Discovery of an Orally Active and Liver-Targeted Prodrug of 5-Fluoro-2′-Deoxyuridine for the Treatment of Hepatocellular Carcinoma

Article abstract of DOI:10.1021/acs.jmedchem.5b01807

We report a series of novel O-(substituted benzyl) phosphoramidate prodrugs of 5-fluoro-2′-deoxyuridine for the treatment of hepatocellular carcinoma. Through structure optimization, the o-methylbenzyl analog (1t) was identified as an orally bioavailable and liver-targeted lead compound. This lead prodrug is well-tolerated at a dose up to 3 g/kg in Kuming mice via oral administration. An efficacy study demonstrated that it possesses good inhibitory effect (61.67% and 72.50%, respectively) on tumor growth in a mouse xenograft model. A metabolism study in Sprague-Dawley rats suggested that 1t can release the desired 5′-monophosphate in the liver with high liver-targeting index.

Full text of DOI:10.1021/acs.jmedchem.5b01807

Article
pubs.acs.org/jmc
Discovery of an Orally Active and Liver-Targeted Prodrug of
5‑Fluoro-2′-Deoxyuridine for the Treatment of Hepatocellular
Carcinoma
Youmei Peng,†,‡,# Wenquan Yu,†,# Ertong Li,† Jinfeng Kang,† Yafeng Wang,§ Qinghua Yang,§ Bingjie Liu,†
Jingmin Zhang,§ Longyu Li,§ Jie Wu,† Jinhua Jiang,‡ Qingduan Wang,‡ and Junbiao Chang*,†
§
†College of Chemistry and Molecular Engineering and School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou
450001, People’s Republic of China
‡Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, People’s Republic of China
S
* Supporting Information
ABSTRACT: We report a series of novel O-(substituted benzyl) phosphoramidate prodrugs of 5-fluoro-2′-deoxyuridine for the
treatment of hepatocellular carcinoma. Through structure optimization, the o-methylbenzyl analog (1t) was identified as an orally
bioavailable and liver-targeted lead compound. This lead prodrug is well-tolerated at a dose up to 3 g/kg in Kuming mice via oral
administration. An efficacy study demonstrated that it possesses good inhibitory effect (61.67% and 72.50%, respectively) on
tumor growth in a mouse xenograft model. A metabolism study in Sprague−Dawley rats suggested that 1t can release the desired
5′-monophosphate in the liver with high liver-targeting index.
phosphates, or nucleotides, cannot themselves be used as viable
drugs due to their poor chemical stability along with high
polarity that vitiates their cellular uptake.7,8 In order to improve
the potency of nucleosides, numerous prodrugs have been
designed by masking the monophosphates thus bypassing the
first-step phosphorylation, which is rate-limiting. Previously,
phosphate and phosphonate prodrugs containing cyclic 1,3-
propanyl esters have been reported. These can be activated via
cytochrome P450-catalyzed oxidative cleavage reaction in the
liver to form higher concentrations of the biologically active
nucleoside triphosphate.6,9−11 However, prodrugs of this type
will also produce a potentially toxic byproduct, aryl vinyl
ketone, which may cause cytotoxicity and genetic toxicity.12,13
Aryloxy phosphoramidates, another type of prodrug, can
partially avoid the dependence of the current drugs on active
transport, saturate efflux transportion and nucleoside kinase-
mediated activation,14,15 and they are also resistant to metabolic
deactivation.15 But once the carboxyl ester is hydrolyzed by
esterases in blood and nontargeted tissues, the phenol group in
the phosphate moiety is cleaved spontaneously by an
1. INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most lethal
cancers and the third most common cause of cancer-related
deaths globally with 626,000 new patients per year.1,2 The
current therapeutic options for HCC include resection,
transplantation, radiofrequency ablation, chemoembolization,
and sorafenib (4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]-
carbamoylamino] phenoxy]-N-methyl-pyridine-2-carboxa-
mide).3 However, most HCC cases are diagnosed in their
advanced stages, which strictly limits the treatment options.4 As
a multikinase inhibitor, sorafenib is the only systemic therapy
for advanced HCC, but its actual benefit is still relatively small.1
The difficulties associated with chemotherapy in HCC may
derive from drug resistance mechanisms, such as rapid drug
metabolism via primary and/or secondary metabolism path-
ways, removal of drugs from tumor cells by drug transport
systems, and inactivation of oncolytic drugs by intracellular
agents such as glutathione. These mechanisms limit the uptake
and retention of the drug molecules in tumor cells and
consequently decrease their overall therapeutic benefit.5
A major reason for the inefficacy of nucleoside antitumor
drugs is that they cannot be efficiently phosphorylated by
specific nucleoside kinases.5,6 On the other hand, nucleoside
Received: November 19, 2015
© XXXX American Chemical Society
A
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intramolecular nucleophilic attack,16,17 resulting in an alanyl
phosphate metabolite, which is a very polar diacid, and will be
poorly transported across cell membranes to the targeted
tissues. In addition, studies have shown that although
teratogenic effects have not been associated with exposure to
phenol, high doses of phenol are fetotoxic.18
This report describes the development of novel phosphor-
amidate prodrugs of 5-fluoro-2′-deoxyuridine (FDUR) for the
treatment of HCC by oral administration. FDUR itself is of
particular value in the clinical treatment of liver cancer,19−21 but
like most nucleosides, it is inactive and requires phosphor-
ylation to the primary bioactive entity, the 5′-monophosphate
(FdUMP).15 FdUMP acts as a potent suicide-type inhibitor of
thymidylate synthase, a key enzyme in DNA synthesis, which
prompts a potent toxic event in the cell.22 In order to reach the
therapeutic goal and minimize systemic toxicities, FDUR in
clinical practice was administered via hepatic arterial infusion
(HAI) to provide direct drug delivery into the tumor feeding
vessels.20,21 Unfortunately, many liver cancer patients cannot
tolerate a catheter inserted into the hepatic artery and a pump
implanted under the skin.
and efficacy of compound 1t were also assessed in Kunming
(KM) mice and in a mouse xenograft model.
2. CHEMISTRY
Prodrug 1 was synthesized as outlined in Scheme 2. Treatment
of a benzyl alcohol (2) with phosphoryl oxychloride (POCl3) in
the presence of triethylamine gave the phosphorodichloride
(3), which is further treated with benzylamine to provide the
chlorophosphoramidate (4). Finally, the reaction of 4 with
FDUR (5) in the presence of N-methylimidazole (NMI)
afforded the desired prodrug 1a−r. The analog 1s was
synthesized by using L-alanine isopropyl ester hydrochloride
in place of benzylamine (Scheme 3).
To synthesize the RP-enantiomer (1t) of the racemic prodrug
1s, (SP)-O-perfluorophenyl phosphoramidate 7 was prepared
by the procedure used for the synthesis of 1s. Treatment of
phosphorochloridate 4s with pentafluorophenol gave diaster-
eomers 6 with the SP-enantiomer as the major isomer. The
enantiomer 7 can be obtained through recrystallization from a
mixture of diisopropyl ether and petroleum ether (Scheme 3).
Selective protection of the 5′- and 3′-hydroxy groups in the
nucleoside 5 with DMTrCl and TBSCl, respectively, gave
intermediate 9, which was treated with TFA to provide the 3′-
TBS protected nucleoside (10). Reaction of compound 10 with
intermediate 7 in the presence of t-butyl magnesium chloride,
followed by recrystallization in a mixture of ethanol and water
afforded prodrug 1t (Scheme 4) as the pure RP-enantiomer
(confirmed by X-ray, see the Supporting Information).
In this paper, we report the design and synthesis of a series of
novel phosphoramidate prodrugs of FDUR bearing O-
(substituted benzyl) moieties (Scheme 1). Through structure
Scheme 1. Lead Optimization of the O-(Substituted Benzyl)
Phosphoramidate Prodrugs of FDUR
3. RESULTS AND DISCUSSION
3.1. In Vitro Evaluation and Lead Optimization. First,
we performed the optimization of the O-benzyl moiety in the
newly designed prodrug structure. In view of the poor stability
of the amino acid ester moiety in rodents,24 to blood circulating
esterases, we initially synthesized the prodrugs (1a−r)
containing a benzylamine group for optimization. These N-
benzyl phosphoramidate prodrugs are all quite stable in rat
plasma (t1/2 > 24 h), the single exception being the O-(o-
methoxy)benzyl analog (1e) for which t1/2 = 3.4 0.5 h. The
activation rate in vitro of the prodrug analogs (1a−r) in rat liver
S9 were determined by measurement of the formation of
FdUMP, the primary bioactive entity, upon incubation for 0,
10, 20, 40, or 60 min with 50 μM of the tested compounds. As
shown in Table 1, the unsubstituted O-benzyl analog (1a)
showed a moderate activation rate for the formation of
FdUMP. In most cases, incorporation of substituents in the
benzene ring of the O-benzyl group resulted in higher rate of
activation. Generally, compounds bearing electron-donating
groups showed better activation rates than those with electron-
withdrawing groups, and ortho-substituted analogs possess
slightly better activation rates than the meta- and para-
optimization, the O-(o-methylbenzyl) analog (1t) was identi-
fied as an orally bioavailable lead compound which targets the
liver.23 Metabolism studies in Sprague−Dawley rats demon-
strated that this phosphoramidate prodrug can release the
desired FdUMP in the liver with a high liver-targeting index
and thus minimize extra hepatic exposure. The acute toxicity
a c
Scheme 2. Synthesis of O-(Substituted Benzyl) Phosphoramidate Prodrugs (1) −
a
b
c
POCl3, Et3N, CH2Cl2, −78 °C. BnNH2, Et3N, DCM, −78 °C to rt. FDUR (5), NMI, CH2CI2, rt, overnight.
B
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Scheme 3. Synthesis of O-(o-Methylbenzyl) Phosphoramidate Prodrug (1s) and (SP)-O-Perfluorophenyl Phosphoramidate
a e
(7) −
a
b
c
d
POCl3, Et3N, CH2Cl2, −78 °C. L-alanine isopropyl ester hydrochloride, Et3N, DCM, −78 °C to rt. 5, Et3N, CH2Cl2, rt. pentafluorophenol, Et3N,
e
CH2Cl2, rt. recrystallization in a mixture of diisopropyl ether and petroleum ether.
a e
Scheme 4. Synthesis of the (SP)-O-(o-Methylbenzyl) Phosphoramidate Prodrug 1t −
a
b
c
d
e
DMTrCl, Py., 0 °C. TBDMSCl, imidazole, DCM, rt. TFA, DCM, rt. 7, t-BuMgCl/THF, 0 °C to rt. TBAF/THF, rt.
substituted analogs. Cytostatic evaluation in vitro of these
prodrugs with the SMMC7721 cell line identified the o-
methylbenzyl (1b) and o-methoxybenzyl analogs (1e) with
potent inhibitory activities. Due to the lower stability of the o-
methoxybenzyl group (1e) in plasma, the o-methylbenzyl
substituent (1b) was selected as the optimal structure design.
Replacement of the N-benzyl group in compound 1b with
the L-alanine isopropyl ester moiety led to the phosphoramidate
1s. Although replacement of the N-benzyl group with the L-
alanine isopropyl ester moiety decreased the in vitro activity,
study of the tissue distribution indicated that the amino acid
ester analog possesses a much higher liver-targeting index (see
Section 3.4). Consistent with the previous data,24 both the
racemic prodrug 1s and its RP-enantiomer 1t have a short half-
life (<5 min) in rat plasma but good stability in human and dog
plasma (t1/2 > 24 h, Table 2). Further evaluation demonstrated
that prodrug 1t exhibited equally good inhibitory activity with
the liver cancer cell line (SMMC7721) and slightly lower
toxicity on a normal liver cell line (LO2) than its racemic form
1s. Besides, considering its superiority in synthesis and
purification (see Section 5.1), we focused our further
development efforts on this RP-phosphoramidate prodrug (1t).
3.2. Assessment of Acute Toxicity in KM Mice. Prior to
the investigation of the in vivo anti-HCC efficacy, an acute
toxicity study of the prodrug (1t) was conducted in KM mice.
A single oral dose of the test compound at 2 and 3 g/kg was
administered to the mice, and this was followed by observation
for 15 days. During the study period, all mice remained alive,
and no significant side effects were observed at either dose.
Only negligible weight loss occurred at 1−5 days post-
treatment (Figure 1). All the animals were sacrificed on day
15, and no damage to the heart, liver, spleen, lung or kidney
was observed. These results demonstrated that orally
administered prodrug 1t was well-tolerated in KM mice at
dosage of up to 3 g/kg in the course of treatment and during
the post-treatment period.
3.3. Assessment of Efficacy in KM Mice. For the
assessment of the antitumor activity in vivo, a mouse xenograft
model established with H22 cells in KM mice was employed.
The tumor-bearing mice were dosed daily by oral gavage with
prodrug 1t at 65, 130, or 260 mg/kg or sorafenib as the positive
control (60 mg/kg) for 8 days (for detailed dose optimization
see Section 5.7). The tumor weight was measured on day 8 and
is given in Table 3. Compared to the control groups, the tested
compound (1t) exhibited good inhibitory effect on the tumor
growth. In particular, prodrug 1t at the dose of 130 or 260 mg/
kg significantly reduced tumor size with inhibitory rates of
61.67% and 72.50%, respectively, which are equally or more
C
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a
Table 1. Stability, Activation, and Cytostatic Activity of Prodrugs 1a−r
a
Data are the means ( SD) of triplicate samples.
a
Table 2. Stability and Cytostatic Activity of Racemic Prodrug (1s) and Its RP-Enantiomer (1t)
a
Data are the means ( SD) of two independent experiments, with each one performed in triplicates.
effective compared to sorafenib at the dose of 60 mg/kg
(effective dose).25 Over the course of this 8 day study,
treatment of 1t did not cause significant toxicity in any of the
three dosed groups (Table 3).
3.4. Assessment of Tissue Distribution in Sprague−
Dawley Rats. Tissue distribution study of prodrug 1t and its
N-benzyl analog 1b was conducted in Sprague−Dawley rats to
assess their liver-targeting indexes, compared to those of
FDUR. Intragastric gavage (i.g.) administration of 52.17 mg/kg
(0.096 mmol/kg) of 1t and 49.87 mg/kg (0.096 mmol/kg) of
1b produced 543 ng/g·h and <155 ng/g·h of FdUMP in the
liver, respectively, while equimolar doses of FDUR (23.62 mg/
kg) resulted in a very low level of the monophosphate (Table 4,
Figure 2). Conversely, only 26 ng/mL·h and 41 ng/mL·h of
D
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mediated by the histidine triad nucleotide-binding protein 1
(Hint1)17 affords the desired monophosphate, FdUMP.
To prove the above putative mechanism, we carried out a
metabolism study of prodrug 1t in the presence of rat liver S9.
Consistent with the previous results in rat plasma (see Table
2), most of the test compound was hydrolyzed to intermediate
A within 20 min (Figure 3), and subsequently, the mono-
phosphate FdUMP is formed. Then the level of this bioactive
entity increased, and the concentration of compound A
decreased both in a time-dependent manner (Figure 3).
However, only a high concentration of A and no FdUMP
were determined in the negative group without NADPH,
indicating that P450-catalyzed oxidation reactions were
involved in this debenzylation step. In addition, HPLC-MS/
MS analysis of the liver and plasma samples indicated that the
metabolite 2-methylbenzoic acid was further conjugated with
glycine to form nontoxic o-methylhippuric acid27 (m/z 192 →
148, also confirmed by running a standard sample). Overall,
during this metabolism process (Scheme 5), only 2-
methylbenzaldehyde possesses little acute toxicity,28 most of
which will be eventually excreted as o-methylhippuric acid in
the urine.27,8
Figure 1. Mean body weight change of KM mice from day 1 to 15
after oral administration of prodrug 1t at a single dose of 2 and 3 g/kg.
FDUR were determined in the plasma of 1t- and 1b-treated
rats, respectively, whereas 308 ng/mL·h was observed in that of
FDUR-treated animals. These results translate to a liver-
targeting index (AUC(inf) FdUMP (liver)/AUC(inf) FDUR
(plasma)) of 20.9 for 1t, <3.8 for 1b, and <0.11 for FDUR.
Overall, the increase in the targeting index for 1t over FDUR is
therefore >190-fold.
4. CONCLUSIONS
In addition, much higher levels of the primary metabolite A
(see Scheme 5) were observed in the liver than in the plasma of
1t-dosed rats (Table 4). The AUC(inf) of A in the liver was 5509
ng/g·h, while the value in the plasma was only 316 ng/mL·h (a
ratio of ∼17.4:1). Formation of FDUR was also a liver-selective
distribution, with AUC(inf) of 1541 ng/g·h in the liver but only
26 ng/mL·h in the plasma (a ratio of ∼59.3:1). Moreover, no
detectable amounts of FdUMP and 5-fluorouracil (5-FU) were
observed in the plasma upon administration of 1t, while a high
level of 5-FU was formed in the plasma of FDUR-dosed
aniamals. The liver-targeting release of the bioactive FdUMP
and related metabolites can significantly reduce their exposure
in nontargeted tissues, therefore diminishing the overall toxicity
in vivo.
3.5. Prodrug Activation Mechanism. A putative
mechanism for the activation of 1t and formation of the
bioactive monophosphate (FdUMP) in the liver is proposed
and is shown in Scheme 5. The metabolism of this prodrug
starts with (a) the hydrolysis of the carboxylic ester moiety by a
carboxyesterase-type enzyme to give a stable intermediate A.17
Then, (b) a plausible metabolite B26 might be formed via a
cytochrome P450-catayzed oxidative hydroxylation at the
benzylic position.6,9 Subsequently, (c) the benzyl moiety is
cleaved via hydrolysis to produce a polar diacid intermediate C
and 2-methylbenzaldehyde which will be further oxidized by
aldehyde dehydrogenase to a nontoxic metabolite, 2-methyl-
benzoic acid.27 Finally, (d) cleavage of the P−N bond of C
We have designed and synthesized a series of novel O-
(substituted benzyl) phosphoramidate prodrugs of FDUR for
the treatment of hepatocellular carcinoma. Evaluation in vitro
allowed identification of the o-methylbenzyl analog (1t) which
is orally bioavailable and liver targeted as a lead compound.
Prodrug 1t is well-tolerated upon oral administration at a dose
up to 3 g/kg in KM mice. During an 8 day efficacy study, this
lead prodrug (130 and 260 mg/kg/d) exhibited a good
inhibitory effect on tumor growth (61.67% and 72.50%,
respectively) in a mouse xenograft model. More importantly,
a metabolism study in Sprague−Dawley rats demonstrated that
this phosphoramidate prodrug can release the desired FdUMP
in the liver with a high liver-targeting index. Further application
of this prodrug strategy to discover more prodrugs of
nucleosides for the treatment of the corresponding diseases is
currently ongoing in our laboratory.
5. EXPERIMENTAL SECTION
1
5.1. Chemistry. H NMR spectra were recorded on a 400 MHz
spectrometer, and 13C NMR spectra were recorded on a 100 MHz
spectrometer. Chemical shift values are given in ppm and referred to
an internal standard of tetramethylsilane. The peak patterns are
indicated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m,
multiplet; dd, doublet of doublets; and dt, doublet of triplets. The
coupling constants (J) are reported in Hertz (Hz). Optical rotations
were measured on a PerkinElmer Polarimeter 341 in a 10 cm cuvette
at 20 °C at 589 nm. The concentration (c) is given in g/100 mL. Flash
a
Table 3. Effect of Tumor Growth in H22 Tumor-Bearing Mice after Treatment for 8 Days
body weight (g)
groups
dosage (mg/kg/d)
pretreatment
post-treatment
tumor weight (g)
1.20 0.40
inhibition rate (%)
control
−
65
22.1 1.0
21.7 1.2
22.3 1.1
22.3 1.4
22.1 0.8
29.4 2.4
25.9 3.6
23.8 3.1
22.3 2.1
26.4 1.8
−
b
1t
0.67 0.32
44.17
61.67
72.50
50.83
b
1t
130
260
60
0.46 0.24
bc
,
1t
0.33 0.17
0.59 0.20
b
sorafenib
a
b
c
Data shown are means SD of tumor weights and mouse body weights for each group of mice (n = 10). P < 0.01 versus control group. P < 0.05
versus sorafenib group.
E
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Table 4. Comparative Tissue Distribution in Rat after Oral Administration of FDUR, 1b, and 1t
plasma
liver
a
compds
FDUR
detected compound
Cmax (ng/mL)
AUC(inf) (ng/mL·h)
Cmax (ng/g)
AUC(inf) (ng/g·h)
b
FDUR
5-FU
180
169
ND
40
308
209
ND
41
ND
ND
232
60
9
c
FdUMP
FDUR
5-FU
<34
838
1b
1t
50
14
ND
ND
316
26
60
ND
c
FdUMP
metabolite A
FDUR
5-FU
ND
234
17
55
<155
3673
747
139
340
5509
1541
257
ND
ND
FdUMP
ND
ND
543
a
b
c
AUC(inf), area under the curve from time zero to infinity. ND, not detected. Lower limit of quantitation and the AUC(inf) were calculated on the
basis of the limit of quantification.
Figure 2. (A) Liver FdUMP AUC(inf) and plasma FDUR AUC(inf) after treating rats with equimolar FDUR (23.62 mg/kg, i.g.), 1b (49.87 mg/kg,
i.g.), and 1t (52.17 mg/kg, i.g.). (B) Time course of liver FdUMP levels after i.g. administration of equimolar FDUR (23.62 mg/kg), 1b (49.87 mg/
kg), and 1t (52.17 mg/kg).
Scheme 5. Proposed Mechanism for the Activation of
Prodrug 1t
Figure 3. Concentration−time curve of prodrug 1t, its metabolite A,
and FdUMP during incubation of 1t (20 μg/mL) with rat liver S9 (10
mg/mL).
CH2Cl2 (2 mL) dropwise. After having been stirred at the same
temperature for 3 h, the reaction mixture was treated slowly with a
mixture of benzylamine (214 mg, 2 mmol) and triethylamine (202 mg,
2 mmol) in anhydrous CH2Cl2 (2 mL). It was stirred at −78 °C for
column chromatography was performed over 200−300 mesh silica gel.
High-resolution mass spectra (HRMS-ESI) were obtained on a Q-
TOF mass spectrometer. All the compounds tested possess a purity of
at least 95%. Analytical HPLC was run on a Thermo Fisher ACCELA
HPLC system equipped with a Phenomenex Luna C8 column (150
mm × 4.6 mm, 5 μm) and UV detection at 267 nm. The mobile phase
consisted of MeOH:H2O = 68:32 (v/v). The flow rate was set at 0.80
mL/min, and the column temperature was maintained at 30 °C.
5.1.1. General Procedure for the Synthesis of Prodrug 1a−r. At−
78 °C, to a solution of phosphoryl chloride (307 mg, 2 mmol) in
anhydrous CH2Cl2 (6 mL) was added a solution of substituted benzyl
alcohol (2 mmol) and triethylamine (202 mg, 2 mmol) in anhydrous
another 1 h and then allowed to warm up to room temperature over 1
h. After being chilled to 0 °C, the above mixture was further treated
with a solution of FDUR (98 mg, 0.4 mmol) and NMI (164 mg, 2
mmol) in anhydrous CH2Cl2 (1 mL) and stirred overnight at 0 °C.
The reaction was quenched with H2O (10 mL) and extracted with
CH2Cl2 (3 × 10 mL). The combined organic layer was washed with
0.5 M dilute HCl (20 mL) and brine (20 mL) in sequence. After
drying over anhydrous Na2SO4, it was concentrated and then purified
through silica gel column chromatography to afford the desired
products 1a−r.
5.1.1.1. ((2R,3S,5R)-5-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methyl 2-methylbenzyl
F
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benzylphosphoramidate (1b). White solid; yield, 33%; 1H NMR
(400 MHz, CD3OD): δ 7.82−7.80 (m, 1H), 7.34−7.15 (m, 9H),
6.22−6.19 (m, 1H), 5.11−4.98 (m, 2H), 4.35−4.31 (m, 1H), 4.20−
4.02 (m, 5H), 2.34 (s, 3H), 2.29−2.21 (m, 1H), 2.07−1.97 (m, 1H);
HRMS (m/z) [M + Na]+ calcd for C24H27FN3O7PNa 542.1463, found
542.1439.
CH2Cl2 (100 mL) was treated with imidazole (11.0 g, 0.162 mol) and
TBSCl (15.3 g, 0.102 mol) in sequence and then stirred at room
temperature for 2 h (TLC indicated that the reaction was complete).
The reaction was quenched with H2O (5 mL), concentrated, treated
with H2O (150 mL), and extracted with EtOAc (3 × 50 mL). The
combined organic layer was washed with brine (100 mL), dried over
anhydrous Na2SO4, and concentrated. The given residue was purified
through silica gel chromatography (EtOAc/PE = 1:3) to afford
intermediate 9 as yellow thick oil. To a solution of compound 9 in
anhydrous CH2Cl2 (200 mL) was added TFA (12 mL) slowly. The
color of the reaction mixture turned to red, then the reaction was
stirred at room temperature until disappearance of compound 9, as
indicated by TLC. It was quenched with MeOH dropwise, until the
color turned to orange, followed by the addition of aqueous ammonia
until the mixture became nearly colorless. The resulting mixture was
concentrated and then purified through silica gel chromatography
(EtOAc/PE = 1:2) to afford intermediate 10 (11.8 g, 81% over three
steps).
5.1.2. Synthesis of Prodrug 1s. At−78 °C, to a solution of
phosphoryl chloride (307 mg, 2 mmol) in anhydrous CH2Cl2 (6 mL)
was added a solution of 2-methylbenzyl alcohol (245 mg, 2 mmol) and
triethylamine (202 mg, 2 mmol) in anhydrous CH2Cl2 (2 mL)
dropwise. After being stirred at the same temperature for 3 h, the
reaction mixture was treated with amino acid ester hydrochloride (2
mmol) in one portion, followed by the addition of triethylamine (405
mg, 4 mmol) dropwise. It was stirred at −78 °C for another 1 h and
then allowed to warm up to room temperature over 1 h. After chilling
to 0 °C, the above mixture was further treated with a solution of
FDUR (98 mg, 0.4 mmol) and NMI (164 mg, 2 mmol) in anhydrous
CH2Cl2 (1 mL) and stirred overnight at 0 °C until TLC indicated that
the FDUR was completely consumed. The reaction was quenched
with H2O (10 mL) and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layer was washed with 0.5 M dilute HCl (20 mL)
and brine (20 mL) in sequence. After dried over anhydrous Na2SO4, it
was concentrated and then purified through silica gel column
chromatography to afford the desired product 1s as colorless
5.1.5. Synthesis of Prodrug 1t. Compounds 10 (5.0 g, 13.9 mmol)
and 7 (10.0 g, 20.8 mmol) were mixed in a 250 mL round-bottom
flask and dried under vacuum at 50 °C for 15 min. Then the mixture
was dissolved in anhydrous THF (50 mL), cooled to 0 °C, and treated
with tBuMgCl (1 M in THF, 27.8 mL, 27.8 mmol). After addition it
was stirred at room temperature for 5 h until TLC indicated that the
reaction was complete. The reaction mixture was concentrated and
then purified through silica gel chromatography (MeOH/CH2Cl2 =
1:40) to afford intermediate 11 as light-yellow thick oil. A solution of
compound 11 in anhydrous THF (150 mL) was treated with TBAF (1
M in THF, 20.8 mL, 20.8 mmol) and stirred at room temperature for
4 h, until TLC indicated that the reaction was complete. The reaction
mixture was concentrated and then purified through silica gel
chromatography (MeOH/CH2Cl2 = 1:30) to afford product 1t (6.8
g, 91% over two steps) as colorless solid. Enantiopure compound 1t
was obtained as white solid through recrystallization in a mixture of
EtOH and H2O (1:2.5). Mp 107−108 °C; [α]2D0 = +14 (c = 0.2,
1
semisolid. Yield, 24%; H NMR (400 MHz, CD3OD): δ 7.85−7.83
(m, 1H), 7.37−7.32 (m, 1H), 7.24−7.15 (m, 3H), 6.25−6.20 (m, 1H),
5.13−5.07 (m, 2H), 4.99−4.92 (m, 1H), 4.42−4.35 (m, 1H), 4.24−
4.16 (m, 2H), 4.06−4.03 (m, 1H), 3.85−3.77 (m, 1H), 2.37−2.35 (m,
3H), 2.31−2.24 (m, 1H), 2.17−2.10 (m, 1H), 1.36−1.32 (m, 3H),
1.23−1.19 (m, 6H); HRMS (m/z) [M + Na]+ calcd for
C23H31FN3O9PNa 566.1674, found 566.1676.
5.1.3. Preparation of (SP)-O-Perfluorophenyl Phosphoramidate 7.
At−78 °C, to a solution of phosphoryl chloride (POCl3, 13.9 g, 0.091
mol) in anhydrous CH2Cl2 (150 mL) was added dropwise a solution
of 2-methylbenzyl alcohol (2b, 11.1 g, 0.091 mol) and triethylamine
(12.6 mL, 0.091 mol) in anhydrous CH2Cl2 (18 mL). After being
stirred at the same temperature for 3 h, the reaction mixture was
treated with L-alanine isopropyl ester hydrochloride (15.2 g, 0.091
mol) in one portion, followed 15 min later by the addition of a
solution of triethylamine (26.4 mL, 0.190 mol) in anhydrous CH2Cl2
(17 mL) dropwise. It was stirred at −78 °C for another 1 h and then
allowed to warm up to room temperature over 1 h. The above mixture
was further treated with a premixed solution of pentafluorophenol
(10.0 g, 0.054 mol) and triethylamine (15.1 mL, 0.109 mol) in
anhydrous CH2Cl2 (15 mL) and stirred overnight at room
temperature. The solid was collected by filtration, and the filtrate
was concentrated. The resulting residue and collected solid were
combined and partitioned between EtOAc (100 mL) and H2O (60
mL). The organic layer was separated, and the aqueous layer was
extracted with EtOAc (2 × 30 mL). The combined organic layer was
washed with brine (80 mL), dried over anhydrous Na2SO4, and then
concentrated. The given residue was treated with a mixture of
diisopropyl ether (60 mL) and petroleum ether (120 mL) and heated
to reflux. After the entire solid was dissolved, it was cooled and kept at
room temperature for 2 days. Pure product 7 (11.7 g, SP:RP > 10:1)
was obtained as white solid by filtration. The filtrate was concentrated
and purified through silica gel chromatography (EtOAc/PE = 1:7−
1:5) to afforded product 7 as a mixture of SP:RP isomers (∼1.3:1),
which was further recrystallized in a mixture of diisopropyl ether (30
mL) and petroleum ether (60 mL) to provide pure product 7 (4.1 g,
SP:RP > 10:1) as white solid. The overall yield was 60%.
1
CHCl3); H NMR (400 MHz, CD3OD): 7.82 (d, J = 6.4 Hz, 1H),
7.34 (d, J = 7.6 Hz, 1H), 7.23−7.13 (m, 3H), 6.22−6.18 (m, 1H), 5.10
(d, J = 7.6 Hz, 2H), 4.96 (heptet, J = 6.4 Hz, 1H), 4.35 (quint, J = 4.0
Hz, 1H), 4.21−4.1 (m, 2H), 4.03−4.00 (m, 1H), 3.83−3.75 (m, 1H),
2.35 (s, 3H), 2.29−2.23 (m, 1H), 2.15−2.08 (m, 1H), 1.31 (dd, J =
7.2, 0.8 Hz, 3H), 1.20 (dd, J = 6.4, 1.6 Hz, 6H); 13C NMR (100 MHz,
CD3OD) δ 173.3 (JC−P = 4.8 Hz), 157.9 (JC−F = 26.2 Hz), 149.2,
140.8 (JC−F = 232.3 Hz), 136.6, 134.1 (JC−P = 7.3 Hz), 129.9, 128.45,
128.38, 125.6, 124.3 (JC−F = 34.2 Hz), 85.4, 85.2 (JC−P = 7.8 Hz), 70.5,
68.7, 66.6 (JC−P = 5.1 Hz), 65.8 (JC−P = 5.4 Hz), 50.1 (JC−P = 1.2 Hz),
39.4, 20.54, 20.48, 19.1 (JC−P = 6.8 Hz), 17.5; IR (KBr) 3414, 2983,
1701, 1465, 1267, 1202, 1107, 1000, 894, 746; HRMS (m/z) [M +
Na]+ calcd for C23H31FN3O9PNa 566.1674, found 566.1672.
5.2. Animals. Female KM mice (20 2 g) and male Sprague−
Dawley rats (220
20 g) were purchased from Hunan SLAC
laboratory animal Co. LTD (Hunan, China). The animals were
maintained in a specific pathogen free (SPF) environment with a 12 h
light/dark cycle at 20−26 °C with a relative humidity of 40−70% and
received sterilized food and water freely available. All animals were
treated according to the procedures outlined in the Guide for the Care
and Use of Laboratory Animals (P. R. China), and experimental
procedures were approved by the Animal Ethics Committee of
Zhengzhou University.
5.3. Stability Assay in Plasma. 5.3.1. Stability of Prodrugs 1a−
r. Stability of prodrugs 1a−r in rat plasma was carried out by adding 4
μL of a 2.5 mM solution to 96 μL of rat plasma, which was incubated
at 37 °C. At the desired times (0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h), the
reaction was stopped by the addition of 400 μL MeOH and
centrifuged at 14000 rpm for 10 min at room temperature. Then 10
μL supernatant was analyzed by HPLC on a Thermo Fisher ACCELA
HPLC system equipped with a Phenomenex Luna C8 column (150
mm × 4.6 mm, 5 μm) and UV detection at 267 nm. The mobile phase
consisted of MeOH:H2O = 68:32 (v/v). The flow rate was set at 0.80
mL/min, and the column temperature was maintained at 30 °C.
5.1.4. Preparation of 3′-TBS Protected Nucleoside 10. At 0 °C, a
solution of compound 5 (10.0 g, 0.041 mol) in anhydrous pyridine
(100 mL) was treated with DMTrCl (27.5 g, 0.081 mol), stirred for 5
h until TLC indicated that the reaction was complete, and then
quenched with H2O (5 mL). The reaction mixture was concentrated,
treated with H2O (150 mL), and extracted with EtOAc (3 × 50 mL).
The combined organic layer was washed with brine (100 mL), dried
over anhydrous Na2SO4, and concentrated to afford intermediate 8 as
thick oil. A solution of compound 8 obtained above in anhydrous
G
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5.3.2. Stability of Prodrugs 1s−t. A stock solution of prodrug 1t
(or 1s) in MeOH was prepared at a concentration of 4 mg/mL, and a
25 μg/mL working solution was obtained by further dilution of the
stock solutions with H2O. 96 μL of rat (or dog, or human) plasma was
used for the test. The reaction was started by adding 4 μL of a 25 μg/
mL solution to give a final concentration of 1 μg/mL and incubated at
37 °C. At the desired times (0, 5, 10, 15, 20, and 25 min for rat plasma
and 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h for dog and human plasma), the
reaction was stopped by the addition of 400 μL ice-cold MeOH
containing internal standard and centrifuged at 14000 rpm for 10 min
at room temperature. Then 400 μL supernatant was evaporated to
dryness under a stream of N2. The residue was reconstituted with 100
μL H2O, briefly vortexed, and again centrifuged. The supernatant was
analyzed by HPLC-MS/MS: The HPLC was performed on a Thermo
Fisher ACCELA LC system equipped with a Phenomenex PFP (2)
column (100 mm × 2.0 mm, 3 μm). The mobile phase consisted of
linear gradients of A (0.5 mM NH4OAc solution) and B (MeOH): 0−
1 min, 95% A (v/v); 1−3 min, 95−2% A; 3−5 min, 2% A; 5−5.1 min,
2−95% A; 5.1−9 min, 95% A. The flow rate was set at 0.18 mL/min,
and the column temperature was maintained at 30 °C. Detection was
achieved with a Thermo Fisher TSQ QUANTUM ULTRA triple-
quadrupole mass spectrometer with an ESI interface in the negative-
ion mode. The parameter settings were as follows: spray voltage,
−3500 V; capillary temperature, 350 °C; sheath gas (N2), 30 arbitrary
units; auxiliary gas (N2), 8 arbitrary units; vaporizer temperature, room
temperature. Detection and quantification of 1t (or 1s) and metabolite
A were performed as described in Section 5.8. The concentrations of
compounds were determined for each time point, and the percentage
remaining was calculated on the basis of the initial amount measured
at 0 h. The half-life of each compound was calculated by using DAS 3.0
program (Mathematical Pharmacology Professional Committee of
China, Shanghai, China).
5.4. Activation in Rat Liver S9. After i.g. administration of
phenobarbital and β-naphthoflavone for 3 days, two Sprague−Dawley
rats were sacrificed by carbon dioxide inhalation, and the liver was
harvested, rinsed with cold saline, and homogenized in 50 mM Tris-
HCl, 150 mM KCl, pH 7.4. The homogenate was clarified by
centrifugation and analyzed for protein content (BCA Protein Assay
Kit). Reaction mixtures consisted of 100 mM KH2PO4, pH 7.4, 50 μM
tested compounds and 5 mg/mL liver S9. After preincubation at 37 °C
for 5 min, the reaction was initiated by adding 2 mM NADPH. The
blank and negative control samples were incubated without prodrug or
NADPH, respectively. At selected times (0, 10, 20, 40, and 60 min),
aliquots were removed and extracted by addition of 5 volumes of
MeOH containing internal standard. The extracts were clarified by
centrifugation and dried under a stream of N2. The residue was
suspended in H2O and analyzed for FdUMP (m/z 325 → 129) by
HPLC-MS/MS.
5.5. Cell Viability Assays. SMMC7721 or a normal liver cell line
(LO2) was cultured in RPMI-1640 medium (Gibco, Milano, Italy)
supplemented with 100 units/mL penicillin G (Gibco), 150 μg/mL
streptomycin (Invitrogen, Milano, Italy), and 10% fetal bovine serum
(Invitrogen). Prodrug stock solutions (350 mM) were obtained by
dissolving in DMSO. Individual wells of a 96-well culture plate were
inoculated with 100 μL of complete medium containing 4 × 103 cells.
The plates were incubated at 37 °C in a humidified 5% CO2 incubator
for 24 h prior to the experiments. After removal of the medium, 100
μL of fresh medium containing the test compound at different
concentrations was added to each well and incubated at 37 °C for 72
h. The percentage of DMSO in the medium never exceeded 0.5%. Cell
viability was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) test as previously described. The IC50
was defined as the compound concentration required to inhibit cell
proliferation by 50%, in comparison with cells treated with the
maximum amount of DMSO (0.5%) and considered as 100% viable.
5.6. Assessment of Acute Toxicity in KM Mice. After overnight
fasting, 10 KM mice were randomly divided into two groups and
dosed orally by gavage with a solution of prodrug 1t in 57%
hydroxypropyl-β-cyclodextrin at a single dose of 2 or 3 g/kg,
respectively. After administration, the mice were observed continu-
ously for the first 4 h for any abnormal behavior and mortality changes,
intermittently for the next 24 h, and occasionally thereafter for 15 days
for weight, appetite, and behavior. All mice were sacrificed by carbon
dioxide inhalation on day 15 after drug administration and were
macroscopically examined for possible damage to the heart, liver,
spleen, lung, and kidneys.
5.7. Assessment of Efficacy in KM Mice. The in vivo antitumor
activity of prodrug 1t was investigated using a syngeneic murine
hepatocellular carcinoma cell line H22 in KM mice. According to the
clinical oral dosage of fluorouracil (300 mg/d), the mouse equivalent
dose of fluorouracil was 60 mg/kg based on body surface area
(BSA),29,30 and the equimolar oral dosage of 1t was 250 mg/kg in
mice. Through further dosage optimization, three oral doses of 1t (60,
130, and 260 mg/kg) were employed. Similarly, the positive control
group was administrated with 60 mg/kg of sorafenib.25 Tumors were
induced by a subcutaneous injection in the axillary region of 2 × 106
cells in 200 μL of 0.9% saline. Fifty KM mice were randomly divided
into five groups, and starting on the second day, they were daily dosed
by oral gavage with 200 μL/10g free vehicle (35% polyethylene glycol
400), prodrug 1t (65, 130, and 260 mg/kg, dissolved in 35%
polyethylene glycol 400), or sorafenib (60 mg/kg, suspended in 0.5%
(w/v) CMC-Na) as the positive control. At the end of the study, the
mice were sacrificed by carbon dioxide inhalation with tumor weights
recorded.
5.8. Assessment of Tissue Distribution in Sprague−Dawley
Rats. The assay was contracted and carried out by Shanghai
ChemPartner (Shanghai) Co., Ltd. Dosing solutions of FDUR and
prodrugs 1b and 1t were prepared in H2O at a drug concentration of
4.72 mg/mL and in 20% hydroxypropyl-β-cyclodextrin at a drug
concentration of 9.97 and 10.43 mg/mL (FDUR equivalents),
respectively. Sprague−Dawley rats (n = 3 per time point) were
treated with 23.62 mg/kg (0.096 mmol/kg) of FDUR, 49.87 mg/kg
(0.096 mmol/kg) of 1b, and 52.17 mg/kg (0.096 mmol/kg) of 1t,
respectively, by gavage. At specified times (0.25, 0.5, 1, 2, 4, 6, 12, and
24 h), blood samples were collected in EDTA-K2 tubes via cardiac
puncture under anesthesia with isoflurane. After centrifugation at 5000
rpm for 5 min at 4 °C, plasma was pipetted and stored at −80 °C. Rats
were sacrificed by carbon dioxide inhalation, and the liver samples
were freeze-clamped and homogenized with 3 volumes (v/w) of
homogenizing solution (20 mM EDTA-K2 in 70% MeOH). After
centrifugation at 5000 rpm for 5 min at 4 °C, the supernatants were
stored at −80 °C until HPLC-MS/MS analysis. 50 μL of plasma and
50 μL of liver homogenate were deproteinated, respectively, by
addition of a solution of 0.1% formic acid in MeOH (200 μL)
containing internal standard, vortexing, and centrifugation (14000
rpm, room temperature, 5 min). 200 μL supernatant was evaporated to
dryness under a stream of N2. The residue was reconstituted with 100
μL H2O (containing 0.1% formic acid), briefly vortexed, and again
centrifuged. The supernatant was analyzed by HPLC-MS/MS: The
HPLC was performed on a Shimadzu LC system equipped with a
Phenomenex PFP column (50 mm × 4.6 mm, 3 μm). The mobile
phase consisted of linear gradients of solution A (0.1% formic acid in
H2O) and solution B (0.1% formic acid in MeOH): 0−0.4 min, 98% A
(v/v); 0.4−1.6 min, 98−2% A; 1.6−2.4 min, 2% A; 2.4−2.41 min, 2−
98% A; 2.41−3.5 min, 98% A. The flow rate was set at 0.8 mL/min,
and the column temperature was maintained at 40 °C. Detection was
achieved using an AB SCIEC API 4000 triple-quadrupole mass
spectrometer with an ESI interface in the negative-ion mode. The
parameter settings were as follows: spray voltage, −4500 V;
temperature 500 °C; curtain gas, 20 psi; collisionally activated
dissociation gas, 5 psi; gas 1, 50 psi; gas 2, 60 psi. Detection and
quantification of the compounds were performed in the selected-
reaction monitoring mode with the transitions of m/z 542 → 314 for
1t, 129 → 42 for 5-FU, 500 → 245 for metabolite A, 245 → 129 for
FDUR, 325 → 129 for FdUMP, and 145 → 42 for 5-chlorouracil (5-
CU, internal standard), respectively.
H
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Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01807.
■
S
Characterization data and NMR spectra of prodrugs 1a−
t (PDF)
X-ray structure and data of prodrug 1t (CIF)
Compound data (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: + 86 (0)371-6778-1588. E-mail: changjunbiao@zzu.
edu.cn.
■
Author Contributions
#These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We acknowledge the National Natural Science Foundation of
China (nos. 81330075 and 81302637) for financial support.
■
ABBREVIATIONS USED
■
HCC, hepatocellular carcinoma; FDUR, 5-fluoro-2′-deoxyur-
idine; FdUMP, 2′-deoxy-5′- fluorouridine-5′-monophosphate;
HAI, hepatic arterial infusion; NMI, N-methylimidazole;
DMTr, 4,4′-dimethoxytrityl; TBS, t-butyldimethylsilyl; TFA,
trifluoroacetic acid; t1/2, elimination half-life; IC50, 50%
inhibitory concentration; CC50, 50% cytotoxic concentration;
SD, standard deviation; 5-FU, 5-fluorouracil; Cmax, maximum
plasma/liver concentration; AUC(inf), area under the curve from
time zero to infinity; ND, not detected; Hint1, histidine triad
nucleotide-binding protein 1; i.g., intragastric gavage; NADPH,
nicotinamide adenine dinucleotide phosphate; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SPF,
specific pathogen free; BSA, body surface area; CMC-Na,
sodium carboxyl methyl cellulose; 5-CU, 5-chlorouracil
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