Synthesis and biological evaluation of deuterated sofosbuvir analogs as HCV NS5B inhibitors with enhanced pharmacokinetic properties

Article abstract of DOI:10.1002/jlcr.3715

A series of deuterated sofosbuvir analogs were designed and prepared with the aim of improving their pharmacokinetic properties. The devised synthetic routes allow for site-selective deuterium incorporation with high levels of isotopic purity. As expected, the deuterated analogs (37-44) are as efficacious as sofosbuvir when tested in vitro inhibition of viral replication (replicon) assays. Compared with sofosbuvir, deuterated analog 40 displays improved in vivo pharmacokinetics profiles in rats and dogs in terms of the metabolite and the prodrug. The Cmax and area under the curve (AUC) of 40 in dogs were increased by 3.4- and 2.7-fold, respectively. Due to the enhanced pharmacokinetic properties and the great synthetic advantage of an inexpensive deuterium source (D₂O) for 40, it was chosen for further investigation.

Full text of DOI:10.1002/jlcr.3715

Zhang Yinsheng (Orcid ID: 0000-0001-5390-671X)
Lawrie Ken (Orcid ID: 0000-0002-8061-3696)
Derdau Volker (Orcid ID: 0000-0002-3767-643X)
Synthesis and biological evaluation of deuterated sofosbuvir analogs as HCV NS5B
inhibitors with enhanced pharmacokinetic properties
Wangwei Ao, Xueqin Ma, Youping Lin, Xiaojing Wang, Wei Song, Qinglin Wang, Xiquan
Zhang, Hongjiang Xu*, Yinsheng Zhang*
Jiangsu Key Laboratory of Targeted Antiviral Research, Chia Tai Tianqing Pharmaceutical
Group Co., LTD, 699-8 Xuanwu Ave. Building #9, Nanjing 210023, Jiangsu Province, China
*Correspondence to email: zys@cttq.com, xuhongjiang@cttq.com;
Address: Jiangsu Key Laboratory of Targeted Antiviral Research, Chia Tai Tianqing
Pharmaceutical Group Co., LTD, 699-8 Xuanwu Ave. Building #9, Nanjing 210023, Jiangsu
Province, China
Abstract
A series of deuterated sofosbuvir analogs were designed and prepared with the aim of
improving their pharmacokinetic properties. The devised synthetic routes allow for site-
selective deuterium incorporation with high levels of isotopic purity. As expected, the
deuterated analogs (37-44) are as efficacious as sofosbuvir when tested in vitro inhibition of
viral replication (replicon) assays. Compared to sofosbuvir, deuterated analog 40 displays
improved in vivo pharmacokinetics profiles in rats and dogs in terms of the metabolite and the
prodrug. The Cmax and AUC of 40 in dogs were increased by 3.4 and 2.7 fold, respectively.
Due to the enhanced pharmacokinetic properties and the great synthetic advantage of an
inexpensive deuterium source (D2O) for 40, it was chosen for further investigation.
Keywords
Hepatitis C virus; NS5B inhibitor; Sofosbuvir; Deuteration; Pharmacokinetic profile
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1. Introduction
Hepatitis C virus (HCV) infection is a global health problem, with over 185 million
infected individuals at risk of serious and potentially life-threatening liver disease. Recent
advances in HCV therapy have been realized through the use of drug combinations with orally
administered direct-acting antivirals (DAAs).[1-2] Highly effective HCV combination therapy
utilizes two or more DAAs targeting multiple HCV drug target proteins, such as NS3/4A,
NS5A, and NS5B that play different roles in the viral life cycle.[3]
Figure 1. Metabolic pathway of prodrug sofosbuvir (GS-7977)
NS5B is one of the non-structural proteins essential for viral RNA replication and has
been found to be a valuable target for directly acting antiviral agents (DAAs).[4] Sofosbuvir
(GS-7977), a uridine nucleotide analog is a phosphoramidate prodrug that has to be
triphosphorylated within the cells to produce its action. The required enzymes for its activation
are present in the human hepatic cells, therefore, it is converted to its active metabolite during
the first-pass metabolism directly at the desired site of action (liver).[5] The metabolic pathway
for activation of the prodrug[6] is shown in Figure 1. The active triphosphate analog (GS-
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461203) then mimics the physiological nucleotide and competitively blocks the NS5B
polymerase, thus inhibiting the HCV-RNA synthesis by RNA chain termination.[4]
Deuteration of compounds is a strategy for discovering me-better drugs.[7,8] Usually,
deuterium substituted compounds can at least retain both biochemical potency and selectivity
for their target, suggesting that deuterium atom substitution does not appreciably alter ligand-
receptor binding in most cases. [7,8] More importantly, replacing hydrogen with deuterium can
enable substantial benefits to the overall pharmacological profile of the compounds.[8] Within
biological systems, numerous competing effects can mask the deuterium kinetic isotope effect
(KIE) such that the observed magnitude and even direction of the KIEs are unpredictable and
depend on a compound’s molecular skeleton, substituents, specific substitution pattern, and the
enzymes responsible for metabolism.[3] As a consequence of the KIE, site-selective deuteration
of compounds can result in altered metabolism patterns including reduced metabolic rates and
metabolic shunting effects.[3] In an effort to develop NS5B inhibitors with improved
pharmacokinetic properties that may allow for reducing the daily dose (and also cutting cost
for patients), as well as minimize the dose-related side effects,[3] we designed and synthesized
a series of specifically deuterated analogs of sofosbuvir, and evaluated the pharmacokinetic
profiles of those analogs.
2. Results and discussion
2.1. Chemistry
Reagents and conditions: (i) chlorotrimethylsilane, L-alanine, microwave (MW) (82%~89%).
(ii) P(O)PhCl2, triethylamine, 2,3,4,5,6-pentafluorophenol, DCM, (48%~55%).
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Scheme 1 Synthesis of deuterated chiral pentafluorophenyl phosphoramidating reagents (7-9)
First of all, the synthesis of the deuterated chiral pentafluorophenyl phosphate
intermediates (7-9) is outlined in Scheme 1. Esterification of L-alanine with deuterated
isopropanol (1-3) in the presence of chlorotrimethylsilane under microwave conditions gave
the deuterated isopropyl L-alanate (4-6). The desired chiral pentafluorophenyl
phosphoramidating reagent (7-9, a single Sp diastereomer) could be readily prepared
(48%~55% yield) in one pot by reacting phenyl dichlorophosphate with the deuterated
isopropyl L-alanate (4-6) in the presence of triethylamine, and then with pentafluorophenol.[9]
A small amount of undesired Rp diastereomers in the reaction mixture were found (5% by
HPLC) and could be removed by solvent trituration. The absolute stereochemistry of 7-9 at
phosphorus atom center was determined based on comparison with 31P and 1H NMR data of
known compounds. [9]
.
Reagents and conditions: (i) a) D2O, P2O5, BF3 Et2O, (93%) or b) CH3CH2OD, Et3N, Pd/C, D2
(57%); (ii) POCl3, triethylamine, diethyl ether, (100%); (iii) L-Alanine isopropyl ester
hydrochloride, triethylamine, 2,3,4,5,6-pentafluorophenol, DCM, (48%~55%).
Scheme 2 Synthesis of deuterated chiral pentafluorophenyl phosphoramidating reagents (16-
17)
Secondary, as shown in Scheme 2, the deuterated phenyl dichlorophosphate (14-15) can be
synthesized in two steps. Phenol (10) can be deuterated using D3PO4 as a catalyst and D2O as
a deuterium resource to give [2,3,4,5,6-D5]phenol (12) in 93% yield. [10] D2 gas reduction of
4-bromophenol (11) in the presence of 10% Pd/C afforded [4-D]phenol (13). The reaction of
deuterated phenol (12-13) with POCl3 gave deuterated phenyl dichlorophosphate (14-15) in
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nearly quantitative yield. Without further purification of 14 and 15, they can be used for the
preparation of the deuterated chiral compounds 16-17 according to the procedure for
preparing the intermediates (7-9). The absolute stereochemistry at phosphorus atom center of
compound 16 & 17 was determined based on comparison with 31P and 1H NMR data of
known compounds. [9]
Reagents and conditions: (i) Jones reagent, acetone, (67%); (ii) BD3-THF, THF, (76%).
Scheme 3. Synthesis of [6’,6’-D2]nucleoside analog 20
Thirdly, the deuterated nucleoside analogs (20, 28 & 35) were synthesized in multiple steps
using simple and commercially available deuterium sources (BD3, D2O, LiAlD4) depicted in
Scheme 3, 4, & 5, respectively.
Briefly, nucleoside analog 18 was first oxidized by Jones reagent to give the acid 19 in
67% yield and then 19 was reduced by a deuterated borane agent (BD3-THF) to afford the
deuterated nucleoside analog 20 in 76% yield (Scheme 3).
Direct H-D exchange of 21 with D2O in the presence of a combination of catalysts[11] Pd/C,
PtO2 and NaBH4 gave [5, 6-D2]4-aminopyrimidin-2(1H)-one (22) in 98% yield. N-
benzoylation of 22 with benzoyl chloride, followed by O-trimethylsilylation of crude 23 with
hexamethyldisilazane afforded 24 in almost quantitative yield. Without isolation of 24, it was
treated with 25 in the presence of titanium tetrachloride giving 26 in 44% yield. The subsequent
two de-protection steps first with 70% AcOH and then 7M methanolic ammonia solution
furnished the desired [5,6-D2]nucleoside analog 28 in 49% yield (Scheme 4).
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Reagents and conditions: (i) D2O, 10% Pd/C, PdO2, NaBH4, (98%); (ii) PhCOCl, Et3N,
DMAP, CDCl3, (43%); (iii) ammonium sulfate, hexamethyldisilazane, (100%); (iv) titanium
tetrachloride, chlorobenzene, 70 C , (44%); (v) 70% AcOH, 110C , (86%); (vi) 7M
methanolic ammonia, (57%)
Scheme 4. Synthesis of [5, 6-D2] nucleoside analog 28
Asimple reduction of lactone (29) with LiAlD4 gave deuterated tetrahydrofuran derivative
30. Without isolation of 30, it was treated with Ac2O in the same pot to afford 31 in 74% yield.
O-Trimethylsilylation
of
31
with
hexamethyldisilazane
afforded
N-(2-
((trimethylsilyl)oxy)pyrimidin-4-yl)benzamide which was directly treated with 32 in the
presence of titanium tetrachloride to offer 33 in 42% yield. Again, the de-protection of 33 first
with 70% AcOH and then 7M methanolic ammonia solution furnished the desired [2-
D]nucleoside analog 35 in 56% yield (Scheme 5).
Reagents and conditions: (i) LiAlD4, THF, t-BuOH, (100%); (ii) Ac2O, DMAP, THF, (74%);
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(iii) N-benzoylcytosine, ammonium sulfate, hexamethyldisilazane, (100%); (iv) titanium
tetrachloride, chlorobenzene, 70 C , (42%); (v) 70% AcOH, 110 C , (84%); (vi) 7M
methanolic ammonia, (56%).
Scheme 5. Synthesis of [2-D]nucleoside analog 35
Finally, as shown in Scheme 6, deuterated sofosbuvir analogs (37-44) can be synthesized
following the similar procedure for preparation of sofosbuvir,[9-12] starting from the
appropriately deuterated intermediates (7-9, 16-17, 20, 28 & 35) or unlabeled intermediates
(18 & 36). The synthetic route depicted in Scheme 6 relies heavily upon the use of site-
selectively deuterated intermediates (7-9, 16-17, 20, 28 & 35), each requiring individual
preparation (Scheme 1-5).
Reagents and conditions: (i) tert-butylmagnesium chloride, THF, (33%~43%).
Scheme 6. Synthesis of deuterated sofosbuvir analogs (37-44)
Nucleoside (18, 20, 28, 35) was then reacted with the chiral reagent (7-9, 16-17, 36) in the
presence of two equivalents of tert-butylmagnesium chloride in THF to afford the deuterated
sofosbuvir analogs (37-44) with a diastereomeric excess of 98.0%~99.0% (HPLC) and a
deuterium enrichment of >98% (NMR & MS). The absolute stereochemistry at phosphorus
atom center of compound 37-44 was determined based on comparison with 31P and 1H NMR
data of known compounds and agents. [9,12]
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2.2. In vitro anti-HCV activity
To determine whether the deuterated sofosbuvir analogs (37-44) are as effective HCV
inhibitors as their parent drug sofosbuvir, we investigated the inhibitory activities of the
synthesized compounds using the GT1b subgenomic replicon assays. As indicated in Table 1,
all of the compounds showed similar potency with sofosbuvir against GT1b. In addition, the
50% cytotoxic concentration (CC50) values of all the compounds tested in an HCV genotype
1b replicon cell line was more than 5000 nM. The similar potency may be attributable to similar
electron clouds and polar surface areas of hydrogen and deuterium atom and therefore the
maintenance of similar biochemical activity.
Table 1. Anti-HCV activities and cytotoxicity of deuterated sofosbuvir analogs on genotype 1b
compd
37
EC50 (nM)a
CC50 (nM)b
compd
EC50 (nM)
125.6
CC50 (nM)
>5000
102.3
>5000
42
43
38
107.0
>5000
145.0
>5000
39
106.9
>5000
44
110.1
>5000
40
119.2
>5000
sofosbuvir
104.9
>5000
41
106.5
>5000
aMean of triplicate well values. All experiments were performed at least twice. EC50 stands for 50%
effective concentration; bCC50 stands for 50% cytotoxic concentration in an HCV genotype 1b replicon
cell line.
2.3. Pharmacokinetic studies
2.3.1. In vitro metabolic stability
Like sofosbuvir, its deuterated analogs should be first hydrolyzed in the hepatic cells to
form the metabolite GS-566500 or its deuterated analogs, and the later was converted to the
monophosphate and then to triphosphate metabolite (Figure 1). The liver S9 fraction was
chosen as a surrogate in vitro model to test for the hydrolysis reaction. Therefore, the stability
of deuterated compounds 37-41 and 44 on exposure to rat, dog, and human liver S9 fraction
was evaluated along with sofosbuvir. As shown in Table 2, all testing compounds were
hydrolyzed more quickly in rat liver S9 than the dog and human liver S9. The remaining
percentages of the deuterated compounds were not significantly different from sofosbuvir,
thus clarifying that the deuterated analogs can be activated in vitro by hydrolytic enzymes in
hepatocytes. In human liver S9, levels of compound 39 & 40 remained slightly higher than
others (Table 2).
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Table 2. The stability of sofosbuvir and deuterated compounds in the rat, dog, and human
liver S9
Remaining % (T = 60 min)
Species
sofosbuvir
1.62
37
0
38
0
39
0
40
0
41
0
44
0
Rat
Dog
79.38
92.59
35.28
95.69
34.15
82.53
42.87
83.39
44.97
96.39
36.06
87.26
40.98
Human
35.16
2.3.2. In vivo PK
To better understand if there was any metabolic benefit in vivo after incorporating
deuterium into sofosbuvir, the in vivo pharmacokinetic profile studies of sofosbuvir, 39 and 40
in rats were generated (Table 3, Figure 2). It should be noticed that because the prodrugs
(sofosbuvir, 39 and 40) were rapidly metabolized in rats, only final metabolite (GS-331007)
was detected. GS-331007 was identified as the primary analyte of interest for clinical
pharmacology studies as it accounted for >90 % of systemic drug-related material exposure,
and provided comparable exposure–response relationships for viral kinetics as observed for
sofosbuvir.[13] The PK for the metabolite GS-331007 may indirectly represent the parent drug’s
PK. As shown in Table 3, the metabolite GS-331007 of deuterated compound 39 and 40
exhibited a 1.33-fold and 1.38-fold increase in AUC0–24h (h*ng/mL) as compared to sofosbuvir,
respectively. Their half-life (t1/2) was also prolonged compared to sofosbuvir (3.29/3.17 vs.
2.71).
Table 3. Rat plasma PK profile after oral administration of 39, 40 and sofosbuvir
PKa
Tmax (h)
Sofosbuvir b
2.67±1.15
873±188
39b
40b
2.67±0.577
1141±218
6178±923
6209±912
4.40±0.316
3.29±0.339
133.14
2.33±0.577
1159±246
6392±1028
6417±1037
4.52±0.200
3.17±0.349
137.75
Cmax (ng/mL)
AUC(0-t) (ng*h/mL)
AUC(0-∞) (ng*h/mL)
MRT(0-t) (h)
t1/2 (h)
4640±1480
4706±1417
4.16±0.637
2.71±0.760
F（%）
b
a
Blood samples collected at 0.25,0.5,1, 2, 3, 4, 6, 8,10, and 24 h postdose. The final
metabolite GS-331007 generated from sofosbuvir, 39 or 40 after a single dose (50 mg/kg).
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2 0 0 0
1 5 0 0
1 0 0 0
5 0 0
0
S o fo s b u v ir
C o m p o u n d 3 9
C o m p o u n d 4 0
0
4
8
1 2
1 6
2 0
2 4
T im e (h )
Figure 2. Mean (±stand deviation) metabolite GS-331007 plasma concentration-profile of
sofosbuvir, 39 and 40 after a single oral doses of sofosbuvir (50 mg/kg), 39 (50 mg/kg) and
40 (50 mg/kg) in rats
Table 4. Dog plasma PK profile after oral administration of 40 and sofosbuvir
PKa
Tmax (h)
sofosbuvirb
0.250±0.00
3260±1952
2882±1565
2896±1578
3808±1573
8112±4420
0.743±0.0368
0.339±0.051
40b
0.250±0.00
10950±1202
7679±2884
7701±2908
1315±412
2797±1056
0.596±0.2051
0.340±0.0226
266.45
Cmax (ng/mL)
AUC(0-t) (ng*h/mL)
AUC(0-∞) (ng*h/mL)
Vd (ml/kg)
Cl (ml/h/kg)
MRT(0-t) (h)
t1/2 (h)
F (%)
b
aBlood samples collected at 0.25,0.5,1, 2, 3, 4, 6, 8,10, and 24 postdose. A single dose 20
mg/kg.
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1 5 0 0 0
1 0 0 0 0
5 0 0 0
0
S o fo s b u v ir
C o m p o u n d 4 0
0
4
8
T im e (h )
Figure 3. Mean (±stand deviation) plasma concentration-profile of sofosbuvir and 40 after a
single oral dose of sofosbuvir (20 mg/kg) and 40 (20 mg/kg) in dogs
Compound 40 was then selected for further pharmacokinetic properties studies in the dog
(Table 4, Figure 3). Comparison of the in vivo PK characteristic in dogs was accomplished by
evaluating plasma exposures upon oral dosing with a 20 mg/kg. The prodrugs, sofosbuvir and
40, were more slowly metabolized in the dog than in rat, therefore, their prototypes were
detected. It is observed that compound 40 showed an improved overall plasma exposure (AUC)
of parent prodrug (2.7 fold) in the dog in comparison to sofosbuvir. The results support further
evaluation of compound 40.
3. Conclusion
In conclusion, we synthesized a series of specifically deuterium substituted sofosbuvir
analogs as potent HCV NS5B inhibitors. All of the prepared analogs (37-44) are as efficacious
as sofosbuvir in vitro inhibition of viral replication (replicon) assays. However, only analogs
39 and 40 showed an improved in vivo PK profile in rats in terms of the final metabolite (GS-
331007) of the prodrugs. The analog 40 was further investigated in dog regarding the PK profile
of the parent prodrug and showed the enhanced PK results compared to sofosbuvir. Collectively,
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analog 40 deserves further evaluation as a pre-clinical candidate for the treatment of HCV
infection.
4. Experimental section
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used
without further purification. NMR spectra were collected on a Bruker-300 FT NMR
spectrometer and a Bruker AVANCE 500 spectrometer with tetramethylsilane (TMS) as an
internal standard. High-resolution mass spectra (HRMS) were recorded on an AB SCIEX
Triplet TOF 4600 Mass Spectrometer. All final compounds were purified to > 98% chemical
purity as determined using a Shimadzu LC-20AT series HPLC instrument at 260 nm, using the
following conditions: BDS HYPERSIL C18 column (4.6 x 250 mm, 5 μm; Thermo.); solvent
A, 0.1% trifluoroacetic acid; solvent B, acetonitrile; A:B = 50:50, 30 min. Microwave reactions
were completed using a Discover SP-Microwave Synthesizer at max power of 250 W.
Sofosbuvir and compound 36 were synthesized according to the reported methods with 99%
purity.[9] Compounds 2 & 3 were prepared according to the reported methods.[14] Compounds
1, 18, 21, 25, 29 and 32 were purchased from Sigma-Aldrich.
4.1.1. L-Alanine [D7]isopropyl ester hydrochloride (4)
To a 35 mL tube was added d8-isopropanol (8.84 g, 131.7 mmol), L-alanine(1 g, 11.2
mmol), chlorotrimethylsilane (7.06 g, 65 mmol). The resulting mixture was stirred at 120 C
under microwave for 10 minutes. The solvent was evaporated under reduced pressure, the
residue was triturated with diethyl ether and collected by filtration to give 1.74 g (89%) of 4
as a white solid. 1H NMR (D2O, 300 MHz) δ: 4.13 (t, J = 7.2 Hz, 1H), 1.54 (d, J = 7.2 Hz ,
3H); 13C NMR (D2O, 75 MHz) δ: 172.9, 74.5 (m, CD), 51.6, 23.5 (m, CD3), 17.7; HRMS
(ESI): m/z [M + H]+ calcd. for C6H7D7NO2: 139.1464; Found 139.1341.
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4.1.2. L-Alanine [1,1,1,3,3,3-D6]isopropyl ester hydrochloride (5)
Compound 5 was prepared as a white solid according to the procedure described for
compound 4. Compound 2 (8.3 g, 125.8 mmol) was treated with L-alanine (1.0 g, 11.2 mmol)
and chlorotrimethylsilane (6.1 g, 56.2 mmol) under microwave conditions to give compound
5 (1.59 g, 82%). 1H NMR (D2O, 300 MHz) δ: 5.07 (s, 1H), 4.08-4.17 (m, 1H), 1.54 (d, J =
7.2 Hz , 3H); 13C NMR (D2O, 75 MHz) δ: 172.9, 74.5, 51.6, 23.5 (m, CD3), 17.7; HRMS
(ESI): m/z [M + H]+ calcd. for C6H8D6NO2: 138.1401; Found 138.1279.
4.1.3. [2-D]Propan-2-yl-L-alaninate hydrochloride (6)
Compound 6 was prepared as a white solid according to the procedure described for
compound 4. Compound 3 (7.0 g, 114.6 mmol) was treated with L-alanine (1.0 g, 11.2 mmol)
and chlorotrimethylsilane (6.1 g, 56.2 mmol) under microwave conditions to give compound
6 (1.61 g, 85%). 1H NMR (D2O, 300 MHz) δ: 4.10-4.17 (m, 1H), 1.54 (d, J = 7.2 Hz , 3H),
1.28 (s, 6H); 13C NMR (D2O, 75 MHz) δ: 172.9, 74.6 (m，D-C)，51.7, 23.2, 17.8; M HRMS
(ESI): m/z [M + H]+ calcd. for C6H13DNO2: 133.1087; Found 133.1077.
4.1.4. [D7]Propan-2-yl- ((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate (7)
To a stirred solution of L-alanine [D7]isopropyl ester hydrochloride (4, 2.52 g, 14.4
mmol) in anhydrous dichloromethane (15 mL) was added dropwise triethylamine (3.07 g, 4.2
mL, 30.3 mmol) at -70 C over 5 minutes. To this mixture was added a solution of phenyl
dichlorophosphate (3.04 g, 14.4 mmol) in anhydrous dichloromethane (15 mL) over 15
minutes. The reaction mixture was stirred at this temperature for an additional 30 minutes and
then allowed to warm to 0 C and stir for 1 hour. To this mixture was added a solution of
2,3,4,5,6-pentafluorophenol (2.65 g, 14.4 mmol) and triethylamine (1.60 g, 2.20 mL, 15.8
mmol) in dichloromethane (10 mL) over 10 minutes. The reaction mixture was allowed to stir
at 0 C overnight, and the white solid (triethylamine hydrochloride) was filtered off and
washed with dichloromethane (5 mL). The filtrate was concentrated under reduced pressure.
After the residue was triturated with TBME (10 mL) the triethylamine hydrochloride salt that
precipitated from the TBME solution was removed by filtration. The filtrate was again
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concentrated to dryness and the resulting crude product was triturated with 20% EtOAc in
hexanes (10 mL). The precipitate was collected by filtration to give 3.17 g (48%) of 7 as a
white solid. There was no other isomer detectable by 31P or 1H NMR. Stereochemistry was
assigned as Sp on the basis of the known stereochemistry of the regent. [9] 1H NMR (CDCl3,
300 MHz) δ: 7.34-7.37 (m, 2H), 7.20-7.25 (m, 3H), 4.10-4.17 (m, 1H, NH), 3.93-3.97 (m,
1H), 1.45 (d, J = 6.9 Hz, 3H); 31P NMR (CDCl3, 121 MHz) δ: -1.54; HRMS (ESI): m/z [M +
H]+ calcd. for C18H11D7F5NO5P: 461.1282; Found 461.1286.
4.1.5. [1,1,1,3,3,3-D6]Propan-2-yl-((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-
alaninate (8)
Compound 8 was prepared as a white solid according to the procedure described for
compound 7. Compound 5 (3.0 g, 21.8 mmol) was treated with phenyl dichlorophosphate (4.25
g, 21.8 mmol), 2,3,4,5,6-pentafluorophenol (4.01 g, 21.8 mmol), Et3N (10 mL) and DCM (38
31
mL) to give compound 8 (5.20 g, 52%). There was no other isomer detectable by P or 1H
NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of the
regent. [9] 1H NMR(CDCl3, 300 MHz) δ: 7.08-7.38 (m, 5H), 5.01 (s, 1H), 4.07-4.17 (m, 1H),
31
3.97-4.01 (m, 1H), 1.45 (d, J = 6.8 Hz, 3H); P NMR (CDCl3, 121 MHz) δ: -1.53; HRMS
(ESI): m/z [M + H]+ calcd. for C18H12D6F5NO5P: 460.1219; Found 460.1235.
4.1.6. [2-D]Propan-2-yl-((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate (9)
Compound 9 was prepared as a white solid according to the procedure described for
compound 7. Compound 6 (2.6 g, 15.4 mmol) was treated with phenyl dichlorophosphate (3.0
g, 15.4 mmol), 2,3,4,5,6-pentafluoro phenol (2.83 g, 15.4 mmol), Et3N (6.8 mL) and DCM (26
31
mL) to give compound 9 (3.84 g, 55%). There was no other isomer detectable by P or 1H
NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of the
regent. [9] 1H NMR (CDCl3, 300 MHz) δ: 7.21-7.38 (m, 5H), 4.09-4.15 (m, 1H), 3.93-4.01 (m,
1H), 1.45 (d, J = 6.8 Hz, 3H), 1.24 (s , 6H); 31P NMR (CDCl3, 121 MHz) δ: -1.54; HRMS (ESI):
m/z [M + H]+ calcd. for C18H17DF5NO5P: 455.0906; Found 460.0918.
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4.1.7. [2,3,4,5,6-D5]Phenol (12)
D2O (10 mL) was slowly added to phosphorus pentoxide (5.4 g, 38 mmol) at 0 C over
15 min to form a D3PO4 aqueous solution. Then, boron fluoride ethyl ether (75 mg, 67 µL,
0.528 mmol) and phenol (10, 1 g, 10.6 mmol ) were added to this solution. The resulting
mixture was stirred at 140 C for 24 hours. The reaction was extracted with DCM (3 x 10 mL).
The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated
to afford the title compound (0.93 g, 93%). 1H NMR（DMSO-d6, 500 MHz）δ: 9.32 (OH, 1H);
13C NMR (DMSO-d6, 125 MHz) δ: 157.7, 129.4 (t, C-D, J = 24.0 Hz), 118.8 (t, C-D, J = 24.0
Hz), 115.3 (t, C-D, J = 24.0 Hz); HRMS (ESI): m/z [M + H]+ calcd. for C6H2D5O: 100.0811;
Found 100.0817.
4.1.8. [4-D]Phenol (13)
To a stirred solution of 4-bromophenol (11, 5.03 g, 28.9 mmol) and triethylamine (8.81g,
12.07 mL, 87mmol) in CH3CH2OD (25 mL) was added 10% Pd/C (0.52 g), followed by
replacing atmosphere of D2, then the mixture was stirred overnight at room temperature. The
suspension was filtered through Celite. The filtrate was concentrated and purified by silica gel
1
chromatography to afford the title compound (1.44 g, 57%). H NMR (CDCl3, 300 MHz) δ:
9.47 (br s, 1H), 7.23 (d, J = 7.5 Hz, 2H), 6.84 (d, J = 7.5 Hz, 2H). 13C NMR (CDCl3, 75 MHz)
δ: 155.2, 129.4, 120.3 (t, C-D, J = 25.0 Hz), 115.1; HRMS (ESI): m/z [M + H]+ calcd. for
C6H6DO: 96.0560; Found 96.0572.
4.1.9. Isopropyl ((S)-(perfluorophenoxy)([ 2,3,4,5,6-D5]phenoxy)phosphoryl)-L-alaninate
(16)
To a solution of compound 12 (3.92 g, 39.5 mmol) in diethyl ether (100 mL), POCl3 (6.67
g, 43.5 mmol) was gradually added and the mixture was cooled to – 5 °C. Et3N (5.20 g, 51.4
mmol) was slowly added. After one hour, the reaction mixture was allowed to warm to room
temperature and then stirred for 3 hours. The reaction mixture was filtered and the filtrate was
evaporated in vacuo to afford 8.53 g (100% yield) of crude 14 as a light yellow liquid. The
yellow liquid was used in the next step without purification.
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To a stirred solution of L-alanine isopropyl ester hydrochloride (6.64 g, 39.6 mmol) in
anhydrous dichloromethane (80 mL) was added dropwise triethylamine (8.82 g, 87.2 mmol) at
-70 C over 5 minutes. To this mixture was added a solution of the crude 14 (8.53 g, 39.5 mmol)
in anhydrous dichloromethane (50 mL) over 15 minutes. The reaction mixture was stirred at -
70 C for an additional 30 minutes and then allowed to warm to 0C and stir for 1 hour. To this
mixture was added a solution of 2,3,4,5,6-pentafluorophenol (7.29 g, 39.6 mmol) and
triethylamine (4.41 g, 43.6 mmol) in dichloromethane (30 mL) over 10 minutes. After the
reaction mixture was allowed to stir at 0 C overnight, the white solid (triethylamine
hydrochloride) in the mixture was filtered off and washed with dichloromethane (5 mL). The
filtrate was concentrated under reduced pressure. After the residue was triturated with TBME
(100 mL) the triethylamine hydrochloride salt that precipitated from the TBME solution was
removed by filtration. The filtrate was again concentrated to dryness and the resulting crude
product was triturated with 20% EtOAc in hexanes (20 mL). The precipitate was collected by
filtration to give 6.76 g (55%) of 16 as a white solid. There was no other isomer detectable by
31P or1H NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry
of sofosbuvir. [9] 1H NMR (CDCl3, 300MHz); δ: 5.01 (m, 1H), 4.09-4.15 (m, 1H), 3.93-4.01
(m, 1H), 1.45 (d, J = 6.8 Hz, 3H), 1.24 (d , J = 6.8 Hz, 6H); 31P NMR(CDCl3, 121 MHz) δ: -
1.54; HRMS (ESI): m/z [M + H]+ calcd. for C18H13D5F5NO5P: 459.1157; Found 459.1163.
4.1.10. Isopropyl ((S)-(perfluorophenoxy)([4-D]phenoxy)phosphoryl)-L-alaninate (17)
Compound 17 was prepared as a white solid according to the procedure described for
compound 16. Compound 13 (1.17 g, 12.3 mmol) was treated with POCl3 (2.12 g, 13.8 mmol)
in Et3N (1.62 g) and diethyl ether (32 mL), and then treated with L-alanine isopropyl ester
hydrochloride (2.06 g, 12.3 mmol) and 2,3,4,5,6-pentafluoro phenol (2.27 g, 12.3 mmol) in
Et3N (2.74 g) and DCM (25 mL) to afford compound 17 (3.15 g, 56%). There was no other
isomer detectable by 31P or 1H NMR. Stereochemistry was assigned as Sp on the basis of the
known stereochemistry of sofosbuvir. [9] 1H NMR (CDCl3, 300 MHz) δ: 7.34 (d, J = 8.0 Hz,
2H), 7.22 (d, J = 8.0 Hz, 2H), 5.01 (m, 1H), 4.09-4.15 (m, 1H), 3.93-4.01 (m, 1H), 1.45 (d, J =
6.8 Hz, 3H), 1.24 (d, J = 6.8 Hz, 6H); 31P NMR (CDCl3, 121 MHz) δ: -1.54; HRMS (ESI): m/z
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[M + H]+ calcd. for C18H17DF5NO5P: 455.0906; Found 455.0910.
4.1.11. (2S,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-
4-methyltetrahydrofuran-2-carboxylic acid (19)
2.5 M Jones reagent (8.5 mL) was added to a stirred mixture of 1-((2R,3R,4R,5R)-3-
fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-
dione (18, 2.00 g, 7.69 mmol) and Celite (2 g) in acetone (120 mL) at 0 C over 5 minutes. The
reaction mixture was stirred at ambient temperature for 12 hours and then allowed to cool to 0
C before adding isopropanol (1.5 mL). The suspension was filtered and the filtrate was
concentrated under reduced pressure, the residue was dissolved in THF (30 mL) and EtOAc
(30 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate,
filtered, concentrated and purified by silica gel chromatography to afford the title compound
(1.42 g, 67%) as an off-white solid. 1H NMR (DMSO-d6, 300 MHz) δ: 11.50 (br s, 1H), 7.97
(d, J = 8.1 Hz, 1H), 6.07 (d, J = 17.7 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 4.24 (d, J = 8.9 Hz,
1H), 4.02 (dd, J1 = 23.6 Hz, J2 = 8.9 Hz, 1H), 1.28 (d, J = 22.6 Hz, 3H); 13C NMR (DMSO-d6,
75 MHz) δ: 171.8 162.8, 150.4, 139.6, 102.2, 101.7, 99.3, 91.9, 79.3, 75.4 (d, J = 17.7 Hz),
16.0 (d, J = 24.8 Hz); HRMS (ESI): m/z [M + H]+ calcd. for C10H12FN2O6: 275.0679; Found
275.0681.
4.1.12. 1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-(hydroxy-[D2]methyl)-3-
methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (20)
0.05 M BD3-THF (45 mL) was added dropwise to a stirred solution of (2S,3R,4R,5R)-5-
(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-
carboxylic acid (19, 1.00 g, 3.65 mmol) in THF (30 mL) at 0 C under N2 atmosphere. The
reaction mixture was stirred at ambient temperature for 48 hours and then quenched with water
and potassium carbonate. The organic layer was separated and dried over anhydrous sodium
sulfate. The salt was filtered, and the filtration was concentrated to dryness. The residue was
purified by silica gel chromatography to afford the title compound (0.73 g, 76%) as an off-
white solid. 1H NMR (DMSO-d6, 300 MHz) δ: 11.46 (s, 1H), 7.97 (d, J = 8.1 Hz, 1H), 5.99 (d,
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J = 18.8 Hz, 1H), 5.64-5.67 (m, 2H), 5.24 (s, 1H), 3.75-3.90 (m, 2H), 1.25 (d, J= 22.4 Hz, 3H);
13C NMR (DMSO-d6, 75 MHz) δ: 162.8, 150.5, 139.6, 102.0 (d, J = 5.3 Hz), 99.7, 81.5, 70.4
(d, J = 17.3 Hz), 16.4 (d, J = 24.8 Hz). HRMS (ESI): m/z [M + H]+ calcd. for C10H12D2FN2O5:
263.1012; Found 263.1009.
4.1.13. 4-Amino-[5,6-D2]pyrimidin-2(1H)-one deuterium chloride (22)
Cytosine (21, 1.50 g, 13.4 mmol), D2O (15 mL), 10% dry Pd/C (0.15 g), PtO2 (15 mg)
and NaBH4 (5 mg) were added to a 35 mL tube. The resulting suspension was stirred at 160
C under microwave for 80 minutes and 37% DCl in D2O solution (3 mL) then was added to
the reaction mixture at 0 C. The resulting suspension was filtered through Celite and the
filtrate was concentrated to give 1.98 g (98%) of 22 as a white solid which was used directly
in the next step without further purification.
4.1.14. N-(2-oxo-1,2-dihydro-[5,6-D2]pyrimidin-4-yl)benzamide (23)
Triethylamine (1.41 g, 1.93 mL, 13.9 mmol) and benzoyl chloride (0.67 g, 0.55 mL, 4.77
mmol) were added to a solution of deuterated cytosine deuterium chloride 22 (0.61 g, 4.10
mmol) and 4-dimethylaminopyridine (0.98 g, 8.02 mmol) in chloroform (50 mL). The resulting
mixture was heated to reflux for 16 hours and then allowed to cool to room temperature before
being filtered. The resulting solid was washed with chloroform (100 mL) and water (100 mL)
before being dried in vacuum to give benzamidocytosine 23 (0.37 g, 43%) as the crude product
which was taken onto the next stage without further purification.
4.1.15. (2R,3R,4R,5R)-5-(4-Benzamido-2-oxo-[5,6-D2]pyrimidin-1(2H)-yl)-2-
((benzoyloxy)methyl)-4-fluoro-4-methyltetrahydrofuran-3-yl benzoate (26)
The silylated base was prepared by heating to reflux a suspension of N-benzoylcytosine
(23, 2.30 g, 10.60 mmol) and ammonium sulfate (42 mg) in hexamethyldisilazane (35 mL) for
6 h and concentrating the resulting solution by vacuum distillation under aspiration, followed
by drying under vacuum for 2 hours at ambient temperature. The oily residue was dissolved in
chlorobenzene (46 mL). To this solution was added compound 25 (2.96 g, 7.11 mmol ) and
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titanium tetrachloride (3.68 mL, 34.0 mmol). After stirring under a nitrogen atmosphere for
2 hours at ambient temperature, the reaction was heated to 70 C for 19 h. The reaction mixture
was cooled to 0 C and solid sodium bicarbonate (16.0 g, 190.5 mmol) and ethyl acetate (70
mL) were added. To the stirred solution was slowly added water (3.4 mL). After the mixture
was stirred for 30 minutes at ambient temperature, the suspension was filtered and the collected
solid was washed with ethyl acetate (20 mL). The filtrate was washed with water (2 x 120 mL),
dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude
product as a pale yellow-brown solid. The crude product mixed with methanol (50 mL) was
heated under reflux for 30 min and then cooled to ambient temperature. The resulting
precipitate was collected by filtration and triturated with methanol (50 mL). The precipitate in
methanol was collected by filtration and dried to give the desired product as a white solid (1.80
g, 44%) which was used directly for the next step without further purification. 1H NMR (CDCl3,
300 MHz) δ: 8.88 (br s, 1H), 8.06-8.10 (m, 4H), 7.90-7.92 (m, 2H), 7.45-7.70 (m, 9H), 6.49
(br d, J = 18.5 Hz, 1H), 5.55 (br dd, J1 = 21.7 Hz, J2 = 9.3 Hz, 1H), 4.88 (d, J = 12.7 Hz, 1H),
4.72 (d, J = 9.4 Hz, 1H), 4.64 (dd, J1 = 12.7 Hz, J2 = 3.2Hz, 1H), 1.48 (d, J = 22.3 Hz, 3H); 13C
NMR (CDCl3, 75 MHz) δ: 165.9, 165.4, 134.0, 133.8, 133.4, 132.8, 130.1, 129.5, 129.4, 129.1,
128.8, 128.6, 128.4, 127.6, 101.3, 98.8, 72.0 (d, J = 15.0 Hz), 61.8, 17.2 (d, J = 24.8 Hz);
HRMS (ESI): m/z [M + H]+ calcd. for C31H25D2FN3O7: 574.1959; Found 574.1963.
4.1.16. ((2R,3R,4R,5R)-3-(Benzoyloxy)-5-(2,4-dioxo-3,4-dihydro-[5,6-D2]pyrimidin-
1(2H)-yl)-4-fluoro-4-methyltetrahydrofuran-2-yl)methyl benzoate (27)
To a stirred solution of water (24 mL) and acetic acid (56 mL) was added (2R,3R,4R,5R)-
5-(4-Benzamido-2-oxo-[5,6-D2]pyrimidin-1(2H)-yl)-2-((benzoyloxy)methyl)-4-fluoro-4-
methyltetrahydrofuran-3-yl benzoate (26) (1.30 g, 2.26 mmol). The resulting mixture was
heated at 110 C for 18 hours. The reaction mixture was cooled to ambient temperature and
water (30 mL) was added. The resulting suspension was filtered and the collected solid was
washed with water (3 mL), and dried to give the desired product as a white solid (913 mg, 86%)
1
which was used directly for the next step without further purification. H NMR (CDCl3, 300
MHz) δ: 8.86 (s, 1H), 8.02-8.11 (m, 4H), 7.46-7.63 (m, 6H), 6.29 (d, J = 18.5 Hz, 1H), 5.55
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(dd, J1 = 21.5 Hz, J2 = 9.4 Hz, 1H), 4.91 (d, J = 12.6 Hz, 1H), 4.65 (d, J = 9.3 Hz, 2H), 4.53
(dd, J1 = 12.7 Hz, J2 = 2.8 Hz, 1H), 1.47 (d, J = 22.3 Hz, 3H); 13C NMR (CDCl3, 75 MHz) δ:
154.7, 113.0, 97.4, 81.6, 81.2, 19.6 (d, J = 16.5 Hz); HRMS (ESI): m/z [M + H]+ calcd. for
C24H20D2FN2O7: 471.1537; Found 471.1541.
4.1.17. 1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-(hydroxymethyl)-3-
methyltetrahydrofuran-2-yl) [5,6-D2]pyrimidine-2,4(1H,3H)-dione (28)
To a stirred 7M methanolic ammonia (26 mL) solution was added ((2R,3R,4R,5R)-3-
(Benzoyloxy)-5-(2,4-dioxo-3,4-dihydro-[5,6-D2]pyrimidin-1(2H)-yl)-4-fluoro-4-
methyltetrahydrofuran-2-yl)methyl benzoate (27, 500 mg, 1.06 mmol). The resulting mixture
was stirred at ambient temperature for 24 hours, then concentrated under reduced pressure to
give crude product as a yellow solid. The crude product was suspended in ethyl acetate (6
mL), filtered and dried to give the the desired product as a white solid (159 mg, 57%). 1H
NMR (DMSO-d6, 300 MHz) δ:11.45 (br s, 1H), 5.99 (d, J = 18.8 Hz, 1H), 5.66 (s, 1H), 5.28
(s, 1H), 3.82-3.85 (m, 3H), 3.61-3.65 (m, 1H), 1.26 (d, J = 22.5 Hz, 3H); 13C NMR (DMSO-
d6, 75 MHz) δ: 162.9, 150.6, 102.1, 99.7, 81.7, 70.3 (d, J = 18.0 Hz), 58.3, 16.4 (d, J = 24.8
Hz). HRMS (ESI): m/z [M + H]+ calcd. for C10H12D2FN2O5: 263.1012; Found 263.1016.
4.1.18. (2R,3R,4R)-5-Acetoxy-2-((benzoyloxy)methyl)-4-fluoro-4-methyl[5-
D]tetrahydrofuran-3-yl benzoate (31)
To a stirred LiAlD4 (5.63g, 134 mmol) in anhydrous tetrahydrofuran (500 mL) was added
a solution of tert-butanol (31.4 g, 40.52 mL, 424 mmol) in anhydrous tetrahydrofuran (50 mL)
under N2 atmosphere over 30 minutes. The resulting solution was warmed to reflux over 2
hours to give the lithium tritert-butylaluminum deuteride reagent.
29 (25.0 g, 67.1 mmol) was dissolved in anhydrous tetrahydrofuran (500 mL) and the
solution was cooled to -20 C under a nitrogen atmosphere. The fresh prepared lithium tritert-
butylaluminum hydride reagent was added in over 15 minutes with stirring while maintaining
the temperature at -20 C. After 5 hours, the reaction was completed to give lactol. To the
reaction mixture were added DMAP (8.2 g, 67.1 mmol) and acetic anhydride (63.1 g, 618
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mmol). After the reaction mixture was stirred at -20 C for 2 hours it was diluted with ethyl
acetate (400 mL) and water (200 mL). The organic layer was separated and the aqueous layer
was extracted with ethyl acetate (2 x 100 mL). The combined organic layer was washed with
water (3 x 150 mL) and brine (150 mL), dried over sodium sulfate, filtered, concentrated and
purified by silica gel chromatography to afford the title compound (20.77 g, 74%). 1H NMR
(DMSO-d6, 300 MHz) δ: 7.95-8.05 (m, 4H), 7.64-7.75 (m, 2H), 7.49-7.60 (m, 4H), 5.63 (br
dd, J1 = 24.2 Hz, J2 = 8.1 Hz, 1H), 4.61-4.69 (m, 2H), 4.42-4.47 (m, 1H), 1.93 (s, 3H), 1.51
(d, J = 22.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz) δ: 168.6, 165.1, 134.0, 133.5, 129.5, 129.4,
129.2, 129.1, 128.8, 128.7, 128.4, 101.0, 98.6, 78.3, 73.1 (d, J = 15.0 Hz), 73.0, 63.0, 20.5,
16.1 (d, J = 23.4 Hz); HRMS (ESI): m/z [M + Na]+ calcd. for C22H21DFNaO7: 440.1232;
Found 440.1238.
4.1.19. (2R,3R,4R,5R)-5-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-2-
((benzoyloxy)methyl)-4-fluoro-4-methyl-[5-D]tetrahydrofuran-3-yl benzoate (33)
The silylated base was prepared by heating to reflux a suspension of N-benzoylcytosine (32,
5 g, 23.2 mmol) and ammonium sulfate (90 mg) in hexamethyldisilazane (75 mL) for 6 hours
and concentrating the resulting solution by vacuum distillation under aspiration, followed by
drying under vacuum for 2 hours at ambient temperature. The oily residue was dissolved in
chlorobenzene (100 mL). To this solution was added (2R,3R,4R)-5-acetoxy-2-
((benzoyloxy)methyl)-4-fluoro-4-methyltetrahydrofuran-3-yl-5-d benzoate (31) (6.45 g, 15.5
mmol) and titanium tetrachloride (13.84 g, 8.0 mL, 73.0 mmol). After stirring under a
nitrogen atmosphere for 2 hours at ambient temperature, the reaction was heated to 70 C for
19 hours. The reaction mixture was cooled to 0 C and solid sodium bicarbonate (34.8 g,
414.3 mmol) and ethyl acetate (150 mL) were added. To the stirred solution was slowly
added water (7.3 mL). After the mixture was stirred for 30 minutes at ambient temperature,
the suspension was filtered and the collected solid was washed with ethyl acetate (30 mL).
The filtrate was washed with water (2 x 250 mL), dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give the crude product as a pale yellow-brown solid.
The crude product mixed with methanol (110 mL) was heated under reflux for 30 minutes
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and then cooled to ambient temperature. The resulting precipitate was collected by filtration,
washed with methanol (110 mL), and dried to give the desired product as a white solid (3.75
g, 42%) which was used directly for the next step without further purification. 1H NMR
(CDCl3 , 300 MHz) δ: 8.05-8.10 (m , 5H) , 7.93-7.95 (m , 2H) , 7.63-7.69 (m , 3H) , 7.45-
7.62 (m , 7H) , 5.56 (br dd , J1 = 24.9 Hz, J2 = 8.7 Hz, 1H) , 4.87 (dd , J1 = 12.6 Hz, J2 = 2.4
Hz, 1H) , 4.72 (d , J = 9.5 Hz, 1H) , 4.65 (dd , J1 = 12.7 Hz, J2 = 3.5 Hz, 1H) , 1.47 (d , J1 =
22.4 Hz, 3H); 13C NMR (CDCl3 , 75 MHz) δ: 166.0, 165.4, 162.95, 143.6 (d , J = 9.0 Hz),
134.0, 133.7, 133.2, 132.9, 130.07, 129.5, 129.3, 128.9, 128.8, 128.6, 128.3, 127.7, 101.3,
98.8, 97.6, 72.1 (d , J = 16 Hz), 61.8, 50.6, 17.2 (d , J = 25 Hz); HRMS (ESI): m/z [M + H]+
calcd. for C31H26DFN3O7: 573.1896; Found 573.1890
4.1.20. ((2R,3R,4R,5R)-3-(Benzoyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
fluoro-4-methyl-[5-D]tetrahydrofuran-2-yl)methyl benzoate (34)
To a stirred solution of water (48 mL) and acetic acid (112 mL) was added (2R,3R,4R,5R)-5-
(4-benzamido-2-oxopyrimidin-1(2H)-yl)-2-((benzoyloxy)methyl)-4-fluoro-4-
methyltetrahydrofuran-3-yl-5-d benzoate (33) (2.6 g, 4.54 mmol). The resulting mixture was
heated at 110 C for 18 hours. The reaction mixture was cooled to ambient temperature and
water (60 mL) was added. The resulting suspension was filtered and the collected solid was
washed with water (6 mL), and dried to give the desired product as a white solid (1.79 g,
84%) which was used directly for the next step without further purification. 1H NMR (CDCl3,
500MHz) δ: 8.06 (m, 4H), 7.48-7.62 (m, 7H), 5.44-5.56 (m, 1H), 4.92 (d, J = 11.7 Hz, 1H),
4.52-4.72 (m, 2H), 1.47 (d, J = 22.2 Hz, 3H); 13C NMR (CDCl3 , 75 MHz) δ: 162.0, 138.8,
134.1, 133.7, 130.1, 129.5, 128.8, 128.7, 103.1, 61.5, 17.3 (d, J = 24.8 Hz); HRMS (ESI): m/z
[M + H]+ calcd. for C31H26DFN3O7: 573.1896; Found 573.1890
4.1.21. 1-((2R,3R,4R,5R)-3-Fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyl-[5-
D]tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (35)
To a stirred 7M methanolic ammonia (80 mL) solution was added ((2R,3R,4R,5R)-3-
(benzoyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-4-
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methyltetrahydrofuran-2-yl-5-d)methyl benzoate (34, 1.56 g, 3.3 mmol). The resulting mixture
was stirred at ambient temperature for 24 hours, then concentrated under reduced pressure to
give crude product as a yellow solid. The crude product was suspended in ethyl acetate (10
1
mL), filtered and dried to give the desired product as a white solid (480 mg, 56%). H
NMR(DMSO-d6, 300 MHz) δ: 11.45 (br s, 1H), 7.97 (d, J = 8.1 Hz, 1H), 5.65-5.67 (m, 2H),
5.29 (s, 1H), 3.82-3.85 (m, 3H), 3.61-3.65 (m, 1H), 1.26 (d, J = 22.5 Hz, 3H); 13C
NMR(DMSO-d6, 75 MHz) δ: 162.8, 150.5, 139.5 (d, J = 7.5 Hz), 102.0, 99.6, 81.7, 70.4 (d, J
= 17.3 Hz), 58.3, 16.4 (d, J = 25.5 Hz); HRMS (ESI): m/z [M + H]+ calcd. for C10H13DFN2O5:
262.0950; Found 262.0960.
4.1.22. [D7]Propan-2-yl-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-
L-alaninate (37)
To a stirred suspension of 1-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-
methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (18, 1.42 g, 5.5 mmol) in anhydrous
THF (30 mL) was added a 1.0 M solution of tert-butylmagnesium chloride in THF (11.4 mL,
11.4 mmol) using an addition funnel over a period of 30 minutes at -5 C. The white suspension
was stirred at this temperature for 30 minutes and then warmed to room temperature at which
time it was stirred for an additional 30 minutes. The reaction mixture was cooled to 5 C and
then a solution of (S)-2-[(S)-(2,3,4,5,6-pentafluorophenoxy)-phenoxyphosphorylamino]
propionic acid isopropyl ester (7, 3.00 g, 6.5 mmol) in THF (20 mL) was added over a period
of 30 minutes. The mixture was stirred at 5 C overnight, cooled to -5 C, and then quenched
with 1 N HCl (5 mL). Toluene (30 mL) was added to the mixture. The organic layer was
separated and washed with water (10 mL), saturated aqueous NaHCO3 (8 mL) and brine (2 x
8 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered,
concentrated and purified by silica gel chromatography to afford the title compound (1.1 g,
38%) as a foam solid. There was no other isomer detectable by 31P or1H NMR. Stereochemistry
was assigned as Sp on the basis of the known stereochemistry of sofosbuvir. [9,12] 1H NMR
(CDCl3, 300 MHz) δ: 8.53 (br s, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.36 (t, 2H), 7.17-7.24 (m , 3H),
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6.18 (br d, J = 19.2 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 4.54 (t , 1H), 4.45 (t, 1H), 4.11 (d, J =
13
9.2 Hz, 1H), 3.90-3.99 (m, 3H), 1.35-1.45 (m, 6H); C NMR (CDCl3, 300 MHz) δ: 162.4,
139.2, 130.0, 125.35, 119.8 (d, J = 4.5 Hz), 103.0, 101.6, 79.86 (d, J = 6.0 Hz), 71.9 (d, J =
18.0 Hz), 63.9 (d, J = 3.8 Hz), 50.4, 29.7, 20.9 (d, J = 5.3 Hz), 16.5 (d, J = 25.5 Hz); 31P NMR
(CDCl3, 121 MHz) δ: 3.58; HRMS (ESI): m/z [M + H]+ calcd. for C22H23D7FN3O9P: 537.2143;
Found 537.2163; HPLC tR 13.79 min, chemical purity = 99.5%.
4.1.23. 1,1,1,3,3,3-D6]Propan-2-yl-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (38)
Compound 38 was prepared as a white solid according to the procedure described for
compound 37. Compound 8 (3.0 g, 6.5 mmol) was treated with compound 18 (1.42 g, 5.5 mmol )
to afford compound 38 (0.76 g, 37%). There was no other isomer detectable by 31P or1H NMR.
Stereochemistry was assigned as Sp on the basis of the known stereochemistry of sofosbuvir.
[9,12] 1H NMR (CDCl3, 300 MHz) δ: 8.74 (br s, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.34 (t, 2H), 7.17-
7.24 (m, 3H), 6.17 (br d, J = 19.2 Hz, 1H), 5.70 (d, J = 7.8 Hz, 1H), 5.00 (s, 1H), 4.41-4.57 (m,
2H), 4.10 (d, J = 9.2 Hz, 1H), 3.97 (m, 3H), 3.66 (br s, 1H), 1.37-1.45 (m, 6H); 31P-
NMR(CDCl3, 121 MHz) δ: 3.56; HRMS (ESI): m/z [M + H]+ calcd. for C22H24D6FN3O9P:
536.2080; Found 536.2107; HPLC tR 14.12 min, chemical purity = 99.5%.
4.1.24. [2-D]Propan-2-yl-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-
L-alaninate (39)
Compound 39 was prepared as a white solid according to the procedure described for
compound 37. Compound 9 (2.6 g, 5.7 mmol) was treated with compound 18 (1.25 g, 4.8
mmol ) to afford compound 39 (0.99 g, 39%). There was no other isomer detectable by 31P or
1H NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of
sofosbuvir. [9,12]1H NMR (CDCl3, 500 MHz) δ: 8.48 (br s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.36
(t, 2H), 7.18-7.24 (m, 3H), 6.18 (br d, J = 18.5 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 4.54 (t, 1H),
4.45 (t, 1H), 4.11 (d, J = 8.9 Hz, 1H), 3.81-3.95 (m, 3H), 1.35-1.44 (m, 6H), 1.24-1.26 (m,
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6H); 31P NMR (CDCl3, 121 MHz): δ 3.58; HRMS (ESI): m/z [M + H]+ calcd. for
C22H29DFN3O9P: 531.1766; Found 531.1782; HPLC tR 14.49 min, chemical purity = 99.0%.
4.1.25. Isopropyl ((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)( [2,3,4,5,6-D5]-
phenoxy)phosphoryl)-L-alaninate (40)
Compound 40 was prepared as a white solid according to the procedure described for
compound 37. Compound 16 (2.50 g, 5.50 mmol) was treated with compound 18 (1.18 g, 4.50
mmol ) to offer compound 40 (0.84 g, 35%). There was no other isomer detectable by 31P or1H
NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of
sofosbuvir. [9,12] 1H NMR (CDCl3, 300 MHz) δ: 8.51 (s, 1H), 7.47 (d, J = 8.1 Hz, 1H), 6.17 (d,
J = 19.3 Hz, 1H), 5.70 (d, J = 8.0 Hz, 1H), 4.97-5.05 (m, 1H), 4.41-4.57 (m, 2H), 4.10 (d, J =
9.3 Hz, 1H), 3.79-4.06 (m, 3H), 1.35-1.45 (m, 6H), 1.23-1.25 (m, 6H); 31P NMR (CDCl3, 121
MHz) δ: 3.54; HRMS (ESI): m/z [M + H]+ calcd. for C22H26D5FN3O9P: 535.2018; Found
535.2037; HPLC tR 14.22 min, chemical purity = 99.6%.
4.1.26. Isopropyl ((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)([4-D]phenoxy)phosphoryl)-L-
alaninate (41)
Compound 41 was prepared as a white solidaccording to the procedure described for
compound 37. Compound 17 (3.08 g, 6.78 mmol) was treated with compound 18 (1.48 g, 5.65
mmol ) to offer compound 41 (0.99 g, 33%). There was no other isomer detectable by 31P or1H
NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of
sofosbuvir. [9,12] 1H NMR (CDCl3, 300 MHz) δ: 8.66 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.35 (d,
J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 6.18 (br d, J = 18.9 Hz, 1H), 5.70 (d, J = 7.9 Hz, 1H),
5.02 (q, 1H), 4.51 (m, 2H), 4.11 (d, J = 8.1 Hz, 1H), 3.92-4.04 (m, 3H), 1.37-1.45 (m, 6H),
13
1.23-1.25 (m, 6H); C NMR (DMSO- d6, 75 MHz) δ: 173.1, 162.5, 150.0, 139.2 (d, J = 3.8
Hz), 129.90 (d, J = 8.3 Hz), 119.75 (d, J = 5.3 Hz), 102.96, 101.56, 99.15, 79.8 (d, J = 6.0 Hz),
71.9 (d, J = 17.3 Hz), 69.7, 63.9 (d, J = 4.5 Hz), 50.4, 21.7 (d, J = 9.0 Hz), 20.8 (d, J = 6.0 Hz),
16.5 (d, J = 25.5 Hz); 31P NMR (CDCl3, 121 MHz) δ: 3.57; HRMS (ESI): m/z [M + H]+ calcd.
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for C22H29DFN3O9P: 531.1766; Found 531.1782; HPLC tR 14.25 min, chemical purity = 99.8%.
4.1.27. Isopropyl ((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) [D2]methoxy)(phenoxy)phosphoryl)-L-
alaninate (42)
Compound 42 was prepared as a white solidaccording to the procedure described for
compound 37. Compound 36 (1.07 g, 2.40 mmol) was treated with compound 20 (520 mg, 1.98
mmol ) to offer compound 42 (423 mg, 40%). There was no other isomer detectable by 31P or
1H NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of
sofosbuvir. [9,12] 1H NMR (CDCl3, 300 MHz) δ: 8.88 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.36 (t,
J = 8.1 Hz, 2H), 7.18-7.24 (m, 3H), 6.17 (d, J = 19.3 Hz, 1H), 5.69 (d, J = 7.9 Hz, 1H), 4.97-
5.06 (m, 1H), 4.09-4.12 (m, 1H), 3.89-4.00 (m, 3H), 1.34-1.44 (m, 6H), 1.23-1.25 (m, 6H); 13C
NMR (DMSO- d6, 75 MHz) δ: 173.1, 162.7, 150.1, 139.3, 130.0, 125.3, 119.8 (d, J = 5.3 Hz),
103.0, 101.6, 79.7, 71.8 (d, J = 17.3 Hz), 69.7, 50.4, 29.7, 21.7 (d, J = 9.0 Hz), 20.8 (d, J = 5.3
Hz), 16.5 (d, J = 24.8 Hz); 31P NMR (CDCl3, 121 MHz) δ: 3.56; HRMS (ESI): m/z [M + H]+
calcd. for C22H28D2FN3O9P: 532.1829; Found 532.1838; HPLC tR 13.86 min, chemical purity
= 99.9%.
4.1.28. Isopropyl ((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydro-[5,6-D2]pyrimidin-
1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-
yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (43)
Compound 43 was prepared as a white solid according to the procedure described for
compound 37. Compound 36 (1.07 g, 2.36 mmol) was treated with compound 28 (517 mg, 1.97
mmol ) to afford compound 43 (450 mg, 43%). There was no other isomer detectable by 31P or
1H NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of
sofosbuvir. [9,12] 1H NMR (CDCl3, 300 MHz) δ: 8.62 (s, 1H), 7.33-7.38 (m, 2H), 7.17-7.24 (m,
3H), 6.17 (br d, J =18.9 Hz, 1H), 4.97-5.05 (m, 1H), 4.41-4.57 (m, 2H), 4.11 (d, J = 9.4 Hz,
1H), 3.79-4.06 (m, 3H), 1.35-1.45 (m, 6H), 1.23-1.25 (m, 6H); 13C NMR (DMSO-d6, 75 MHz)
δ: 162.5, 150.0, 13.0, 125.3, 119.8 (d, J = 5.3 Hz), 101.6, 99.2, 79.9 (d, J = 6.0 Hz), 71.9 (d, J
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= 18.0 Hz), 69.7, 64.0 (d, J = 5.3 Hz), 50.4, 21.7 (d, J = 9.0 Hz), 20.8 (d, J = 6.0 Hz), 16.5 (d,
31
J = 25.5 Hz); P NMR (CDCl3, 121 MHz) δ: 3.56; HRMS (ESI): m/z [M + H]+ calcd. for
C22H28D2FN3O9P: 532.1829; Found 532.1853; HPLC tR 15.13 min, chemical purity = 99.4%.
4.1.29. Isopropyl ((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
fluoro-3-hydroxy-4-methyl-[5-D]tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl) -
L-alaninate (44)
Compound 44 was prepared as a white solidaccording to the procedure described for
compound 37. Compound 36 (2.53 g, 5.58 mmol) was treated with compound 35 (1.21 g, 4.65
mmol ) to offer compound 44 (1.06 g, 43%). There was no other isomer detectable by 31P or1H
NMR. Stereochemistry was assigned as Sp on the basis of the known stereochemistry of
sofosbuvir. [9,12] 1H NMR (CDCl3, 300 MHz) δ: 9.05 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.36 (t,
J = 7.2 Hz, 2H), 7.18-7.24 (m, 3H), 5.69 (d, J = 8.1 Hz, 1H), 4.97-5.05 (m, 1H), 4.41-4.57 (m,
13
2H), 4.04-4.12 (m, 2H), 3.90-3.98 (m, 3H), 1.34-1.43 (m, 6H), 1.23-1.25 (m, 6H); C NMR
(CDCl3, 75 MHz) δ: 162.8, 150.3 (d, J = 12.0 Hz), 139.2 (d, J = 6.0 Hz), 129.9, 125.3, 119.8
(d, J = 5.3 Hz), 106.4, 102.9, 101.5, 99.1, 79.8 (d, J = 6.0 Hz), 71.9 (d, J = 5.3 Hz), 69.7, 64.0,
50.4, 29.7, 21.7 (d, J = 9.0 Hz), 20.8 (d, J = 6.0 Hz), 16.5 (d, J = 25.5 Hz); 31P NMR (CDCl3,
121 MHz) δ: 3.52; HRMS (ESI): m/z [M + H]+ calcd. for C22H29DFN3O9P: 531.1766; Found
531.1785; HPLC tR 14.33 min, chemical purity = 99.9%.
4.2 Biological studies
4.2.1. Measurement of the anti-HCV activities of compounds using HCV replicon
4.2.1.1. Cell line and cell culture
The Huh7 cell line was kindly provided by AppTec company (America). HCV GT1b NS5B
chimeric replicons were provided by WuXi AppTec. Huh7 cells were grown in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum. The replicon RNAs
were transcribed in vitro using the replicon plasmid DNAs.
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4.2.1.2 Antiviral activity assay test with HCV replicon
Compounds were added to 96-well plates by Echo at 9 concentrations with a serial 3-fold
dilution, in triplicate. The final concentration of DMSO in the cell culture medium was 0.5%.
The Huh7 cells were transiently transfected with the replicon RNAs by electroporation and
seeded at a density of 10,000 cells/well in 96-well plates. And then the cells were cultured and
treated with the compounds at 37 °C and 5% CO2 for 3 days. The cell viability in GT1b replicon
assay was tested using CellTiter-Fluor following the manufacturer's instruction. For antiviral
activity detection, the supernatants were removed from the wells, then Bright-Glo will be added
to detect luciferase luminescent signal. The raw data (RLU) will be used for calculating the
antiviral activity (% inhibition) of the compounds. EC50 and CC50 values were calculated using
the GraphPad Prism software.
4.2.2. PK assessment
4.2.2.1 Compound Stability in Human Liver S9 Fraction
Stock solutions of the test compounds (10mM) were prepared in DMSO and stored at -
20 °C. Rat, dog, and human liver S9 fractions were obtained from the Research Institute for
Liver Diseases (Shanghai) Co LTD. A typical standard reaction mixture was prepared in a total
volume of 300 µL containing 5 mM MgCl2, 100 mM potassium phosphate buffer (pH 7.4) and
1 µM of the test compounds. The reactions were initiated by the addition of 1 mg/mL liver S9
fractions to the reaction mixture and incubation at 37 °C in a shaking metal bath. At the desired
time (0, 0.5, and 1h), 50 µL aliquots were taken and the reaction was stopped by adding 300
µL amounts of ice-cold acetonitrile containing internal standards. The samples were
centrifuged at 13 000 rpm for 10 minutes at 4 °C. Then 100 μL of the supernatant was mixed
with 100 μL of LC/MS solvent A (70% water, 30% acetonitrile, and 0.1% formic acid) and
storage at 4 °C.
The standard solutions of each compound were prepared from 5 mg/mL of stock solution
and diluted to a final concentration of 1 μg/mL in methanol. The standard solutions were
injected into the LC/MS to determine the daughter ion, cone voltage, and collision energy in
order to develop the LC/MS/MS method.
Each compound was separated by using an Acquity UPLC HSS T3 1.8 μm (2.1×50 mm)
column with a Waters UPLC system. The mobile phase consisted of solvent A (70% water and
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0.1% formic acid ) and solvent B (30% acetonitrile, and 0.1% formic acid). Elution was
performed using a linear gradient of solvent B from 30% to 90% for 3.0 min.
The MS/MS detection was performed by using Waters TQ-S settled in the positive ion
mode (ESI+), with a capillary voltage of 3.0 kV, a source temperature of 150 °C and a
desolvation temperature of 400 °C. The nitrogen gas flow was 800 L/h and 50 L/h for
desolvation. The samples collected at the desired time points were injected 1 μL aliquots for
LC/MS/MS analysis. The amount of parent compound was determined on the basis of the peak
area for each time point, and the percentage remaining was calculated on the basis of the initial
amount measured at 0 hour.
4.2.2.2 In vivo PK assessment in rats and dogs
Adult male rats and male beagle dogs, provided by Sino-British SIPPR/BK Lab Animal
Ltd, were housed at the centralized animal facilities with a 12 h light-dark cycle. The housing
temperature and relative humidity were controlled at 22 °C and 55%, respectively. The animals
fasted 16 hours before drug administration had free access to water. Test compounds were
dosed orally by gavage at a dose of 50 mg/kg for rats and 20 mg/kg for dogs in 0.5%
carboxymethylcellulose sodium with a single dose. Dose-volume was 10 mL/kg. Serial blood
samples were collected at 0.25,0.5,1, 2, 3, 4, 6, 8,10, and 24 hour postdose after dose
administration. All blood samples were obtained by venipuncture into polypropylene tubes
containing K2EDTA and kept on ice for processing by centrifugation. Plasma samples were
quick-frozen over dry ice and kept at -70 °C until LC/MS/MS analysis.
The content of test compounds was quantified by LC/MS/MS. LC conditions were as
follows: Waters UPLC instrument; column, Acquity UHPLC HSS T3 1.8 μm (2.1×50 mm);
40 °C column temperature; mobile phase of (A) 0.1% formic acid in water, (B) 0.1% formic
acid in acetonitrile; flow rate of 0.3 mL/min; gradient of 0-4 min; 0 min-0.6min at 95% A and
5%B, 2.0min-2.6 min at 10% A and 90% B, 2.7min-4.0 min at 95% A and 5% B. Mass
spectrometer conditions were as follows: Waters TSQ, electrospray ionization, MRM positive
mode, capillary voltage 3.0 kV, source temperature of 150 °C , desolvation temperature of
400 °C, nitrogen gas flow 800 L/h. Plasma versus time data was analyzed by non-
compartmental approaches using the DAS software program, version 3.2.5.
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Acknowledgments
Authors are thankful to Mr. Meng Guo for helpful discussion during the implementation of this
project.
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