Synthesis and characterization of a novel liver-targeted prodrug of cytosine-1-β-D-arabinofuranoside monophosphate for the treatment of hepatocellular carcinoma

Article abstract of DOI:10.1021/jm0607449

Cytotoxic nucleosides have proven to be ineffective for the treatment of hepatocellular carcinoma (HCC) due, in part, to their inadequate conversion to their active nucleoside triphosphates (NTP) in the liver tumor and high conversion in other tissues. Th

Full text of DOI:10.1021/jm0607449

J. Med. Chem. 2006, 49, 7711-7720
7711
Synthesis and Characterization of a Novel Liver-Targeted Prodrug of
Cytosine-1-â-D-arabinofuranoside Monophosphate for the Treatment of Hepatocellular
Carcinoma
Serge H. Boyer,* Zhili Sun, Hongjian Jiang, Julie Esterbrook, Jorge E. Go´mez-Galeno, William Craigo, K. Raja Reddy,
Bheemarao G. Ugarkar, Deidre A. MacKenna, and Mark D. Erion
Departments of Medicinal Chemistry and Biosciences, Metabasis Therapeutics, Inc., 11119 North Torrey Pines Road,
La Jolla, California 92037
ReceiVed June 22, 2006
Cytotoxic nucleosides have proven to be ineffective for the treatment of hepatocellular carcinoma (HCC)
due, in part, to their inadequate conversion to their active nucleoside triphosphates (NTP) in the liver tumor
and high conversion in other tissues. These characteristics lead to poor efficacy, high toxicity, and a drug
class associated with an unacceptable therapeutic index. Cyclic 1-aryl-1,3-propanyl phosphate prodrugs
selectively release the monophosphate of a nucleoside (NMP) into CYP3A4-expressing cells, such as
hepatocytes, while leaving the prodrug intact in plasma and extrahepatic tissues. This prodrug strategy was
applied to the monophosphate of the well-known cytotoxic nucleoside cytosine-1-â-D-arabinofuranoside
(cytarabine, araC). Compound 19S (MB07133), in mice, achieves good liver targeting compared to araC,
generating >19-fold higher cytarabine triphosphate (araCTP) levels in the liver than levels of araC in the
plasma and >12-fold higher araCTP levels in the liver than in the bone marrow, representing a >120-fold
and >28-fold improvement, respectively, over araC administration.
Chart 1
Introduction
Hepatocellular carcinoma (HCC)a remains a poorly treated
cancer and, as the fifth most lethal malignancy worldwide,
afflicts >500,000 people each year with survival rates of only
23% and <5% at 1 and 5 years, respectively.1 One reason for
the high mortality rate is that treatment options are limited with
no chemotherapeutic regimen currently approved for the treat-
ment of unresectable HCC, which represents >80% of cases.
The only truly successful therapy is liver transplantation, which
is limited due to the paucity of available organs. The difficulties
associated with the chemotherapeutic treatment of HCC may
stem from the presence of a multitude of drug resistance
mechanisms that exist in the liver and correspondingly the
primary liver tumor. These include mechanisms for rapid drug
metabolism, including both primary and secondary metabolism
pathways. Moreover, a vast array of xenobiotics and drug
transport systems exist in the liver which are well-known to
transport certain drugs out of the tumor (e.g., p-glycoprotein).
Last, there exists a high concentration of agents (e.g., gluta-
thione) that inactivate certain oncolytic drugs. These properties
limit the uptake and retention of oncolytic drugs in the primary
liver tumor and therefore result in the need for very high doses
to achieve an antitumor response, which then leads to extra-
hepatic toxicity.
kinases in hepatocytes, whether normal or tumorigenic, which
leads to poor conversion of oncolytic nucleosides to biologically
active nucleoside triphosphates (NTPs). Accordingly, bypassing
the nucleoside kinase may result in high intracellular NTP levels,
since NTPs are usually not subject to metabolism (other than
dephosphorylation) or export from cells.
Cytosine-1-â-D-arabinofuranoside (cytarabine, araC, Chart 1)
is a nucleoside oncolytic currently used clinically for the
treatment of acute myelocytic leukemia (AML). Like most
nucleosides, araC is inactive and requires phosphorylation to
its triphosphate, araCTP, to exert its antineoplastic activity by
inhibiting DNA polymerase2 or by incorporation into elongating
DNA strands3 in a cell cycle dependent manner. However, the
use of araC is not suitable to treat other types of cancers, as it
is inactive against solid tumors due to poor phosphorylation
and conversion into its triphosphate, araCTP.4 In addition, any
attempts to reach therapeutic levels of araCTP in tissues with
low kinase activity, such as the liver, by raising the dose are
thwarted by dose-limiting myelosuppresion, which arises from
rapid phosphorylation and conversion into araCTP in the
otherwise healthy bone marrow cells.
One class of drugs that typically is ineffective against solid
tumors and, more specifically, HCC is the nucleoside oncolytic
drug class. The reason for their inability to inhibit cell
proliferation may stem from the low levels of specific nucleoside
* To whom correspondence should be addressed. Telephone: 858 622
5576. Fax: 858 622 5573. E-mail: boyer@mbasis.com.
a Abbreviations: HCC, hepatocellular carcinoma; cytarabine, araC,
cytosine-1-â-D-arabinofuranoside; araCMP, cytosine-1-â-D-arabinofurano-
side monophosphate; araCTP, cytosine-1-â-D-arabinofuranoside triphos-
phate; CYP3A4, cytochrome P450-3A4; adefovir, 9-(2-phosphonylmethoxy-
ethyl)adenine; pradefovir, 9H-purin-6-amine-9-[2-[[(2R,4S)-4-(3-chlorophenyl)-
2-oxido-1,3,2-dioxaphosphorinan-2-yl]methoxy]ethyl]; HBV, hepatitis B
virus.
Recently we have reported our findings on a new class of
prodrugs (1, Figure 1), which have proven capable of selectively
delivering nucleoside monophosphates (NMPs) to the liver.5,6
These prodrugs are activated to their NMPs 4, via hydroxylation
at the benzylic carbon by the cytochrome P450 isozyme-3A4
10.1021/jm0607449 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2006

7712 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Boyer et al.
Figure 1. Activation mechanism of cyclic 1-(aryl)-1,3-propanyl prodrugs.
Scheme 1a
a Reagents: (a) LDA, EtOAc, THF, -78 °C; (b) LiAlH4, ether, 0 °C or NaBH4, EtOH, reflux, 24-83% for 2 steps; (c) (i) Cl2P(O)O(4-NO2-Ph), Et3N,
THF, rt; (ii) NaO(4-NO2-Ph), rt, 23-92%; (d) t-BuMgCl, 2′,3′-di-O-TBS-araC (9),5 THF, rt; (e) Et4NF, THF or TBAF, 13-59% for 2 steps; (f) Pd(PPh3)4,
CO, MeOH, Et3N, sealed bomb, 85 °C, 66-87%.
(CYP3A4) and subsequent elimination of the â-nucleoside-
monophosphate. In this report, the cyclic 1-(3-chlorophenyl)-
1,3-propanyl prodrug (10f) of cytarabine monophosphate
(araCMP) was shown to generate more than 80-fold more
araCTP in rat hepatocytes compared to araC.5 As the successful
application of this prodrug strategy to araC for the treatment of
HCC is highly dependent on the presence of CYP3A4 in tumor
cells, there are numerous reports showing that CYP3A4 is
retained in primary HCC tumors.7 Moreover, delivery of araCTP
to the normal, healthy liver, as well as the diseased liver, is not
expected to be cytotoxic because normal hepatocytes proliferate
at a much slower rate than liver tumor cells and araCTP exerts
its efficacy during cell replication.
Intracellular araCTP is relatively short-lived. Consequently,
cytarabine is administered to leukemic patients by continuous
intravenous (i.v.) infusion, which ensures the presence of araCTP
within individual cells during the time that they enter into the
proliferative phase.8 Similarly, we sought an i.v. formulation
of our liver-targeted prodrug of araCMP and therefore set out
to identify a prodrug with good aqueous solubility and stability
suitable for parenteral administration. In this report we describe
the identification of the liver-targeted prodrug of araCMP, 19S
(MB07133), with good aqueous solution characteristics, high
production of araCTP in the liver, and reduced extrahepatic
exposure.
epimerization of the cis-phosphate to the thermodynamically
more stable trans-phosphate10 with sodium-nitrophenoxide
afforded trans-phosphate reagents 8b-l in 23-92% yield.
Phosphorylation of the 5′-magnesium-alkoxide of protected
cytarabine 95 with trans-phosphate reagents 8b-l, followed by
fluoride or acid deprotection, stereospecifically gave cis-
prodrugs 10b-l as a mixture of diastereomers at the C4-carbon
of the dioxaphosphorinane ring and with minimal epimerization
at the phosphorus center (<5%) in 13-59% overall yield.
Prodrug 10i was selected as the lead for further testing.
However, before proceeding toward the evaluation of single
stereoisomers, a more efficient synthesis was needed. While
prodrug formation with the other phosphorylating reagents was
accomplished in good yields, phosphorylation with the 4-pyridyl
phosphate reagent 8i was slow and yields decreased significantly
as the scale increased. Addition of excess phosphorylating
reagent and further increasing the temperature of the reaction
further decreased the yield due to the formation of diphos-
phorylated cytarabine nucleoside 12 (Scheme 2). The phos-
phoramidate moiety could be selectively cleaved with 70%
aqueous trifluoroacetic acid (TFA) at 60 °C, with concomitant
removal of the silyl groups, to produce the desired prodrug 10i
in 50% yield, but this process was judged unsatisfactory. A
significant improvement was made by protecting the 4-amino
group as a dimethylformamidine (DMF dimethylacetal, 99%),
which led to the smooth formation of prodrug 10i, after removal
of all the protecting groups with TFA, in 50-65% overall yield.
Results
Chemistry. Cyclic cis-1-(aryl)-1,3-propanyl prodrugs of
cytarabine 10b-l (Scheme 1) were stereospecifically prepared
by an SN2-like reaction between 2′,3′-di-O-tert-butyldimethyl-
silyl-cytarabine (9, 2′,3′-di-O-TBS-araC) and the corresponding
trans-(4-nitrophenyl)-phosphate reagents 8b-l as previously
described.5,9 1-Aryl-1,3-propane diols 7a-j were synthesized
as racemic mixtures via the two-step sequence aldol condensa-
tion/reduction in 24-83% overall yield. Palladium-catalyzed
carbonylation of bromophenyl diols 7a,d gave the corresponding
methyl benzoate diols 7k,l in 66-87% yield. Reaction of diols
7b-l with 4-nitrophenyl phosphorodichloridate followed by
Synthesis of both stereoisomers of prodrug 10i was ac-
complished by first resolving racemic diol 7i. While high optical
purity was obtained in the resolution of diol 7f with (-)-
menthone,5 chromatographic separation of acetals of diol 7i
proved to be very difficult. Alternatively, esterification of
racemic â-hydroxy ester 14 with N,N-dimethyl-phenylalanine
(15, Scheme 3) led to an easier separation of both diastereomers
in high optical purities. Reduction of phenylalanates 16R and
16S gave desired diols 17R and 17S, respectively. Stereo-
chemical assignments were initially made on the basis of the
comparison of the sign of the optical rotation of 17S with the

LiVer-Targeted Prodrug of AraCMP for HCC
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7713
Scheme 2a
a Reagents: (a) t-BuMgCl, THF, rt, 11 (13%), 12 (20%); from 13 (70-80%); (b) DMF-dimethyl acetal, pyridine, rt, 99%; (c) Et4NF, THF, 40%; (d) 70%
aq TFA, 60 °C, 80%.
Scheme 3a
a Reagents: (a) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide‚HCl (EDCI), 4-dimethylaminopyridine (DMAP), CH2Cl2, 16R (77%), 16S (83%); (b)
LiAlH4, ether, 17R (33%), 17S (66%); (c) (i) Cl2P(O)O(4-NO2-Ph), pyridine, THF, rt; (ii) NaO(4-NO2-Ph), rt, 18R (60%), 18S (63%); (d) t-BuMgCl, 13,
THF, rt; (e) 70% aq TFA, 60 °C, 19R (53%), 19S (65%) for 2 steps.
one of commercially available (-)-(S)-1-phenyl-1,3-propanediol.
While formation of phosphorylating reagents 18R and 18S using
the previously described reaction conditions led to a significant
loss of optical purity at the carbon stereocenter, high optical
purity was achieved by substituting pyridine for triethylamine.
Phosphorylation of protected cytarabine 13 with phosphate 18R
or 18S followed by TFA deprotection produced cis-prodrugs
19R and 19S in high optical purities. An unambiguous stereo-
chemical assignment was made following single-crystal X-ray
structure determination of compound 19S. An ORTEP diagram
is shown in Figure 2. The absolute stereochemistry of the
benzylic carbon on the prodrug moiety was established as S, as
initially assigned. Most importantly, the X-ray crystal structure
clearly establishes the relative stereochemistry between the
pyridyl ring and the nucleoside as cis.
Biological Results. Cytarabine and prodrugs of cytarabine
were evaluated in freshly isolated suspended rat hepatocytes
over 4 h for production of araCTP as previously described.5
Cytarabine produced very little araCTP while prodrugs 10b-l
produced substantially higher levels of araCTP with relative
amounts depending on the aryl substitution (Table 1). However,
no correlation was observed between the electronic effects of
the substituents and the amounts of araCTP produced.
The aqueous stability was clearly related to the electronic
effects of the substituents. As the electron density of the aryl
group decreased, the aqueous stability increased. This effect was

7714 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Boyer et al.
Figure 2. ORTEP Drawing of compound 19S. Atoms are represented by 50% probability anisotropic thermal ellipsoids.
Table 1. Generation of AraCTP in Freshly Isolated Rat Hepatocytes and Aqueous Prodrug Stability and Solubility
hepatocyte activation
stability, % remainingc
at 24 h at 72 h
water solubility (mg/mL)
cmpd
aryla
AUC0-4h araCTP (nmol‚h/g) b
pH 4e
pH 7.4f
araC
10b
10c
10d
10e
10fd
10g
10h
10i
10j
10k
10l
19R
19S
5
44
44
159
197
398
434
558
95
84
170
33
86
99
3-Me-Ph
4-F-Ph
4-Br-Ph
4-Cl-Ph
9
5
63
74
82
92
99
60
84
4
4
21
52
76
90
83
23
61
3-Cl-Ph
3.7
6.7
1.1
1.7
3.2
0.5
3-Cl-4-F-Ph
3,5-di-Cl-Ph
4-pyridyl
>200
172
10.3
>200
176
7.2
3-pyridyl
3-Ph-COOMe
4-Ph-COOMe
(R)-4-pyridyl
(S)-4-pyridyl
7.4
37.9
23.9
5.7
16.7
7.1
>99
>99
>97
>95
a Diols 10b-l are racemic. b Activation of 100 µM araCMP prodrugs or araC to araCTP in primary rat hepatocytes: area under the curve (AUC) 0-4
h. c Stability of a 100 µM solution of araCMP prodrugs in aqueous buffer at pH 7.4 at 37 °C. d See ref 5. e 100 mM citrate buffer pH 4. f 100 mM phosphate
buffer pH 7.4.
Table 2. Comparative Tissue Distribution in Mice after i.p. Administration of 100 mg/kg araC Equivalents
liver araCTP
AUC0-4h (nmol/g‚h)
plasma araC
AUC0-4h (µM‚h)
bone marrow
cmpd
peak (nmol/g)
peak (µM)
peak (µM)
AUC0-4h araCTP (nmol/g)c
19R
19S
27
70
7.6
68.0
148.8
1.6
3
150
3.5
7.6
121
<3b
<3b
19
<12b
<12b
46
araCa
<19.2b
a See ref 6. b Lower limit of quantitation (LOQ). c Area under the curve (AUC) 0-4 h.
further compounded by the position of the substituents on the
ring. Compounds with electron-donating groups at the meta-
position (e.g., 10f, 63% prodrug remaining at 24 h) had greater
stability than prodrugs with similar substitutions at the para-
position (e.g., 10e, 5% at 24 h), while compounds with electron-
withdrawing groups at the para-position had improved stability
compared to that of prodrugs with similar substitutions at the
meta-position (e.g., 10l, 84% at 24 h vs 10k, 60%).
The aqueous solubility of relatively stable prodrugs was low
(Table 1), and lowering the pH of the buffer solution to 4, to
take advantage of the basic character of the cytosine base of
cytarabine, showed only a small improvement. Addition of
another chloro substituent to prodrug 10f further decreased the
solubility (10h) while addition of a fluoro substituent increased
the solubility (10f vs 10g). The pyridyl prodrugs 10i and 10j,
on the other hand, showed very high solubility at both pH 4
and pH 7.4, presumably due to their greater hydrophilicity.
Interestingly, the solubilities of the individual isomers 19R and
19S were significantly reduced relative to that observed with
10i. This is not unduly surprising, since 10i is a mixture of
diastereomers (and likely amorphous), whereas 19R and 19S
are isomerically pure (and likely crystalline). Most importantly,
both 19R and 19S display solubility sufficient for intravenous
administration.
Both diastereomeric cis-prodrugs 19R and 19S were tested
in ViVo in mice to compare their tissue distribution and assess
their liver targeting indices relative to those of cytarabine.
Intraperitoneal (i.p.) administration of 100 mg/kg araC equiva-
lents of prodrugs 19R and 19S, respectively, produced 68 and
149 nmol‚h/g of araCTP in the liver, over 4 h, while administra-
tion of araC produced only 19.2 nmol‚h/g6 (Figure 3; Table 2).
Plasma araC exposure was 3.5 µM‚h for 19R, 7.6 µM‚h for
19S, and 121 µM‚h over 4 h following araC administration.6
AraCTP levels in the bone marrow, the primary organ of araC
toxicity, were below the limit of quantitation (<3 nmol‚h/g) at
all time points following prodrug administration and 46 nmol‚
h/g for araC.6

LiVer-Targeted Prodrug of AraCMP for HCC
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7715
Table 3. Liver Targeting Indexes
AUC liver araCTP
AUC plasma araC
AUC liver araCTP
cmpd
AUC bone marrow araCTP
araC
19R
19S
<0.16a
19.4
19.6
<0.42a
>7a
>12a
a
Ratio estimated on the basis of the LOQ.
and primary liver tumor while simultaneously limiting araC
exposure to the bone marrow and gut epithelial cells.
Efficient cleavage of cyclic 1-(aryl)-1,3-propanyl prodrugs
of cytarabine was observed in freshly isolated rat hepatocytes.
No correlation existed between the different rates of activation
and the electronic properties of the activating aryl group. In
contrast, a good correlation was observed between the electronic
properties and the stability of the prodrug in aqueous media.
Prodrugs bearing electron-withdrawing groups were more stable
than prodrugs with electron-donating substituents. Furthermore,
moving the electron-donating substituent from the para-position
to the meta-position further improved the stability while the
opposite effect was observed with electron-withdrawing groups.
This is consistent with the SN1 solvolysis of the benzylic
phosphate bond being the primary source of prodrug instability
in aqueous medium.13 Aqueous solubility was poor for all
substituted phenyl prodrugs. However, the pyridyl prodrugs 10i
and 10j showed very good solubility, making them suitable for
parenteral administration. On the basis of the superior stability
of prodrug 10i compared with 10j, the diastereomeric mixture
of 4-pyridyl prodrugs 10i was selected for further characteriza-
tion of its individual diastereomers.
Contrary to our previous experience with other prodrug single
isomers of araC,5 the two diastereomeric prodrugs 19R and 19S
showed no major differences in their hepatocyte activation,
aqueous stability, and aqueous solubility. However, a distinct
advantage was observed for the S-isomer in liver araCTP levels
in ViVo. Liver araCTP exposure after administration of prodrug
19S was twice the exposure generated by prodrug 19R. Both
prodrugs generated small amounts of araC in the plasma, likely
due to dephosphorylation of araCMP in the liver and leakage
into the bloodstream. No detectable amount of araCTP was
observed in the bone marrow upon administration of either
prodrug while large amounts were found upon administration
of araC.6 Comparison of the liver-targeting indices, relative to
historical araC levels,6 clearly demonstrated the in ViVo advan-
tage of both prodrugs compared with araC in terms of both
hepatic araCTP production and reduction of extrahepatic
exposure to araC (Table 3).
Figure 3. Tissue distribution studies in mice for compounds 19R and
19S. (A) AraCTP levels were measured in liver, (B) araC and (C)
prodrug levels were measured in plasma at times up to 4 h after i.p.
administration of 100 mg/kg of araC equivalents.
Due to the higher levels of araCTP produced in the liver,
prodrug 19S was selected over prodrug 19R as a lead compound
for further evaluation of its potential as a parentally administered
nucleoside oncolytic prodrug for the treatment of HCC.
Discussion
Conclusions
Cyclic 1-(aryl)-1,3-propanyl prodrugs of NMPs are used to
increase NTP levels in the liver while decreasing NTP levels
in extrahepatic tissues. Liver targeting has been demonstrated
with a variety of structurally different compounds, including
both nucleotide and nucleoside analogues. Pradefovir, a prodrug
of the antihepatitis B virus (HBV) drug adefovir, is currently
in Phase 2 clinical trials. Pradefovir showed excellent liver
targeting in rats and monkeys11 and, most recently, in humans12
with chronic HBV infections. In this work, we have applied
this prodrug strategy to araCMP in order to bypass the
nucleoside kinase that is expressed at low levels in the liver
A series of cyclic 1-(aryl)-1,3-propanyl prodrugs of cytarabine
was synthesized and optimized for activation in freshly isolated,
suspended rat hepatocytes and for aqueous stability and solubil-
ity. Both cis-isomeric prodrugs contained in the diastereomeric
mixture of cis-prodrugs 10i were synthesized in high optical
purity and characterized in ViVo for their ability to produce
araCTP in the liver and minimize extrahepatic exposure to araC.
Prodrug 19S generated hepatic araCTP levels >12-fold higher
than the araCTP levels generated in the bone marrow and >19-
fold higher than the levels of araC in the plasma, representing
a >28-fold and >120-fold improvement, respectively, over araC

7716 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Boyer et al.
(c) 1-(4-Fluorophenyl)-1,3-propanediol (7c). LiAlH4 reduction
gave an oil (5.28 g, 77% for 2 steps): 1H NMR (300 MHz, CDCl3)
δ 7.40-7.34 (m, 2H), 7.07 (t, J ) 8.9 Hz, 2H), 4.98 (dd, J ) 8.4,
4.2 Hz, 1H), 3.89 (t, J ) 5.9 Hz, 2H), 2.10-1.85 (m, 2H).
(d) 1-(4-Bromophenyl)-1,3-propanediol (7d). LiAlH4 reduction
gave an oil (3.3 g, 24% for 2 steps): 1H NMR (200 MHz, CDCl3)
δ 7.55 (m, 2H), 7.3 (m, 2H), 4.99 (m, 1H), 3.85 (m, 2H), 2.64 (bs,
2H, exchangeable with D2O), 1.98 (m, 2H).
(e) 1-(4-Chlorophenyl)-1,3-propanediol (7e). LiAlH4 reduction
gave an oil: 1H NMR (200 MHz, CDCl3) δ 7.40-7.15 (m, 4H),
4.95-4.85 (dd, J ) 6.8, 5.0 Hz, 1H), 3.82 (t, J ) 5.0 Hz, 2H),
2.32 (s, 3H), 2.00-1.75 (m, 2H).
(f) 1-(3-Chlorophenyl)-1,3-propanediol (7f). LiAlH4 reduction
gave an oil (42.07 g, 63% for 2 steps): 1H NMR (200 MHz, CDCl3)
δ 7.18-7.40 (m, 4H), 5.00-4.90 (dd, J ) 6.8, 6.1 Hz, 1H), 3.85
(t, J ) 5.8 Hz, 2H), 2.40 (s, 2H), 2.00-1.90 (m, 2H).
(g) 1-(3-Chloro-4-fluorophenyl)-1,3-propanediol (7g). NaBH4
reduction gave an oil (18 g, 55% for 2 steps): 1H NMR (300 MHz,
DMSO-d6) δ 7.53-7.48 (m, 1H), 7.40-7.35 (m, 3H), 5.33 (d, J )
4.8 Hz, 1H exchangeable with D2O), 4.75-4.65 (m, 1H), 4.46 (t,
J ) 5.1 Hz, 1H exchangeable with D2O), 3.60-3.35 (m, 2H), 1.83-
1.65 (m, 2H).
administration, and was selected for further characterization as
a potential drug candidate for the treatment of HCC.
Experimental Section
General Information. Glassware for moisture sensitive reactions
was flame dried and cooled to room temperature in a desiccator,
and all reactions were carried out under an atmosphere of nitrogen.
Anhydrous solvents were purchased from Aldrich or Acros. Thin
layer chromatography was performed on EM Science silica gel 60
F254 plates and was visualized with a UV lamp (254 nm) or cerium
stain. Column chromatography was performed on 230-400 mesh
EM Science Silica gel 60. Melting points were recorded on a
Thomas Hoover capillary melting point apparatus and are uncor-
1
rected. H and 13C NMR were obtained on a Varian Gemini-200
operating at 200 and 50 MHz, respectively, or a Varian Mercury-
300 operating at 300 and 75 MHz, respectively. 1H and 13C NMR
spectra were recorded in δ units using the solvent’s chemical shift
as the reference line. C, H, N microanalyses were performed by
Robertson Microlit Laboratories, Inc., Madison, NJ, or NuMega
Resonance Labs, Inc., San Diego, CA. Single-crystal structure
analysis for compound 19S was performed by SSCI Inc., West
Lafayette, IN. All protocols involving animal experimentation were
reviewed and approved by the Metabasis Therapeutics IACUC
(Institution Animal Care and Use Committee) and follow the
guidelines established by the NRC “Guide for the Care and Use of
Laboratory Animals”.
General Procedure for Preparation of 1-Aryl Substituted
Propane-1,3-diols 7a-j. (A) Step A. To a solution of diisopropyl-
amine (2 mmol) in THF (0.7 mL/mmol diisopropylamine) at -78
°C was slowly added a solution of n-BuLi (2 mmol, 2.5 M in
hexanes). The reaction was then stirred for 15 min at -78 °C before
a solution of ethyl acetate (2 mmol) in THF (0.14 mL/mmol ethyl
acetate) was slowly introduced. After stirring an additional 30 min
at -78 °C, a THF solution containing the aryl aldehyde (1.0 mmol
in 0.28 mL of THF) was added. The reaction was then stirred at
-78 °C for 30 min, warmed to room temperature, and stirred an
additional 2 h. After aqueous workup (0.5 M HCl), the organic
layer was concentrated to a crude oil and purified by either
chromatography or distillation.
(h) 1-(3,5-Dichlorophenyl)-1,3-propanediol (7h). NaBH4 re-
duction gave an oil (47 g, 83% for 2 steps): bp 135-137 °C/0.75
1
mmHg; H NMR (300 MHz, DMSO-d6) δ 7.43 (s, 1H), 7.32 (s,
2H), 5.40 (d, J ) 4.5 Hz, 1H exchangeable with D2O), 4.75-4.60
(m, 1H), 4.44 (t, J ) 5.1 Hz, 1H exchangeable with D2O), 3.55-
3.31 (m, 2H), 1.77-1.60 (m, 2H).
(i) 1-(Pyridin-4-yl)-1,3-propanediol (7i). LiAlH4 reduction gave
1
a tan solid (45 g, 61%): mp 98-100 °C; H NMR (300 MHz,
DMSO-d6) δ 8.47 (d, J ) 6.3 Hz, 2H), 7.30 (d, J ) 6.3 Hz, 2H),
5.37 (d, J ) 4.5 Hz, 1H exchangeable with D2O), 4.72-4.62 (m,
1H), 4.48 (t, J ) 5.1 Hz, 1H exchangeable with D2O), 3.60-3.38
(m, 2H), 1.75-1.65 (m, 2H).
(j) 1-(Pyridin-3-yl)-1,3-propanediol (7j). NaBH4 reduction gave
an oil (11 g, 77% for 2 steps): 1H NMR (300 MHz, DMSO-d6) δ
8.49 (d, J ) 2.1 Hz, 1H), 8.25-8.20 (m, 1H), 7.72-7.65 (m, 1H),
7.45-7.30 (m, 1H), 5.28 (d, J ) 4.5 Hz, 1H exchangeable with
D2O), 4.75-4.65 (m, 1H), 4.45 (t, J ) 5.1 Hz, 1H exchangeable
with D2O), 3.55-3.33 (m, 2H), 1.85-1.62 (m, 2H).
(k) 1-(3-Methoxycarbonylphenyl)-1,3-propanediol (7k). A
pressure vessel was charged with 1-(3-bromophenyl)-1,3-propane
diol (7a, 2 g, 8.6 mmol), methanol (30 mL), triethylamine (5 mL),
and bis(triphenylphosphine)palladium dichloride (0.36 g, 0.5 mmol).
The sealed vessel was pressurize with carbon monoxide at 55 psi
and heated at 85 °C for 24 h. The cooled vessel was opened, and
the reaction mixture was filtered through Celite and rinsed with
methanol. The combined filtrates were concentrated under reduced
pressure, and the residue was purified by column chromatography
(silica gel, hexanes/ethyl acetate 1/1) to provide diol 7k (1.2 g,
66%): 1H NMR (200 MHz, CDCl3) δ 8.02 (s, 1H), 7.95 (d, J )
7.6 Hz, 1H), 7.70-7.35 (m, 2H), 5.07-4.97 (m, 1H), 3.91 (s, 3H),
3.90-3.80 (m, 2H), 2.05-1.90 (m, 2H).
(l) 1-(4-Methoxycarbonylphenyl)-1,3-propanediol (7l). Pal-
ladium catalyzed carbonylation of diol 7d using the procedure
described for the synthesis of diol 7k gave diol 7l (1.58 g, 87%):
1H NMR (200 MHz, CDCl3) δ 8.02 (dd, J ) 8.4, 1.8 Hz, 2H),
7.44 (d, J ) 8.0 Hz, 2H), 5.05 (t, J ) 6.3 Hz, 1H), 3.91 (s, 3H),
3.90-3.80 (m, 2H), 2.05-1.90 (m, 2H).
General Procedure for the Synthesis of trans-4-(Aryl)-2-(4-
nitrophenoxy)-2-oxido-1,3,2-dioxaphosphorinanes. A solution of
1-aryl-1,3-propane diol (7b-l, 2.5 g, 13.4 mmol) and triethylamine
(6.25 mL, 44.2 mmol) in THF was added to a solution of
4-nitrophenyl-phosphorodichloridate (3.77 g, 14.7 mmol) in THF
at room temperature, and the resulting solution was heated at reflux.
After 2 h, TLC indicated complete consumption of the starting diol
and formation of the cis and trans isomers in a 50/50 to 60/40
ratio (HPLC). The clear yellow solution was cooled to 30 °C,
sodium 4-nitrophenoxide (5.6 g, 40.2 mmol) was added, and the
reaction mixture was heated at reflux. After 90 min the reddish
reaction mixture was cooled to room temperature, quenched with
(B) Step B: Reduction of â-Hydroxy Esters. The reduction
of the â-hydroxy esters was achieved using either LiAlH4 or NaBH4.
(1) Using LiAlH4. A solution of hydroxyester (1 mmol) in ether
(1 M) was added to a suspension of LiAlH4 (3 mmol) in ether (1
M) at 0 °C. The reaction was stirred, allowing the cooling bath to
melt and the reaction to reach room temperature. Once the reaction
was complete (TLC), the reaction mixture was cooled back to 0
°C and quenched with ethyl acetate. Aqueous workup (0.5 M HCl)
afforded the crude diol, which was purified by either chromatog-
raphy or distillation.
(2) Using NaBH4. NaBH4 was added portionwise to a solution
of hydroxy ester in EtOH at room temperature. After 20 min the
heterogeneous white reaction mixture was heated to reflux until
all starting material was consumed (3-5 h, TLC). The cooled
mixture was concentrated under reduced pressure and partitioned
between water and EtOAc. The layers were separated, and the
aqueous layer was back-extracted with EtOAc. The combined
extracts were dried (Na2SO4), concentrated under reduced pressure,
and purified by chromatography on silica gel to give the corre-
sponding diol.
(a) 1-(3-Bromophenyl)-1,3-propanediol (7a). NaBH4 reduction
gave an oil (45.1 g, 72% for 2 steps): 1H NMR (300 MHz, DMSO-
d6) δ 7.53 (s, 1H), 7.45-7.40 (m, 1H), 7.35-7.25 (m, 2H), 5.30
(d, J ) 4.5 Hz, 1H exchangeable with D2O), 4.73-4.63 (m, 1H),
4.47 (t, J ) 5.1 Hz, 1H exchangeable with D2O), 3.60-3.35 (m,
2H), 1.85-1.65 (m, 2H).
(b) 1-(3-Methylphenyl)-1,3-propanediol (7b). LiAlH4 reduction
gave an oil: 1H NMR (200 MHz, CDCl3) δ 7.30-7.02 (m, 4H),
4.95 (dd, J ) 6.0, 4.0 Hz, 1H), 3.85 (t, J ) 6.0 Hz, 2H), 2.32 (s,
3H), 2.10-1.85 (m, 2H).

LiVer-Targeted Prodrug of AraCMP for HCC
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7717
a saturated solution of NH4Cl, and diluted with ethyl acetate. The
layers were separated, and the organics were washed four times
with a 0.3 N solution of sodium hydroxide, followed by a brine
solution, dried (Na2SO4), and concentrated under reduced pressure.
The residue was purified by column chromatography.
(a) trans-4-(3-Methylphenyl)-2-(4-nitrophenoxy)-2-oxido-1,3,2-
dioxaphosphorinane (8b). Isomerization was conducted with
4-nitrophenol (3 equiv) and DBU (4 equiv) to give phosphate 8b:
1H NMR (200 MHz, CDCl3) δ 8.26 (d, J ) 9.0 Hz, 2H), 7.55 (d,
J ) 9.0 Hz, 2H), 7.35-7.10 (m, 4H), 5.58 (d, J ) 11.7 Hz, 1H),
4.70-4.45 (m, 2H), 2.60-2.30 (m, 1H), 2.38 (s, 3H), 2.18-2.00
(m, 1H). TLC: hexanes/ethyl acetate 6/4; Rf: cis-phosphate ) 0.2;
trans-phosphate ) 0.5.
J ) 11.6, 2.2 Hz, 1H), 4.75-4.50 (m, 2H), 3.93 (s, 3H), 2.55-
2.25 (m, 1H), 2.20-2.05 (m, 1H).
General Procedure for the Synthesis of araC Prodrugs. (A)
Step A: Phosphorylation Reaction. A solution of t-BuMgCl (1.55
mL, 1.55 mmol) in THF was added to a solution of 2′,3′-di-O-
tert-butyldimethylsilyl-cytosine-â-D-arabinofuranoside5 (9, 0.5 g,
1.03 mmol) in THF (30 mL) at room temperature. The tan solution
was stirred at room temperature for 30 min, and the respective trans-
phosphate reagent 8b-l (1.3 equiv) was added in one portion. After
stirring at room temperature for 18 h, the tan reaction mixture was
quenched with a saturated solution of NH4Cl (20 mL) and extracted
twice with ethyl acetate. The combined organic extracts were
washed three times with a 1 N solution of sodium hydroxide and
twice with a brine solution, dried (Na2SO4), and concentrated under
reduced pressure. The residue was purified by flash column
chromatography to give the protected prodrug
(b) trans-4-(4-Fluorophenyl)-2-(4-nitrophenoxy)-2-oxido-1,3,2-
dioxaphosphorinane (8c). White solid (1.8 g, 43%): mp 123-
1
125 °C; H NMR (200 MHz, CDCl3) δ 8.24 (d, J ) 9.0 Hz, 2H),
(B) Step B: Deprotection of the Protected Prodrug. Tetra-
ethylammonium fluoride hydrate (TEAF, 272 mg, 1.83 mmol) or
a 1 M solution of tetrabutylammonium fluoride (TBAF, 1.83 mL,
1.83 mmol) was added to a solution of the protected prodrug (428
mg, 0.61 mmol) in THF (6 mL) at room temperature. After stirring
at room temperature for 18 h, the reaction mixture was concentrated
under reduced pressure and the residue was purified by column
chromatography to provide prodrugs 10b-l.
(a) 5′-O-cis-[4-(3-Methylphenyl)-2-oxido-1,3,2-dioxaphospho-
rinan-2-yl]-cytosine-â-D-arabinofuranoside (10b). Phosphoryl-
ation step (150 mg, 52%). Deprotection with TEAF provided
prodrug 10b as a white solid, (40 mg, 40%): mp 189-192 °C; 1H
NMR (200 MHz, CD3OD) δ 7.80-7.65 (m, 1H), 7.35-7.10 (m,
4H), 6.21 (d, J ) 5.5 Hz, 1H), 5.80-5.50 (m, 2H), 4.80-4.00 (m,
76H), 2.50-2.10 (m, 2H), 2.33 (s, 3H). Anal. (C19H24 N3O7 P‚
0.5H2O) C, H, N.
7.50-7.20 (m, 4H), 7.10 (t, J ) 8.8 Hz, 2H), 5.57 (d, J ) 11.4
Hz, 1H), 4.75-4.45 (m, 2H), 2.60-2.35 (m, 1H), 2.20-2.00 (m,
1H).
(c) trans-4-(4-Bromophenyl)-2-(4-nitrophenoxy)-2-oxido-1,3,2-
dioxaphosphorinane (8d). White solid (2.15 g, 60%): mp 123-
1
125 °C; H NMR (200 MHz, CDCl3) δ 8.26 (d, J ) 9.0 Hz, 2H),
7.55 (d, J ) 9.0 Hz, 2H), 7.45 (d, J ) 9.0 Hz, 2H), 7.27 (d, J )
9.0 Hz, 2H), 5.58 (d, J ) 11.7 Hz, 1H), 4.80-4.50 (m, 2H), 2.55-
2.25 (m, 1H), 2.20-2.00 (m, 1H).
(d) trans-4-(4-Chlorophenyl)-2-(4-nitrophenoxy)-2-oxido-1,3,2-
dioxaphosphorinane (8e). Off-white powder (4.0 g, 51%): mp
102-105 °C; 1H NMR (200 MHz, CDCl3) δ 8.30 (d, J ) 9.0 Hz,
2H), 7.55-7.25 (m, 6H), 5.60 (d, J ) 11.7 Hz, 1H), 4.80-4.50
(m, 2H), 2.55-2.25 (m, 1H), 2.20-2.00 (m, 1H).
(e) trans-4-(3-Chlorophenyl)-2-(4-nitrophenoxy)-2-oxido-1,3,2-
dioxaphosphorinane (8f). White solid (4.5 g, 91%): mp 115-
(b) 5′-O-cis-[4-(4-Fluorophenyl)-2-oxido-1,3,2-dioxaphospho-
rinan-2-yl]-cytosine-â-D-arabinofuranoside (10c). Phosphoryl-
ation step (170 mg, 57%). Deprotection with TBAF provided
1
116 °C; H NMR (200 MHz, CDCl3) δ 8.26 (d, J ) 9.0 Hz, 2H),
7.50-7.20 (m, 6H), 5.56 (d, J ) 11.7 Hz, 1H), 4.70-4.40 (m,
2H), 2.60-2.20 (m, 1H), 2.20-2.00 (m, 1H).
1
prodrug 10c as a white solid (86 mg, 86%): mp 190 °C, dec; H
(f) trans-4-(3-Chloro-4-fluorophenyl)-2-(4-nitrophenoxy)-2-
oxido-1,3,2-dioxaphosphorinane (8g). White solid (4.16 g,
73%): mp 115-116 °C; 1H NMR (300 MHz, CDCl3) δ 8.28 (d, J
) 9.0 Hz, 2H), 7.55-7.45 (m, 3H), 7.35-7.28 (m, 1H), 7.23 (t, J
) 8.4 Hz, 1H), 5.60 (dd, J ) 11.7, 2.1 Hz, 1H), 4.70-4.55 (m,
2H), 2.55-2.37 (m, 1H), 2.20-2.10 (m, 1H).
(g) trans-4-(3,5-Dichlorophenyl)-2-(4-nitrophenoxy)-2-oxido-
1,3,2-dioxaphosphorinane (8h). White solid (5.4 g, 42%): mp
124-126 °C; 1H NMR (300 MHz, CDCl3) δ 8.28 (d, J ) 9.0 Hz,
2H), 7.44 (d, J ) 9.0 Hz, 2H), 7.38 (s, 1H), 7.31 (s, 1H), 5.56 (dd,
J ) 11.7, 2.1 Hz, 1H), 4.70-4.55 (m, 2H), 2.55-2.37 (m, 1H),
2.20-2.10 (m, 1H).
NMR (200 MHz, DMSO-d6) δ 7.60-7.40 (m, 7H), 7.30-6.95 (m,
4H, 2H exchangeable with D2O), 6.08 (d, J ) 3.4 Hz, 1H), 5.80-
5.45 (m, 4H, 2H exchangeable with D2O), 4.60-4.10 (m, 4H),
4.00-3.80 (m, 3H), 2.35-2.00 (m, 2H). Anal. (C18H21FN3O8P‚
H2O) C, H, N.
(c) 5′-O-cis-[4-(4-Bromophenyl)-2-oxido-1,3,2-dioxaphospho-
rinan-2-yl]-cytosine-â-D-arabinofuranoside (10d). Phosphoryl-
ation step (200 mg, 51%). Deprotection with TBAF provided
prodrug 10d as a white solid (105 mg, 87%): mp 196 °C, dec; 1H
NMR (200 MHz, DMSO-d6) δ 7.65-7.30 (m, 5H), 7.20-6.95 (m,
2H, exchangeable with D2O), 6.08 (d, J ) 3.4 Hz, 1H), 5.75-5.45
(m, 4H, 2H exchangeable with D2O), 4.60-4.15 (m, 4H), 4.00-
3.80 (m, 3H), 2.30-2.05 (m, 2H). Anal. (C18H21BrN3O8P‚0.8H2O)
C, H, N.
(h) trans-2-(4-Nitrophenoxy)-2-oxido-4-(pyridin-4-yl)-1,3,2-
dioxaphosphorinane (8i). White solid (8.9 g, 92%): mp 140-
1
142 °C; H NMR (300 MHz, DMSO-d6) δ 8.61 (d, J ) 4.8 Hz,
(d) 5′-O-cis-[4-(4-Chlorophenyl)-2-oxido-1,3,2-dioxaphospho-
rinan-2-yl]-cytosine-â-D-arabinofuranoside (10e). Phosphoryl-
ation step (390 mg, 65%). Deprotection with TBAF provided
prodrug 10e as a white solid (130 mg, 64%): mp 190-192 °C; 1H
NMR (200 MHz, CD3OD) δ 7.80-7.65 (m, 1H), 7.50-7.30 (m,
4H), 6.30-6.17 (m, 1H), 5.80-5.60 (m, 2H), 4.75-4.00 (m, 7H),
2.50-2.10 (m, 2H). Anal. (C18H21ClN3O8P‚1.2H2O) C, H, N.
(e) 5′-O-cis-[4-(3-Chlorophenyl)-2-oxido-1,3,2-dioxaphospho-
rinan-2-yl]-cytosine-â-D-arabinofuranoside (10f). Phosphorylation
step (4.48 g, 62%). Deprotection with TEAF provided prodrug 10f
2H), 8.33 (d, J ) 9.6 Hz, 2H), 7.57 (d, J ) 9.6 Hz, 2H), 7.42 (d,
J ) 4.8 Hz, 2H), 6.00-5.85 (m, 1H), 4.80-4.50 (m, 2H), 2.6-2.1
(m, 2H). TLC conditions: 3/2 acetone/hexanes; diol: Rf ) 0.2;
trans-phosphate: Rf ) 0.6; cis-phosphate: Rf ) 0.5.
(i) trans-2-(4-Nitrophenoxy)-2-oxido-4-(pyridin-3-yl)-1,3,2-di-
1
oxaphosphorinane (8j). White solid: mp 165-167 °C. H NMR
(200 MHz, CDCl3) δ 8.70-8.60 (m, 2H), 8.28 (d, J ) 9.0 Hz,
2H), 7.90-7.80 (m, 1H), 7.44 (d, J ) 9.0 Hz, 2H), 7.45-7.38 (m,
1H), 5.65 (d, J ) 11.0 Hz, 1H), 4.80-4.50 (m, 2H), 2.60-2.37
(m, 1H), 2.22-2.07 (m, 1H).
1
as a white powder (1.62 g, 65%): mp >200 °C; H NMR (200
(j) trans-4-(3-Methoxycarbonylphenyl)-2-(4-nitrophenoxy)-2-
oxido-1,3,2-dioxaphosphorinane (8k). White solid (1.2 g, 54%):
1H NMR (200 MHz, CDCl3) δ 8.26 (dd, J ) 9.2, 2.2 Hz, 2H),
7.80-8.00 (m, 1H), 7.63 (d, J ) 7.6 Hz, 1H), 7.60-7.40 (m, 4H),
5.66 (dd, J ) 11.6, 2.2 Hz, 1H), 4.80-4.50 (m, 2H), 3.93 (s, 3H),
2.60-2.30 (m, 1H), 2.20-2.05 (m, 1H).
(k) trans-4-(4-Methoxycarbonylphenyl)-2-(4-nitrophenoxy)-
2-oxido-1,3,2-dioxaphosphorinane (8l). White solid (0.7 g,
23%): 1H NMR (200 MHz, CDCl3) δ 8.24 (dd, J ) 9.2, 2.2 Hz,
2H), 8.09 (dd, J ) 8.4, 2.0 Hz, 2H), 7.55-7.37 (m, 4H), 5.66 (dd,
MHz, DMSO-d6) δ 7.60-7.37 (m, 5H), 7.07 (br s, 2H, exchange-
able with D2O), 6.10 (d, J ) 3.3 Hz, 1H), 5.78-5.65 (m, 1H),
5.65-5.50 (m, 3H, 2H exchangeable with D2O), 4.60-4.20 (m,
4H), 4.00-3.85 (m, 3H), 2.30-2.10 (m, 2H). Anal. (C18H21N3O8-
ClP‚H2O) C, H, N.
(f) 5′-O-cis-[4-(3-Chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxa-
phosphorinan-2-yl]-cytosine-â-D-arabinofuranoside (10g). Phos-
phorylation step (180 mg, 58%). Deprotection with TBAF provided
prodrug 10g as a white solid (101 mg, 83%): mp 128-131 °C; 1H
NMR (200 MHz, DMSO-d6) δ 7.70-7.40 (m, 3H), 7.2-6.95 (m,

7718 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Boyer et al.
2H, exchangeable with D2O), 6.08 (d, J ) 3.4 Hz, 1H), 5.80-5.50
(m, 4H, 2H exchangeable with D2O), 4.60-4.20 (m, 4H), 4.00-
3.80 (m, 2H), 2.30-2.10 (m, 2H). Anal. (C18H21ClFN3O8P‚0.9H2O)
C, H, N.
(g) 5′-O-cis-[4-(3,5-Dichlorophenyl)-2-oxido-1,3,2-dioxaphos-
phorinan-2-yl]-cytosine-â-D-arabinofuranoside (10h). Phospho-
rylation step (440 mg, 63%). Deprotection with TBAF provided
prodrug 10h as a white solid (240 mg, 81%): mp 195-197 °C;
1H NMR (200 MHz, DMSO-d6) δ 7.70-7.43 (m, 4H), 7.15-6.95
(m, 2H, exchangeable with D2O), 6.09 (d, J ) 3.0 Hz, 1H), 5.80-
5.65 (m, 1H), 5.65-5.50 (m, 3H, 2H exchangeable with D2O),
4.80-4.20 (m, 4H), 4.00-3.80 (m, 3H), 2.30-2.10 (m, 2H). Anal.
(C18H20Cl2N3O8P‚1.2H2O) C, H, N.
CH2Cl2 (200 mL) was added to a stirring solution of EDCI (35 g,
183 mmol), DMAP (3.7 g, 30.5 mmol), and L-N,N-dimethyl-
phenylalanine (26 g, 135 mmol) in CH2Cl2 (800 mL) at room
temperature. After stirring for 16 h at room temperature, water was
added and the layers were separated. The aqueous phase was back-
extracted with CH2Cl2, and the combined organic extracts were
dried (Na2SO4) and concentrated under reduced pressure. Chro-
matography on silica gel with hexane/acetone 80/20 to 50/50
removed the unreacted starting material. The diastereomers were
then separated by a succession of chromatographies with hexane/
ethyl acetate (1%TEA) 60/40 to 30/70 to get pure fractions.
(a) Ethyl 3-(L-2-N,N-Dimethylamino-3-phenyl-propanoyloxy)-
3-(pyridin-4-yl)-propanoate (16S). Faster moving product (18.78
g. 83%): 1H NMR (200 MHz, CD3OD) δ 8.27 (d, J ) 6.4 Hz,
2H), 7.20-6.95 (m, 5H), 6.84 (d, J ) 6.4 Hz, 2H), 6.03 (t, J ) 6.8
Hz, 1H), 4.07 (q, J ) 7.3 Hz, 2H), 3.52 (t, J ) 7.9 Hz, 1H), 2.90
(d, J ) 7.8 Hz, 2H), 2.77 (d, J ) 7.6 Hz, 2H), 2.33 (s, 6H), 1.15
(t, J ) 7.3 Hz, 3H).
(h) 5′-O-cis-[2-Oxido-4-(pyridin-4-yl)-1,3,2-dioxaphosphori-
nan-2-yl]-cytosine-â-D-arabinofuranoside (10i). Phosphorylation
step (1.44 g, 52%). Deprotection with TEAF provided prodrug 10i
1
as a white solid (391 mg, 40%): mp 150 °C, dec; H NMR (200
MHz, DMSO-d6) δ 8.65-8.50 (m, 2H), 7.60-7.25 (m, 3H), 7.20-
6.90 (m, 2H, exchangeable with D2O), 6.15-6.00 (m, 1H), 5.80-
5.65 (m, 1H), 5.65-5.50 (m, 3H, 2H exchangeable with D2O),
4.65-4.10 (m, 4H), 4.05-3.80 (m, 3H), 2.35-2.00 (m, 2H). Anal.
(C17H21N4O8P‚0.5H2O) C, H, N.
(b) Ethyl 3-(L-2-N,N-Dimethylamino-3-phenyl-propanoyloxy)-
3-(pyridin-4-yl)-propanoate (16R). Slower moving product (17.5
g, 77%): 1H NMR (200 MHz, CD3OD) δ 8.43 (d, J ) 6.4 Hz,
2H), 7.29 (d, J ) 6.4 Hz, 2H), 7.23-7.07 (m, 5H), 5.98 (t, J ) 6.8
Hz, 1H), 3.99 (q, J ) 7.3 Hz, 2H), 3.52 (t, J ) 7.9 Hz, 1H), 2.96
(d, J ) 7.8 Hz, 2H), 2.80-2.55 (m, 2H), 2.27 (s, 6H), 1.10 (t, J )
7.3 Hz, 3H).
(i) 5′-O-cis-[2-Oxido-3-(pyridin-3-yl)-1,3,2-dioxaphosphori-
nan-2-yl]-cytosine-â-D-arabinofuranoside (10j). Phosphorylation
step (240 mg, 34%). Deprotection with TEAF provided prodrug
1
10j as a white solid (60 mg, 38%): mp 184-188 °C; H NMR
(c) (-)-(S)-1-(Pyridin-4-yl)-1,3-propanediol (17S). A three-neck
flask was flame dried and charged with LiAlH4 (10.4 g, 170.6
mmol). Ether (750 mL) was added slowly. The slurry was cooled
to -20 °C, and a solution of ester 16S (16.97 g, 45.8 mmol) in
ether (250 mL) was added dropwise via addition funnel. The
reaction progressed for 1 h and then was quenched with EtOAc
(150 mL) followed by a saturated solution of Na2SO4 (50 mL) and
MeOH (100 mL). This mixture was stirred for 1 h and filtered,
and the solid was rinsed with MeOH. The filtrate was concentrated
under reduced pressure and purified by chromatography (95/5
CH2Cl2/MeOH + 1% Et3N) to give a brown oil that crystallized
upon standing. Recrystallization from ethyl acetate gave tan crystals
(4.64 g, 66%): mp 98-100 °C. ee ) 96% S-isomer determined
by HPLC: Chiralpak AD, 4.6 × 250 mm2; 0:90, ethanol/hexane,
isocratic; 1.5 mL/min; detection at 254 nm; room temperature:
(200 MHz, CD3OD) δ 8.65 (s, 1H), 8.60-8.50 (m, 1H), 7.95 (d, J
) 5.7 Hz, 1H), 7.75-7.65 (m, 1H), 7.55-7.40 (m, 1H), 6.25-618
(m, 1H), 5.90-5.60 (m, 2H), 4.80-4.25 (m, 4H), 4.25-4.00 (m,
3H), 2.60-2.10 (m, 2H). Anal. (C17H21N4O8P‚1.4H2O) C, H, N.
(j) 5′-O-cis-[4-(3-Methoxycarbonylphenyl)-2-oxido-1,3,2-di-
oxaphosphorinan-2-yl]-cytosine-â-D-arabinofuranoside (10k).
Phosphorylation step (220 mg, 72%). Phosphorylation step (120
mg, 80%). Deprotection with TBAF provided prodrug 10k as a
white solid (120 mg, 80%;): mp 130-132 °C; 1H NMR (200 MHz,
DMSO-d6) δ 8.00 (s, 1H), 7.93 (d, J ) 7.8 Hz, 1H), 7.70 (d, J )
7.6 Hz, 1H), 7.60-7.45 (m, 2H), 7.05 (d, J ) 9.2 Hz, 2H,
exchangeable with D2O), 6.08 (d, J ) 3.4 Hz, 1H), 5.90-5.70 (m,
1H), 7.65-5.50 (m, 3H, 2H exchangeable with D2O), 4.65-4.20
(m, 4H), 4.00-3.80 (m, 3H), 3.84 (s, 3H), 2.30-2.10 (m, 2H).
Anal. (C20H24N3O10P‚0.9 H2O) C, H, N.
(k) 5′-O-cis-[4-(4-Methoxycarbonylphenyl)-2-oxido-1,3,2-di-
oxaphosphorinan-2-yl]-cytosine-â-D-arabinofuranoside (10l). Phos-
phorylation step (190 mg, 62%). Deprotection with TBAF provided
prodrug 10l as a white solid (105 mg, 95%): mp 137-140 °C; 1H
NMR (200 MHz, DMSO-d6) δ 7.97 (d, J ) 8.4 Hz, 2H), 7.60-
7.45 (m, 2H), 7.15-6.95 (m, 2H, exchangeable with D2O), 6.08
(d, J ) 3.4 Hz, 1H), 5.85-5.70 (m, 1H), 5.70-5.45 (m, 3H, 2H
exchangeable with D2O), 4.65-4.20 (m, 4H), 4.00-3.80 (m, 3H),
3.83 (s, 3H), 2.25-2.15 (m, 2H). Anal. (C20H24N3O10P‚0.7H2O)
C, H, N.
(l) 2′,3′-di-O-tert-Butyldimethylsilyl-4-N-(N,N-dimethylform-
amidine)-cytosine-â-D-arabinofuranoside (13). DMF-dimethyl
acetal (3.4 mL, 25.7 mmol) was added dropwise to a stirred solution
of 2′,3′-di-O-TBS-ara-C (9, 9.7 g, 20.56 mmol) in pyridine (100
mL) at room temperature. After stirring the clear solution at room
temperature for 16 h, the volatiles were removed under reduced
pressure. The crude product was purified by column chromatog-
raphy on silica gel, eluting with 2-5% ethanol in dichloromethane
to afford the desired nitrogen-protected intermediate 13 as a white
foam (9.75 g, 90% yield): 1H NMR (200 MHz, CDCl3) δ 8.80 (s,
1H), 7.78 (d, J ) 7.2 Hz, 1H), 6.25 (d, J ) 2.5 Hz, 1H), 6.02 (d,
J ) 7.2 Hz, 1H), 4.30-4.22 (m, 1H), 4.10-4.02 (m, 2H), 3.87-
3.75 (m, 2H), 3.12 (s, 3H), 3.10 (s, 3H), 0.87 (s, 9H), 0.77 (s, 9H),
0.10 (s, 3H), 0.08 (s, 3H), 0.02 (s, 3H), -0.18 (s, 3H).
25
R-diol ) 12.7 min; S-diol ) 14 min. [R]D ) -50.48° (c 1.00,
MeOH).
(d) (+)-(R)-1-(Pyridin-4-yl)-1,3-propanediol (17R). Reduction
of ester 16R (17.5 g) provided diol 17R (2.4 g, 33%): mp 98-100
°C, ee ) 98% R-isomer (determined by HPLC).
(e) (-)-(4S)-trans-2-(4-Nitrophenoxy)-2-oxido-4-(pyridin-4-yl)-
1,3,2-dioxaphosphorinane (18S). To a stirred solution of 4-nitro-
phenyl phosphorodichloridate (7.35 g, 28.7 mmol) in THF (80 mL)
at 0 °C was slowly added pyridine (6.3 mL, 78.3 mmol) over 1 h.
The reaction mixture was stirred for 5 min at 0 °C and slowly added
to a solution of (-)-(S)-1-(pyridin-4-yl)-1,3-propanediol (17S, 97%
ee, 4.0 g, 26.1 mmol) in THF (240 mL) at 0 °C over 1 h. The
reaction mixture was allowed to warm to room temperature and
was stirred for 2.5 h. Sodium 4-nitrophenoxide (10.91 g, 78.3 mmol)
was added, and the heterogeneous orange reaction mixture was
heated at 40 °C for 4 h. The reaction was cooled to 0 °C, quenched
with a saturated solution of NH4Cl (250 mL), and extracted with
ethyl acetate (100 mL × 2). The combined organic extracts were
washed with a 0.3 N solution of sodium hydroxide (100 mL × 4)
and dried (MgSO4). The filtered solution was concentrated under
reduced pressure to give an oil that crystallized upon standing. The
yellow solid was recrystallized from 100 mL of 2-propanol to afford
trans-phosphate 18S as a white solid (4.74 g, ee 99.4%). A second
crop was obtained by concentrating the mother liquor (780 mg,
98% ee) to bring the total yield to 5.52 g, 63%: mp 140-142 °C.
(m) Ethyl 3-Hydroxy-3-(pyridin-4-yl)-propanoate (14). Oil (87
g, 97%): 1H NMR (CDCl3) δ 8.53 (d, J ) 6.6 Hz, 2H), 7.31 (d,
J ) 6.6 Hz, 2H), 5.19-5.11 (m, 1H), 4.18 (q, J ) 7.0 Hz, 2H),
2.80-2.65 (m, 2H), 1.22 (s, 3H).
Resolution of Diols 17R and 17S. A solution of methyl
3-hydroxy-3-(pyridin-4-yl)-propanoate (14, 23.9 g, 122 mmol) in
20
[R]D -80.9 (c 1.0, MeOH). de ) 99% trans by HPLC: Zorbax
Rx-C18 (4.6 × 250 mm2); 35% acetonitrile/ 65% 20 mM phosphate
buffer pH 7.95; 0.5 mL/min; detection at 250 nm; retention times:
cis isomer ) 9.39 min, trans isomer ) 10.11 min; ee ) 99.4% by

LiVer-Targeted Prodrug of AraCMP for HCC
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7719
HPLC: Chiral Pak AD; 1:1 2-propanol/hexanes; 1.0 mL/min;
detection at 254 nm; retention times in min: trans-phosphate )
7.02.
previously.9 Compounds were incubated at 100 µM in suspended
rat hepatocytes, shaken for 4 h under oxygen at 37 °C, and then
extracted by brief centrifugation (10 000 rpm in microfuge) through
a mineral/silicon (4:1) oil layer into 10% perchloric acid (PCA).
The perchloric acid layer was centrifuged for 10 min (14 000 rpm).
The resulting supernatant was neutralized with 0.3 volumes of 3
M KOH/3 M KHCO3. Neutralized hepatocyte extracts were treated
with sodium periodate to remove interfering ribonucleoside tri-
phosphates and analyzed by ion exchange chromatography for
araCTP levels on a Whatman Partisil SAX column (4.6 × 250 mm2)
eluted with a linear gradient of buffer A (10 mM ammonium
phosphate pH 3.5 and 6% v/v ethanol) and buffer B (1 M
ammonium phosphate pH 3.5 and 6% v/v ethanol) [30-80% buffer
B over 0-25 min] at a flow rate of 1.25 mL/min and with detection
at 280 nm. AraCTP eluted at approximatively 12 min and was
quantified relative to a known araCTP standard spiked into
neutralized extracts.
(B) Stability Assay. Prodrug stability studies were performed
using 100 µM concentrations of prodrugs in 10 mM phosphate
buffer, pH 7.4 at 37 °C. Intact prodrug was quantified by HPLC.
(C) Aqueous Solubility. Solubility measurements of prodrugs
were conducted by dissolving 10 mg/mL of prodrug in 100 mM
Ki, pH 7.4, or 100 mM citrate buffer, pH 4.0 (prodrugs 10i, 10j,
19R, and 19S were dissolved at 200 mg/mL). Samples were then
sonicated for 20 min in a sonication bath and allowed to stand at
room temperature for ∼10 min before being filtered through a 0.45
µm, nylon membrane, 4 mm syringe filter. Samples were then
diluted 1:100 or 1:10000 in DMSO before being analyzed by HPLC.
(D) HPLC Analysis. Samples were analyzed on a HP1100
(Hewlett-Packard) using a C-18 Phenomenex Luna column, 150
mm × 4.6 mm, catalog # 00F-4252-E0, with a C-18 Alltech
Econosphere guard column, 7.5 mm × 4.6 mm, catalog # 96121.
Fifty microliters of filtrate was injected onto the column with mobile
phase buffer, 20 mM potassium phosphate buffer, pH 6.2. Samples
were eluted with an acetonitrile gradient: 10-80% over 10 min.
The column was allowed to equilibrate for 5 min before the next
injection. The flow rate was 1 mL/min with a column temperature
of 40 °C. Prodrugs eluted around 3-7 min and were monitored at
270 nm.
(E) Mouse Tissue Distribution. Normal nonfasted male Swiss
Webster mice (25-35 g body weight, Harlan Sprague Dawley,
Indianapolis, IN) were injected i.p. with 19R or 19S at a molar-
equivalent dose of 100 mg/kg of araC. At specified times after
injection, mice were anesthetized and blood drawn. The liver was
removed, rapidly snap-frozen in liquid nitrogen, and homogenized
using a Polytron homogenizer PT 10/35 (Brinkmann Instruments,
Westbury, NY) in three volumes of 10% (v/v) PCA. After a 5 min
centrifugation at 2500g, 1 mL of supernatant was neutralized using
0.3 mL of 3 M KOH/3 M KHCO3 and mixed thoroughly. One
hundred microliters of tissue extract was incubated with 4 µL of
0.5 M sodium periodate and 10 µL of 1.8 M methylamine for 30
min at room temperature. The reaction was stopped with the
addition of 2 µL of 1 M L-Rhamnose. Resulting samples were
analyzed by HPLC as described above. The bone marrow samples
were flushed from the marrow cavities of femurs with 1.2 mL of
saline into preweighed Eppendorf tubes. After centrifugation for
20-30 s (Eppendorf microfuge, 14 000 rpm) and removal of the
supernatant, 12 volumes of 3% PCA (v/v) were added to the bone
marrow cell pellets. Samples were then vortexed until the pellet
was well dissolved and centrifuged as above for 10 min. Ninety
microliters of extracted supernatant was neutralized to pH 7-8 using
30 µL of 1 M KOH/1 M KHCO3 and again centrifuged. Resulting
supernatants were periodate-treated, and araCTP levels were
determined by HPLC as above.
(f) (+)-(4R)-trans-2-(4-Nitrophenoxy)-2-oxido-(4-pyridinyl)-
1,3,2-dioxaphosphorinane (18R). Starting from 5.0 g of (+)-(R)-
1-(pyridin-4-yl)-1,3-propanediol (17R, 96% ee) gave an oil that
crystallized upon standing. The yellow solid was recrystallized from
100 mL of 2-propanol to afford trans-phosphate 18R as a white
20
solid (6.67 g, 60%, 95% ee, [R]D + 74.2° (c 1.0, MeOH)).
(g) (4S)-5′-O-cis-[2-Oxido-4-(pyridin-4-yl)-1,3,2-dioxaphos-
phorinan-2-yl]-cytosine-â-D-arabinofuranoside (19S). To a stirred
solution of 2′,3′-di-O-TBS-4-N-(N,N-dimethylformamidine)-cyti-
dine-â-D-arabinofuranoside (13, 1.8 g, 3.42 mmol) in THF (40 mL)
at room temperature was slowly added a solution of t-BuMgCl (1
M in THF, 4.44 mL, 4.44 mmol). After 30 min, trans-phosphate
18S (1.94 g, 5.77 mmol) was added and the reaction mixture was
stirred at room temperature for 16 h. The reaction was cooled to 0
°C, quenched with a saturated aqueous solution of ammonium
chloride, and extracted with ethyl acetate (50 mL × 2). The
combined organic layers were dried (MgSO4), filtered, and con-
centrated under reduced pressure. The crude product was purified
by column chromatography on silica gel, eluting with acetone to
afford the desired protected prodrug as an off-white solid (1.92 g,
78%): mp 162-164 °C. 1H NMR (200 MHz, CDCl3) δ 8.80
(s, 1H), 8.64 (dd, J ) 4.4, 1.4 Hz, 2H), 7.78 (d, J ) 7.2 Hz, 1H),
7.26 (dd, J ) 4.4, 1.4 Hz, 2H), 6.34 (d, J ) 2.8 Hz, 1H), 6.03 (d,
J ) 7.2 Hz, 1H), 5.70-5.60 (m, 1H), 4.80-4.15 (m, 6H), 4.04 (s,
1H), 3.14 (s, 3H), 3.12 (s, 3H), 2.40-2.05 (m, 2H), 0.89 (s, 9H),
0.77 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.01 (s, 3H), -0.19 (s,
3H).
A stirred solution of the protected prodrug (4.96 g, 2.64 mmol)
in 70% aqueous TFA (50 mL) was heated at 60 °C for 16 h, and
the solvent was removed under reduced pressure. To the residue
was added methanol (20 mL), and the mixture was made slightly
basic with sodium bicarbonate. The cloudy solution was dried
(MgSO4), filtered, and concentrated under reduced pressure, and
the crude product was purified by column chromatography on silica
gel, eluting with methanol/acetone (1:1) to afford 19S as an off-
white solid (2.55 g, 83%): mp 153-155 °C; 1H NMR (200 MHz,
DMSO-d6) δ 8.58 (dd, J ) 4.7, 1.8 Hz, 2H), 7.51 (d, J ) 7.6 Hz,
1H), 7.41 (dd, J ) 4.7, 1.8 Hz, 2H), 7.13 (s, 1H, exchangeable
with D2O), 7.05 (s, 1H, exchangeable with D2O), 6.09 (d, J ) 3.6
Hz, 1H), 5.80-5.70 (m, 1H), 5.63 (d, J ) 2.7 Hz, 1H exchangeable
with D2O), 5.62 (d, 1H exchangeable with D2O), 5.57 (d, J ) 7.6
Hz, 1H), 4.60-4.20 (m, 4H), 4.00-3.85 (m, 3H), 2.25-2.15 (m,
2H). 13C NMR (50 MHz, DMSO-d6) δ 164.9, 154.4, 149.3, 147.3
(d, J ) 8.0 Hz), 142.2, 119.6, 91.9, 85.7, 81.7 (d, J ) 6.8 Hz),
77.7 (d, J ) 5.0 Hz), 75.6, 73.5, 67.1 (d, J ) 6.5 Hz), 66.6 (d, J
) 5.7 Hz), 31.5 (d, J ) 6.8 Hz). Anal. (C17H21N4O8P‚0.1 H2O) C,
H, N.
(h) (4R)-5′-O-cis-[2-Oxido-4-(pyridin-4-yl)-1,3,2-dioxaphos-
phorinan-2-yl]-cytosine-â-D-arabinofuranoside (19R). Phospho-
rylation of 2′,3′-di-O-tert-butyldimethylsilyl-4-N-(N,N-dimethyl-
formamidine)-cytidine-â-D-arabinofuranoside (13, 2.4 g, 4.55 mmol)
with trans-phosphate 18R (1.84 g, 5.50 mmol) afforded the
protected prodrug as an off-white solid (2.19 g, 66%, mp 142-
1
145 °C); H NMR (200 MHz, CDCl3) δ 8.78 (s, 1H), 8.63 (dd, J
) 4.4, 1.4 Hz, 2H), 7.73 (d, J ) 7.2 Hz, 1H), 7.26 (dd, J ) 4.4,
1.4 Hz, 2H), 6.33 (d, J ) 2.8 Hz, 1H), 5.99 (d, J ) 7.2 Hz, 1H),
5.70-5.55 (m, 1H), 4.80-4.1 (m, 6H), 4.04 (s, 1H), 3.14 (s, 3H),
3.11 (s, 3H), 2.30-2.10 (m, 2H), 0.90 (s, 9H), 0.77 (s, 9H), 0.12
(s, 3H), 0.10 (s, 3H), 0.01 (s, 3H), -0.19 (s, 3H).
Deprotection as for 19S provided prodrug 19R as an off-white
solid (1.06 g, 80%): mp 178-180 °C; 1H NMR (DMSO-d6) δ 8.61
(d, J ) 6.0 Hz, 2H), 7.52 (d, J ) 7.4 Hz, 1H), 7.44 (d, J ) 5.8 Hz,
2H), 7.15-7.00 (m, 2H, exchangeable with D2O), 6.11 (d, J ) 3.6
Hz, 1H), 5.80-5.70 (m, 1H), 5.70-5.65 (m, 3H, 2H exchangeable
with D2O), 4.65-4.20 (m, 4H), 4.00-3.85 (m, 3H), 2.25-2.15 (m,
2H). Anal. (C17H21N4O8P‚0.7 H2O) C, H, N.
Blood was transferred to heparinized microcollection tubes and
centrifuged in an Eppendorf microfuge at 14 000 rpm for 2 min to
collect plasma, which was placed on dry ice and subsequently stored
at -20 °C. On the day of analysis, proteins were precipitated by
adding 1 mL of acetonitrile to 100 µL of plasma. After a 10 min
centrifugation (Eppendorf microfuge, 14 000 rpm), the supernatant
was removed and dried in a Savant SpeedVac Plus SC110A.
Biological Methods. (A) Hepatocyte Activation Assay. Rat
hepatocytes were isolated using standard procedures described

7720 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
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Samples were reconstituted with 110 µL of mobile phase buffer
(20 mM KH2PO4, pH 4.5), sonicated for 5 min, and centrifuged
for 30 s. Supernatants were analyzed by reverse-phase HPLC:
Alltech C18 column (5 µm, 4.6 × 250 mm2); acetonitrile gradient
0-10% over 10 min, then to 50% over 15 min. Elution times for
araC and prodrugs were around 3 and 19 min, respectively. AraC
and prodrugs were detected by absorbance at 280 nm.
Acknowledgment. We thank Ms. Annika Montag, Dr. Sanna
Rosengren, Mr. Paul Rolzin, Ms. Barbara Treash-Osio, Mr
Minghua Liu, Miss Kristin Ollis, and Dr. Michael C. Matelich
for their scientific contributions.
Supporting Information Available: Microanalysis data for all
final compounds; crystallographic data for compound 19S (packing
diagrams, tables of crystal data and data collection parameters,
positional parameters and their estimated standard deviations,
anisotropic temperature factor coefficients, bond distances, bond
angles, and torsion angles). This material is available free of charge
via the Internet at http://pubs.acs.org.
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