Discovery of matrinic thiadiazole derivatives as a novel family of anti-liver fibrosis agents via repression of the TGFβ/Smad pathway

Article abstract of DOI:10.3390/molecules23071644

A series of novel matrinic thiadiazole derivatives were designed, synthesized and evaluated for their inhibitory effect on COL1A1 promotor. The SAR indicated that: (i) the introduction of a thiadiazole on the 11-side chain was beneficial for activity; (ii) a 12-N-benzyl moiety was favorable for activity. Among them, compound 6n displayed a high activity with an inhibitory rate of 39.7% at a concentration of 40 μM. It also effectively inhibited the expression of two representative collagen proteins (COL1A1 and α-SMA) on both the mRNA and protein levels and showed a high safety profile in vivo, indicating its great promise as an anti-liver fibrosis agent. Further study indicated that it might repress hepatic fibrogenesis via the TGFβ/Smad pathway. This study provided powerful information for further strategic optimization and the top compound 6n was selected for further study as an ideal liver fibrosis lead for next investigation.

Full text of DOI:10.3390/molecules23071644

molecules
Article
Discovery of Matrinic Thiadiazole Derivatives as
a Novel Family of Anti-Liver Fibrosis Agents via
Repression of the TGFβ/Smad Pathway
†
†
Tianyu Niu , Weixiao Niu , Yunyang Bao, Ting Liu, Danqing Song, Yinghong Li * and
Hongwei He *
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China; niutianyubasn@163.com (T.N.); niuweixiao@126.com (W.N.); 17714333891@163.com (Y.B.);
lutyliu@126.com (T.L.); songdanqingsdq@hotmail.com (D.S.)
*
Correspondence: liyinghong@imb.pumc.edu.cn (Y.L.); hehwei@imb.pumc.edu.cn (H.H.);
Tel.:+86-10-6303-3012 (Y.L.); +86-10-8316-6673 (H.H.)
†
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 14 June 2018; Accepted: 30 June 2018; Published: 5 July 2018
Abstract: A series of novel matrinic thiadiazole derivatives were designed, synthesized and evaluated
for their inhibitory effect on COL1A1 promotor. The SAR indicated that: (i) the introduction of a
thiadiazole on the 11-side chain was beneficial for activity; (ii) a 12-N-benzyl moiety was favorable
for activity. Among them, compound 6n displayed a high activity with an inhibitory rate of 39.7% at
a concentration of 40
proteins (COL1A1 and
profile in vivo, indicating its great promise as an anti-liver fibrosis agent. Further study indicated that
it might repress hepatic fibrogenesis via the TGF /Smad pathway. This study provided powerful
µM. It also effectively inhibited the expression of two representative collagen
α
-SMA) on both the mRNA and protein levels and showed a high safety
β
information for further strategic optimization and the top compound 6n was selected for further
study as an ideal liver fibrosis lead for next investigation.
Keywords: liver fibrosis; matrinic; thiadiazole; structure-activity relationship; COL1A1;
TGFβ/Smad pathway
1
. Introduction
Liver fibrosis is a general stage in the process of cirrhosis, which is characterized by the excessive
accumulation of extracellular matrix (ECM) and abnormal hyperplasia of intrahepatic connective
tissue [ ]. Fibrillation is the scarring response to chronic liver injury [ ]. It can be controlled in
1
2
the early phase after effective treatment, but most often, fibrous septa, pseudolobules or nodules
would form and eventually develop into liver cirrhosis. Unfortunately, to date, treatments for liver
fibrosis are quite limited and therefore, there is still an urgent need to develop new effective anti-liver
fibrosis candidates.
The activation and proliferation of hepatic stellate cells (HSCs) leads to the excess production
and abnormal deposition of ECM components [
fibrosis [ ].Transforming growth factor 1 (TGF
the most important fibrotic cytokines known so far [
mainly by activating HSCs and up-regulating the secretion of collagen, especially collagen type I
COL1) and -smooth muscle actin ( -SMA). Since the transcription of key gene collagen type I
chain (COL1A1) for liver fibrosis could be activated by TGF 1 through TGF /Smad pathway [
high-throughput drug screening cell model based on COL1A1 promotor was established earlier in our
3
,
4
], as indicated in the cytological basis for liver
1) is closely related to liver fibrosis, and it is one of
]. It promotes the production of liver fibrosis
5
β
β
6
(
α
α
α1
β
β
7], a
Molecules 2018, 23, 1644; doi:10.3390/molecules23071644
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1644
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group, in which the activity of COL1A1 promotor and luciferase reporter gene could be elevated by
TGFβ1, and inhibited by candidate agents [8].
Our group has been working on the discovery of innovative candidates agents with novel
structure skeletons from Chinese natural products such as matrine (MT) [
anti-liver fibrosis monomer [12], the matrinic acid compound library constructed in our lab [
9
–
11]. Since MT is an
10 13
9
, , ]
was screened by the high-throughput screening model based on COL1A1 promotor as mentioned
above, taking epigallocatechin gallate (EGCG) as the positive control [14]. To our great delight, the hit
compound 12-N-p-methyl benzenesulfonyl matrinic acid (
a reasonable inhibitory effect against COL1A1 at a rate of 18.5% at the concentration of 40
also worth mentioning that compound possesses a special tricyclic flexible scaffold, and appealing
druglike characteristics. These studies prompted our interest in continuing our structure-activity
1, Figure 1) [15] had been identified to display
µM. It was
1
relationship (SAR) study of this compound class taking compound
and discover novel anti-liver fibrosis candidates.
1 as the lead, in an effort to develop
Figure 1. Chemical structures of matrine and the lead compound 1, and structure modification strategy.
The 1,3,4-thiadiazole group is recognized as a bioisostere of the carboxyl group [16], and also a
privileged chemical scaffold [17 19], therefore, in the present study, the carboxyl was replaced with
,3,4-thiadiazole group on the 11-side chain, whereby a series of novel tricyclic matrinic thiadiazole
–
1
derivatives was generated and evaluated for their anti-COL1A1 activity. Herein, we describe the
synthesis of 19 novel thiadiazole derivatives, SAR analysis, as well as in vivo safety profile and primary
mechanism of action of key compounds.
2
. Results and Discussion
2
.1. Chemistry
The semi-synthetic routes of all target compounds were outlined in Scheme 1, taking MT as the
starting material. 12-N-substituted matrinic acids
four-step procedure including hydrolysis, esterification, 12-N substitution and hydrolysis instead of
the original diphenyldiazomethane protection method [ 15]. Then a series of matrinic thiadiazole
derivatives 6a were obtained by the cyclization of or 5a with thiosemicarbazide or various of
N-substituted thiosemicarbazides in the presence of phosphorus pentoxide/methanesulfonic acid (1:5)
in yields of 28–58% [20 21]. The acylation of 6a 6f 6i and 6l in triethylamine generated compounds
with yields of 93–96%. The desired products were purified with flash column chromatography
on silica gel using dichloromethane/methanol or petroleum/ethyl acetate as eluents.
1, 5a–d were obtained in high yields of 42–66% by a
9,
–
o
1
–d
,
,
,
7a–d

Molecules 2018, 23, 1644
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Scheme 1. Reagents and conditions: (a) 5 N NaOH, reflux, 9 h; 10 N HCl, pH = 3–5; (b) 2 N MeOH/HCl,
reflux, 2 h; (c) Substituted benzensulfonyl chloride or benzyl bromide, TEA or K CO , CH Cl or MeCN,
2
3
2
2
r.t., 6–8 h; (d) 5 N NaOH, reflux, 6 h; 10 N HCl, pH = 7–7.5; (e) P O /MsOH (1:5), thiosemicarbazide,
2
5
◦
7
0 C, 8 h; (f) Acetyl chloride, TEA, THF, r.t., 0.5 h.
2
.2. Inhibition of COL1A1 Promotor in Human Hepatic Stellate LX-2 Cells by Target Compounds
A single luciferase reporter gene detection model was applied to screen the inhibitory
effects towards COL1A1 promotor of all target compounds in human hepatic stellate LX-2
cells at the concentration of 40 M, taking EGCG and compound as the positive controls.
The LX2-COL monoclone cell line was treated simultaneously with TGF 1 and a target compound [13].
The cytotoxicity was determined using MTT assay in HepG2 cells. The structures and inhibitory effects
%) of all target compounds are shown in Table 1.
Taking as the lead, the carboxyl group was retained, and the SAR investigation was initiated
with the variation of 12-N substitution, by which 4 matrinic acid derivatives 5a were obtained.
As depicted in Table 1, the replacement of the methyl group on the benzene ring with a trifluoromethyl
µ
1
β
(
1
–
d
group (compound 5a) or the replacement of a benznesulfonyl with a benzyl motif (compounds 5b–d)
caused a significant decrease in activity.
The carboxyl group at the end of the 11-side chain was then replaced by its 1,3,4-thiadiazole
bioisostere, and a series of target matrinic thiadiazole compounds was prepared and evaluated.
To start, the 12-N-p-methylbenzenesulfonyl was retained, and five matrinic aminothiadiazoles 6a
–e
were obtained. Compound 6a showed higher inhibitory effects than 6b and the lead , which
–d
1
indicated that 5-amino-1,3,4-thiadiazole motif was a beneficial group, and the introduction of an
extra alkyl group on the amino group was not helpful for activity in this series. In the meantime,
the introduction of a phenyl group into the amino (compound 6e) resulted in an obvious increase in
activity. It was indicated that the aminothiadiazole motif was more beneficial, which might result
from the smaller spatial hindrance. Then, the 12-N-substituent was replaced with a p-trifluoromethyl
benzenesulfonyl moiety to prepare target compounds 6f
–h, which displayed decreased activities in
varied degrees, as anticipated.
Next, the aminothiadiazole motif was retained and the 12-
-benzyl group to generate the corresponding compounds 6i
showed a significant improvement in activity, and compounds 6i
N
–
-benzenesulfonyl was replaced with a
1
2-N
o
. To our great delight, most of them
,
6l and 6n gave inspiring inhibitory
rates of 47.8%, 38.7% and 39.7%, respectively, significantly higher than that of
27.5%). These results suggested that benzyl was a favorable 12-N substitution.
1 (18.5%) and EGCG
(

Molecules 2018, 23, 1644
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Finally, an acyl group was condensed to the amino group of 6a
, 6f, 6i and 6l to generate 7a–d,
respectively, and 12- -benzenesulfonyl compounds 7a and 7b displayed higher activity than their
N
amino counterparts 6a and 6f, while the 12-
than their counterparts 6i and 6l.
N-benzyl compounds 7c and 7d displayed lower activity
Five target compounds 6a
representative compounds to further evaluate their cytotoxicities. As indicated in Table 1, compounds
6i 6l and 6n displayed high cellular safety with the concentration of half cellular toxicity (CC )
value of over 320
with a CC50 value of over 82.6
, 6e, 6i, 6l and 6n with the highest activity were selected as the
6
a
,
,
50
µM, comparable to that of
1, while compound 6e displayed a high cellular toxicity
µM, indicating that the introduction of a benzylamino to the thiadiazole
motif might contribute to a high cytotoxicity, which might result from its high lipophilicity.
Table 1. Inhibition of COL1A1 promotor of target compounds and cytotoxicity of key compounds.
Code
1
R1
R2
Inhibition Percentage a
CC50 (µM) b
p-CH C H SO
2
-
-
-
-
-
18.5% ± 1.1%
−3.7% ± 3.0%
−33.6% ± 10.6%
−116.2% ± 45.7%
6.4%± 0.9%
>320
3
6
4
c
5
a
p-CF C H SO
NT
NT
NT
NT
3
6
4
2
c
5
5
b
c
C H CH
6
5
2
c
p-CH C H CH
3
6
4
2
c
5
6
d
a
p-BrC H CH
6
4
2
p-CH C H SO
NH2
21.2% ± 10.4%
12.8% ± 7.3%
−84.2% ± 63.1%
−36.7% ± 17.3%
69.0% ± 13.5%
−19.0% ± 7.8%
6.1% ± 0.8%
>320
3
6
4
2
c
6
6
b
c
p-CH C H SO
NHCH3
NHCH(CH3)2
N(CH3)2
NHPh
NH2
NT
NT
NT
3
6
4
2
2
2
2
c
p-CH C H SO
3
6
4
c
6
6
d
e
p-CH C H SO
3
6
4
p-CH C H SO
82.55
3
6
4
c
6
f
p-CF C H SO
NT
NT
NT
3
6
4
2
c
6
g
p-CF C H SO
NHCH3
N(CH3)2
NH2
NHCH3
N(CH3)2
NH2
NHCH3
N(CH3)2
NH2
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
-
3
6
4
2
2
c
6
h
p-CF C H SO
−35.2% ± 2.1%
47.8% ± 29.0%
29.5% ± 6.1%
15.2% ± 7.1%
38.7% ± 4.4%
−29.6% ± 13.6%
39.7%± 12.3%
7.8% ± 3.0%
3
6
4
6
6
i
j
C H CH
>320
6
5
2
2
2
c
C H CH
NT
NT
6
5
c
6
6
k
l
C H CH
6
5
p-CH C H CH
>320
3
6
4
2
2
2
c
6
6
m
n
p-CH C H CH
NT
3
6
4
p-CH C H CH
>320
3
6
4
c
6
7
o
a
p-BrC H CH
NT
NT
NT
NT
6
4
2
c
p-CH C H SO
33.9% ± 9.9%
−6.0% ± 5.0%
12.9% ± 10.6%
27.0% ± 9.5%
27.5% ± 7.9%
2.9% ± 0
3
6
4
2
c
7
7
b
c
p-CF C H SO
3
6
4
2
c
C H CH
6
5
2
c
7
d
p-CH C H CH
NT
3
6
4
2
EGCG
DMSO
-
-
-
-
-
a
b
The inhibition rate of COL1A1 promotor’s activity in LX2 cells; Cytotoxic concentration required to inhibit
HepG2 cells cell growth by 50%; c Not tested.
2
.3. Inhibition Effects of COL1A1 on RNA and Protein Levels by Key Compounds
Next, compounds 6i
effects against COL1A1. LX-2 cells were stimulated with TGF
compounds 6i 6l and 6n (80 M) respectively, taking as the control. Their anti-COL1A1 effects
were evaluated on RNA level by real-time PCR amplification. As indicated in Figure 2A, all three
compounds 6i 6l and 6n could significantly reduce COL1A1 mRNA level with the inhibitory rates of
,
6l and 6n were chosen as the key compounds to verify their inhibitory
β1 (2 ng/mL), and then treated with
,
µ
1
,

Molecules 2018, 23, 1644
5 of 16
46.1%,58.2% and 78.4%, respectively, prevailing over that of
1
(41.9%) to varying degrees. Then their
anti-COL1A1 effects were investigated on the protein level by western blot assays. As indicated in
Figure 3A, 6i 6l and 6n could significantly reduce the COL1A1 protein level with inhibition rates
of 65.0%, 99.3% and 47.6%, respectively, significantly higher than that of (16.2%). These results
,
1
suggested that these matrinic thiadiazole compounds could effectively reduce COL1A1 on both the
mRNA and protein levels.
Figure 2. Effects of compounds 6i
,
6l and 6n inhibiting (
A
) COL1A1, (
B)
α
-SMA mRNA levels in LX-2
cells. Data were analyzed by Real-time PCR and presented as the mean
to that of control group; (*) p < 0.05, (**) p < 0.01 as compared to that of TGF-β1 group. The mRNA
±
SEM, (#) p < 0.05 as compared
expression levels were normalized against GAPDH.
Figure 3.
(A
) Effects of compounds 6i
,
6l and 6n on inhibiting fibrogenetic COL1A1 and
α-SMA
protein levels in LX-2 cells; (B) Effects of compounds 6i
,
6l and 6n on p-smad2 and smad2 levels in
LX-2 cells. Data were analyzed by western blot assay. The protein expression levels were normalized
against GAPDH.
2
.4. Inhibition Effects of α-SMA on RNA and Protein Levels by Key Compounds
α
-SMA is a more commonly recognized indicator for liver fibrosis, and it could be induced by
a stimulation of TGF 1 [22], therefore, the amounts of -SMA were also evaluated. In the same
experiments as illustrated above, 6i 6l and 6n significantly reduced -SMA mRNA levels with
inhibitory rates of 75.1%, 82.5% and 87.6% respectively, superior to that of (69.7%), as indicated in
Figure 2B. Compounds 6i 6l and 6n significantly reduced the -SMA protein level with inhibition
rates of 64.9%, 93.8% and 104.1%, respectively, significantly superior to that of 1 (28.9%), as indicated
β
α
,
α
1
,
α
in Figure 3A. These results disclosed that these kind of compounds also inhibited fibrogenetic
on both mRNA and protein levels.
α-SMA
2
.5. Action on TGFβ/Smad Pathway of Key Compounds
Considering the key role of TGF /Smad pathway in activating COL1A1, the effects of the three
compounds on Smad and phosphorylated Smad were then evaluated. As indicated in Figure 2B, 6i
and 6n did not affect the Smad level, while they repressed the phosphorylation level of Smad2
significantly, therefore, it was speculated that they might exert anti-liver fibrotic activity via repressing
β
,
6
l

Molecules 2018, 23, 1644
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the TGFβ/Smad pathway, as depicted in Figure 4. Among them, compound 6n showed the highest
inhibition rate of 49.8% as anticipated, and it was selected for the next investigation.
Figure 4. Compound 6n exerted the anti-liver fibrotic activity via repressing the Smad/TGFβ pathway.
2
.6. Safety Profile of Key Compounds
To evaluate the safety profile of 6n, an acute toxicity test was performed in Kunming mice.
−
1
Compound 6n was given orally in a single-dosing experiment at 125, 250 or 500 mg kg , respectively.
·
The mice were closely monitored for 7 days. To our delight, it gave the median lethal dose (LD )
value over 500 mg·kg− , indicating a good safety profile in vivo.
50
1
3
. Experimental Section
3
.1. Apparatus, Materials, and Analysis Reagents
All chemical reagents and anhydrous solvents were obtained from commercial sources and
used without further purification. Melting points (mp) were obtained with a MP90 melting point
apparatus and was uncorrected (Mettler-Toledo, Greifensee, Switzerland). Specific rotations were
obtained with Rudolph Autopol IV automatic polarimeter (Jasco Products Company, Oklahoma City,
1
13
1
OK, USA). H- and C-NMR spectra were recorded on Avance (600 MHz and 500 MHz for H-NMR;
51 MHz and 126 MHz for 13C-NMR) spectrometers (Bruker, Zürich, Switzerland). The H chemical
shifts were referenced to the solvent peak: DMSO-d (2.49 ppm) and CDCl (7.26 ppm) and the
1
1
13
C
6
3
chemical shifts were referenced to the solvent peak: DMSO-d (40.5 ppm) and CDCl (77.4 ppm).
6
3
ESI high-resolution mass spectra (HRMS) were recorded on an AutospecUltima-TOF spectrometer
Micromass UK Ltd., Manchester, UK). The method of the HPLC analysis was as follows: column,
Inertsustain C18, 250 mm 4.6 mm, id; flow rate, 1.0 mL/min; mobile phase, acetonitrile/water
KH PO in water, pH = 4.0) = 5:95; Flash column chromatography was performed on
(
×
−
L
1
(
0.01 mol
Combiflash Rf 200 (Teledyne, Lincoln, NE, USA), particle size 0.038 mm. The target compounds
were reported previously [ 15 23], but and 5b were obtained in this manuscript by a new
method with improved yields.
·
2
4
1
and
5
a
–
d
9,
,
1
–d
3
.2. Chemistry
3
.2.1. General Procedure for the Synthesis of Compounds 1, 5b–d
Matrine (5.0 g, 20 mmol) was added to 5 N NaOH solution (30 mL), and the reaction mixture was
refluxed for 9 h, cooled to room temperature and then acidified with HCl (2 N) to pH 2–3. The solvent
was removed in vacuo and the residue was dissolved with 2% HCl in methanol and then refluxed for

Molecules 2018, 23, 1644
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2
h. The solvent was removed under reduced pressure to give a crude product, which was purified by
recrystallization from ethanol to achieve intermediate 3 (6.6 g, 93.1%). To a suspension of compound
3
(5.0 g, 14 mmol) in dichloromethane (50 mL), triethylamine (4.34 g, 43 mmol) and substituted
benzenesulfonyl chloride (17 mmol) or substituted benzyl bromide (17 mmol) were added, the reaction
mixture was stirred at room temperature for 6 h until TLC analysis showed completion of the reaction.
The reaction mixture was then washed by water (50 mL), saturated aqueous ammonium chloride
(
50 mL) and saturated aqueous sodium chloride (50 mL) subsequently, dried over anhydrous sodium
sulfate and concentrated under reduced pressure. The residue was purified by recrystallization from
methanol to give . Compound (10 mmol) were refluxed in 5 N NaOH solution (30 mL) for 4 h,
4
4
cooled and acidified with HCl (2 N) to pH 4–5. The solvent was removed by condensation, and the
residue was suspended in methanol, and the precipate was filtered off. The filtrate was evapotared
to give a crude. The desired compounds (
1 or 5b–d) were gained by flash column chromatography
purification on silica gel with dichloromethane/methanol as the eluent.
◦
1
2
2-N-p-Methylbenzenesulfonyl matrinic acid (
39–241 C [15]).
1
). Total yield: 64%, m.p.: 239–240 C (Total yiled: 6%, m.p.:
◦
◦
◦
1
2-N-Benzyl matrinic acid (5b). Total yield: 41%, m.p.: 95–96 C (Total yield: 15%, m.p.: 95–96 C [15]).
◦
1
2-N-p-Methylbenzyl matrinic acid (5c). Total yield: 46%, m.p.: 106–108 C (Total yield: 34%, m.p.:
◦
1
06–108 C [9]).
◦
1
2-N-p-Bromobenzyl matrinic acid (5d). Total yield: 59%, m.p.: 134–135 C (Total yield: 27%, m.p.:
◦
1
33–135 C [9]).
3
.2.2. General Procedure for the Synthesis of Compounds 6a–o
A mixture of
1 or 5a–d (2.5 mmol), thiosemicarbazide or N-substituted thiosemicarbazide
(
7
2.5 mmol), methanesulfonic acid (12.5 mmol) and phosphorus pentoxide (2.5 mmol) were heated at
0 C for 8 h. The reaction mixture was poured into ice water (40 mL) and neutralized with aqueous
◦
ammonia to pH 8. The solution was extracted with dichloromethane (50 mL), and the organic layer
was washed by saturated aqueous sodium chloride (50 mL), dried over anhydrous sodium sulfate,
and concentrated under reduced pressure. The residue was purified by flash column chromatography
on silica gel with dichloromethane/methanol or petroleum ether/ethyl acetate as the eluent.
0
1
2-N-p-Methlybenzenesulfonyl-3 -(5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6a). Compound
1
(1.04 g,
2
.5 mmol) was treated with thiosemicarbazide (0.23 g, 2.5 mmol) according to the general procedure,
then purified by flash column chromatography with dichloromethane/methanol as the eluent to give
◦
25
1
the desired product 6a as a white solid, yield: 40.9%; m.p.: 237 C (dec.); [α]5
=
−
6.16; H-NMR
89
(500 MHz, DMSO-d ) δ 7.68 (d, J = 8.2 Hz, 2H, 16-CH, 20-CH), 7.38 (d, J = 8.2 Hz, 2H, 17-CH, 19-CH),
6
0 0
.00 (s, 2H, 9 -NH ), 3.42–3.36 (m, 2H, 3 -CH ), 3.16–3.08 (m, 1H, 6-CH), 2.73–2.69 (m, 1H, 5-CH),
2 2
7
2
0
.65–2.61 (m, 1H, 7-CH), 2.59–2.56 (m, 1H, 11-CH), 2.38 (s, 3H, Ph-CH ), 1.95–1.90 (m, 1H, 1 a-CH),
3
0
1
1
.87–1.80 (m, 2H, 2 -CH ), 1.76–1.61 (m, 6H, 2-CH , 10-CH , 13-CH ), 1.51–1.45 (m, 2H, 4-CH ),
.39–1.23 (m, 7H, 3-CH , 8-CH , 9-CH , 1 b-CH); C-NMR (126 MHz, DMSO-d6)
2
2
2
2
2
0
13
0
δ 168.2 (7 -C), 158.2
2
2
2

Molecules 2018, 23, 1644
8 of 16
0
(
4 -C), 142.9 (18-C), 136.7 (15-C), 129.5 (17-CH, 19-CH), 127.2 (16-CH, 20-CH), 62.3 (6-CH), 56.6 (11-CH),
0 0
6.2 (2-CH ), 56.1 (10-CH ), 47.1 (13-CH ), 38.6 (7-CH), 34.0 (5-CH), 29.7 (1 -CH ), 29.6 (3 -CH ), 27.6
2 2 2 2 2
5
(
0
4-CH ), 27.5 (8-CH ), 24.8 (2 -CH ), 21.1 (Ph-CH ), 20.3 (3-CH ), 20.2 (9-CH ); ESI-HRMS: m/z Calcd
2
2
2
3
2
2
+
for C H O N S [M + H] , 476.2148; Found, 476.2149; HPLC: t = 10.29 min, normalization method
2
3
34
2
5 2
R
purity 97.11%.
2-N-p-Mmethlybenzenesulfonyl-3 -(N-methyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic
Compound (1.04 g, 2.5 mmol) was treated with 4-methyl-3-thiosemicarbazide (0.26 g, 2.5 mmol)
0
1
propane
(6b).
1
according to the general procedure, then purified by flash column chromatography with
dichloromethane/methanol as the eluents to give to give the desired product 6b as a white
◦
25
7.98; 1H-NMR (500 MHz, DMSO-d )
solid, yield: 33.4%; m.p.: 169–170 C; [α]5
(
1
=
−
δ
7.68
89
6
0
d, J = 7.9 Hz, 2H, 16-CH, 20-CH), 7.49 (q, J = 4.8 Hz, 1H, 9 -NH), 7.38 (d, J = 7.9 Hz, 2H, 17-CH,
0 0
9-CH), 3.43–3.35 (m, 2H, 3 -CH ), 3.14–3.09 (m, 1H, 6-CH), 2.83 (d, J = 4.8 Hz, 3H, 9 -NCH ), 2.76–2.70
2 3
(
1
3
m, 1H, 5-CH), 2.67–2.61 (m, 1H, 7-CH), 2.57 (m 1H, 11-CH), 2.38 (s, 3H, Ph-CH ), 1.95–1.90 (m, 1H,
3
0
0
a-CH), 1.86–1.80 (m, 2H, 2 -CH ), 1.75–1.62 (m, 6H, 2-CH , 10-CH , 13-CH ), 1.49–1.23 (m, 9H,
2 2 2 2
0
13
0
0
-CH , 4-CH , 8-CH , 9-CH , 1 b-CH ); C-NMR (126 MHz, DMSO-d6)
δ
169.1 (7 -C), 157.6 (4 -C),
2
2
2
2
1
5
2
42.9 (18-C), 136.7 (15-C), 129.5 (17-CH, 19-CH), 127.2 (16-CH, 20-CH), 62.3 (6-CH), 56.6 (11-CH),
0
0
6.1 (2-CH ), 56.0 (10-CH ), 47.0 (13-CH ), 38.6 (7-CH), 34.0 (5-CH), 31.2 (9 -NCH ), 29.7 (1 -CH ),
2
2
2
3
2
0
0
+
9.6 (3 -CH ), 27.5 (4-CH ), 27.4 (8-CH ), 24.9 (2 -CH ), 21.0 (Ph-CH ), 20.3 (3-CH ), 20.2 (9-CH );
2
2
2
2
3
2
2
ESI-HRMS: m/z Calcd for C H O N S [M + H] , 490.2304; Found, 490.2304; HPLC: t = 10.78 min,
24
36
2
5 2
R
normalization method purity 98.74%.
0
1
2-N-p-Methylbenzenesulfonyl-3 -(N-iso-propyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane
(6c).
Compound
1 (1.04 g, 2.5 mmol) was treated with 4-iso-propyl-3-thiosemicarbazide (0.33 g,
2.5 mmol) according to the general procedure, then purified by flash column chromatography with
dichloromethane/methanol as the eluents to give the desired product 6d as a white solid, yield: 34.1%;
◦
25
1
m.p.: 142–143 C; [α]5
=
−
15.79; H-NMR (600 MHz, DMSO-d )
δ
7.68 (d, J = 8.1 Hz, 2H, 16-CH,
89
6
0
2
9
0-CH), 7.44 (d, J = 7.2 Hz, 1H, 9 -NH), 7.38 (d, J = 8.1 Hz, 2H, 17-CH, 19-CH), 3.77–3.69 (m, 1H,
-NCH), 3.42–3.38 (m, 2H, 3 -CH ), 3.13 (t, J = 11.6 Hz, 1H, 6-CH), 2.74–2.70 (m, 1H, 5-CH), 2.66–2.61
m, 1H, 7-CH), 2.58–2.53 (m, 1H, 11-CH), 2.38 (s, 3H, Ph-CH ), 1.94 (m, 1H, 1 a-CH), 1.86–1.80 (m, 2H,
-CH ), 1.75–1.61 (m, 6H, 2-CH , 10-CH , 13-CH ), 1.49–1.46 (m, 2H, 4-CH ), 1.39–1.23 (m, 7H, 3-CH ,
-CH , 9-CH , 1 b-CH), 1.17 (d, J = 6.4 Hz, 6H, NC(CH3)2); C-NMR (151 MHz, DMSO-d6)
7 -C), 157.2 (4 -C), 142.9 (18-C), 136.7 (15-C), 129.5 (17-CH, 19-CH), 127.2 (16-CH, 20-CH), 62.3 (6-CH),
0
0
2
0
(
2
8
3
0
2
2
2
2
2
2
0
13
δ
167.4
2
2
0
0
(
5
6.6 (11-CH), 56.1 (2-CH ), 56.0 (10-CH ), 47.0 (13-CH ), 46.5 (NCH), 38.6 (7-CH), 34.0 (5-CH), 29.8
2
2
2
0 0 0
1 -CH ), 29.5 (3 -CH ), 27.5 (4-CH ), 27.4 (8-CH ), 24.9 (2 -CH ), 22.2 (NC(CH ) ), 21.0 (Ph-CH ), 20.3
2 2 2 2 2 3 2 3
(
(
+
3-CH ), 20.2 (9-CH ); ESI-HRMS: m/z Calcd for C H O N S [M + H] , 518.2617; Found, 518.2607;
2
2
26 40
2
5 2
HPLC: t = 12.11 min, normalization method purity 97.99%.
R
0
1
2-N-p-Methylbenzenesulfonyl-3 -(N,N-dimethyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane
(6d).
Compound
1 (1.04 g, 2.5 mmol) was treated with 4,4-dimethyl-3-thiosemicarbazide (0.31 g,
2.5 mmol) according to the general procedure, then purified by flash column chromatography with
dichloromethane/methanol as the eluents to give the desired product 6e as a white solid, yield: 24.2%;
◦
25
1
m.p.: 171–172 C; [α]5
2
=
−
9.66; H-NMR (500 MHz, DMSO-d )
δ
7.68 (d, J = 8.2 Hz, 2H, 16-CH,
89
6
0
0-CH), 7.39 (d, J = 8.2 Hz, 2H, 17-CH, 19-CH), 3.45–3.36 (m, 2H, 3 -CH ), 3.18–3.07 (m, 1H, 6-CH), 3.03
2
(
2
1
1
s, 6H, N(CH ) ), 2.81–2.72 (m, 1H, 5-CH), 2.72–2.63 (m, 1H, 7-CH), 2.57 (d, J = 11.4 Hz, 1H, 11-CH),
3 2
0
0
.38 (s, 3H, Ph-CH ), 1.94 (m, 1H, 1 a-CH), 1.90–1.79 (m, 2H, 2 -CH ), 1.78–1.60 (m, 6H, 2-CH , 10-CH ,
3
2
2
2
0
13
3-CH ), 1.54–1.18 (m, 9H, 3-CH , 4-CH , 8-CH , 9-CH , 1 b-CH); C-NMR (151 MHz, DMSO-d6)
71.2 (7 -C), 158.5 (4 -C), 142.8 (18-C), 136.8 (15-C), 129.5 (17-CH, 19-CH), 127.1 (16-CH, 20-CH), 62.3
δ
2
2
2
2
2
0
0
(
(
(
6-CH), 56.6 (11-CH), 56.1 (2-CH ), 56.0 (10-CH ), 47.0 (13-CH ), 41.1 (N(CH ) ), 38.7 (7-CH), 34.0
2 2 2 3 2
0
0
0
5-CH), 29.7 (1 -CH ), 29.6 (3 -CH ), 27.5 (4-CH ), 27.4 (8-CH ), 25.1 (2 -CH ), 21.0 (Ph-CH ), 20.3
2
2
2
2
2
3
+
3-CH ), 20.2 (9-CH ); ESI-HRMS: m/z Calcd for C H O N S [M + H] , 504.2441; Found, 504.2447;
2
2
25 38
2
5 2
HPLC: t = 11.59 min, normalization method purity 98.45%.
R

Molecules 2018, 23, 1644
9 of 16
0
1
2-N-p-Methylbenzenesulfonyl-3 -(N-phenyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic
propane
(6e).
Compound
1 (1.04 g, 2.5 mmol) was treated with 4-phenyl-3-thiosemicarbazide (0.42 g, 2.5 mmol)
according to the general procedure, then purified by flash column chromatography with petroleum
ether/ethyl acetate as the eluents to give the desired product 6c as a white solid, yield: 48.2%; m.p.:
◦
25
1
0
1
2
7
3
1
2
93–194 C; [α]5 = −13.89; H-NMR (500 MHz, DMSO-d ) δ 10.26 (s, 1H, 9 -NH), 7.68 (d, J = 8.0 Hz,
89
6
0
H, 16-CH, 20-CH), 7.60 (d, J = 8.0 Hz, 2H, 17-CH, 19-CH), 7.38 (d, J = 7.5 Hz, 2H, 2
.33 (t, J = 7.5 Hz, 2H, 2
.13 (t, J = 11.6 Hz, 1H, 6-CH), 2.87–2.81 (m, 1H, 5-CH), 2.79–2.72 (m, 1H, 7-CH), 2.57 (d, J = 11.1 Hz,
H, 11-CH), 2.35 (s, 3H, Ph-CH ), 1.93 (m, 1H, 1 a-CH), 1.86–1.83 (m, 2H, 2 -CH ), 1.75–1.68 (m, 6H,
×
9 -NCHarom),
0
0
0
×
9 -NCHarom), 6.97 (t, J = 7.5 Hz, 1H, 9 -NCHarom ), 3.43–3.38 (m, 2H, 3 -CH ),
2
0
0
3
2
13
0
-CH , 10-CH , 13-CH ), 1.56–1.23 (m, 9H, 3-CH , 4-CH , 8-CH , 9-CH , 1 b-CH); C-NMR (151 MHz,
2
2
2
2
2
2
2
0
0
0
DMSO-d ) δ 163.9 (7 -C), 159.5 (4 -C), 142.8 (18-C), 140.8 (9 N-Ph), 136.8 (15-C), 129.5 (17-CH, 19-CH),
6
0
0
0
1
(
(
29.0 (9 N-Ph), 127.1 (16-CH, 20-CH), 121.6 (9 N-Ph), 117.1 (9 N-Ph), 62.3 (6-CH), 56.6 (11-CH), 56.1
0 0
2-CH ), 56.0 (10-CH ), 46.9 (13-CH ), 38.8 (7-CH), 34.0 (5-CH), 29.8 (1 -CH ), 29.4 (3 -CH ), 27.6
2 2 2 2 2
0
4-CH ), 27.5 (8-CH ), 24.9 (2 -CH ), 21.0 (Ph-CH ), 20.3 (3-CH ), 20.2 (9-CH ); ESI-HRMS: m/z Calcd
2
2
2
3
2
2
+
for C H O N S [M + H] , 552.2461; Found, 552.2462; HPLC: t = 13.39 min, normalization method
2
9
38
2
5 2
R
purity 96.69%.
2-N-p-Ttrifluoromethlybenzenesulfonyl-3 -(5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6f). Compound
(1.19 g, 2.5 mmol) was treated with thiosemicarbazide (0.23 g, 2.5 mmol) according to the general
procedure, then purified by flash column chromatography with dichloromethane/methanol as the
0
1
5
a
◦
25
eluents to give the desired product 6f as a white solid, yield: 48.0%; m.p.: 206 C (dec.); [α] = −0.65
;
589
1
H-NMR (500 MHz, CDCl )
δ
7.96 (d, J = 8.2 Hz, 2H, 16-CH, 20-CH), 7.73 (d, J = 8.2 Hz, 2H, 17-CH,
9-CH), 5.56 (s, 2H, 9 -NH ), 3.67–3.57 (m, 1H, 3 a-CH), 3.53–3.45 (m, 1H, 3 b-CH), 3.22 (t, J = 11.5 Hz,
H, 6-CH), 2.93–2.75 (m, 2H, 5-CH, 7-CH), 2.54 (d, J = 11.5 Hz, 1H, 13a-CH), 2.45 (d, J = 11.5 Hz, 1H,
3b-CH), 1.96 (m, 2H, 2 -CH ), 1.89–1.62 (m, 8H, 2-CH , 4-CH , 10-CH , 11-CH, 1 a-CH), 1.49–1.26
0 0 0
m, 7H, 3-CH , 8-CH , 9-CH , 1 b-CH); C-NMR (126 MHz, CDCl ) δ 168.2 (7 -C), 161.2 (4 -C), 143.9
2 2 2 3
15-C), 133.9 (18-C), 128.1 (17-CH, 19-CH), 125.9 (16-CH, 20-CH), 123.4 (Ph-CF ), 62.7 (6-CH), 57.5
11-CH), 56.6 (2-CH ), 56.5 (10-CH ), 46.9 (13-CH ), 39.7 (7-CH), 34.4 (5-CH), 31.6 (1 -CH ), 30.3
3
0
0
0
1
1
1
(
(
(
(
2
0
0
2
2
2
2
13
3
0
2
2
2
2
0
0
3 -CH ), 28.3 (4-CH ), 27.9 (8-CH ), 25.8 (2 -CH ), 20.9 (3-CH ), 20.6 (9-CH ); ESI-HRMS: m/z Calcd
for C H O N F S [M + H] , 530.1865; found, 530.1864; HPLC: t = 11.41 min, normalization method
2
2
2
2
2
2
+
23
31
2
5
3 2
R
purity 98.85%.
0
1
2-N-p-Trifluoromethlybenzenesulfonyl-3 -(N-methyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6g).
Compound 5a (1.19 g, 2.5 mmol) was treated with 4-methyl-3-thiosemicarbazide (0.26 g, 2.5 mmol)
according to the general procedure, then purified by flash column chromatography with
◦
dichloromethane/methanol to give the desired product 6g as a white solid, yield: 45.0%; m.p.: 181 C
dec.); [α]2
5
=
−
1.89; H-NMR (500 MHz, CDCl )
1
δ
7.97 (d, J = 8.2 Hz, 2H, 16-CH, 20-CH), 7.73
d, J = 8.2 Hz, 2H, 17-CH, 19-CH), 5.79 (s, 1H, 9 -NH), 3.68–3.57 (m, 1H, 3 a-CH ), 3.53–3.46 (m, 1H,
b-CH), 3.24 (t, J = 11.4 Hz, 1H, 6-CH), 3.01 (s, 3H, 9 -NCH ), 2.91–2.77 (m, 2H, 5-CH, 7-CH), 2.54 (d,
(
(
3
589
3
0
0
0
0
3
0
J = 11.4 Hz, 1H, 13a-CH), 2.46 (d, J = 11.4 Hz, 1H, 13b-CH), 2.02–1.91 (m, 2H, 2 -CH ), 1.86–1.63 (m, 8H,
2
0 0
-CH , 4-CH , 10-CH , 11-CH, 1 a-CH), 1.51–1.31 (m, 7H, 3-CH , 8-CH , 9-CH , 1 b-CH); C-NMR
2 2 2 2 2 2
13
2
0 0
δ 171.4 (7 -C), 159.2 (4 -C), 144.1 (15-C), 133.9 (18-C), 128.1 (17-CH, 19-CH), 125.8
(126 MHz, CDCl3)
(
3
(
16-CH, 20-CH), 123.4 (Ph-CF ), 62.7 (6-CH), 57.6 (11-CH), 56.6 (2-CH ), 56.5 (10-CH ), 47.0 (13-CH ),
3
2
2
2
0 0 0
9.8 (7-CH), 34.5 (5-CH), 33.3 (9 -NCH ), 31.6 (1 -CH ), 30.4 (3 -CH ), 28.3 (4-CH ), 28.0 (8-CH ), 25.9
3 2 2 2 2
0
+
2 -CH ), 20.9 (3-CH ), 20.7 (9-CH ); ESI-HRMS: m/z Calcd for C H O N F S [M + H] , 544.2022;
2
2
2
24 33
2
5 3 2
found, 544.2019; HPLC: t = 11.89 min, normalization method purity 98.90%.
R
0
1
2-N-p-Trifluoromethlybenzenesulfonyl-3 -(N,N-dimethyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane
(
2
6h). Compound 5a (1.19 g, 2.5 mmol) was treated with 4,4-dimethyl-3- thiosemicarbazide (0.31 g,
.5 mmol) according to the general procedure, then purified by flash column chromatography with
petroleum ether/ethyl acetate to give the desired product 6h as a white solid, yield: 33.3%; m.p.:
◦
25
1
1
47–148 C; [α]5
=
−
3.30; H-NMR (500 MHz, CDCl ) δ 7.97 (d, J = 8.2 Hz, 2H, 16-CH, 20-CH), 7.72
3
89

Molecules 2018, 23, 1644
10 of 16
0 0
d, J = 8.2 Hz, 2H, 17-CH, 19-CH), 3.66–3.62 (m, 1H, 3 a-CH), 3.51 (dd, J = 12.6, 6.2 Hz, 1H, 3 b-CH),
(
0
.27–3.22 (m, 1H, 6-CH), 3.09 (s, 6H, 9 -N(CH ) ), 2.90–2.71 (m, 2H, 5-CH, 7-CH), 2.56–2.53 (m, 1H,
3 2
3
0
3a-CH), 2.49–2.45 (m, 1H, 13b-CH), 1.99–1.94 (m, 2H, 2 -CH ), 1.86–1.61 (m, 8H, 2-CH , 4-CH ,
2 2 2
1
1
δ
0
0
13
0-CH , 11-CH, 1 a-CH), 1.52–1.29 (m, 7H, 3-CH , 8-CH , 9-CH , 1 b-CH); C-NMR (126 MHz, CDCl3)
2
2
2
2
0
0
172.0 (7 -C), 159.3 (4 -C), 144.2 (15-C), 133.8 (18-C), 128.1 (17-CH, 19-CH), 125.8 (16-CH, 20-CH), 123.4
0
(
(
Ph-CF ), 62.8 (6-CH), 57.6 (11-CH), 56.6 (2-CH ), 56.5 (10-CH ), 47.1 (13-CH ), 41.5 (9 N(CH ) ), 39.8
3
2
2
2
3 2
0 0 0
7-CH), 34.5 (5-CH), 31.5 (1 -CH ), 30.4 (3 -CH ), 28.3 (4-CH ), 28.0 (8-CH ), 25.9 (2 -CH ), 20.9 (3-CH ),
2 2 2 2 2 2
+
2
0.7 (9-CH ); ESI-HRMS: m/z Calcd for C H O N F S [M + H] , 558.2178; found, 558.2169; HPLC:
2 25 35 2 5 3 2
tR = 12.71 min, normalization method purity 98.60%.
0
1
2-N-Benzyl-3 -(5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6i) Compound 5b (1.19 g, 2.5 mmol) was
treated with thiosemicarbazide (0.23 g, 2.5 mmol) according to the general procedure, then purified
by flash column chromatography with dichloromethane/methanol to give the desired product 6i
◦
25
1
as a white solid, yield: 34.7%; m.p.: 170–171 C; [α]5
=
−
29.79; H-NMR (600 MHz, CDCl ) δ
3
89
7
2
3
2
.30 (dt, J = 15.0, 7.2 Hz, 4H, 16-CH, 17-CH, 19-CH, 20-CH), 7.20 (t, J = 7.2 Hz, 1H, 18-CH), 5.67 (s,
H, 9 -NH ), 4.00 (d, J = 13.4 Hz, 1H, 14a-CH), 3.12 (d, J = 13.4 Hz, 1H, 14b-CH), 2.94–2.72 (m, 5H,
-CH , 5-CH, 7-CH, 11-CH ), 2.64 (t, J = 11.9 Hz, 1H, 6-CH), 2.33 (m, 1H, 1 a-CH), 1.95–1.57 (m, 12H,
-CH , 4-CH , 8-CH , 10-CH , 13-CH , 2 -CH ), 1.45–1.28 (m, 5H, 3-CH , 9-CH , 1 b-CH); C-NMR
0
2
0
0
2
0
0
13
2
2
2
2
2
2
2
2
0
0
(
151 MHz, CDCl3)
δ
168.3 (7 -C), 161.6 (4 -C), 140.6 (15-C), 128.7 (17-CH, 19-CH), 128.3 (16-CH, 20-CH),
1
3
26.7 (18-CH), 64.6 (6-CH), 57.6 (11-CH), 57.4 (14-CH ), 57.3 (13-CH ), 56.7 (2-CH ), 52.4 (10-CH ),
2
2
2
2
0
0
0
7.9 (7-CH), 34.0 (5-CH), 30.8 (1 -CH ), 28.3 (3 -CH ), 28.2 (4-CH ), 27.4 (8-CH ), 23.9 (2 -CH ), 21.7
2
2
2
2
2
+
(3-CH ), 21.4 (9-CH ); ESI-HRMS: m/z Calcd for C H N S [M + H] , 412.2529; found, 412.2530;
2 2 23 34 5
HPLC: t = 8.26 min, normalization method purity 98.65%.
R
0
1
2-N-Benzyl-3 -(N-methyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6j) Compound 5b (1.19 g,
2.5 mmol) was treated with 4-methyl-3-thiosemicarbazide (0.26 g, 2.5 mmol) according to the general
procedure, then purified by flash column chromatography with dichloromethane/methanol to give
◦
25
1
the desired product 6j as a white solid, yield: 32.3%; m.p.: 137–138 C; [α]5
=
−
28.80; H-NMR
89
(
600 MHz, CDCl )
δ
7.32–7.27 (m, 4H, 16-CH, 17-CH, 19-CH, 20-CH), 7.20 (t, J = 7.2 Hz, 1H, 18-CH),
.86 (m, 1H, 9 -NH), 4.01 (d, J = 13.0 Hz, 1H, 14a-CH), 3.12 (d, J = 13.0 Hz, 1H, 14b-CH), 2.97–2.60 (m,
H, 3 -CH , 5-CH, 6-CH, 7-CH, 9 -NCH , 11-CH), 2.34 (d, J = 11.6 Hz, 1H, 1 a-CH), 1.99–1.52 (m, 12H,
-CH , 4-CH , 8-CH , 10-CH , 13-CH , 2 -CH ), 1.47–1.29 (m, 5H, 3-CH , 9-CH , 1 b-CH); C-NMR
3
0
5
9
2
0
0
0
2
3
0
0
13
2
2
2
2
2
2
2
2
0
0
(
151 MHz, CDCl3)
δ
171.4 (7 -C), 159.7 (4 -C), 140.6 (15-C), 128.7 (17-CH, 19-CH), 128.3 (16-CH, 20-CH),
1
3
(
26.7 (18-CH), 64.6 (6-CH), 57.7 (11-CH), 57.4 (14-CH ), 57.3 (13-CH ), 56.7 (2-CH ), 52.3 (10-CH ),
2
2
2
2
0
0
0
7.9 (7-CH), 34.0 (5-CH), 33.3 (9 -NCH ), 30.8 (1 -CH ), 28.3 (3 -CH ), 28.2 (4-CH ), 27.4 (8-CH ), 24.0
3
2
2
2
2
0
2 -CH ), 21.7 (3-CH ), 21.4 (9-CH ); ESI-HRMS: m/z Calcd for C H N S [M + H] , 426.2686; found,
2 2 2 24 36 5
+
4
26.2685; HPLC: t = 9.14 min, normalization method purity 98.91%.
R
0
1
2-N-Benzyl-3 -(N,N-dimethyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane hydrochloride
(6k).
Compound 5b (1.19 g, 2.5 mmol) was treated with 4,4-dimethyl-3-thiosemicarbazide (0.31 g, 2.5 mmol)
according to the general procedure, then purified by flash column chromatography with petroleum
ether/ethyl acetate to give the crude product and treated with 2 N hydrochloride/ether (3 mL) to
◦
25
1
give the desired product 6k as a white solid, yield: 16.3%; m.p.: 189–190 C; [α]
=
−
14.64; H-NMR
589
+
(500 MHz, DMSO-d6)
δ
11.81 (s, 1H, 1-NH ), 7.66–7.62 (m, 2H, 16-CH, 20-CH), 7.47–7.43 (m, 3H,
1
2
2
7-CH, 18-CH, 19-CH), 5.00 (d, J = 12.7 Hz, 2H, 14-CH ), 4.26 (t, J = 10.3 Hz, 1H, 6-CH), 3.99–3.93 (m,
2
0
0
H, 13-CH ), 3.61 (d, J = 10.3 Hz, 1H, 7-CH), 3.32–3.20 (m, 2H, 3 -CH ), 3.05–2.87 (m, 6H, 9 -N(CH ) ),
2
2
3 2
0
0
.66–2.60 (m, 2H, 5-CH, 11-CH), 2.57–2.52 (m, 1H, 1 a-CH), 2.18–1.92 (m, 6H, 2-CH , 10-CH , 2 -CH ),
2
13
2
2
0
.89–1.55 (m, 8H, 3-CH , 4-CH , 8-CH , 9-CH ), 1.43 (d, J = 13.6 Hz, 1H, 1 b-CH); C-NMR (126 MHz,
2 2 2 2
1
0
0
DMSO-d6)
δ
171.0 (7 -C), 158.7 (4 -C), 132.0 (18-C), 130.7 (15-C), 123.0 (17-CH, 19-CH), 129.4 (16-CH,
2
(
0-CH), 61.0 (6-CH), 60.8 (14-CH ), 58.1 (11-CH), 54.8 (2-CH ), 54.7 (10-CH ), 49.3 (13-CH ), 43.0
2 2 2 2
0 0 0
9 -N(CH ) ), 36.5 (7-CH), 30.5 (5-CH), 29.9 (1 -CH ), 28.2 (3 -CH ), 26.4 (4-CH ), 24.7 (8-CH ), 24.2
3 2 2 2 2 2

Molecules 2018, 23, 1644
11 of 16
0
+
(
2 -CH ), 18.5 (3-CH ), 18.4 (9-CH ); ESI-HRMS: m/z Calcd for C H N S [M-HCl+H] , 440.2842;
2
2
2
25 38
5
found, 440.2839; HPLC: t = 9.40 min, normalization method purity 98.49%.
R
0
1
2-N-p-Methylbenzyl-3 -(5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6l) Compound 5c (0.93 g,
2
.5 mmol) was treated with thiosemicarbazide (0.23 g, 2.5 mmol) according to the general procedure,
then purified by flash column chromatography with dichloromethane/methanol to give the desired
◦
25
1
product 6l as a white solid, yield: 41.4%; m.p.:167–168 C; [α]
=
−
30.62; H-NMR (600 MHz, CDCl )
589
3
0
δ
3
7
7.19 (d, J = 7.7 Hz, 2H, 16-CH, 20-CH), 7.09 (d, J = 7.7 Hz, 2H, 17-CH, 19-CH), 5.68 (s, 2H, 9 -NH ),
.96 (d, J = 13.4 Hz, 1H, 14a-CH), 3.08 (d, J = 13.4 Hz, 1H, 14b-CH), 2.89–2.73 (m, 4H, 3 -CH , 5-CH,
-CH), 2.62 (t, J = 11.9 Hz, 1H, 6-CH), 2.35–2.32 (m, 1H, 11-CH), 2.31 (s, 3H, Ph-CH ), 2.04–2.00 (m, 1H,
2
0
2
3
0
0
1
9
a-CH), 1.92–1.60 (m, 12H, 2-CH , 4-CH , 8-CH , 10-CH , 13-CH , 2 -CH ), 1.43–1.29 (m, 5H, 3-CH ,
2
2
2
2
2
2
2
0
13
0 0
-CH , 1 b-CH ); C-NMR (151 MHz, CDCl3)
δ
168.3 (7 -C), 161.6 (4 -C), 137.3 (18-C), 136.2 (15-C),
2
1
5
29.0 (17-CH, 19-CH), 128.7 (16-CH, 20-CH), 64.6 (6-CH), 57.6 (14-CH ), 57.4 (11-CH), 56.4 (2-CH ),
2 2
0
0
2.2 (10-CH ), 37.9 (13-CH ), 33.9 (7-CH), 30.8 (5-CH), 28.3 (1 -CH ), 28.2 (3 -CH ), 27.4 (4-CH ), 23.9
2
2
2
2
2
0
(
8-CH ), 21.7 (2 -CH ), 21.4 (3-CH ), 21.2 (9-CH ), 21.1 (Ph-CH ); ESI-HRMS: m/z Calcd for C H N S
2
2
2
2
3
24 36
5
+
[M + H] , 426.2686; found; 426.2685; HPLC: t =8.78 min, normalization method purity 97.53%.
R
0
1
2-N-p-Methylbenzyl-3 -(N-methyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6m). Compound 5c
(
0.93 g, 2.5 mmol) was treated with 4-methyl-3-thiosemicarbazide (0.26 g, 2.5 mmol) according to the
general procedure, then purified by flash column chromatography with dichloromethane/methanol
◦
25
to give the desired product 6m as a white solid, yield: 34.1%; m.p.: 163–164 C; [α]5
=
−
35.69;
89
1
H-NMR (600 MHz, CDCl )
δ
7.19 (d, J = 7.6 Hz, 2H, 16-CH, 20-CH), 7.08 (d, J = 7.6 Hz, 2H, 17-CH,
3
0
1
(
(
1
δ
6
9-CH), 6.28 (s, 1H, 9 -NH), 3.96 (d, J = 13.3 Hz, 1H, 14a-CH), 3.06 (d, J = 13.3 Hz, 1H, 14b-CH), 2.94
0 0
s, 3H, 9 -NCH ), 2.89–2.77 (m, 4H, 3 -CH , 5-CH, 7-CH), 2.61 (t, J = 12.0 Hz, 1H, 6-CH), 2.35–2.32
3 2
0
m, 1H, 11-CH), 2.30 (s, 3H, Ph-CH ), 2.01 (m, 1H, 1 a-CH), 1.95–1.55 (m, 12H, 2-CH , 4-CH , 8-CH ,
3 2 2 2
0 0
0-CH , 13-CH , 2 -CH ), 1.44–1.29 (m, 5H, 3-CH , 9-CH , 1 b-CH); C-NMR (151 MHz, CDCl3)
2 2 2 2 2
13
0
0
171.6 (7 -C), 159.5 (4 -C), 137.4 (18-C), 136.1 (15-C), 128.9 (17-CH, 19-CH), 128.6 (16-CH, 20-CH),
4.6 (6-CH), 57.7 (14-CH ), 57.4 (11-CH), 56.4 (2-CH ), 52.2 (10-CH ), 38.0 (13-CH ), 34.0 (7-CH), 33.3
2
2
2
2
0 0 0 0
9 -NCH ), 30.8 (5-CH), 28.3 (1 -CH ), 28.2 (3 -CH ), 27.4 (4-CH ), 24.0 (8-CH ), 21.7 (2 -CH ), 21.4
3 2 2 2 2 2
(
+
(3-CH ), 21.2 (9-CH ), 21.1 (Ph-CH ); ESI-HRMS: m/z Calcd for C H N S [M + H] , 440.2842; found,
2 2 3 25 38 5
4
40.2845; HPLC: t =10.09 min, normalization method purity 95.47%.
R
0
1
2-N-p-Methylbenzyl-3 -(N,N-dimethyl-5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane hydrochloride (6n).
Compound 5c (0.93 g, 2.5 mmol) was treated with 4,4-dimethyl-3-thiosemicarbazide (0.31 g, 2.5 mmol)
according to the general procedure, then purified by flash column chromatography with petroleum
ether/ethyl acetate to give the crude product and treated with 2 N hydrochloride/ether (3 mL) to
◦
25
1
give the desired product 6n as a white solid, yield: 21.1%; m.p.: 199–200 C; [α]5
=
−
17.19; H-NMR
89
(
500 MHz, DMSO-d ) δ 7.51 (d, J = 7.6 Hz, 2H, 16-CH, 20-CH), 7.25 (d, J = 7.6 Hz, 2H, 17-CH, 19-CH),
6
5
.76 (s, 1H, 12-NH), 4.94 (m, 1H, 5-CH), 4.24 (m, 1H, 6-CH), 3.98–3.84 (m, 2H, 14-CH ), 3.62 (m,
2
0
1
(
2
H, 11-CH), 3.24 (m, 2H, 13-CH ), 3.17 (s, 6H, 9 -N(CH ) ), 3.05–2.86 (m, 4H, 2-CH ,10-CH ), 2.62
2
3 2
2
2
0 0
d, J = 10.3 Hz, 2H, 3 -CH ), 2.32 (s, 3H, Ph-CH ), 2.17–2.07 (m, 1H, 7-CH), 1.98 (m, 3H, 1 a-CH,
2 3
0
0
-CH ), 1.87–1.55 (m, 8H, 3-CH , 4-CH , 8-CH , 9-CH ), 1.43 (d, J = 13.8 Hz, 1H, 1 b-CH).; C-NMR
2 2 2 2 2
13
0 0
δ 170.5 (7 -C), 158.0 (4 -C), 138.8 (18-C), 131.4 (15-C), 129.4 (17-CH, 19-CH),
(126 MHz, DMSO-d6)
1
26.9 (16-CH, 20-CH), 60.3 (6-CH), 60.2 (14-CH ), 57.2 (11-CH), 55.0 (2-CH ), 54.2 (10-CH ), 48.5
2
2
2
0 0
13-CH ), 42.3 (N(CH ) ), 35.9 (7-CH), 29.9 (5-CH), 29.2 (1 -CH ), 27.6 (3 -CH ), 25.8 (4-CH ), 24.1
2 3 2 2 2 2
(
(
[
0
+
8-CH ), 23.5 (2 -CH ), 20.9 (3-CH ), 17.9 (9-CH ), 17.8 (Ph-CH ); ESI-HRMS: m/z Calcd for C H N S
M − HCl + H] , 454.2999 found, 454.2996; HPLC: t = 9.84 min, normalization method purity 96.00%.
2
2
2
2
3
26 40
5
R
0
12-N-p-Bromobenzyl-3 -(5-amino-1,3,4-thiadiazol-2-yl)-matrinic propane (6o) Compound 5d (1.09 g,
2
.5 mmol) was treated with thiosemicarbazide (0.23 g, 2.5 mmol) according to the general procedure,
then purified by flash column chromatography with dichloromethane/methanol to give the desired
◦
25
1
product 6o as a white solid, yield: 44.1%; m.p.: 193–194 C; [α]
=
89
−
19.58; H-NMR (500 MHz,
5

Molecules 2018, 23, 1644
12 of 16
CDCl )
δ 7.39 (d, J = 8.0 Hz, 2H, 16-CH, 20-CH), 7.19 (d, J = 8.0 Hz, 2H, 17-CH, 19-CH), 5.61 (s,
3
0
2
3
2
H, 9 -NH ), 3.92 (d, J = 13.8 Hz, 1H, 14a-CH), 3.06 (d, J = 13.8 Hz, 1H,14b-CH), 2.91–2.75 (m, 5H,
2
0
0
-CH , 5-CH, 7-CH, 11-CH), 2.64 (t, J = 11.8 Hz, 1H, 6-CH), 2.27 (m, 1H, 1 a-CH), 1.94–1.55 (m, 12H,
2
0
0
13
-CH , 4-CH , 8-CH , 10-CH , 13-CH , 2 -CH ), 1.45–1.30 (m, 5H, 3-CH , 9-CH , 1 b-CH); C-NMR
2
2
2
2
2
2
2
2
0 0
(
1
(
126 MHz, CDCl3)
20.3 (18-C), 64.5 (6-CH), 57.6 (11-CH), 57.4 (14-CH ), 57.2 (13-CH ), 55.9 (2-CH ), 52.3(10-CH ), 37.9
δ 168.2 (7 -C), 161.5 (4 -C), 139.6 (15-C), 131.4 (17-CH, 19-CH), 130.3 (16-CH, 20-CH),
2
2
2
2
0 0 0
7-CH), 33.9 (5-CH), 30.7(1 -CH ), 28.2 (3 -CH ), 28.1 (4-CH ), 27.4 (8-CH ), 23.8 (2 -CH ), 21.6 (3-CH ),
2 2 2 2 2 2
+
21.4 (9-CH ); ESI-HRMS: m/z Calcd for C H N BrS [M + H] , 490.1635; found, 490.1635; HPLC:
2 23 33 5
tR = 9.88 min; normalization method purity 97.24%.
3.2.3. General Procedure for the Synthesis of Compounds 7a–d
To a solution of 6a
,
6f
,
6i or 6l (0.40 mmol) in dichloromethane (10 mL), triethylamine (0.25 g,
◦
2
.5 mmol) and acetyl chloride (0.16 g, 2 mmol) were added at 0 C and stirred at room temperature,
until the TLC analysis showed completion of the reaction. The reaction mixture was washed by
saturated aqueous ammonium chloride (10 mL 2) and saturated aqueous sodium chloride (10 mL),
×
dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel with dichloromethane/methanol as the eluents
to give 7a–d.
0
1
2-N-p-Methylblenzenesulfonyl-3 -(5-acetylamino-1,3,4-thiadiazol-2-yl)-matrinic propane (7a). White solid,
◦
25
1
0
yield: 93.7%; m.p.: 273 C (dec,); [α]5
.68 (d, J = 8.0 Hz, 2H, 16-CH, 20-CH), 7.37 (d, J = 8.0 Hz, 2H, 17-CH, 19-CH), 3.46–3.38 (m, 2H, 3 -CH ),
=
−
4.83; H-NMR (500 MHz, DMSO-d )
δ
12.39 (s, 1H, 9 -NH),
89
6
0
7
2
3
.14 (t, J = 11.7 Hz, 1H, 6-CH), 2.92–2.84 (m, 1H, 5-CH), 2.83–2.74 (m, 1H, 7-CH), 2.62–2.51 (m, 1H,
0 0
1-CH), 2.37 (s, 3H, Ph-CH ), 2.17 (s, 3H, 11 CH ), 1.96–1.92 (m, 1H, 1a’-CH), 1.89–1.81 (m, 2H, 2 -CH ),
3 3 2
1
1
0
.77–1.66 (m, 8H, 2-CH , 4-CH , 10-CH , 13-CH ), 1.58–1.22 (m, 7H, 3-CH , 8-CH , 9-CH , 1 b-CH);
2
2
2
2
2
2
2
13
0 0 0
C-NMR (126 MHz, DMSO-d6) δ 168.4 (7 -C), 163.7 (10 -CO), 158.1 (4 -C), 142.9 (18-C), 136.8 (15-C),
1
29.5 (17-CH, 19-CH), 127.2 (16-CH, 20-CH), 62.4 (6-CH), 56.7 (11-CH), 56.2 (2-CH ), 56.1 (10-CH ), 47.2
2
2
0
0
0
(
13-CH ), 38.7 (7-CH), 34.2 (5-CH), 29.6 (1 -CH ), 29.0 (3 -CH ), 27.5 (4-CH ), 27.4 (8-CH ), 25.1(2 -CH ),
2
2
2
2
2
2
0
2.4 (11 -CH ), 21.0 (Ph-CH ), 20.4 (3-CH ), 20.3 (9-CH ); ESI-HRMS: m/z Calcd for C H O N S
3 3 2 2 25 36 3 5 2
2
+
[M + H] , 518.2254; found, 518.2252; HPLC: t = 11.66 min, normalization method purity 95.35%.
R
0
1
2-N-p-Trifluoromethlybenzenesulfonyl-3 -(5-acetylamino-1,3,4-thiadiazol-2-yl)-matrinic propane
(
7b).
1.30; H-NMR (600 MHz, CDCl ) δ 13.27 (s,
3
◦
25
1
White solid, yield: 99.1%; m.p.: 197 C (dec,); [α]
H, 9 -NH), 7.98 (d, J = 8.2 Hz, 2H, 16-CH, 20-CH), 7.73 (d, J = 8.2 Hz, 2H, 17-CH, 19-CH), 3.66–3.57 (m,
H, 3 a-CH), 3.53–3.42 (m, 1H, 3 b-CH), 3.25 (t, J = 11.6 Hz, 1H, 6-CH), 3.03–2.91 (m, 2H, 5-CH, 7-CH),
=
−
589
0
1
1
2
1
1
0
0
0
0
.54 (d, J = 11.4 Hz, 1H, 13a-CH), 2.49–2.42 (m, 4H, 13b-CH, 11 -CH ), 1.99–1.96 (m, 2H, 2 -CH ),
3
2
0
.88–1.74 (m, 8H, 2-CH , 4-CH , 10-CH , 11-CH, 1 a-CH), 1.52–1.29 (m, 7H, 3-CH , 8-CH , 9-CH ,
2 2 2 2 2 2
0
13
0 0 0
b-CH); C-NMR (151 MHz, CDCl3)
δ 168.9 (7 -C), 164.6 (10 -CO), 160.5 (4 -C), 144.1 (15-C), 134.0
(
(
(
18-C), 128.1 (17-CH, 19-CH), 125.8 (16-CH, 20-CH), 123.4 (Ph-CF ), 62.7 (6-CH), 57.5 (11-CH), 56.7
3
0
0
2-CH ), 56.6 (10-CH ), 46.9 (13-CH ), 39.8 (7-CH), 34.4 (5-CH), 31.8 (1 -CH ), 29.8 (3 -CH ), 28.4
2
2
2
2
2
0
0
4-CH ), 28.0 (8-CH ), 25.8 (2 -CH ), 23.3 (11 -CH ), 20.9 (3-CH ), 20.7 (9-CH ); ESI-HRMS: m/z Calcd
2
2
2
3
2
2

Molecules 2018, 23, 1644
13 of 16
+
for C H O N F S [M + H] , 572.1971; found, 572.1970; HPLC: t =11.91 min, normalization method
25
33
3
5
3 2
R
purity 99.53%.
0
1
1
1
(
6
2
(
(
(
(
2-N-Benzyl-3 -(5-acetylamino-1,3,4-thiadiazol-2-yl)-matrinic propane (7c). White solid, yield: 93.5%; m.p.:
◦
25
1
0
27.65; H-NMR (500 MHz, CDCl ) δ 13.31 (s, 1H, 9 -NH), δ 7.32–7.27 (m, 4H,
3
88–189 C; [α]5
=
−
89
6-CH, 17-CH, 19-CH, 20-CH), 7.20 (t, J = 7.2 Hz, 1H, 18-CH), 3.98 (d, J = 13.4 Hz, 1H, 14a-CH), 3.10
d, J = 13.4 Hz, 1H, 14b-CH), 2.97–2.73 (m, 5H, 3 -CH , 5-CH, 7-CH, 11-CH), 2.63 (t, J = 12.0 Hz, 1H,
-CH), 2.42 (s, 3H, 11 -CH ), 2.37–2.31 (m, 4H, 2-CH ,10-CH ), 2.06–1.78 (m, 8H, 4-CH , 8-CH , 13-CH ,
-CH ), 1.47–1.22 (m, 6H, 3-CH , 9-CH , 1 -CH ); C-NMR (126 MHz, CDCl3) δ 169.0 (7 -C), 165.0
10 -CO), 160.5 (4 -C), 140.4 (15-C), 128.6 (17-CH, 19-CH), 128.4 (16-CH, 20-CH), 126.8 (18-CH), 64.6
6-CH), 57.6 (11-CH), 57.4 (14-CH ), 56.9 (13-CH ), 56.7 (2-CH ), 52.4 (10-CH ), 37.9 (7-CH), 34.0
5-CH), 30.2 (1 -CH ), 28.4 (3 -CH ), 28.2 (4-CH ), 27.4 (8-CH ), 23.9 (11 -CH ), 23.3 (2 -CH ), 21.6
0
2
0
3
2
13
2
2
2
0
2
0
0
2
2
2
2
0
0
2
2
2
2
0
0
0
0
2
2
2
2
3
2
+
3-CH ), 21.4 (9-CH ); ESI-HRMS: m/z Calcd for C H ON S [M + H] , 454.2662; found, 454.2662;
2
2
25 36
5
HPLC: t = 8.96 min, normalization method purity 98.36%.
R
0
1
9
(
1
1
2-N-p-Methylbenzyl-3 -(5-acetylamino-1,3,4-thiadiazol-2-yl)-matrinic propane (7d). White solid, yield:
◦
25
1
0
31.74; H-NMR (500 MHz, CDCl ) δ 13.34 (s, 1H, 9 -NH), 7.19
3
1.5%; m.p.: 226 C (dec.); [α]5
d, J = 7.6 Hz, 2H, 16-CH, 20-CH), 7.09 (d, J = 7.6 Hz, 2H, 17-CH, 19-CH), 3.98 (d, J = 13.3 Hz, 1H,
4a-CH), 3.10 (d, J = 13.3 Hz, 1H, 14b-CH), 2.95–2.86 (m, 2H, 3 -CH ), 2.83 (m, 1H, 5-CH), 2.76 (m,
H, 7-CH), 2.63 (t, J = 12.0 Hz, 1H, 6-CH), 2.42 (s, 3H, 11 -CH ), 2.36 (d, J = 11.0 Hz, 1H, 11-CH), 2.31
s, 3H, Ph-CH ), 2.03 (s, 1H, 1 a-CH), 1.92–1.78 (m, 6H, 2-CH , 4-CH , 8-CH ,), 1.75–1.62 (m, 4H,
0 0
0-CH , 2 -CH ), 1.48–1.30 (m, 6H,3-CH , 9-CH , 13-CH ), 1.25 (s, 1H, 1 b-CH); C-NMR (126 MHz,
2 2 2 2 2
=
−
89
0
2
0
3
0
(
1
3
2
2
2
13
0
0
0
CDCl3)
δ
169.0 (7 -C), 165.1 (10 -CO), 160.5 (4 -C), 137.4 (18-C), 136.2 (15-C), 129.0 (17-CH, 19-CH), 128.6
(
3
16-CH, 20-CH), 64.6 (6-CH), 57.7 (14-CH ), 57.5 (11-CH), 56.7 (2-CH ), 52.4 (10-CH ), 38.0 (13-CH ),
2
2
2
2
0 0 0
4.1 (7-CH), 30.2 (5-CH), 29.8 11 -CH ), 28.5 (1 -CH ), 28.2 (3 -CH ), 27.4 (4-CH ), 24.0 (8-CH ), 23.3
3 2 2 2 2
0
+
(
2 -CH ), 21.7 (3-CH ), 21.5 (9-CH ), 21.2 (Ph-CH ); ESI-HRMS: m/z Calcd for C H ON S [M + H] ,
2
2
2
3
26 38
5
4
68.2792; found, 468.2789; HPLC: t =9.34 min, normalization method purity 98.77%.
R
3
.3. Biology Assay
3
.3.1. Cell Culture and Screening of Compound
Cells were laid on 96-well plate, cultured in Dulbecco’s Modified Eagle’s medium (DMEM),
◦
containing 10% fetal bovine serum (FBS) in a 5% CO atmosphere at 37 C. Moreover, serum-free
2
culture was required until the cells at 90–95% confluence. After 24 h, cells were treated with a matrinic
derivative (80
µM) for 24 h. The COL1A1 promotor activity was determined using the Bright-Glo
luciferase assay system (Promega Corporation, Madison, WI, USA) [8].
3
6
.3.2. Cell Survival Assay
The cell survival was evaluated by MTT assay. HepG2 cells were seeded at density of
3
× 10 cells/well in 96-well plate. Cells were treated with various concentrations of matrinic derivate
until the cells at 50% confluence. After 24 h of incubation, 20 µL of the MTT (5 mg/mL) solution was
added into each plate and incubated for 4 h at 37 C, 5% CO . Subsequently, the culture supernatant
◦
2
was replaced with 150
µL DMSO to dissolve the formazan crystal. The absorbance at 570 nm was
measured using a microplate reader (ELx800, BioTek Instruments, Winooski, VT, USA).
3
.3.3. RT-qPCR Assay
LX-2 cells were seeded in 6-well plate, cultured in DMEM, containing 10% fetal bovine serum
◦
(
FBS) in a 5% CO atmosphere at 37 C. And serum-free culture was required until the cells at 90–95%
2
confluence. After 24 h, cells were treated with TGFβ1 (2 ng/mL) and matrinic derivatives (80 µM)
for 24 h. Total RNA from the LX-2 cells was extracted using Trizol reagent, purified by NucleoSpin
RNA Clean-up. Reverse transcription was performed with a Transcriptor first strand cDNA synthesis

Molecules 2018, 23, 1644
14 of 16
kit. The cDNA was then analysis by ABI 7500 Fast Real-Time PCR System (ThermoFisher Scientific,
Singapore) using TaqMan probes of TGF 1, COL1A1, -SMA, and GAPDH (sequence reserved by
ABI, Foster City, CA ,USA) and FastStart Universal Probe master mix (Roche, Indianapolis, IN, USA).
β
α
3
.3.4. Western Blot
LX-2 cells were cultured as described above. Briefly, cells were washed with phosphate-buffered
◦
saline (PBS) and were lysed in radioimmunoprecipitation assay (RIPA) lysis for 30 min in 4 C; the
◦
supernatant was collected after centrifugation at 12,000
×
g, 4 C for 15 min. Equal amounts of protein
were quantified with Bradford assay, separated by SDS-PAGE and transferred to polyvinylidene
difluoride membrane. The membranes were blocked for one hour at room temperature in PBST
containing 5% milk and probed with specific first antibodies overnight at room temperature. Membrane
was washed 3 times by PBST, followed by horseradish peroxidase-conjugated secondary antibodies
and GAPDH. The proteins were visualized using chemiluminescence reagents.
3
.4. Acute Toxicity
Female Kunming mice with weight of 20.0 ( 1.0 g) were obtained from the Institute of Laboratory
±
Animal Science (Beijing, China). Animals were cared according to the institutional guidelines of
the Institute of Materia Medica, CAMS&PUMC (00000364). The mice were fed with regular rodent
chow and housed in an air conditioned room. The mice were randomly divided into different groups
with 6 mice each. Each compound was given orally in a single-dosing experiment at 0, 250, 500,
−
kg (ddH O as control), respectively. The mice were closely monitored for 7 days.
2
1
7
50 or 1000 mg
·
Body weight as well as survival was monitored.
4
. Conclusions
To summarize, a series of novel matrinic thiadiazole derivatives were designed, synthesized
and evaluated for their inhibitory effect on the COL1A1 promotor. The SAR indicated that: (i) the
introduction of a thiadiazole on the 11-side chain was beneficial for the activity; (ii) a 12-N-benzyl
was favorable for activity. Among the new derivatives, compound 6n gave a high inhibitory effect
on COL1A1 promotor at a rate of 39.7% on the cellular level at a concentration of 40
µM. It also
effectively inhibited the expression of two representative fibrogenetic collagen proteins (COL1A1 and
α
-SMA) on both the mRNA and protein levels as well as a high safety profile with the LD value
50
−
1
of over 500 mg
via repressing the TGF
·
kg in vivo. Further study indicated that it might exert anti-liver fibrotic activity
/Smad pathway. Overall, this results offer powerful information for further
β
structure optimization, in which compound 6n has been chosen as an ideal anti-liver fibrosis lead for
further investigation by means of computer aided drug and other design strategies.
Author Contributions: T.N. performed part of synthetic experiments and wrote the paper, W.N. and Y.B.
performed the biological assay, T.L. was responsible for literature search, D.S. conceived and designed the
chemistry experiments, Y.L. designed the target compounds and chemistry experiments, H.H. conceived and
designed the biology experiments.
Funding: This work was supported by the National Natural Science Foundation of China (81321004 and 81473248),
and CAMS initiative for innovative medicine (2017-12M-3-012).
Acknowledgments: The authors thank center for analysis and testing of Institute of Materia Medica and Institute
of Medicinal Biotechnology, Chinese Academy of Medical Sciences for their contributions to the determination
1
13
of HR-MS, H-NMR and C-NMR, thank Animal Center of the Institute of Materia Medica, CAMS&PUMC for
providing the animal experimental facilities, and thank Ms. Yanbin Wu from Institute of Medicinal Biotechnology
for performing the animal experiment.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 1644
15 of 16
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Sample Availability: Samples of the compounds 1, 5a–d, 6a–o and 7a–d are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(