90, 121.06, 119.17, 114.04, 113.29, 109.68, 60.33. The
1
13
dimethylamine signals (RN(CH
were not apparently shown.
4.1.2 The synthetic procedures and structural determination of all target compounds were present as following.
.1.3 1-ethyl-1H-indole-5-carbonitrile (13)
0% NaH (200.2 g, 5.0 mol) was suspended in DMF (800 mL) at ambient temperature. 1H-indole-5-carbonitrile (284.0
3
)
2
) in both H and C spectrum were both sheltered by the DMSO solvent signal which
4
6
g, 2.0 mol) was added when the temperature reached to 40 ºC. After the gas escaped, the solution was cooled to 25ºC. 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 (1500 mL). After stirring for 0.5 h, the mixture was filtered to obtain the solid 13 in 88%
yield.
4
.1.4 1-ethyl-1H-indole-5-carbothioamide (14)
To a solvent of DMF (600 mL) was added NaHS (85.0 g, 3.5 mol), MgCl
2
·6H O (406.0 g, 2 mol) and 13(170 g, 0.7 mol)
2
under stirring. The reaction mixture was stirred at 25ºC 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
1
resulting solid was washed with plenty of water to afford the title compound 14 as a yellow solid 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).
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.1.5 4-(chloromethyl)-2-(1-ethyl-1H-indol-5-yl)thiazole (15)
At room temperature, the 1,3-dichloropropan-2-one (63 g, 0.5 mol) and 14 (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
1
with water. The resulting precipitate was filtered, washed with water to give a yellow solid after drying in 78.9% yield. H
0
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0
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).
.1.6 1-(5-(4-(chloromethyl)thiazol-2-yl)-1-ethyl-1H-indol-3-yl)-2,2,2-trifluoroethan-1-one(16)
4
At room temperature, 15 (108 g, 0.4 mol) was dissolved in DMF (800 mL). 2,2,2-trifluoroacetic anhydride (0.6 mol)
was added dropwise at 0ºC . 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.
1
The solution was filtered to give 16 as a solid 113.5 g in 78.1% yield. H NMR (600 MHz, DMSO) δ 8.80 (d, J = 1.4 Hz,
1
H), 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
1
3
=
7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H). C 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. MS (ESI) m/z (%): 373.37 [(M+H) , 100].
4.1.7 General procedure for preparation of intermediate 17 (17a-17l)
Amine (0.2 mol) and K
2
CO (0.25 mol) were added to a stirred solution of 16 (0.1mol) in MeCN (350 mL) at room
3
temperature. After a 2 h stirring, the reaction mixture was filtered and the filtrate was evaporated. The residue was taken
up in water, stirred for 30 min, filtered to give orange solids 17a-17l in 80.1% to 83% yield.
1
1
-(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethan-1-one(17i): H NMR
(
600 MHz, DMSO) δ 8.78 (d, J = 1.5 Hz, 1H), 8.69 (d, J = 1.6 Hz, 1H), 7.97 (dd, J = 8.6, 1.7 Hz, 1H), 7.86 (d, J = 8.7 Hz,
H), 7.47 (s, 1H), 4.56 (d, J = 3.9 Hz, 1H), 4.45 (q, J = 7.2 Hz, 2H), 3.64 (s, 2H), 3.46 (d, J = 3.7 Hz, 1H), 2.79 (d, J = 10.5
Hz, 2H), 2.16 (s, 2H), 1.73 (d, J = 9.5 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H), 1.44–1.39 (m, 2H).
1
(
(
(
S)-1-(1-ethyl-5-(4-((2-(hydroxymethyl)pyrrolidin-1-yl)methyl)thiazol-2-yl)-1H-indol-3-yl)-2,2,2-trifluoroethan-1-one
1
17j): H NMR (600 MHz, DMSO) δ 8.78 (d, J = 1.6 Hz, 1H), 8.69 (d, J = 1.6 Hz, 1H), 7.97 (dd, J = 8.6, 1.7 Hz, 1H), 7.86
d, J = 8.6 Hz, 1H), 7.48 (s, 1H), 4.45 (q, J = 7.2 Hz, 2H), 4.15 (d, J = 14.3 Hz, 1H), 3.72 (d, J = 14.3 Hz, 1H), 3.47 (dd, J
=
10.5, 4.4 Hz, 1H), 3.32 (dd, J = 10.5, 6.5 Hz, 1H), 3.03 (dt, J = 9.3, 4.6 Hz, 1H), 2.74–2.66 (m, 1H), 2.39 (q, J = 8.5 Hz,
13
1
H), 1.84 (dq, J = 12.2, 8.2 Hz, 1H), 1.70–1.61 (m, 2H), 1.58 (dt, J = 19.3, 6.5 Hz, 1H), 1.46 (t, J = 7.3 Hz, 3H). C NMR
(101 MHz, DMSO) δ 167.16, 156.57, 141.13, 137.67, 129.72, 127.27, 123.23, 119.40, 118.65, 116.48, 115.75, 112.97,
1
08.78, 64.98, 64.58, 54.60, 54.50, 42.60, 28.45, 23.14, 15.61.
4
.1.8 General procedure for preparation of intermediate 18 (18a-18l)
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Intermediates 17a-17l (0.08 mol) were added to 120 mL 4 M aqueous NaOH and refluxed for 2 h. The reaction mixture
was cooled to room temperature and the products 18a-18l were obtained in quantitative yield through a principle filtration
6
7
8
9
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in reduced pressure.
4
.1.9 General procedure for preparation of intermediate 19 (19a-19l)
0
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Intermediates 18a-18l (0.05 mol) were dissolved in 200 mL MeOH followed by slowly addition of concentrated H
2
SO
4
(
5 mL). The mixture was refluxed for 2-5 h until the completely consumption of the raw materials. The solvent was removed
in reduced pressure, 200 mL water was introduced and the mixture was extracted with EA(150 mLⅹ2). The organic layers
were combined, dried with anhydrous Na SO , and concentrated in vacuum to give oil materials which were used in the
following step without further purification.
2
4
The oil materials obtained (0.02 mol) and 15 mL hydrazine hydrate were dissolved in 150 mL EtOH and the system was
refluxed for 2.5 h. After completion, the solvent was discarded in vacuum followed by extraction with DCM in water. The
organic phase was concentrated to give a raw material which was further purified in column chromatography (DCM:
MeOH= 30~10:1) to give 19a-19l as yellow solids in reasonable yields (40% ~70%).
4.1.10 General procedure for preparation of target compounds 20-52
Intermediates 19a-19k (1 mmol) and aldehydes/ketones (1.2 mmol) were added to 15 mL EtOH and refluxed for 3 h.
For 20~23, the final product precipitated from the solvent which could be accessed through direct filtration with high
purities. For compounds 24~52, the solvents were purged to give the crude products which were further purified with
column chromatography/p-TLC (DCM: MeOH= 40~15:1) in acceptable yields.
(
E)-1-ethyl-N'-(4-fluorobenzylidene)-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (20). mp 212-
1
2
14 ºC. H NMR (400 MHz, CDCl
3
) δ 8.87 (s, 1H), 8.44 (s, 1H), 7.84 (s, 1H), 7.59 (s, 2H), 7.33 (s, 1H), 7.29 (s, 2H), 6.98
1
3
(s, 2H), 4.15 (s, 2H), 3.95 (s, 2H), 3.86 (s, 4H), 2.85 (s, 4H), 1.48 (s, 3H). C NMR (101 MHz, CDCl
3
) δ 169.81, 164.89,
1
62.40, 136.88, 134.40, 132.29, 130.64, 130.61, 129.01, 128.93, 127.37, 121.55, 121.00, 120.86, 118.10, 117.91, 115.94,
+
115.72, 110.15, 65.88, 57.69, 52.96(2C), 41.81, 29.71, 15.11. MS (ESI) m/z (%): 492.24 [(M+H) ,65].
(
E)-1-ethyl-N'-(4-methoxybenzylidene)-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (21). mp 222-
1
2
24 ºC. H NMR (400 MHz, DMSO) δ 11.45 (s, 1H), 8.85 (s, 1H), 8.33 (s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.70 (s, 2H), 7.68
(s, 1H), 7.45 (s, 1H), 7.04 (d, J = 7.9 Hz, 2H), 4.35 (s, 2H), 3.82 (s, 3H), 3.66 (s, 2H), 3.61 (s, 3H), 2.51 (s, 3H), 1.47 (t, J
13
=
6.6 Hz, 3H). C NMR (101 MHz, DMSO) δ 168.46, 160.98, 154.32, 132.27, 130.44, 128.85(2C), 127.70, 127.13, 124.28,
1
21.24, 119.97, 118.18, 116.64, 116.01, 114.83(2C), 111.60, 109.14, 66.65(2C), 58.46, 55.75, 53.65(2C), 41.62, 15.64. MS
+
(ESI) m/z (%): 504.23 [(M+H) ,55].
(
E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-ethyl-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide
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(
22). mp 208-210 ºC. H NMR (400 MHz, DMSO) δ 11.47 (s, 1H), 8.85 (s, 1H), 8.31 (s, 2H), 7.83 (s, 1H), 7.69 (s, 1H),
7
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.44 (s, 1H), 7.33 (s, 1H), 7.16 (s, 1H), 7.01 (s, 1H), 6.11 (s, 2H), 4.34 (s, 2H), 3.65 (s, 2H), 3.61 (s, 4H), 2.49 (m, 4H),
13
.47 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, DMSO) δ 168.45, 161.16, 154.33, 149.20, 148.48, 129.57, 128.78, 127.16,
25.58, 123.37, 121.24, 120.03, 116.62, 111.58, 109.03, 108.94, 106.45, 105.46, 102.18, 101.95, 66.65(2C), 58.46,
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+
3.65(2C), 41.60, 15.60. MS (ESI) m/z (%): 518.18 [(M+H) ,45].
(
E)-1-ethyl-N'-(1-(4-fluorophenyl)ethylidene)-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (23).
1
mp 211-213 ºC. H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 8.84 (s, 1H), 8.43 (s, 1H), 7.93 – 7.87 (m, 2H), 7.84 (d, J =
8
4
1
6
.5 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.45 (s, 1H), 7.28 (t, J = 8.7 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.66 (s, 2H), 3.61 (s,
13
H), 2.49 (s, 3H), 2.39 (s, 3H), 1.46 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, DMSO) δ 168.47, 164.36, 161.91, 154.37,
36.94, 135.56, 135.54, 128.89(2C), 128.80, 128.22, 127.16, 121.22, 120.05, 116.61, 115.84(2C), 115.63, 111.58, 108.76,
+
6.67(2C), 58.46, 53.65(2C), 41.60, 15.60, 14.44. MS (ESI) m/z (%): 506.20 [(M+H) , 100].
(
E)-1-ethyl-N'-(4-fluorobenzylidene)-5-(4-(piperidin-1-ylmethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (24). mp 201-
1
2
03 ºC. H NMR (400 MHz, DMSO) δ 11.60 (s, 1H), 8.84 (s, 1H), 8.42 (s, 1H), 7.84 (dd, J = 8.6, 1.5 Hz, 1H), 7.80 (dd, J
= 8.5, 5.7 Hz, 2H), 7.70 (d, J = 8.6 Hz, 1H), 7.43 (s, 1H), 7.31 (t, J = 8.8 Hz, 2H), 4.35 (d, J = 6.3 Hz, 2H), 3.68 (s, 2H),
13
3.38 (s, 4H), 1.54 (d, J = 5.1 Hz, 3H), 1.47 (t, J = 7.2 Hz, 3H), 1.40 (d, J = 4.3 Hz, 2H). C NMR (101 MHz, DMSO) δ
159.55, 159.12, 155.78, 155.10, 152.27, 138.67, 138.57, 135.34, 131.90, 131.33, 129.28, 124.93, 123.89, 123.55, 123.37,
122.43, 108.86, 104.49, 101.85, 55.88, 55.40, 45.99, 45.23, 37.00, 29.45, 15.32, 8.93. MS (ESI) m/z (%): 490.17 [(M+H)+,
1
00].
(
E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-ethyl-5-(4-(piperidin-1-ylmethyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (25). mp 196-198 ºC. H NMR (400 MHz, DMSO) δ 11.56 (s, 1H), 8.84 (s, 1H), 8.38 (s, 2H), 7.84 (d, J =
8
2
1
.6 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.44 (s, 1H), 7.33 (s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.11 (s,
13
H), 4.34 (s, 2H), 3.68 (s, 2H), 1.55 (s, 4H), 1.47 (t, J = 7.2 Hz, 3H), 1.40 (s, 2H). C NMR (101 MHz, DMSO) δ 168.40,
60.66, 154.44, 149.20, 148.48, 145.42, 143.09, 137.17, 132.26, 129.59, 127.98, 127.15, 123.37, 121.24, 120.02, 116.74,
+
1
11.61, 108.96, 105.45, 101.95, 58.59, 54.26, 41.60, 25.74, 24.19, 15.62. MS (ESI) m/z (%): 516.70 [(M+H) , 100].
(
E)-1-ethyl-N'-(4-methoxybenzylidene)-5-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide
(26).
1
mp182-184 ºC. H NMR (400 MHz, DMSO) δ 11.55 (s, 1H), 8.87 (s, 1H), 8.36 (s, 2H), 7.86 (d, J = 8.7 Hz, 1H), 7.72 (d,
J = 8.6 Hz, 1H), 7.64 (s, 1H), 7.62 (s, 2H), 7.29 (d, J = 7.9 Hz, 2H), 4.35 (s, 2H), 4.12 (s, 1H), 2.93 (s, 3H), 2.36 (s, 3H),
1
1
4
.83 (s, 3H), 1.48 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 162.76, 159.02, 157.52, 155.78, 155.11, 152.20,
38.63, 138.15, 135.33, 131.34, 124.81, 123.95, 123.61, 123.43, 122.83, 119.81, 108.85, 104.57, 101.80, 55.89, 55.37,
+
2.76, 41.58, 40.29, 36.24, 31.22, 15.31. MS (ESI) m/z (%): 488.15 [(M+H) , 100].
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E)-N'-(4-chlorobenzylidene)-1-ethyl-5-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (27). mp
4
5
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8
9
1
1
1
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1
1
1
1
1
2
2
2
2
2
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5
93-195 ºC. H NMR (400 MHz, DMSO) δ 11.81 (s, 1H), 8.88 (s, 1H), 8.49 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.75 (d, J =
.2 Hz, 2H), 7.67 (d, J = 8.6 Hz, 1H), 7.51 (d, J = 8.2 Hz, 2H), 7.47 (s, 1H), 4.32 (d, J = 4.4 Hz, 3H), 3.86 (d, J = 21.1 Hz,
1
3
H), 2.69 (s, 5H), 1.74 (s, 5H), 1.46 (t, J = 7.1 Hz, 4H). C NMR (101 MHz, DMSO) δ 168.55, 164.55, 161.10, 154.38,
0
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37.08, 134.46, 134.14, 129.37(2C), 128.87(2C), 128.67, 127.17, 121.31, 120.03, 116.72, 111.61, 110.11, 108.85, 55.09,
+
3.89(2C), 41.64, 23.58(2C), 15.58. MS (ESI) m/z (%): 492.38 [(M+H) , 100].
(
E)-1-ethyl-N'-(1-(4-fluorophenyl)ethylidene)-5-(4-(pyrrolidin-1-ylmethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (28).
1
mp 194-196 ºC. H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 8.84 (s, 1H), 8.44 (s, 1H), 7.89 (dd, J = 8.3, 5.8 Hz, 2H),
7
.85 (dd, J = 8.7, 1.5 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.51 (s, 1H), 7.29 (t, J = 8.8 Hz, 2H), 4.33 (q, J = 7.2 Hz, 2H), 3.93
+
(
s, 2H), 2.73 (s, 4H), 2.39 (s, 3H), 1.77 (s, 4H), 1.46 (t, J = 7.2 Hz, 3H). MS (ESI) m/z (%): 490.17 [(M+H) , 100].
(
E)-1-ethyl-N'-(1-(4-fluorophenyl)ethylidene)-5-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (29). mp 213-215 ºC. H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 8.82 (s, 1H), 8.44 (s, 1H), 7.89 (s, 2H),
7
.83 (s, 1H), 7.71 (s, 1H), 7.42 (s, 1H), 7.29 (s, 2H), 4.32 (s, 2H), 3.65 (s, 2H), 2.39 (m, 8H), 2.17 (s, 3H), 1.46 (t, J = 7.1
+
Hz, 3H), 1.23 (s, 3H). MS (ESI) m/z (%): 519.70 [(M+H) , 100].
(E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-ethyl-5-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (30). mp 204-206 ºC. H NMR (400 MHz, DMSO) δ 11.44 (s, 1H), 8.84 (s, 1H), 8.32 (s, 2H), 7.83 (s, 1H),
7.71 (s, 1H), 7.39 (s, 1H), 7.33 (s, 1H), 7.18 (s, 1H), 7.02 (s, 1H), 6.11 (s, 2H), 4.35 (s, 2H), 3.62 (s, 2H), 2.51 (s, 4H), 2.46
13
(
s, 4H), 1.52 (s, 3H), 1.49 (t, J = 7.1 Hz, 3H). C NMR (1 01 MHz, DMSO) δ 168.23, 160.92, 155.19, 149.20, 148.48,
1
5
45.39, 137.02, 129.58, 127.23, 123.35, 121.23, 120.02, 116.11, 113.66, 111.58, 108.95, 108.43, 105.48, 104.43, 101.94,
+
8.98, 54.43(2C), 41.60, 26.03(2C), 24.43, 15.61. MS (ESI) m/z (%): 531.27 [(M+H) , 100].
(
(
E)-1-ethyl-N'-(4-fluorobenzylidene)-5-(4-((4-methylpiperidin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-carbohydrazide
1
31). mp 210-212 ºC. H NMR (400 MHz, DMSO) δ 11.63 (s, 1H), 8.86 (s, 1H), 8.44 (s, 2H), 7.85 (dd, J = 8.7, 1.4 Hz,
1
3
H), 7.80 (dd, J = 8.6, 5.7 Hz, 2H), 7.71 (d, J = 8.6 Hz, 1H), 7.52 (s, 1H), 7.32 (t, J = 7.2 Hz, 2H), 4.35 (d, J = 6.6 Hz, 2H),
.83 (s,2H), 3.03 (d, J = 11.0 Hz, 2H), 2.27 (s, 2H), 1.64 (d, J = 12.0 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H), 1.40 (m, 1H) 1.24
(
d, J = 11.0 Hz, 2H), 0.90 (d, J = 6.4 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 168.69, 164.55, 162.10, 154.64, 153.10,
1
5
32.58, 132.49, 131.76, 129.44, 129.36, 127.10, 121.34, 120.10, 117.74, 116.47, 116.25, 116.12, 115.90, 111.67, 57.53,
+
3.36, 41.66, 33.51, 30.10, 22.03, 15.62. MS (ESI) m/z (%): 504.60 [(M+H) , 100].
(
E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-ethyl-5-(4-((4-methylpiperidin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (32). mp 210-212 ºC. H NMR (400 MHz, DMSO) δ 11.42 (s, 1H), 8.83 (s, 1H), 8.31 (s, 2H), 7.83 (d, J =
8
.2 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.38 (s, 1H), 7.33 (s, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.10 (s,
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H), 4.34 (d, J = 5.7 Hz, 2H), 3.62 (s, 2H), 2.90 (d, J = 10.8 Hz, 2H), 2.02 (t, J = 11.0 Hz, 2H), 1.58 (d, J = 11.7 Hz, 2H),
.47 (t, J = 7.1 Hz, 3H), 1.32 (s, 1H), 1.17 (dd, J = 22.0, 10.2 Hz, 2H), 0.89 (d, J = 6.3 Hz, 3H). MS (ESI) m/z (%): 530.59
+
[(M+H) ,45].
(
E)-1-ethyl-5-(4-((4-ethylpiperazin-1-yl)methyl)thiazol-2-yl)-N'-(4-fluorobenzylidene)-1H-indole-3-carbohydrazide (33).
0
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mp170-172 ºC. H NMR (400 MHz, DMSO) δ 11.57 (s, 1H), 8.84 (s, 1H), 8.39 (s,2H), 7.84 (dd, J = 8.7, 1.7 Hz, 1H), 7.80
(dd, J = 8.7, 5.6 Hz, 2H), 7.70 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.31 (t, J = 8.8 Hz, 2H), 4.35 (q, J = 6.8 Hz, 2H), 3.65 (s,
13
2
H), 2.41 (m, 6H), 2.32 (dd, J = 14.3, 7.2 Hz, 4H), 1.47 (t, J = 7.2 Hz, 3H), 0.98 (t, J = 7.2 Hz, 3H). C NMR (101 MHz,
DMSO) δ 168.32, 164.55, 162.09, 154.80, 137.07, 132.36, 132.28, 131.77, 129.44, 129.36, 127.25, 121.29, 120.02, 116.47,
16.38, 116.25, 115.68, 115.45, 111.62, 58.14, 53.08(2C), 52.79(2C), 52.04, 41.65, 15.62, 12.41. MS (ESI) m/z (%): 519.12
1
+
[(M+H) , 100].
(
E)-N'-(4-chlorobenzylidene)-1-ethyl-5-(4-((4-ethylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (34).
1
mp 200-202 ºC. H NMR (400 MHz, DMSO) δ 11.60 (s, 1H), 8.84 (s, 1H), 8.39 (s, 2H), 7.84 (dd, J = 8.6, 1.7 Hz, 1H),
7
.76 (d, J = 8.5 Hz, 2H), 7.70 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.41 (s, 1H), 4.35 (q, J = 6.8 Hz, 2H), 3.65 (s,
1
3
2H), 2.43 (m, 6H), 2.34 (dd, J = 14.0, 6.9 Hz, 4H), 1.47 (t, J = 7.2 Hz, 3H), 0.99 (t, J = 7.2 Hz, 3H). C NMR (151 MHz,
DMSO) δ 168.31, 154.75, 137.16, 134.48, 134.12, 132.54, 130.01, 129.40(2C), 128.91(2C), 127.27, 121.32, 120.01, 119.96,
119.09, 116.44, 111.66, 102.11, 58.10, 52.98(2C), 52.75(2C), 52.03, 41.66, 15.62, 12.34. MS (ESI) m/z (%): 534.74
+
[(M+H) , 50].
(
E)-1-ethyl-5-(4-((4-ethylpiperazin-1-yl)methyl)thiazol-2-yl)-N'-(1-(4-fluorophenyl)ethylidene)-1H-indole-3-
1
carbohydrazide (35). mp 214-216 ºC. H NMR (400 MHz, DMSO) δ 10.38 (s, 1H), 8.82 (s, 1H), 8.42 (s, 1H), 7.92 – 7.86
(m, 2H), 7.84 (d, J = 8.5 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.43 (s, 1H), 7.28 (t, J = 8.8 Hz, 2H), 4.33 (q, J = 7.3 Hz, 2H),
3
.67 (s, 2H), 2.52 (s, 4H), 2.47 – 2.42 (m, 6H), 2.39 (s, 3H), 1.46 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 6.7 Hz, 3H). MS (ESI)
+
m/z (%): 533.21 [(M+H) , 100].
(
E)-N'-(4-fluorobenzylidene)-1-methyl-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (36). mp 221-
1
2
23 ºC. H NMR (600 MHz, DMSO) δ 11.57 (s, 1H), 8.84 (s, 1H), 8.36 (s, 2H), 7.86 (dd, J = 8.6, 1.6 Hz, 1H), 7.80 (dd, J
=
8.2, 5.8 Hz, 2H), 7.66 (d, J = 8.6 Hz, 1H), 7.46 (s, 1H), 7.31 (t, J = 8.6 Hz, 2H), 3.94 (s, 4H), 3.66 (s, 3H), 3.61 (m, 6H).
+
MS (ESI) m/z (%): 478.32 [(M+H) , 100].
(
E)-N'-(4-chlorobenzylidene)-1-methyl-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide (37). mp 241-
1
2
43 ºC. H NMR (400 MHz, DMSO) δ 11.57 (s, 1H), 8.84 (s, 1H), 8.31 (s, 2H), 7.86 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 8.3
Hz, 2H), 7.65 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.45 (s, 1H), 3.94 (s, 3H), 3.65 (s, 2H), 3.61 (m, J = 4.0 Hz,
13
4
H), 2.50–2.45 (m, 4H). C NMR (151 MHz, DMSO) δ 168.42, 154.36, 144.07, 142.52, 137.94, 136.62, 134.47, 134.08,
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31.63, 129.39(2C), 128.96(2C), 128.79, 127.24, 121.34, 119.88, 116.67, 111.66, 66.66(2C), 58.47, 53.65(2C), 33.84. MS
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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4
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4
4
4
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5
5
5
5
5
5
5
5
5
6
+
(
(
(
ESI) m/z (%): 494.33 [(M+H) , 75].
E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-methyl-5-(4-(morpholinomethyl)thiazol-2-yl)-1H-indole-3-carbohydrazide
1
38). mp173-175 ºC. H NMR (600 MHz, DMSO) δ 11.47 (s, 1H), 8.82 (s, 1H), 8.28 (m, 2H), 7.85 (dd, J = 8.6, 1.6 Hz,
0
1
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0
1
H), 7.65 (d, J = 8.6 Hz, 1H), 7.45 (s, 1H), 7.33 (d, J = 1.4 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), 6.10
+
(
(
(
s, 2H), 3.93 (s, 3H), 3.65 (s, 2H), 3.62–3.59 (m, 4H), 2.49–2.47 (m, 4H). MS (ESI) m/z (%): 504.02 [(M+H) , 100].
E)-N'-(4-fluorobenzylidene)-1-methyl-5-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-carbohydrazide
1
39). mp 201-203 ºC. H NMR (400 MHz, DMSO) δ 11.55 (s, 1H), 8.84 (s, 1H), 8.33 (s, 2H), 7.85 (dd, J = 8.6, 1.6 Hz,
1
2
1
5
H), 7.81 (dd, J = 8.5, 5.7 Hz, 2H), 7.65 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.31 (p, J = 7.2 Hz, 2H), 3.94 (s, 3H), 3.65 (s,
13
H), 2.50–2.46 (s, 3H), 2.38 (m, 4H), 2.18 (m, 4H). C NMR (101 MHz, DMSO) δ 167.80, 163.99, 161.54, 154.22, 137.39,
31.86, 131.19, 128.93, 128.85, 126.68, 120.75, 119.51, 119.41, 115.90(2C), 115.69, 115.29, 111.06, 57.54, 54.54(2C),
+
2.36(2C), 45.52, 33.27. MS (ESI) m/z (%): 490.67 [(M+H) , 43].
(
E)-N'-(1-(4-fluorophenyl)ethylidene)-1-methyl-5-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (40). mp 226-228 ºC. H NMR (400 MHz, DMSO) δ 10.36 (s, 1H), 8.82 (s, 1H), 8.36 (s, 1H), 7.89 (dd, J
= 8.6, 5.6 Hz, 2H), 7.85 (dd, J = 8.6, 1.7 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.42 (s, 1H), 7.28 (t, J = 8.9 Hz, 2H), 3.92 (s,
1
3
3H), 3.82 (s, 2H), 3.65 (s, 3H), 2.39 (m, 4H), 2.38 (s, 3H), 2.19 (m, 4H). C NMR (101 MHz, DMSO) δ 167.84, 154.15,
137.28, 134.98, 131.08, 128.37, 128.28, 128.09, 126.62, 120.67, 119.50, 119.38, 118.43, 115.90, 115.29, 115.08, 111.03,
+
1
10.19, 101.31, 57.48, 54.48(2C), 52.24(2C), 45.41, 33.21, 13.92. MS (ESI) m/z (%): 505.30 [(M+H) , 100].
(
E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-methyl-5-(4-((4-methylpiperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (41). mp 228-230 ºC. H NMR (400 MHz, DMSO) δ 11.46 (s, 1H), 8.83 (s, 1H), 8.27 (s, 2H), 7.85 (dd, J
=
8.6, 1.7 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.34 (s, 1H), 7.17 (d, J = 8.1 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H),
6
1
.10 (s, 2H), 3.93 (s, 3H), 3.64 (s, 2H), 2.50–2.48 (m, 4H), 2.37 (m, 4H), 2.17 (s, 3H). MS (ESI) m/z (%): 517.14 [(M+H)+,
00].
(
(
E)-1-ethyl-N'-(4-fluorobenzylidene)-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-carbohydrazide
1
42). mp163-165 ºC. H NMR (600 MHz, DMSO) δ 11.59 (s, 1H), 8.86 (s, 1H), 8.37 (s, 2H), 7.84 (dd, J = 8.6, 1.6 Hz,
1
2
2
1
H), 7.80 (dd, J = 8.5, 5.7 Hz, 2H), 7.70 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.32 (t, J = 8.7 Hz, 2H), 4.58 (s, 1H), 4.35 (s,
H), 3.65 (s, 2H), 3.47 (s, 1H), 2.81 (s, 2H), 2.18 (s, 2H), 1.74 (d, J = 9.7 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H), 1.45–1.41 (m,
13
H). C NMR (151 MHz, DMSO) δ 167.75, 163.61, 161.97, 154.54, 152.44, 136.51, 131.24, 128.87(2C), 128.82(2C),
26.74, 120.75, 119.52, 115.86(2C), 115.72(2C), 111.04, 66.09, 57.62, 50.81(2C), 41.10, 34.26(2C), 15.05. MS (ESI) m/z
+
(%): 506.43 [(M+H) , 100].
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 41
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9
1
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(
(
(
E)-N'-(4-chlorobenzylidene)-1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-carbohydrazide
1
43). mp143-145 ºC. H NMR (600 MHz, DMSO) δ 11.66 (s, 1H), 8.84 (s, 1H), 8.39 (s, 2H), 7.84 (d, J = 8.4 Hz, 1H), 7.77
d, J = 6.9 Hz, 2H), 7.70 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 6.9 Hz, 2H), 7.42 (s, 1H), 4.58 (s, 1H), 4.35 (s, 2H), 3.67 (s, 2H),
3
.48 (s, 1H), 2.82 (s, 2H), 2.20 (s, 2H), 1.73 (s, 2H), 1.47 (t, J = 7.1 Hz, 3H), 1.43 (s, 2H). MS (ESI) m/z (%): 522.00
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
[(M+H) , 100].
(
E)-1-ethyl-N'-(1-(4-fluorophenyl)ethylidene)-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (44). mp 191-193 ºC. H NMR (600 MHz, DMSO) δ 10.40 (s, 1H), 8.83 (s, 1H), 8.43 (s, 1H), 7.90 (dd, J =
7
1
1
1
5
.7, 6.0 Hz, 2H), 7.84 (dd, J = 8.6, 1.7 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.28 (t, J = 8.8 Hz, 2H), 4.58 (s,
H), 4.33 (q, J = 7.2 Hz, 2H), 3.65 (s, 2H), 3.47 (s, 1H), 2.81 (s, 2H), 2.39 (s, 3H), 2.19 (s, 2H), 1.74 (d, J = 9.8 Hz, 2H),
13
.46 (t, J = 7.2 Hz, 3H), 1.40 (m, 2H). C NMR (101 MHz, DMSO) δ 168.35, 164.35, 161.90, 154.99, 136.92, 135.56,
35.53, 128.88, 128.80, 128.25, 127.19, 121.21, 120.04, 116.34, 116.31, 115.84, 115.62, 111.57, 108.74, 66.62, 58.10,
+
1.35, 41.60, 34.77, 15.60, 14.32. MS (ESI) m/z (%): 520.42 [(M+H) , 100].
(
E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (45). mp166-168 ºC. H NMR (600 MHz, DMSO) δ 11.52 (s, 1H), 8.83 (s, 1H), 8.34 (s, 2H), 7.84 (d, J =
8.5 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.46 (s, 1H), 7.33 (s, 1H), 7.16 (d, J = 7.4 Hz, 1H), 7.01 (d, J = 7.9 Hz, 1H), 6.10 (s,
2H), 4.63 (s, 1H), 4.34 (s, 2H), 3.72 (s, 2H), 3.51 (s, 1H), 3.35 (s, 2H), 2.87 (s, 2H), 2.26 (s, 2H), 1.75 (s, 2H), 1.47 (t, J =
+
7.2 Hz, 3H). MS (ESI) m/z (%): 532.45 [(M+H) , 100].
(
S,E)-1-ethyl-N'-(4-fluorobenzylidene)-5-(4-((2-(hydroxymethyl)pyrrolidin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (46). mp 147-149 ºC. H NMR (600 MHz, DMSO) δ 11.56 (s, 1H), 8.83 (s, 1H), 8.36 (s, 2H), 7.84 (dd, J
=
8.6, 1.7 Hz, 1H), 7.80 (dd, J = 8.6, 5.6 Hz, 2H), 7.70 (d, J = 8.7 Hz, 1H), 7.42 (s, 1H), 7.32 (t, J = 8.8 Hz, 2H), 4.35 (s,
2
3
1
1
H), 4.13 (d, J = 14.2 Hz, 1H), 3.71 (d, J = 14.2 Hz, 1H), 3.47 (dd, J = 10.6, 4.7 Hz, 1H), 3.32 (dd, J = 10.6, 6.4 Hz, 1H),
.03 (dt, J = 9.2, 4.6 Hz, 1H), 2.71 (s, 1H), 2.40 (dd, J = 16.5, 8.2 Hz, 1H), 1.91 (s, 1H), 1.84 (dq, J = 12.2, 8.2 Hz, 1H),
.69 – 1.62 (m, 2H), 1.58 (dt, J = 19.1, 6.5 Hz, 1H), 1.47 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz, DMSO) δ 168.20,
64.14, 162.51, 156.23, 147.63, 144.36, 137.01, 131.77, 129.42, 129.37, 127.30, 121.27, 120.05, 120.01, 116.42, 116.27,
+
1
15.70, 115.51, 111.60, 65.00, 64.55, 54.59, 54.51, 41.64, 28.45, 23.14, 15.61. MS (ESI) m/z (%): 506.43 [(M+H) , 100].
(
S,E)-1-ethyl-N'-(1-(4-fluorophenyl)ethylidene)-5-(4-((2-(hydroxymethyl)pyrrolidin-1-yl)methyl)thiazol-2-yl)-1H-
1
indole-3-carbohydrazide (47). mp 185-187 ºC. H NMR (400 MHz, DMSO) δ 10.39 (s, 1H), 8.83 (s, 1H), 8.43 (s, 1H),
7
.93 – 7.87 (m, 2H), 7.84 (d, J = 8.7 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.45 (s, 1H), 7.28 (t, J = 8.7 Hz, 2H), 4.39 – 4.28
(m, 2H), 4.17 (d, J = 13.7 Hz, 1H), 3.76 (d, J = 12.3 Hz, 1H), 3.49 (d, J = 6.0 Hz, 1H), 3.48 (d, J = 4.3 Hz, 1H), 3.06 (s,
H), 2.78 (s, 1H), 2.39 (s, 3H), 1.85 (d, J = 9.7 Hz, 1H), 1.67 (s, 2H), 1.63 – 1.55 (m, 1H), 1.46 (t, J = 7.1 Hz, 3H). 13
1
C
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Journal of Medicinal Chemistry
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NMR (101 MHz, DMSO) δ 168.20, 164.18, 161.73, 155.70, 155.53, 136.75, 135.38, 135.36, 128.72, 128.64, 128.03,
4
5
6
7
8
9
1
1
1
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1
1
1
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2
2
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5
27.01, 121.03, 119.89, 116.00, 115.67, 115.46, 111.41, 108.60, 65.04, 64.07, 54.43, 54.21, 41.43, 28.15, 22.92, 15.45,
+
+
4.28.MS (ESI) m/z (%): 520.42 [(M+H) , 100]. HRMS (ESI) m/z calculated for C28
H31FN
5
O S [M+H] : 520.2104. Found:
2
20.2187.
0
1
2
3
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(
S,E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-ethyl-5-(4-((2-(hydroxymethyl)pyrrolidin-1-yl)methyl)thiazol-2-yl)-1H-
1
indole-3-carbohydrazide (48). mp128-130 ºC. H NMR (400 MHz, DMSO) δ 11.49 (s, 1H), 8.85 (s, 1H), 8.35 (s, 1H), 7.85
(d, J = 8.5 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.44 (s, 1H), 7.34 (s, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H),
6
.11 (s, 2H), 4.33 (s, 2H), 4.18 (d, J = 14.2 Hz, 1H), 3.76 (d, J = 14.1 Hz, 1H), 3.50 (dd, J = 10.6, 4.5 Hz, 1H), 3.40 – 3.32
(m, 1H), 3.10 – 3.01 (m, 1H), 2.78 (s, 1H), 2.47 (d, J = 8.3 Hz, 1H), 1.92 (s, 1H), 1.85 (dd, J = 19.0, 8.2 Hz, 1H), 1.65 (dd,
13
J = 20.8, 6.7 Hz, 2H), 1.64 – 1.54 (m, 1H), 1.47 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, DMSO) δ 172.48, 168.36, 155.61,
1
1
49.20, 148.47, 137.03, 129.59, 127.20, 123.34, 121.23, 120.38, 120.09, 120.03, 116.10, 111.56, 110.71, 108.93, 107.14,
+
05.48, 101.94, 65.21, 64.24, 54.59, 54.38, 41.60, 28.31, 23.09, 15.58. MS (ESI) m/z (%): 532.45 [(M+H) , 100].
(
E)-1-ethyl-N'-(4-fluorobenzylidene)-5-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (49). mp 133-135 ºC. H NMR (400 MHz, DMSO) δ 11.55 (s, 1H), 8.84 (s, 1H), 8.38 (s, 2H), 7.83 (m,
1H), 7.80 (m, 2H), 7.70 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.31 (s, 2H), 4.36 (m, 2H), 4.34 (m, 2H), 3.65 (m, 2H), 3.49 (t, J
+
=6.7 Hz, 2H), 2.50 (m, 4H), 2.42 (m, 4H) 1.47 (t, J = 6.9 Hz, 3H). MS (ESI) m/z (%): 535.41 [(M+H) , 100].
(E)-N'-(4-chlorobenzylidene)-1-ethyl-5-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (50). mp 157-159 ºC. H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 8.83 (s, 1H), 8.42 (s, 2H), 7.84 (d, J =
8
2
.2 Hz, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.41 (s, 1H), 4.36 (s, 2H), 4.34 (m,
H), 3.64 (s, 2H), 3.48 (s, 2H), 2.45 (m, 4H), 2.38 (t, J = 6.2 Hz, 4H), 1.47 (t, J = 7.1 Hz, 3H). MS (ESI) m/z (%): 551.42
+
[(M+H) , 100].
(
E)-1-ethyl-N'-(1-(4-fluorophenyl)ethylidene)-5-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)thiazol-2-yl)-1H-indole-3-
1
carbohydrazide (51). mp131-133 ºC. H NMR (600 MHz, DMSO) δ 10.39 (s, 1H), 8.82 (s, 1H), 8.43 (s, 1H), 7.89 (dd, J =
8
4
.1, 5.8 Hz, 2H), 7.83 (dd, J = 8.6, 1.7 Hz, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.42 (s, 1H), 7.30–7.27 (m, 2H), 4.44 (s, 1H),
.33 (q, J = 7.2 Hz, 2H), 3.66 (s, 2H), 3.50 (s, 2H), 3.33 (s, 2H), 2.52 (m, 4H), 2.48–2.42 (m, 4H), 2.39 (s, 3H), 1.46 (t, J
+
=
7.2 Hz, 3H). MS (ESI) m/z (%): 549.46 [(M+H) , 100].
(
E)-N'-(benzo[d][1,3]dioxol-5-ylmethylene)-1-ethyl-5-(4-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)thiazol-2-yl)-1H-
1
indole-3-carbohydrazide (52). mp 157-159 ºC. H NMR (600 MHz, DMSO) δ 11.47 (s, 1H), 8.82 (s, 1H), 8.31 (s, 2H),
7
.83 (dd, J = 8.6, 1.7 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.42 (s, 1H), 7.32 (s, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.01 (d, J = 8.0
Hz, 1H), 6.10 (s, 2H), 4.45 (s, 1H), 4.34 (s, 2H), 3.66 (s, 2H), 3.50 (s, 2H), 3.34 (s, 2H), 2.52 (m, 4H), 2.49–2.37 (m, 4H),
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