6-Methylpurine derived sugar modified nucleosides: Synthesis and in vivo antitumor activity in D54 tumor expressing M64V-Escherichia coli purine nucleoside phosphorylase

Article abstract of DOI:10.1016/j.ejmech.2015.12.029

Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cyt

Full text of DOI:10.1016/j.ejmech.2015.12.029

Accepted Manuscript
6-methylpurine derived sugar modified nucleosides: Synthesis and in vivo
antitumor activity in D54 tumor expressing M64V-Escherichia coli purine nucleoside
phosphorylase
Abdalla E.A. Hassan, Reham A.I. Abou-Elkhair, William B. Parker, Paula W. Allan,
John A. Secrist, III
PII:
S0223-5234(15)30415-3
10.1016/j.ejmech.2015.12.029
EJMECH 8269
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2015
Revised Date: 24 November 2015
Accepted Date: 14 December 2015
Please cite this article as: A.E.A. Hassan, R.A.I. Abou-Elkhair, W.B. Parker, P.W. Allan, J.A. Secrist
III., 6-methylpurine derived sugar modified nucleosides: Synthesis and in vivo antitumor activity in
D54 tumor expressing M64V-Escherichia coli purine nucleoside phosphorylase, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.029.
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ACCEPTED MANUSCRIPT
Selective delivery of
M64V-E.coli PNP to
tumor cells
Cell produces
E. coil PNP protein
MeP can diffuse into
neighboring cells that do not
express E. coil PNP
Bystander
killing

ACCEPTED MANUSCRIPT
6-Methylpurine Derived Sugar Modified Nucleosides: Synthesis and
in vivo Antitumor Activity in D54 Tumor Expressing M64V-
Escherichia coli Purine Nucleoside Phosphorylase
Abdalla E. A. Hassan,[a,c]* Reham A. I. Abou-Elkhair,[a,c] William B. Parker,[a,b] Paula W. Allan[a]
John A. Secrist III [a] *
aSouthern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, (USA). bPNP Therapeutics Inc., 15
Richard Arrington Jr. Blvd North Birmingham, AL 35203, (USA). cApplied Nucleic Acids Research Centre, Zagazig
University, Zagazig, PO Box 44519
Abstract. Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express E.
coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-
Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered
systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The
prototype MeP releasing prodrug 9-(2-deoxy-β-D-ribofuranosyl)-6-methylpurine (1) [MeP-dR] has demonstrated good
activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the
generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a
combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best
MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5’-
position of adenosine and its analogues, were 9-(6-deoxy-α-L-talofuranosyl)-6-methylpurine (3) [methyl(talo)-MeP-R]
and 9-(α-L-lyxofuranosyl)6-methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-
MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is
described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-
MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The
observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these
agents could be useful for the treatment of cancer.
Keywords: 6-Methylpurine • 2-Fluoroadenine • Purine nucleoside phosphorylase • Prodrugs • Cancer gene therapy.
Introduction.
Suicide gene therapy is a promising strategy that attempts to limit the systemic toxicity
inherent to cancer chemotherapy by selective delivery of an exogenous gene to a tumor whose
expression product converts a nontoxic prodrug into a highly cytotoxic agent.[1, 2] We have
developed a suicide gene therapy approach for the treatment of solid tumors that is based on
selective activation of none-toxic prodrugs of toxic adenine analogs such as 2-fluoroadenine and 6-
methylpurine with E. coli purine nucleoside phosphorylase (PNP).[3-5] E.coli PNP differs from
human PNP in its ability to cleave the glycosidic bond of adenosine and adenosine analogs in
addition to 6-oxopurine derivatives.[6, 7] Excellent in vivo antitumor activity has been
demonstrated with this strategy against human tumor xenografts in mice using a variety of
prodrugs,[8-11] including 9-[2-deoxy-β-D-ribofuranosyl]-6-methylpurine (MeP-dR; 1), 9-[β-D-
arabinofuranosyl]-2-fluoroadenine (F-araA; 2), and 2-fluoro-2’-deoxyadenosine (F-dAdo). The
best in vivo activity has been observed with F-araA,[12] and a phase I clinical has been initiated to
evaluate its safety and efficacy.[13] However, the antitumor activity of this approach is still

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limited by the inherent toxicity of the prodrugs. Although MeP-dR is much less toxic than F-araA
in vitro, it is much more toxic to mice due to its cleavage by PNP that is expressed in intestinal
bacteria,[14] which results in the systemic liberation of 6-methylpurine (MeP), which is known not
to have selective in vivo antitumor activity.[15] In spite of this toxicity, good in vivo antitumor
activity has been observed with MeP-dR, and it is possible that MeP prodrugs could be effectively
used in a gene therapy system utilizing E. coli PNP, if they were not cleaved by intestinal bacteria.
Therefore, we have initiated a program to change the substrate specificity of E. coli PNP to
identify an enzyme/prodrug combination that can cleave MeP containing prodrugs that are poor
substrates for the endogenous bacterial phosphorylases.[16]
Our studies have led to the identification of an E. coli PNP mutant (M64V) that is able to
cleave numerous 5’-modified nucleoside analogs with much greater efficiency than the wild-type
enzyme.[16] We have determined the crystal structure of the M64V mutant and have evaluated its
activity with a few nucleoside analogs.[16] The two best MeP-based substrates were 9-(6-deoxy-
α-L-talofuranosyl)-6-methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-L-lyxofuranosyl)-6-
methylpurine (4) [lyxo-MeP-R] (Figure 1).
Insert Figure 1
Methyl(talo)-MeP-R demonstrated good activity against D54 tumors that express M64V
PNP.[16] However, cytotoxicity (MTD) of the (M64V-PNP/methyl(talo)-MeP-R) combination
was observed and is attributed to the cleavage of methyl(talo)-6-MeP by unidentified bacterial
enzymes.[14] To circumvent this toxicity problem, in this study, we have determined the
intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined
the in vivo bystander activity of methyl(talo)-MeP-R in mice bearing D54 tumors that express
M64V E.coli-PNP. In continuation of our efforts to identify a better (MeP-prodrug/phosphorylase)
combination, we have determined the substrate activity of 3 and 4 against several other enzymes
known to cleave adenosine that should not be located in the gut bacteria. The detailed synthesis of
methyl(talo)-MeP-R and lyxo-MeP-R is described.
Results and Discussion
Chemistry. Synthesis of methyl(talo)-MeP-R, 3. Vorbrüggen glycosylation of 1,2,3,5-tetra-O-
acyl-ribofuranosides with 6-alkyl/arylpurines has been reported by us and others to selectively
produce 9-(β-D-ribofuranosyl)-6-alkyl/arylpurine nucleosides.[14, 17-20]
The anomeric
configuration is predominantly trans with respect to the 1′-heterocyclic moiety and 2′-O-acyl
group.[14, 18, 19, 21] The key intermediate needed for the coupling reaction; α-L-talofuranoside
derivative 9 could be attained by the inversion of the configuration at the C-5 position of 6-deoxy-
D-allofuranose derivative 6 followed by the application of the standard procedures of glycosyl
donor protection and activation (Scheme 1). 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (5)
was converted in six steps to 6-deoxy-D-allofuranose derivative 6 , with minor modification of the
literature procedure, in 44% total overall yield.[22, 23] Esterification of 6 under Mistunobu
reaction conditions (BzOH/PPh3/DEAD/THF) gave the corresponding 5-O-benzoate derivative 7
with inversion of the configuration at the C-5 position in 86% yield. Treatment of 7 with HCl in
MeOH/H2O, followed by benzoylation of the 2-hydroxyl group gave the corresponding methyl

ACCEPTED MANUSCRIPT
glycoside derivative 8 in 94% yield. Acetolysis of 8 gave the corresponding 1-O-acetyl-α-L-
talofuranose derivative 9 in 94% yield. Coupling of silylated 6-methylpurine with α-L-
talofuranose derivative 9 in the presence of SnCl4 in dry CH3CN gave the corresponding 9-(2,3,5-
tri-O-benzoyl-β-D-talofuranosyl)-6-methylpurine (10) in 96% yield. NOE correlation between H-
1’ and H-4’ confirms the β-configuration of the nucleoside 10. Irradiation at the H-1’ signal
showed NOE enhancement at H-4’ signal (3%) and irradiation at H-4’ signal showed enhancement
at H-1’ signal (3%). Debenzoylation of 10 with NH3/MeOH gave 11 in 94% yield. Pd/C
catalyzed hydrogenation of 11 gave 9-(6-deoxy-β-D-talofuranosyl)-6-methylpurine (3) in 87%
yield. The β-stereochemistry of 3 was further confirmed by an NOE correlation between H-8 and
H-3’. Irradiation at H-3’ signal showed NOE enhancement at H-8 signal (1%) and irradiation at
H-8 signal showed enhancement at H-3’ signal (1%).
Insert Scheme 1
Synthesis of Lyxo-MeP-R, 4[24]. Starting with the commercially available L-lyxose, the
glycosyl donor 1-O-acetyl-2,3,5-tri-O-benzoyl-L-lyxofuranose (12) was prepared in three steps
with minor modification of the literature procedures.[25-27] Treatment of L-lyxose with acetyl
chloride in MeOH at 0 °C gave a mixture of the 1-O-Me-lyxofuranoside and 1-O-Me-
lyxopyranoside derivatives in a 5:1 ratio as determined by 1H-NMR. A pure β-anomer of 1-O-Me-
lyxofuranoside was isolated by crystallization of the mixture from MeOH at -20°C. Benzoylation
of methyl lyxofuranoside with BzCl in dry pyridine followed by acetolysis of the corresponding
methyl 2,3,4-tri-O-benzoyl-lyxofuranoside derivative with Ac2O/AcOH in the presence of conc.
H2SO4 gave predominantly the corresponding 1-O-acetyl-2,3,5-tri-O-benzoyl-β-L-lyxofuranose
(12)[27] in 89% yield. Coupling of silylated 6-methyl purine with 12 in the presence of SnCl4 in
dry CH3CN to give the desired 6-MeP-lyxoside derivative 13 in 88% yield. Deprotection of 13
with NaOMe/MeOH gave then 9-(α-L-lyxofuranosyl)-6-methylpurine (4) in 86% yield.
Insert Scheme 2
Biology
Previous experiments have demonstrated that methyl(talo)-MeP-R had good activity against
D54 tumors that express M64V PNP.[12] In order to determine the in vivo bystander activity of
methyl(talo)-MeP-R, mice bearing tumors in which 10% of the cells expressed M64V were treated
ip with methyl(talo)-MeP-R. Three days of administration of compound (days 17, 18, and 19 after
tumor implantation) resulted in prolonged inhibition of tumor growth at the two highest doses of
methyl(talo)-MeP-R (Figure. 2). We also determined that the terminal half-life of methyl(talo)-
MeP-R was 21 minutes (Figure 3), which was a little longer than that of MeP-dR (14 minutes,
calculated from the change in plasma concentration between 30 and 60 minutes).
In an effort to identify better enzymes for activating these agents, we determined the substrate
activity of both methyl(talo)-MeP-R and lyxo-MeP-R against several other enzymes known to
cleave the glycosidic bond of adenosine and which should not be located in gut bacteria (Table 1).
Methyl(talo)-MeP-R showed approximately 8-fold less substrate activity against T. vaginalis PNP
compared with the M64V PNP. Interestingly, moderate substrate activity was observed with lyxo-

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MeP-R against T. vaginalis PNP and the mutant, E. coli M64V PNP. Poor substrate activity of
both methyl(talo)-MeP-R and lyxo-MeP-R was observed with E. coli SAH/MTA hydrolase and F
tulorensis PNP. Although considerable cleavage activity was observed with these enzymes, it was
not better than that observed with M64V PNP, which suggests that the antitumor activity of these
two prodrugs with any of these enzymes would not be superior to that seen with methyl(talo)-MeP-
R and M64V.
Insert Table 1
Excellent in vivo bystander activity was observed with methyl(talo)-MeP-R (T-C of > 32 days
at the maximally tolerated dose). In the experiment shown in Figure 2, the enzyme activity of
M64V PNP using methyl(talo)-MeP-R as substrate in tumor extracts removed from mice at the
initiation of treatment (day 17) was 365 ± 155 nmoles of compound cleaved/mg-hr (N = 3),
whereas in previous studies[8-11] the enzyme activity of E. coli PNP was 40 nmoles and 11,000
nmoles of MeP-dR cleaved/mg-hr of tumor tissue, respectively. In D54 tumors in which 20% of
the cells expressed low levels of E. coli PNP[3] there was also a good antitumor response (T-C of
26 days at the maximally tolerated dose of 67 mg/kg) , which was similar to the antitumor activity
of MeP-dR (T-C of 22 days at the maximally tolerated dose of 33 mg/kg) against D54 in which
10% of the tumors expressed high levels of E. coli PNP.[16] Note that the dose of MeP-dR needed
to be reduced in mice bearing tumors expressing high levels of E. coli PNP due to increased
toxicity of MeP-dR in these mice. The total dose of methyl(talo)-MeP-R used in the current
experiments (300 mg/kg) was approximately 3-fold greater than MeP-dR used against tumor
expressing high levels of E. coli PNP.[16]
Insert figure 2
These results indicate that the antitumor activity of methyl(talo)-MeP-R was as good as MeP-
dR, even though there was much less expression of activating enzyme in the tumor cells.[16]
Although there was not a correlation of antitumor activity with E. coli PNP expression with MeP-
dR, due to having to decrease MeP-dR administered, it is possible that better antitumor activity
with methyl-(talo)-MeP-R would be observed, if D54 tumors expressed higher levels of activating
enzyme (M64V).
Insert Figure 3
Conclusions.
Methyl(talo)-MeP-R and Lyxo-MeP-R were synthesized efficiently via Vorbrüggen glycosylation
procedure. Their substrate activity with newly tested adenosine cleaving enzymes were not
superior to that with the mutant enzyme M64V-E. Coli PNP. Although methyl(talo)-MeP-R at its
maximally tolerated dose (MTD) demonstrated very good in vivo antitumor activity against D54
tumors that express M64V, it did not demonstrate better antitumor activity than that seen with F-
araA.[11, 16, 28] Therefore, these results suggest that better MeP-prodrugs are still needed.

ACCEPTED MANUSCRIPT
Efforts to identify MeP-based prodrugs with a better combination of pharmacological properties is
ongoing.
13
Experimental Section. 1H NMR and C NMR spectra were recorded on a Nicolet NT 300 NB
spectrometer operating at 300.635 MHz (1H) or 75.6 MHz (13C). Chemical shifts are expressed in
parts per million from tetramethylsilane. The hydrogen-decoupled 13C-NMR spectra were
assigned by comparison of the JCH values obtained from hydrogen-coupled 13C-NMR spectra.
When necessary, selective hydrogen decoupling was performed in order to confirm the
assignments. Ultraviolet absorption spectra were determined on Perkin-Elmer Lambda 19
spectrometer by dissolving each compound in methanol or water and diluting appropriately with
0.1N HCl, pH 7 buffer, or 0.1N NaOH. Values are in nanometers, and numbers in parentheses are
extinction coefficients (ε x 10-3). Mass spectra were recorded on a Varian/MAT 311A double-
focusing mass spectrometer in the fast atom bombardment (FAB) mode (glycerol matrix). CHN
elemental analysis was carried out on Perkin-Elmer 2400 elemental analyzer. HPLC analysis was
carried out on a Hewlett-Packard 1100 series liquid chromatograph with a Phenomenex
Sphenclone 5 µM ODS (1) column (4.6 mm x 25 cm) with UV monitoring (254 nm). All flash
column chromatography used 230-400 mesh silica gel from E. Merck. TLC was done on Analtech
pre-coated (250 µm) silica gel (GF) plates.
5-O-Benzoyl-3-O-benzyl-1,2-O-isopropylidene-α-L-talofuranose (7). A mixture of DEAD (22
mL, 0.139 mol) and BzOH (17 g, 0.139 mol) in THF (75 mL) was added to a solution of 6 (16.4 g,
57 mmol) and Ph3P (36.5 g, 0.139 mol) in THF (150 mL) at 0 °C under Ar atmosphere. The
mixture was stirred for 10 h at rt, quenched with EtOH (10 mL) and the solvents were evaporated
under reduced pressure. The residue was dissolved in EtOAc (250 mL), washed with H2O, dried
over MgSO4 and evaporated. The residue was purified by a flash silica gel column (eluate; 10%
EtOAc/Hexanes) to give 7 (19.5 g, 86% yield) as a pale yellow syrup. MS [FAB] m/z 405.1
1
[M+Li]+ , m/z 447.1 [M+LiCl]+; m/z 803 [2M+Li]+, H NMR (300 MHz, CDCl3): δH = 7.97-7.94
(2H, m, Bz), 7.54-7.37 (3H, m, Bz),7.29-7.11 (5H, m, Bn-Ar), 5.80 (1H, d, J = 3.5 Hz, H-1). 5.32
(1H, ddd, H-5, J = 3.1, J = 6.6, J = 9.7 Hz), 4.73 (1H, d, PhCH2a, J = 12.1 Hz), 4.58 (1H, H-2, dd,
J = 4.2, J = 3.5 Hz), 4.52 (1H, d, J = 12.1 Hz, PhCH2b), 4.16 (1H, dd, H-4, J = 3.1, J = 9.0 Hz),
3.68 (1H, dd, H-3, J = 4.4, J = 9.0 Hz), 1.62 (3H, s, i-pr), 1.44 (3H, d, 5-CH3, J = 6.6 Hz), 1.38
(3H, s, i-pr).
Methyl 3-O-benzyl-2,5-di-O-benzoyl-α-L-talofuranoside (8). Compound 7 (6.45 g, 16.1 mmol)
was dissolved in 20% HCl in MeOH-H2O [prepared from MeOH (150 mL), H2O (42 mL) and
AcCl (27 mL)] and the mixture was stirred for 6 h at rt. The mixture was neutralized with Et3N
and diluted with EtOAc. The whole was washed with H2O (300 mL) and NaHCO3 (300 mL). The
organic phase was dried over MgSO4 and evaporated. The residue was dried under vacuum for 3
hr, then dissolved in dry pyridine (150 mL) and treated with BzCl (4.2 mL, 31.5 mmol) at 0 °C
under Ar atmosphere. The mixture was stirred for 30 min. at room temperature. Ice-H2O was
added to the mixture and the solvents were evaporated in vacuo. The residue was purified by a
flash silica gel column (eluate; 5% EtOAc/Hexanes) to give 8 (7.2 g, 94% yield) as a colorless
1
syrup. MS [FAB] m/z 494 [M+NH4]+ , m/z 499 [M+Na]+; m/z 445 [M-OMe]+; H NMR (300

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MHz, CDCl3): δH = 8.11-8.02 (4H, m, Ar), 7.59-7.40 (6H, m, Ar),7.19-7.12 (5H, m, Ar), 5.80
(1H, d, H-2, J = 3.74 Hz). 5.22 (1H, m, H-5), 5.04 (1H, s, H-1), 5.64 (1H, d, PhCH2a, J = 11.2 Hz),
4.43 (1H, d, J = 11.2 Hz), 4.32-4.23 (2H, m, H-3 and H-4, J = 4.2, J = 3.5 Hz), 3.44 (3H, s, OMe),
1.62 (3H, s, i-pr), 1.43 (3H, d, 5-CH3, J = 6.6 Hz).
9-(3-O-Benzyl-2,5-di-O-benzoyl-β-L-talofuranosyl)-6-methylpurine (10). 6-Methylpurine (5.1
g, 37.5 mmol) was suspended in a mixture of DCE (150 mL) and HMDS (150 mL) and treated
with TMSCl (4.5 mL). The mixture was heated for 2 h at 80 °C whereupon a complete dissolution
was observed. The solvents were evaporated and co-evaporated with toluene (100 mL x 3 times),
under residue pressure, to give a white solid. A suspension of the solid residue and 9 (12.75 g,
25.5 mmol) in dry CH3CN (600 mL) was treated with SnCl4 (1M in CH2Cl2, 126 mL) at -10 °C.
The mixture was stirred for 10 min whereupon the starting material was completely consumed.
NaHCO3 (1M, 450 mL) was added to the mixture, diluted with CHCl3 (1 L), the organic phase was
separated, dried over MgSO4 and evaporated. The residue was purified by a silica gel column
(eluate: 3% MeOH in CHCl3) to give 10 (13.5 g, 94%) as a white foam: MS [FAB] m/z 579 [M +
H]+; UV λmax pH 1: 264.3 nm; 233.7nm, pH 7: 237.1 nm, pH 13, 260.4 nm; 1H NMR (300 MHz,
CDCl3): δH = 8.67 (1H, s, H-2), 8.22 (1H, s, H-8,), 8.11-7.12 (10H, m, Bz and Bn-Ar), 6.28 (1H, d,
J = 2.6 Hz, H-1’), 6.22 (1H, dd, H-2’, J = 2.4, J = 5.3 Hz), 5,42 (1H, m, H-5’), 4.86 (1H, dd, H-3’,
J = 5.3, J = 7.7 Hz), 4.69 (1H, d, CH2a-Bn, J = 11.2 Hz), 4.69 (1H, d, CH2b-Bn, J = 11.2 Hz), 4.56
(1H, d, CH2b-Bn, J = 11.2 Hz), 4.36 (1H, dd, H-4’, J = 7.9, J = 3.1 Hz), 2.87 (3H, s, 6-CH3), 1.43
(3H, d, 5’-CH3, J = 6.6 Hz); NOE: irradiation at H-1’ signal showed enhancement at H-4’
signal(3%) and irradiation at H-4’ signal showed enhancement at H-1’ signal (3%); Anal. Calcd.
for C30H30N4O6ꢀ0.5 H20: C, 67.67; H, 5.42; N, 9.42. Found: C, 67.69; H, 5.46; N, 9.29.
9-(3-O-Benzyl-α-L-talofuranosyl)-6-methylpurine (11). A solution of 10 (6 g, 10.4 mmol) was
dissolved in saturated NH3/MeOH (130 mL) and the mixture was kept stirring at rt for 2 days. The
solvent was evaporated and the residue was purified over a silica gel column (eluate: 7 % MeOH
1
in CHCl3) to give 11 (3.6 g, 96%) as a white glassy solid: H NMR (300 MHz, CDCl3): δH = 8.80
(1H, s, H-2), 8.79 (1H, s, H-8), 7.42-7.31 (5H, m, Bn-Ar), 6.08 (1H, d, H-1’, J = 6.1 Hz), 5.64
(1H, d, 2’-OH, J = 6.4 Hz, exchangable with D2O), 5.17 (1H, d, J = 6.3 Hz, exchangable with
D2O, 5’-OH), 4.78 (1H, m, H-2’), 4.76 (1H, d, CH2a-Bn, J = 12.2 Hz), 4.63 (1H, d, J = 12.2 Hz,
CH2b-Bn), 4.09 (1H, dd, H-3’, J = 3.4, J = 4.7 Hz), 4.02(1H, t, H-4’, J = 3.3 Hz), 3.82 (1H, m, H-
5’), 2.47(3H, s, 6-CH3), 1.13 (3H d, 5’-CH3, J = 6.5 Hz); NOE irradiation at H-1’ signal showed
enhancement at H-4’ signal (1%) and irradiation at H-4’ signal showed enhancement at H-1’ signal
(2%); UV λmax pH 1: 263.7 nm; 233.7 nm, pH 7: 244.1 nm, 260.2; pH 13, 260.7 nm; MS [FAB]
m/z 371 [M + H]+ , 377.1 [M + Na]+; Anal. Calcd. for C19H22N4O4ꢀ0.5 H20: C, 60.46; H, 5.99; N,
14.73. Found: C, 60.29; H, 6.11; N, 14.77.
9-(6-Deoxy-α-L-talofuranosyl)-6-methylpurine (3). A mixture of 11 (2.3 g, 6.1 mmol) and 10%
Pd/C (0.5 g) in EtOH (95%, 100 mL) was stirred for 6 h at room temperature under H2 atmosphere.
The mixture was filtered over Celite pad and the filtrate was evaporated under reduced pressure.
The residue was purified over a silica gel column (eluate: 10 % MeOH in CHCl3) to give 3 (1.5 g,
87%) as a white solid: MS [FAB] m/z 280.9 [M + H]+; UV λmax pH 1: 263.9 nm; 233.8 nm, pH 7:
244.5 nm, 260.9; pH 13; 260.8 nm; HPLC [100%; RT 14.918 min; 0.01M NH4H2PO4 : MeOH (85

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; 15); 20 min linear gradient from 10-90% B]; 1H NMR (300 MHz, DMSO-d6) δ = 8.79 (1H, s, H-
2), 8.78 (1H, s, H-8), 6.02 (1H, d, H-1’, J = 5.7 Hz), 5.50 (1H, d, 2’-OH, J = 5.9 Hz, exchangable
with D2O), 5.18 (1H, d, 3’-OH, J = 5.1 Hz, exchangable with D2O), 5.09 (1H, d, 5’-OH, J = 5.9
Hz, exchangable with D2O), 4.54 (1H, m, H-2’), 4.14 (1H, m, H-3’), 3.85-3.81 (2H, m, H4’ and H-
5’), 2.74 (1H, s, 6-CH3), 1.14 (3H, d, 5’-CH3, J = 6.15 Hz); 13C-NMR (75 MHz, DMSO-d6) δC =
158.62 (C-6), 151.46 (C-2), 149.93 (C-4), 144.35 (C-8), 133.07 (C-5), 92.08 (C-4 ̀), 87.23 (C-1’),
73.7 (C-2’), 69.78 (C-3’), 69.67 (C-5’), 26.42 (5’-CH3), 26.08 (5’-CH3). NOE: irradiation at H-3’
signal showed enhancemnet at H-8 signal (1%) and irradiation at H-8 signal showed enhancemnet
at H-3’ signal (1%); Anal. Calcd. for C12H16N4O4: C, 42; H, 75; N, 19.98. Found: C, 51.37.; H,
5.73; N, 19.74.
1-O-Acetyl-2,3,5-tri-O-benzoyl-α-L-lyxofuranose (12).[27] L-Lyxose (5 g, 33.3 mmol) was
dissolved in dry MeOH (40 mL) and treated with conc. H2SO4 (40 µL) at 0 °C under Ar.
atmosphere and the mixture was stirred for 72 h at rt. Pyridine (2 mL) was added and the solvents
were evaporated and coevaporated with toluene under reduced pressure. The residue was purified
by a silica gel column (eluate: 7% MeOH in CHCl3) to give Methyl α-L-lyxofuranoside[26] (3.34 g,
1
61 % yield) as a mixture of α and β anomers, which was crystalized from cold MeOH: H NMR
(300 MHz, DMSO-d6,) δH = 4.85 (1H, d, H-1, J = 3.73 Hz). 4.23 (1H, m, H-3), 4.15 (1H, dt, H-4, J
= 4.2, J = 6.8 Hz), 4.02 (1H, dd, H-2, J = 3.7, J = 4.8 Hz), 3.74 (1H, dd, H-5ª, J = 4.3, J = 11.9
Hz), 3.64 (1H, dd, H-5b, J = 6.8, J = 11.9 Hz), 3.24 (3H, s, OCH3). Benzoyl chloride (6.3 mL,
54.9 mmol) was added to a solution of 1-O-Methyl-α-L-lyxofuranose (2 g, 12.2 mmol) in dry
pyridine (50 mL) at 0 °C. The mixture was stirred for 30 min. at 0 °C, MeOH (2 ml) was added
and the mixture was stirred further for 10 min. The solvents were evaporated and the residue was
partitioned between EtOAc and H2O. The organic phase was dried (MgSO4) and evaporated. The
residue was purified by a flash silica gel column (eluate: 25% EtOAc in hexanes) to give Methyl
2,3,5-tri-O-benzoyl-α-L-lyxofuranoside (4.1 g, 73% yield) as a white solid: MS [FAB] m/z 477.5
1
[M + H]+; H NMR (CDCl3, 300 MHz) δH = 7.99-7.96 (6H, m, O-Bz), 7.53-7.47 (3H, m, p-Bz),
7.40-7.28 (6H, m, m-Bz), 6.03 (1H, t, H-3, J = 5.6 Hz), 5.63 (1H, dd, H-2, J = 1.43, J = 5.3 Hz),
5.23 (1H, d, H-1, J = 1.43 Hz), 4.84 (1H, dd, H-4, J = 5.9, J = 6.1 Hz), 4.72-4.60 (2H, m, H5a,b),
3.49 (3H, s, O-CH3). Methyl 2,3,5-tri-O-benzoyl-α-L-lyxofuranoside (1.2 g, 2.52 mmol) was
dissolved in AcOH (36 mL), Ac2O (3.6 mL) and the mixture was cooled to 0 °C. conc. H2SO4
(150 µL) was added dropwise to the mixture at 0 °C over 30 min. The mixture was stirred for 2 h
at rt. The mixture was pured into a cold saturated NaHCO3 solution and diluted with EtOAc. The
organic phase was separated, dried over (MgSO4) and evaporated. The residue was purified by a
flash silica gel column (eluate; 30% EtOAc/Hexanes) to give 12 (1.14 g, 89% yield) as a white
1
solid: MS [FAB] m/z 505 [M + H]+; H NMR (300 MHz, CDCl3) δH = 7.97-7.88 (6H, m, o-Bz),
7.53-7.52 (3H, m, p-Bz’s), 7.40-7.32 (6H, m, m-Bz’s), 6.50 (1H, d, H-1, J = 1.9 Hz), 6.09 (1H, dd,
H-3, J = 5.2, J = 5.8 Hz), 5.78 (1H, dd, H-2, J = 1.9, J = 5.2 Hz), 4.94 (1H, dd, H-4, J = 5.8, J =
6.0 Hz), 4.67- 4.65 (2H, m, H5a,b), 2.17 (3H, s, CH3).
9-(2,3,5-tri-O-Benzoyl-α-L-lyxofuranosyl)-6-methylpurine (13). 6-Methylpurine (0.66 g, 4.8
mmol) was suspended in a mixture of DCE (30 mL) and HMDS (6 mL) and treated with TMSCl
(0.6 mL). The mixture was heated for 3 h at 80 °C whereupon a complete dissolution was

ACCEPTED MANUSCRIPT
observed. After cooling down to room temperature, the solvents were evaporated and co-
evaporated with toluene (60 mL x 3 times), under reduced pressure to give a white solid.
Compound 12 (1.14 g, 2.22 mmol) in dry CH3CN (40 mL) was added to the silylated 6-MeP, the
mixture was then treated with SnCl4 (1M in CH2Cl2, 11.1 mL) at -10 °C. The mixture was stirred
for 4 h whereupon the starting material was completely consumed. Cold NaHCO3 (1M, 140 mL)
was added to the mixture, diluted with CHCl3 (150 mL), the organic phase was separated, dried
over MgSO4 and evaporated. The residue was purified by a silica gel column (eluate: 1.5:1 EtOAc
: hexanes) to give 13 (1.17 g, 88%) as a white foam: MS [FAB] m/z 579 [M + H]+; UV λmax pH 1:
1
265.1 nm; 232.7 nm, pH 7: 245.6 nm, pH 13, 260.6 nm;224.2 nm; H NMR (300 MHz, CDCl3)
δ = 8.91 (1H, s, H-2), 8.15 (1H, s, H-8), 8.07-7.25 (15H, m, Bz), 6.78 (1H, t, H-2’, J = 5.2 Hz),
6.44-6.40 (2H, m, H-2’ and H-3’), 5.50 (1H, m, H-4’), 4.80 (1H, dd, H-5’a, J = 8.94, J = 11.8 Hz),
13
4.71 (1H, dd, H-5’b, J = 5.2, J = 11.8 Hz), 2.88 (3H, s, 6-CH3,); C NMR (75 MHz, CDCl3) δ =
166.06 (C=O, Bz), 165.13 (C=O, Bz), 165.03 (C=O, Bz), 159.95 (C-6), 152.45 (C-2), 150.33 (C-
4), 143.12 (C-8), 133.98 (C-5), 133.75 (C-Ph), 133.68 (C-Ph), 133.21 (C-Ph), 129.79 (C-Ph),
129.72 (C-Ph), 129.30 (C-Ph), 128.73 (C-Ph), 128.62 (C-Ph), 128.40 (C-Ph), 128.33 (C-Ph),
128.15 (C-Ph), 88.03 (C-1’), 79.47 (C-4’), 74.88 (C-2’), 72.40 (C-3’), 62.33 (C-5’), 19.52 (6-CH3);
Anal. Calcd. for C32H26N4O7: C, 66.43; H, 4.53; N, 9.68. Found: C, 66.13; H, 5.66; N, 9.08.
9-(α-L-Lyxofuranosyl)-6-methylpurine (4). To a solution of 13 (0.54 g, 1.59 mmol) in dry
MeOH (15 mL) was added NaOMe (1M soln., 0.8 mL) dropwise at 0 °C. The mixture was stirred
for 2 h at rt, then neutralized with Amperlyst 50 (H+, weakly acidic). The mixture was filtered off
and the filtrate was evaporated under reduced pressure. The residue was purified by a flash silica
gel column (eluate: 10%MeOH in CHCl3) to give 4 (0.12 g, 86%) as a white solid: MS [FAB] m/z
1
267 [M + H]+; UV λmax pH 1: 264.5 nm; pH 7: 260.5 nm, pH 13, 260.6 nm; H NMR (300 MHz,
DMSO-d6) δ = 8.81 (1H, s, H-2), 8.79 (1H, s, H-8), 6.00 (1H, d, H-1’, J = 7.3 Hz), 5.53 (1H, d, 2’-
OH, J = 6.77 Hz, exchangable with D2O), 5.22 (1H, d, 3’-OH, J = 4.1 Hz, exchangable with D2O),
5.05 (1H, m, H-2’, J = 4.18, J = 7.3 Hz), 4.68 (1H, t, 5’-OH, J = 5.6 Hz, exchangable with D2O),
4.54 (1H, m, H-4’), 4.21 (1H, m, H-3’), 3.68 (1H, m, H-5’a, J = 5.3, J = 11.43 Hz), 3.55 (1H, m,
H-5’b, J = 6.6, J = 11.43 Hz,), 2.74 (3H, s, 6-CH3). 13C NMR (75 MHz, DMSO-d6) δ = 158.30 (C-
6), 151.69 (C-2), 150.28 (C-4), 144.82 (C-8), 133.1 (C-5), 87.73 (C-1’), 82.85 (C-4’), 74.67 (C-2’),
70.87 (C-3’), 59.84 (C-5’), 19.11 (6-CH3); Anal. Calcd. for C11H14N4O4ꢀ0.2 CH3OH: C, 49.34; H,
5.47; N, 20.55. Found: C, 49.43; H, 5.47; N, 20.56.
Methods for Biological Section.
In vivo studies. Parental and M64V PNP expressing D54MG (human glioma) tumor cells were
mixed so that 10% of the cells expressed M64V PNP. This mixture was injected subcutaneously
(2 x 107 total cells) into the flanks of nude mice (nu/nu) purchased from Charles River Laboratories
(Wilmington, MA, USA). When the tumors reached the appropriate size (approximately 300 mg),
the mice were treated with methyl(talo)-MeP-R. The compound was administered in the
peritoneal cavity 5 times a day (every 2 hours) for 3 consecutive days at doses of 5, 10, or 20
mg/kg. There were 6 mice per treatment group. The effect of compound on tumor size was
determined by measuring the tumors twice a week with calipers. Tumor weight was calculated
from the formula: [length (mm) x width2 (mm)]/2 = mg, assuming unit density and assuming that
the tumor takes the general shape of a prolate ellipsoid. In all in vivo experiments, mice were

ACCEPTED MANUSCRIPT
examined daily for evidence of mortality or other gross clinical signs. All procedures were
performed in accordance with a protocol that was approved by the IACUC of Southern Research
Institute.
Enzyme assay and HPLC analysis of purine nucleosides and bases. Enzymes were incubated
with various substrates in 1000 ꢁl reactions containing 50 mM potassium phosphate (pH 7.4), 100
ꢁM of substrate, and an appropriate amount of enzyme so that linear increase in product formation
could be followed over time. After incubation for 0, 0.25, 0.5, 1, and 2 hours at 25°C, 150 µl of
the reaction was removed and mixed with 150 µl of water, and the reaction was stopped by
boiling. The precipitated proteins were removed by filtration (0.2 ꢁm syringe filter), and the
sample was injected onto a 5 ꢁm BDS Hypersil C-18 column (150 x 4.6 mm) (Keystone Scientific
Inc., State College, PA). The mobile phase was a 50 mM ammonium dihydrogen phosphate buffer
(pH 4.5) containing acetonitrile (flow rate of 1 ml/minute). The appropriate amount of acetonitrile
(1.25 to 5%) was determined for each compound to optimize separation of substrate and product.
The nucleosides and their respective bases were detected as they eluted from the column by their
absorbance at their 254 nm. The percent conversion of substrate to product was calculated and the
specific activity was determined by dividing the number of nmoles of product formed by the mg
protein and the time of incubation.
Determination of plasma concentration of MeP-dR and methyl(talo)-MeP-R. Groups (4
mice/group) of nontumored animals were euthanized at various times (5, 15, 30, 60, and 120
minutes) after a single intraperitoneal injection of 100 mg/kg of [3H]-methyl(talo)-MeP-R or 67
mg/kg [3H]MeP-dR. The plasma was collected into heparinized tubes and frozen until the amount
of methyl(talo)-MeP-R or MeP-dR in each plasma sample could be determined using reverse phase
HPLC analysis as described above. The plasma samples were centrifuged through a Centrifree
Ultrafiltration Device (Millipore) prior to analysis by HPLC.
Acknowledgements. This investigation was supported by a National Cooperative Drug Discovery
Grant (U19CA67763) from the National Cancer Institute. We thank M.D. Richardson, and J.C.
Bearden of the Molecular Spectroscopy Laboratory of Southern Research Institute for analytical
and spectral data and S. Campbell for HPLC analyses. We are grateful to M. Kirk, University of
Alabama at Birmingham Comprehensive Cancer Center Shared Mass Spectrometry Facility, for
supplying some of the mass spectral data.
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Figure Captions
Figure 1. 6-Methylpurine and 2-fluoroadenine based prodrug structures.
Figure 2. Effect of methyl(talo)-MeP-R on D54 tumors in which 10% of the cells express M64V-
PNP.
Figure 3. Plasma concentrations after IP administration of methyl(talo)-MeP-R and MeP-dR.

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Table 1. Activity of sugar modified nucleosides with various phosphorylase enzymes.
Compd.
Human
PNP
Human
MTAP
E. coli
PNP
E. coli
M64V-PNP
T. vag
PNP
S. sul
MTAP
SAH/MTA
Hydrolyase
F. tul
PNP
(nmol/mg/hr) (nmol/mg/hr) (nmol/mg/hr) (nmol/mg/hr) (nmol/mg/hr) (nmol/mg/hr) (nmol/mg/hr) (nmol/mg/hr)
Adenosine
Inosine
MeP-dR
MeP-R
Lyxo-MeP-
R
-
596
-
<1
-
398,000*
342,000*
528,000*
96,000*
218
-
-
501,000
154,000
484,000
155,000
10,000
57,000
84,000
12,000
4,000
21,00
-
30
290
36
2,100
2000
3,900
4,800
180
391,000*
35
12
-
593,000
176,000
10,000**
<1
1,400
methyl(talo)- <1
MeP-R
4
915
86,000**
8,400
1500
51
100
Enzymes were incubated at 25°C with 100 µM of each compound, 50 mM potassium phosphate (pH 7.4), and an
appropriate amount of enzyme to measure a linear rate of cleavage. Substrate activity was measured using reverse phase
HPLC to separate product from substrate [8]. The enzyme concentration was changed based on substrate activity and the
lower limit for detection of activity is 1 nmoles/mg/hr. Each value shown is the average of at least two experiments which
were in good agreement. All enzymes were recombinants except for tularemia PNP which was isolated from crude extract
from tularemia. M64V PNP was prepared as described [12]. T. vaginalis PNP, S. solfataricus MTAP, and E. coli
SAH/MTA hydrolase was obtained for Dr. Steve Ealick at Cornell University. * Reference [5]. **Reference [12,16].

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Figure 1. Prodrug structures for 6-methylpurine and 2-fluoroadenine

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Figure 2. Effect of methyl(talo)-MeP-R on D54 tumors in which 10% of the cells express M64V
PNP.
Vehicle
2000
5 mg/kg
10 mg/kg
1000
20 mg/kg
0
0
20
40
60
Days post implant
Wild-type D54 tumor cells were mixed with D54 tumor cells that had been transduced with the M64V PNP gene so
that 10% of the mixture contained cells that expressed M64V PNP. This 90/10 mixture was injected sc into the flanks
of nude mice. Mice were treated with methyl(talo)-MeP-R at the doses shown in the figure 5 times daily (every 2
hours) for 3 consecutive days beginning on day 17 when tumors were approximately 300 mg. Values shown are the
mean and SEM of 6 measurements (10 in the vehicle treated group). The experiment has been repeated with similar
results.

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Figure 3. Plasma concentrations after IP administration of methyl(talo)-MeP-R and MeP-dR.
1000
100
5'-methyl(talo)-MeP-riboside
MeP-dR
10
1
0
30
60
90
120
150
Minutes after injection of 5'-methyl(talo)-MeP-riboside or MeP-dR
Mice were injected ip with 100 mg/kg [3H]5’-methyl(talo)-MeP-riboside or 67 mg/kg [3H]MeP-dR . Four mice were
sacrificed 5, 15, 30, 60, and 120 minutes after injection of drug, and the plasma was collected and frozen until the
amount of 5’-methyl(talo)-MeP-riboside (open circles) or MeP-dR (filled squares) could be determined using reverse
phase HPLC analysis. The figure shows the result of two experiments for each compound. The results are the mean
and standard deviation.

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aReagents and conditions. a) ref. 11. b) DEAD, TPP, BzOH, THF, 0 °C, 10 h, 86%; c) 20% HCl/MeOH-H2O, 6 h , rt,
then BzCl, dry pyr., 30 min, rt, 94%; d) Ac2O, AcOH, conc. H2SO4, 4 h, rt, 94%; e) 6-Mep, HMDS, DCE, TMSCl, 3
h, 80 °C, then SnCl4, CH3CN, -10 °C, 10 min, 96%; f) NH3/MeOH, 48 h, rt, 94%; g) 10% Pd/C , 95% EtOH, H2, 6 h,
rt, 87%.

ACCEPTED MANUSCRIPT
aReagents and conditions. a) i) conc. H2SO4, MeOH, 72 h, rt; ii) BzCl, dry Pyr., 30 min, 0 °C; iii) Ac2O, AcOH, conc.
H2SO4, 0 °C, 30 min, 39.6%; b) 6-MeP, HMDS, DCE, TMSCl, 3 h, 80 °C, then SnCl4, CH3CN, -10 °C, 40 min, 88%;
c) NaOMe, MeOH, 2 h, rt, 86%.

ACCEPTED MANUSCRIPT
Highlights

Methyl(talo)-MeP-R; 3 and lyxo-MeP-R; 4 were synthesized and evaluated as potential
prodrugs.
 The substrate activity 3 and 4 were evaluated with several phosphorylase enzymes.
In conjunction with M64V-E. coli PNPm, 3 showed good in vivo bystander activity.
