Synthesis and biological evaluation of 12-benzyl matrinic amide derivatives as a novel family of anti-HCV agents

Article abstract of DOI:10.1016/j.cclet.2016.03.006

A new series of 12-benzyl matrinic amide/ethanamide derivatives were synthesized from matrinine (1) and evaluated for their anti-HCV activity, taking compound 2 as the lead. SAR revealed that the introduction of a suitable substituent at the N′-end of matrinic amide might greatly enhance the potency. Among them, matrinic acid 17 and N′-substituted matrinic amides 18a–d exhibited promising potency with low micromolar EC₅₀values ranging from 1.03?μmol/L to 7.54?μmol/L, and better therapeutic window with SI from 66 to 132. Moreover, compound 17 displayed an excellent PK and safety profile in vivo, demonstrating good drug-like characteristics. Thus, it has been selected for further investigation, with an advantage of decreased chances of inducing drug-resistance mutations.

Full text of DOI:10.1016/j.cclet.2016.03.006

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Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis and biological evaluation of 12-benzyl matrinic amide
derivatives as a novel family of anti-HCV agents
Sheng Tang, Zong-Gen Peng, Xin Zhang, Xin-Yue Cheng, Wen-Jing Li, Jian-Dong Jiang,
*
Ying-Hong Li, Dan-Qing Song
Beijing Key Laboratory of Anti-infective Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College,
Beijing 100050, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A new series of 12-benzyl matrinic amide/ethanamide derivatives were synthesized from matrinine (1)
and evaluated for their anti-HCV activity, taking compound 2 as the lead. SAR revealed that the
introduction of a suitable substituent at the N0-end of matrinic amide might greatly enhance the potency.
Among them, matrinic acid 17 and N0-substituted matrinic amides 18a–d exhibited promising potency
Received 30 November 2015
Received in revised form 4 February 2016
Accepted 4 March 2016
Available online xxx
with low micromolar EC50 values ranging from 1.03
mmol/L to 7.54 mmol/L, and better therapeutic
window with SI from 66 to 132. Moreover, compound 17 displayed an excellent PK and safety profile in
vivo, demonstrating good drug-like characteristics. Thus, it has been selected for further investigation,
with an advantage of decreased chances of inducing drug-resistance mutations.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Keywords:
Matrinic acid
Matrinic amide
Hepatitis C virus
Structure–activity relationship
1. Introduction
We have successfully identified 12-benzyl matrinic acid
analogs to be a novel class of anti-HCV agents from matrine (1,
Currently, hepatitis C virus (HCV) infection has become a major
public health problem and heavy burden worldwide [1–3].
Recently, the direct-acting antivirals (DAAs), which specifically
target HCV proteins had made great progresses. Telaprevir and
Fig. 1), a Chinese natural product [11]. The representative
compound, 12-p-methoxylbenzyl matrinic acid (2, Fig), demon-
strated a moderate activity with EC50 of 118
mmol/L and SI of
over 22 [12–14]. Its mode of action is down-regulating host
heat-stress cognate 70 (Hsc70) protein expression at the post
Boceprevir in combination with peg IFN-
a plus RBV, as the first
generation of NS3/4A HCV protease inhibitors, have been widely
used in clinic for the treatment of HCV infections. However the side
effects, drug interactions and resistance mutations have limited
their clinical use [4–7]. Several new HCV DAAs and their
combinations have been approved since the end of 2013, such
as NS3/4A inhibitors Simeprevir and Asunaprevir, NS5A inhibitors
Ledipasvir and Daclatasvir and NS5B polymerase inhibitors
Sofosbuvir and Dasabuvir [8]. Especially, some new HCV DAAs
and their triple therapies show significant promise in some drug-
resistant HCV in clinical patients. However, their chemotherapeu-
tic pressure is on viral components; thus, antiviral therapies
targeting specific viral enzyme might cause drug-resistant
mutations [9]. Therefore, novel antiviral therapeutic strategies
against HCV with no or decreased chance of inducing drug-
resistance are highly desirable [10].
transcriptional level through destabilizing Hsc70 mRNA, a
distinctly different action mode from the currently used drugs
such as Telaprevir and Simeprevir [12]. As the target is not on
the viral component, this kind of antiviral agents might inhibit
viral replication with decreased chance of causing drug-resistant
mutations [14].
This unique action mode and special scaffold of compound
2 strongly provoked our interest to continuously explore the
structure–activity relationship (SAR) of this kind of compounds,
in an effort to discover novel anti-HCV candidates with an
advantage of overcoming the drug resistance. Therefore, in the
present study, taking compound 2 as the lead, SAR studies were
further conducted with the variations of the 40-carboxyl group
and the shortening length of the 11-side chain. Based on this
strategy, a series of new 12-benzyl matrinic amide/ethanamide
and matrinic acid/amine derivatives were constructed and
evaluated for their in vitro anti-HCV activity as well as the in
vivo pharmacokinetic (PK) and safety profile of the representa-
tive compounds.
*
Corresponding author.
E-mail address: songdanqingsdq@hotmail.com (D.-Q. Song).
http://dx.doi.org/10.1016/j.cclet.2016.03.006
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: S. Tang, et al., Synthesis and biological evaluation of 12-benzyl matrinic amide derivatives as a novel
family of anti-HCV agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.006

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General procedures for other title compounds and their
physical and spectroscopic characterization data were given in
Supporting information.
2.2. Biological methods
Human liver cell line Huh7.5 cells (kindly provided by Vertex
Pharmaceuticals, Inc., Boston, MA) were cultured in Dulbecco’s
modified eagle medium (DMEM) supplemented with 10% inacti-
vated fetal bovine serum and 1% penicillin–streptomycin (Invitro-
gen). Cells were digested with 0.05% trypsin–ethylene diamine
tetraacetic acid (EDTA) and split twice a week.
Fig. 1. Chemical structures of matrinine (1), matrinic acid and 12-p-methoxylbenzyl
matrinic acid (2).
2. Experimental
2.2.1. Anti-HCV effect in vitro [15]
2.1. Chemistry
Huh7.5 cells were seeded into 96-well or 6-well plates (Costar)
at a density of 3.0 ꢂ 104 cells/cm2. After 24 h of incubation, the
cells were infected with HCV viral stock (recombination virus
strain J6/JFH/JC, 45 IU/cell) and simultaneously treated with the
test compounds at various concentrations. The culture medium
was removed after 72 h of incubation, and the intracellular total
RNA (in 96-well plates) was extracted with RNeasy Mini Kit
(Qiagen), and total intracellular proteins (in 6-well plates) were
extracted with Cyto-Buster Protein Extraction Reagent added with
1 mmol/L protease inhibitor cocktail. The intracellular HCV RNA
was quantified with a real time one-step reverse-transcription
polymerase chain reaction (RT-PCR).
Melting points (mp) were obtained using a CXM-300 melting
point apparatus. 1H NMR and 13C NMR spectra were recorded on a
Bruker Avance 400 spectrometer or Bruker Avance III 500
spectrometer (Varian, San Francisco, CA) respectively, in CDCl3,
DMSO or CD3OD using Me4Si as the internal standard. Flash
chromatography was performed on Combiflash Rf 200 (Teledyne,
Nebraska, USA). All the target compounds were synthesized using
commercially available sophocarpine (3) and matrine (1) with
purity over 98% as the starting material, which were purchased
from the Yanchi Dushun Biological and Chemical Co., Ltd. (Shanxi,
China).
2.2.2. Cytotoxicity assay [15]
2.1.1. General procedures for methyl N-substituted matrinic acetate
(16a–b)
Huh7.5 cells were seeded into 96-well plates (Costar) at a density
of 3.0 ꢂ 104 cells/cm2. After 24 h of incubation, fresh culture
medium containing test compounds at various concentrations were
added. 72 h later, cytotoxicity was evaluated with 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
Matrine (1, 1.0 g, 4 mmol) was added to 5 mol/L NaOH (20 mL),
and the reaction mixture was heated under reflux for 9 h, cooled in
an ice bath and then acidified with HCl (6 mol/L) to pH 4–5. The
solvent was removed under reduced pressure, and the residue was
dissolved in methanol (100 mL). The suspension was filtered and
the filtrate was concentrated. The residue was dissolved in 2 mol/L
HCl/MeOH (20 mL) and the mixture was refluxed for 2 h. The crude
compound 15 was obtained by evaporation and used in the next
step without further purification. Anhydrous K2CO3 (3.5 equiv.)
and benzyl bromide (1.5 equiv.) were added to a solution of
compound 15 (1.0 equiv.) in MeCN (20 mL), and the reaction
solution was stirred at room temperature until TLC analysis
showed completion of the reaction. The reaction mixture was
filtered, and the filtrate was concentrated under reduced pressure.
The residue was dissolved in CH2Cl2 and washed by water and
brine, dried with anhydrous Na2SO4, filtered, and concentrated to
afford crude compounds 16a–b. The title compounds 16a–b were
obtained by purifying with flash column chromatography on silica
gel with dichloromethane and methanol as the eluents in yields of
50%–60%.
2.2.3. Pharmacokinetic studies
Animals were purchased from the Institute of Laboratory
Animal Sciences, Chinese Academy of Medical Sciences, Beijing,
China. All experimental procedures were approved by the
Biomedical Ethics Committee of the Chinese Academy of Medical
Sciences for Animal Use and Protection. Three male SD rats with
weights in the range of 180–220 g were used in each study. Each
rat was administered orally with compounds at a dose of 25 mg/kg.
Blood samples (0.3 mL) were collected at 0, 0.25, 0.5, 1.0, 1.5, 2.0,
4.0, and 6.0 h and were immediately centrifuged to separate the
plasma fraction. The plasma samples obtained were stored at
S20 8C until analysis. Concentration–time profile was obtained for
each analyte, and standard noncompartmental analysis was
performed on the data using WinNonlin, Version 5.3 to determine
the AUC and other noncompartmental parameters.
3. Results and discussion
2.1.2. 12-(4-Pyridylmethyl) matrinic acid dihydrochloride (17)
Compound 16b (1.5 mmol) was refluxed in 3 mol/L HCl (15 mL)
for 1 h, then cooled, and adjusted to pH 5–6 with 3 mol/L KOH. The
solution was extracted with ethyl acetate, and the aqueous layer
was evaporated to dryness, and the residue was purified through
flash chromatography over silica gel to give the title compounds
17. White solid yield: 83%, mp: 175–177 8C; 1H NMR (400 MHz,
As depicted in Scheme 1, the matrinic acetic acid 7 was obtained
from compound 3 using a four-step sequence including oxidation,
esterification, 12-substitution and ester hydrolysis with overall
yields of 30%–35%. The desired matrinic ethanamides (8a–b) were
prepared using
7 and NH4HCO3 via the mixed anhydride
procedures in 70%–80% yields [16]. N0-Substituted matrinic
ethanamides (9a–c) were obtained by the coupling of 7 with
different amines [17] with good yields of 65%–70%.
DMSO-d6):
d 12.16 (s, 1H), 12.01 (s, 1H), 11.08 (s, 1H), 8.68 (d, 2H,
J = 6.0), 7.73 (d, 2H, J = 6.0), 5.02 (s, 1H), 4.22 (s, 1H), 4.05–3.95 (m,
2H), 3.58 (s, 1H), 3.24 (t, 2H, J = 14.0), 2.84–2.96 (m, 2H), 2.78 (s,
As shown in Scheme 2, the matrinic amide products 14a–e
were acquired from compound 1 using a five-step sequence
including ring-opening, 12-protection using tert-butoxycarbonyl
(Boc), amidation of carboxyl, de-protection of Boc and 12-
substitution with 30%–40% overall yields. The key intermediates
1H), 2.65 (s, 1H), 2.50 (s, 1H), 2.34 (s, 2H), 1.51–2.07 (m, 12H); 13
C
NMR (101 MHz DMSO-d6): d 174.3, 149.5 (2), 139.4, 126.0 (2), 60.7,
60.2, 56.0, 54.2 (2), 49.1, 35.9, 32.8, 29.9, 27.9, 23.9, 23.5, 21.6, 17.9
(2); HRMS: calcd. for C21H32N3O2ꢀ2HCl [Mꢁ2HCl+H]+: 358.2495,
found: 358.2482.
(16a–b) were gained from
1 in ideal yields as described
Please cite this article in press as: S. Tang, et al., Synthesis and biological evaluation of 12-benzyl matrinic amide derivatives as a novel
family of anti-HCV agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.006

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Scheme 1. Synthetic route of matrinic ethanamide derivatives. (a) 10% H2SO4, KMnO4, reflux, 2 h; (b) 2 N MeOH/HCl, reflux, 2 h; (c) RCH2Br, K2CO3, MeCN, r.t., 8 h; (d) 3 mol/L
HCl, reflux, 1 h; (e) Boc2O, NH4HCO3, pyridine, MeCN, r.t., 8 h; (f) EDCI, HOBt, TEA, NH2R1, CH2Cl2, r.t. 4 h.
Scheme 2. Synthetic route of matrinic acid/amide and N0-acyl matrinic amine derivatives. (a) 5 mol/L NaOH, reflux, 9 h; (b) Boc2O, MeOH, r.t., 4 h, 10% citric acid, pH 4–5; (c)
Boc2O, NH4HCO3, pyridine, MeCN, r.t., 8 h; (d) 2 mol/L HCl/Et2O, 30 min; (e) RCH2Br, K2CO3, MeCN, r.t., 8 h; (f) 2 mol/L MeOH/HCl, reflux, 2 h; (g) RCH2Br, K2CO3, MeCN, r.t.,
8 h; (h) 3 mol/L HCl, reflux, 1 h; (i) EDCI, HOBt, TEA, NHR1R2, CH2Cl2, r.t. 8 h; or 2 mol/L HCl/Et2O, 30 min; (j) LiAlH4, THF, r.t., 30 min; (k) DMSO, oxalyl chloride, TEA, CH2Cl2,
S78 8C, 30 min; (l) o-phthalimide, ClCH2CH2Cl, reflux, 4 h, STAB, r.t., overnight; (m) hydrazine hydrate, EtOH; (n) EDCI, HOBt, TEA, NHR1R2, CH2Cl2, r.t. 8 h; or 2 mol/L HCl/
Et2O, 30 min.
previously [12,18]. The desired product 17 was obtained by the
hydrolysis of compound 16b in 83% yield. The products 18a–e
were synthesized from compound 2 using standard coupling
protocols with yields of 60%–70%, and 18f was produced after
the removal of N-Boc of 18d in high yield. The target compounds
23a–b were generated via a five-step procedure, including ester
reduction by LiAlH4, selective oxidization of hydroxy group,
reductive amination using sodium triacetoxyborohydride [19]
(STAB), hydrazinolysis and amino-acylation from 16a with
overall yields of 20%–30%. All the final products were purified
by flash column chromatography on silica gel with CH2Cl2/
CH3OH as gradient eluents.
Please cite this article in press as: S. Tang, et al., Synthesis and biological evaluation of 12-benzyl matrinic amide derivatives as a novel
family of anti-HCV agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.006

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All the newly synthesized compounds were examined for their
anti-HCV activity (EC50) and cytotoxicity (TC50) in human Huh7.5
cells using the specific real-time RT-PCR assay. As an important
indicator, the selectivity index (SI) was calculated as a ratio of TC50
to EC50. Anti-HCV ability of each compound was estimated by
combining its EC50 and SI values. Nineteen matrinic amide/
ethanamide and acid/amine derivatives and their anti-HCV effects
are shown in Table 1.
better than those of 2. Second, 12-pyridylmethylene and 12-
methoxylbenzyl were retained, and SAR investigation was
focused on the influence of 40-carboxyl and shortening length
of the 11-side chain, with which a series of new matrinic amide
(14a–e) and matrinic ethanamide (8a–b) derivatives were
constructed and measured. As described in Table 1, compounds
8a, 14a and 14d–e displayed a reasonable activity with EC50
ranging from 9.6
mmol/L to 97.8 mmol/L and SI from 10 to over
First, the p-methoxylphenyl on the nitrogen-12 atom was
replaced with a p-pyridyl. The resultant compound 17 exerted an
29. These data suggested that replacing carboxyl with amido
group might not greatly improve the potency against HCV,
regardless of the length of the 11-side chain.
activity with EC50 of 7.54
mmol/L and SI of over 66 (Table 1),
Table 1
SAR of matrinic derivatives for anti-HCV activity.
Compd.
8a
R
R1
H
R2
H
CC50
(
m
mol/L)
EC50
(
m
mol/L)
SIa
>29
>500
>500
17.0 ꢃ 0.86
97.8 ꢃ 47.6
8b
H
H
>5.1
9a
9b
H
H
27.8 ꢃ 2.19
16.3 ꢃ 0.96
5.09 ꢃ 1.56
2.18 ꢃ 1.69
5.0
7.0
9c
H
42.7 ꢃ 0.39
2.12 ꢃ 2.02
20
14a
14b
H
H
H
H
>500
52.2 ꢃ 7.08
>10
>3.2
>500
150.3 ꢃ 2.45
14c
14d
14e
H
H
H
H
H
H
>500
>500
90.9 ꢃ 37.1
24.0 ꢃ 2.32
9.59 ꢃ 2.56
>5.5
>21
240.2 ꢃ 86.2
>500
161.0 ꢃ 12.9
25
17
7.54 ꢃ 2.20
1.22 ꢃ 0.32
>66
18a
H
132
18b
18c
H
H
492.2 ꢃ 7.00
222.7 ꢃ 11.8
4.89 ꢃ 3.15
2.76 ꢃ 0.64
101
81
18d
98.3 ꢃ 8.99
1.03 ꢃ 0.22
96
18e
18f
23a
23b
>500
14.1 ꢃ 3.37
5.26 ꢃ 0.39
14.4 ꢃ 5.49
5.00 ꢃ 0.42
>35
58.4 ꢃ 11.6
116.0 ꢃ 5.31
112.1 ꢃ 14.7
11
8.0
22
Telaprevir
47.6 ꢃ 0.61
0.02 ꢃ 0.016
1950
a
Selectivity index (SI) value equaled to CC50/EC50
.
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Table 2
PK parametersa of the key compounds in rats after single oral dosing (n = 3).
Compd
Tmax (h)
Cmax
(
m
mol/L)
AUC0–t
(
mmol/L h)
AUC0-1
(
mmol/L h)
MRT (h)
t1/2 (h)
14a
17
0.75
1.17
0.25
0.33
1.47
3.97
0.11
0.84
4.89
16.69
0.21
–
2.77
2.94
2.75
3.59
–
16.71
0.23
1.24
2.70
4.43
5.15
18a
18b
1.21
a
PK parameters were calculated by non-compartmental analysis using WinNonlin, version 5.3.
In addition, several substituents were introduced to the N0-end
of 8a, and three new N0-substituted matrinic ethanamide (9a–c)
analogs were designed and tested. As described in Table 1, all three
compounds afforded good potency with EC50 values between
treatment with 17 showed no effect on body weight of mice as well
(data not shown). The results suggested that compound 17 was
reasonably safe in vivo.
2.12
m
mol/L and 5.09
m
mol/L, but a high cytotoxicity with CC50
4. Conclusion
values between 16.3 and 42.7
m
mol/L. Then, several groups were
also added to the same position of 14d, and a series of N0-
substituted matrinic amides 18a–f were also obtained. As
anticipated, most of them (18a–d) exhibited promising anti-HCV
Taken together, 19 matrinic amide/ethanamine and matrinic
acid/amine derivatives were synthesized and evaluated for their
antiviral activity against HCV using compound 2 as the lead with
novel mechanism against HCV. The SAR study indicated that the
introduction of a suitable group at the N0-end of matrinic amide
could greatly improve the anti-HCV potency. Among the newly
synthesized compounds, matrinic acid analog 17 demonstrated
good potency against HCV with SI of over 66, much better than that
of the lead 2. Moreover, it also displayed an excellent PK and safety
profile in vivo, indicating good drug-like properties. Thus,
compound 17 has been chosen as a novel anti-HCV agent for
further investigation with the advantage of decreased chance of
causing drug-resistant mutations.
activity with low micromolar EC50 values from 1.03
mmol/L to
4.89 mol/L, and better therapeutic windows with SI from 81 to
m
132. The results indicated that the introduction of a proper
substituent at the N0-end of matrinic amides could significantly
enhanced the activity against HCV. In addition, two N0-acyl
matrinic amine derivatives 23a–b were also designed and tested.
As depicted in Table 1, both of them had moderate activity with
EC50 values of 14.4 and 5.0
mmol/L, and SI of 8.0 and 22,
respectively. The results suggested that introducing a suitable
group at the formyl group of matrinic amine derivatives might
keep the antiviral activity against HCV.
Fourrepresentativecompounds14a, 17and18a–b withdifferent
structural features were chosen to examine their PK parameters in
SD rats at a single dosage of 25 mg/kg via oral route. As indicated in
Table 2 and Fig. 2, 18a and 18b had a poor PK profile with the area
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Nos. 21472246 and 81321004) and the
Beijing Natural Science Foundation (No. 7152097) and National
Mega-Project for Innovation Drugs (No. 2012ZX09103101-037).
under the curve (AUC) of 0.21 and 1.21
Similarly, compound 14a demonstrated a moderate PK parameter
withAUC of4.89 mol/L h. Asanticipated, 17displayedexcellentPK
properties with the maximum concentration (Cmax) and AUC of
3.97 mol/L and 16.7 mol/L h, respectively, indicating a good
mmol/L h, respectively
m
Appendix A. Supplementary data
m
m
stability to metabolism in vivo. Compared with 17, compounds 14a
and 18a–b displayed an unsatisfying PK profile, which might be
resulted from metabolically labile amido group at the N0-end.
Furthermore, single dose toxicity test for 17 was also carried out
in mice. After compound 17 was given orally at a dose of 250, 500
or 1000 mg/kg, respectively, the mice were closely monitored for 7
days. No mouse died in the experiment, indicating that the LD50
value for 17 via oral route was over 1000 mg/kg. In addition, this
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2016.03.006.
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family of anti-HCV agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.006

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Please cite this article in press as: S. Tang, et al., Synthesis and biological evaluation of 12-benzyl matrinic amide derivatives as a novel
family of anti-HCV agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.03.006