Design, Synthesis, and Anti-HBV Activity of New Bis(l -amino acid) Ester Tenofovir Prodrugs

Article abstract of DOI:10.1021/acsmedchemlett.9b00184

A series of bis(l-amino acid) ester prodrugs of tenofovir (TFV) were designed and synthesized as new anti-HBV agents in this work. Four compounds 11, 12a, 12d, and 13b displayed better anti-HBV activity (IC₅₀: 0.71-4.22 μM) than the parent drug TFV. The most active compound 11 (IC₅₀: 0.71 μM), a bis(l-valine) ester prodrug of TFV, was found to have obviously greater AUC_0-∞, C_max, and F% than tenofovir disoproxil fumarate (TDF), and potent in vivo efficacy which is not inferior to TDF in a duck HBV (DHBV) model and a HBV DNA hydrodynamic mouse model, and it may serve as a promising lead compound for further anti-HBV drug discovery.

Full text of DOI:10.1021/acsmedchemlett.9b00184

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Letter
Design, synthesis and anti-HBV activity of new
bis(L-amino acid) ester tenofovir prodrugs
Apeng Wang, Shuo Wu, Zeyu Tao, Xiaoning Li, Kai Lv, Chao Ma, Yu-Huan Li, Linhu Li, and Mingliang Liu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00184 • Publication Date (Web): 16 May 2019
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Design, synthesis and anti-HBV activity of new bis(L-amino acid)
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a, †
b,c, †
, Zeyu Tao , Xiaoning Li , Kai Lv , Chao Ma , Yuhuan Lib,c*, Linhu Lia,
a
a
a
a
Apeng Wang , Shuo Wu
a,**
Mingliang Liu
aInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China
bCAMS Key Laboratory of Antiviral Drug Research, Institute of Medicinal Biotechnology, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing, China
cBeijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing, China
KEYWORDS: Tenofovir, Anti-HBV activity, L-Amino acid, Prodrugs.
ABSTRACT: A series of bis(L-amino acid) ester prodrugs of tenofovir (TFV) were designed and synthesized as new anti-HBV
agents in this work. Four compounds 11, 12a, 12d and 13b displayed better anti-HBV activity (IC50: 0.71–4.22 μM) than the parent
drug TFV. The most active compound 11 (IC50: 0.71 μM), a bis(L-valine) ester prodrug of TFV, was found to have obviously
greater AUC0-∞, Cmax and F% than tenofovir disoproxil fumarate (TDF), and potent in vivo efficacy which is not inferior to TDF in a
duck HBV (DHBV) model and a HBV DNA hydrodynamic mouse model, and it may serve as a promising lead compound for
further anti-HBV drug discovery.
Hepatitis B virus (HBV) infection is a major public health
problem. According to the World Health Organization
[12, 13]. Although newly approved tenofovir-alafenamide (TAF,
Fig. 1) shows little to no nephrotoxicity and more potent
antiviral activity than TDF at 1/10 the dose
high cost limits its widespread use.
[
14]
(WHO), an estimated over 250 million people are chronically
, the
infected with HBV currently. About one quarter of these
individuals will be likely to develop serious liver diseases such
as cirrhosis and hepatocellular carcinoma (HCC). The end
stage of liver diseases caused by HBV infection claims the
[1]
lives of approximately 700,000 patients annually . Current
therapies include nucleos(t)ide analogues [lamivudine (3TC),
adefovir, tenofovir (TFV), and entecavir (ETV) etc.] and
interferons (IFNs). Unfortunately, neither of them can result in
a high rate of clinical cure, which is defined as the loss of
Figure 1. Structures of tenofovir (TFV) and its prodrugs
TDF and TAF
[2,3]
HBV surface antigen (HBsAg)
. As such, there are huge
unmet medical needs to discover and develop novel agents
[4,5]
with differentiated mechanisms of action
. Several of such
We are impressed by L-amino-acid based prodrug esters
such as the antiviral agents valacyclovir and valganciclovir,
both of which significantly improve antiviral activity and oral
bioavailability of the corresponding parent drugs acyclovir and
candidates are currently in clinical trials, but none of them has
[6]
been approved by FDA . Therefore, a more practical
approach seems to modify the structures of existing drugs to
increase safety and potency.
[15, 16]
ganciclovir, respectively
. This property was ascribed to
Tenofovir (TFV, Fig. 1), an acyclic nucleoside, displays
their advantages of being able to be efficiently delivered via
[7]
[8]
[9]
[17, 18]
potent antiviral activity against HBV , HIV and HSV-2
the hPEPT1
. To the best of our knowledge, none of such
[10]
but nephrotoxicity and poor bioavailability . Several lipid
prodrug strategies of TFV have been developed for this
L-amino acid ester prodrug strategies have been reported in
the case of TFV phosphonates. Initially, therefore, we
intended to design and synthesize a series of novel bis(L-
amino acid) ester prodrugs of TFV as anti-HBV agents in this
work. Our primary objective was to find promising prodrugs
with improved bioavailability and anti-HBV activity than
TDF, to facilitate the further development of these
compounds.
[11]
purpose and were extensively reviewed
. As the first
prodrug of TFV, tenofovir disoproxil fumarate (TDF, Fig. 1)
manufactured by Gilead Sciences, has been widely used
in treating HBV infection, but it was reported to induce lactic
acidosis, Fanconi syndrome, acute renal failure, and bone loss
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Detailed synthetic pathways to target compounds 11-13 are
shown below in Scheme 1. According to known procedures
NH2
N
[19-
N
N
O
21]
N
,
condensation of N-Boc-protected L-amino acids 1-4 with
NH2
O
chloromethyl chlorosulfate using n-Bu NHSO as a phase
4
4
O
P
O
O
( )n
R
transfer catalyst (PTC) gave chloromethyl ester (5), or with 2-
bromoethanol and 3-bromoporpan-1-ol yielded 2-bromoethyl
and 3-bromopropyl esters (6a-d, 7a-c), respectively, in the
presence of dicyclohexylcarbodiimide (DCC) and 4-
dimethylaminopyridine (DMAP). The resulting esters (5-7)
O
(
O
)n NH2
R
O
11-13
’
were then coupled with TFV using N,N -dicyclohexyl-4-
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morpholine carboxamidine (DCMC) as an acid scavenger in
dimethyl formamide (DMF) to give intermediates 8-10. The
target compounds 11-13 were obtained by removal of the Boc-
protecting group of the corresponding 8-10 in hydrochloride-
1,4-dioxane solution.
Scheme 1. Synthesis of target compounds 11-13.
a
Obtained as hydrochloride salts. b IC50: 50% inhibitory
concentration of cytoplasmic HBV-DNA synthesis. CC50
0% cytotoxic concentration on HepG 2.2.15 cells.
selective index (CC50/IC50). The cytotoxicity of compounds on
HepG 2.2.15 cells was assayed by CPE (cytopathic effect)
method. Both CC50 and IC50 were determined by Reed &
Muench method. Each experiment was performed at least
twice separately. TFV: Tenofovir; TDF: tenofovir disoproxil
fumarate; 3TC: lamivudine.
c
:
d
5
SI:
The most active compound 11 was further evaluated for its
in vivo pharmacokinetic (PK) profiles in SD rats, following a
single oral dose administration, in comparison with the parent
TFV and prodrug TDF. For TFV the oral dose was calculated
to be 20 mg/kg (0.07 mol/kg) while the intravenous injection
was 5 mg/kg. Considering that both 11 and TDF quickly
undergoes hydrolysis to TFV, we compared the PK parameters
of TFV obtained after oral administrations of equimolar (0.07
mol/kg) 11, TFV and TDF. As shown in Table 2, compound
11 displayed the shortest T1/2 of 1.30 h, but significantly
greater AUC0-∞ of 6524 h.ng/mL and Cmax of 2882 ng/mL than
both TFV and TDF. More importantly, the oral bioavailability
(F, 10.30%) of 11 was found to be about 7 and 3 times than
TFV and TDF, respectively. These results indicated that
compound 11 had promising PK properties to support in vivo
efficacy studies in animal models.
Reagents and conditions: i) n = 1, chloromethyl chlorosulfate,
n-Bu
4
NHSO
4
, NaHCO
3
, CH
2
Cl
2
-H
2
O, rt, 5h, 71%; n = 2 or 3,
2
Cl ,
2
-bromoethanol / 3-bromoporpan-1-ol, DCC, DMAP, CH
2
rt, 12 h, 79.3%; ii) TFV, DCMC, DMF, 80 ℃ , 2.5h,
microwave, 25%-51%; iii) HCl (g), dioxane solution, rt, 0.5h,
1-90%.
8
The synthesized compounds 11-13 were initially screened
for in vitro inhibitory effect on the replication of HBV in
HepG 2.2.15 cell lines according to the reference [22]. The IC50
,
CC50 and SI values of them along with TFV, TDF and 3TC for
comparison are presented in Table 1. Four target compounds
1
1, 12a, 12d, and 13b displayed more potent anti-HBV
activity (IC50: 0.71–4.22 μM) than the parent drug TFV (IC50
5.98 μM). Among them, compound 11 (IC50: 0.71 μM) was
:
1
roughly comparable to TDF (IC50: 0.85 μM), and three and
twenty two times more potent than lamivudine (IC50: 2.22 μM)
and TFV, respectively. The SI value of compound 11 was
about three times higher than those of TFV and TDF. On the
other hand, all of the bis(L-amino acid) ester prodrugs 11-13
possessed significantly reduced cell toxicity compared with
that of TDF. In general, anti-HBV activity and cell toxicity of
these prodrugs are related to kinds of the L-amino acids and
length of the carbon chain linker (n), although the structure-
activity relationship (SAR) is difficult to summarize.
Table 2. Pharmacokinetics properties of 11 in SD rats
Mean value (n = 3 per group)
To determine whether compound 11 shows an antiviral
effect in vivo, DHBV-infected ducks were first treated with
.1, 0.2 or 0.4 mol/kg/day of compound 11 orally. Blood
Table 1. In vitro anti-HBV activity of bis(L-amino acid)
ester prodrugs 11-13 of TFV
0
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samples were collected at day 0 (before treatment, T0), 7 (T7),
DNA was observed in animals treated with ETV. There was
no statistically significant difference of serum HBV DNA
between the 20% Kolliphor HS 15 treatment group and water
group. The twice daily oral administration of compound 11
resulted in reduction in serum HBV DNA in a dose-dependent
manner (Fig. 3). The 4-day treatment of compound 11 and
TDF either at the dose of 0.10 mol/kg or 0.50 mol/kg
produced a similar reduction of approximate 1.1 and 1.7 log10
respectively in serum HBV DNA (Fig. 3D). However, the
groups treated with these two dosing groups of compound 11
at 2 day posttreatment performed better than the TDF-treated
groups (Fig. 3C) although this was only statistically significant
(P＜0.05) for the group treated with 0.50 mol/kg of compound
11. In summary, compound 11 also exerts a potent antiviral
activity in a HBV DNA hydrodynamic mouse model.
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and 14 (T14) treatment and at day 3 after the termination of
treatment (P3) to quantify DHBV DNA. No statistically
significant weight change during treatment was observed (data
not shown). As negative controls, the serum DHBV DNA
level in the normal ducks was less than 100 IU/ml (data not
shown). There were also no statistically significant differences
of serum DHBV DNA level between the two vehicle controls
[0.5% CMC-Na (sodium carboxyl methyl cellulose) and
water] at each time point (Fig. 2). The serum DHBV DNA in
ducks treated with 0.4mol/kg of 3TC was significantly
reduced. Except at T7, both compound 11 and TDF treatment
induced a dose-dependent reduction of serum DHBV DNA up
to 3.2 log10 compared to vehicle controls (Fig. 2). Noticeably,
the reduction level of the serum DHBV DNA in ducks treated
with compound 11 was similar to that treated with TDF at the
same dose and treatment time. These data demonstrate that
oral administration of compound 11 results in a potent anti-
HBV activity in a duck HBV model.
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Figure 3. In vivo antiviral activity of compound 11 in a
HBV hydrodynamic mouse model.
Figure 2. In vivo antiviral activity of compound 11 in a
duck HBV model.
One day after hydrodynamic injection of HBV 1.3mer
plasmid (day 0), mice were treated with three doses of TDF or
compound 11 at 0.10 mol/kg, and 0.50 mol/kg or the vehicle
via oral gavage twice-daily. ETV was administered orally
once-daily (n =5-6 per group). (A) Blood was collected at 0, 2,
DHBV-infected ducks were treated with three doses of TDF
or compound 11 at 0.1 mol/kg, 0.2 mol/kg, and 0.4 mol/kg or
the vehicle via oral gavage twice-daily. 3TC was administered
orally once-daily. Six duck were included in each group. (A)
Blood was collected on day 0 (T0, before treatment), 7 (T7)
and 14 (T14) days posttreatment and 3 days after treatment
cessation (P3) and serum DHBV DNA was extracted and
analyzed by a real-time PCR assay. Mean values ± SD are
plotted for each group. (B C D) DHBV DNA in serum on day
T7 (B), T14 (C) and P3 (D) of DHBV-infected ducks with
various doses of TDF or compound 11. 3TC treatment serves
as positive controls. ***, P＜0.001; **, P＜0.01; *, P＜0.05
(compared with the vehicle control).
4
and 6 days posttreatment and serum HBV DNA was
extracted and analyzed by a real-time PCR assay. Mean values
SD are plotted for each group. (B) The mice body weight
during 6 days of treatment. (C D) HBV DNA in serum on day
(C) and 4 (D) of HBV hydrodynamic injection mice with
±
2
various doses of TDF or compound 11. ETV treatment serves
as positive controls. ***, P＜0.001; **, P＜0.01; *, P＜0.05
(compared with the vehicle control).
In summary, a series of bis(L-amino acid) ester prodrugs of
TFV were designed and synthesized as new anti-HBV agents.
Four compounds 11, 12a, 12d and 13b displayed more potent
anti-HBV activity (IC50: 0.71–4.22 μM) than the parent drug
TFV. Especially, the most active compound 11 (IC50: 0.71
μM) with improving SI, a bis(L-valine) ester prodrug of TFV,
was found to show excellent PK properties and potent in vivo
efficacy in the DHBV model and HBV DNA hydrodynamic
mouse model, and it may serve as a promising lead compound
for further anti-HBV drug discovery. Studies to determine the
in vivo efficacy of 11 in an adeno-associated virus-mediated
mouse HBV replication models are ongoing in our lab.
To further determine whether compound 11 effectively
exerts an antiviral effect in mice, C57BL/6 mice were
hydrodynamically injected with an HBV 1.3mer plasmid to
establish HBV replication in mice hepatocytes. As shown in
Fig. 3B, no significant weight change was observed during
treatment. Five mice injected with PBS through tail vein
without compound treatment were included as negative
controls and the serum HBV DNA level was less than 100
IU/ml (data not shown). A significant reduction in serum HBV
ASSOCIATED CONTENT
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Supporting Information
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Supporting Information available: The Supporting Information is
available free of charge on the ACS Publications website.
Experiments procedures and analytic data of the synthesized
compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: lmllyx@126.com. Phone: 86-010-63030965.
*E-mail: yuhuanlibj @126.com (Y. Li).
(10) Giesler, K. E.; Marengo, J.; Liotta, D. C.; Reduction Sensitive
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Author Contributions
(
11) Pradere, U.; Garnier-Amblard, E. C.; Coats, S. J.; Amblard, F.;
Schinazi, R. F. Synthesis of Nucleoside Phosphate and
Phosphonate Prodrugs. Chem. Rev. 2014, 114, 9154−9218.
†These authors contributed equally. The manuscript was written
through contributions of all authors. All authors have given
approval to the final version of the manuscript.
(12) Lewis, W.; Day, B. J.; Copeland, W. C. Mitochondrial Toxicity
of Nrti Antiviral Drugs: An Integrated Cellular Perspective. Nat.
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Funding Sources
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13) Herlitz, L. C.; Ohan, M. S.; Stokes, M. B.; Radhakrishnan, J.;
D’Agati, V. D.; Markowitz, G. S. Tenofovir Nephrotoxicity:
Acute Tubular Necrosis with Distinctive Clinical, Pathological,
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The work was financially supported by CAMS Initiative for
Innovative Medicine CAMS-2018-I2M-3-004, National Mega-
project for Innovative Drugs (2018ZX09711001-007-002),
National Science and Technology Major Project of the Ministry of
Science and Technology of China (2018ZX09101003-003-003)
and Science Fund for Creative Research Groups of the National
Natural Science Foundation of China (81621064).
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14) Ruane, P. J.; DeJesus, E.; Berger, D.; et al. Antiviral Activity,
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Alafenamide as 10-Day Monotherapy in Hiv-1-Positive Adults.
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M. E. Transport of Valganciclovir, a Ganciclovir Prodrug, via
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
Thank you for the financial supporting from CAMS Initiative for
Innovative Medicine, National Mega-project for Innovative Drugs,
National Science and Technology Major Project of the Ministry of
Science and Technology of China and Science Fund for Creative
Research Groups of the National Natural Science Foundation of
China.
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ABBREVIATIONS
4
34.
Cl, clearance; Cmax, maximum serum concentration; Tmax, time to
maximum concentration; AUC0- ∞ , area under curve from time
zero to infinity; t1/2, plasma elimination half-life; F, oral
bioavailability.
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synthesis of novel bis(L-amino acid) ester prodrugs of 9-[2-
(phosphonomethoxy)ethyl]adenine (PMEA) with improved
anti-HBV activity. Bioorg. Med. Chem. Lett. 2007, 17, 465–470
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6) Feng, S.; Gao, L.; Han, X.; Hu, T.; Hu, X.; Liu, H.; Thomas, A.
W.; Yan, Z.; Yang, S.; Young, J.; Yun, H.; Zhu, W.; Shen, H.
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(7) Delaney, W. E.; Ray, A. S.; Yang, H.; Qi, X.; Xiong, S.; Zhu,
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ACS Medicinal Chemistry Letters
Table of Contents Graphic (For Table of Contents Use Only)
Design, synthesis and anti-HBV activity of new bis(L-amino acid) ester tenofovir prodrugs
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ACS Medicinal Chemistry Letters
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Fig2. DHBV
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ACS Medicinal Chemistry Letters
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