Synthesis and biological activity of 2-fluoro adenine and 6-methyl purine nucleoside analogs as prodrugs for suicide gene therapy of cancer

Article abstract of DOI:10.1081/NCN-200059237

A novel series of 6-methylpurine nucleoside derivatives with substitutions at 5′-position have been synthesised. These compounds bear a 5′-heterocycle such as triazole or a imidazole with a two carbon chain, and an ether, thio ether or amine. To extend the SAR study of 2-fluoroadenine and 6-methyl purine nucleosides, their corresponding α-linker nucleosides with L-xylose and L-lyxose were also synthesized. All of these compounds have been evaluated for their substrate activity with E. coli PNP. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200059237

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SYNTHESIS AND BIOLOGICAL ACTIVITY OF 2-FLUORO
ADENINE AND 6-METHYL PURINE NUCLEOSIDE ANALOGS
AS PRODRUGS FOR SUICIDE GENE THERAPY OF CANCER
A. V. Silamkoti a , P. W. Allan a , A. E. A. Hassan a , A. T. Fowler a , E. J. Sorscher a , W. B.
Parker a & J. A. Secrist a
a Southern Research Institute, Birmingham, AL, USA
Published online: 15 Nov 2011.
To cite this article: A. V. Silamkoti , P. W. Allan , A. E. A. Hassan , A. T. Fowler , E. J. Sorscher , W. B. Parker & J. A.
Secrist (2005) SYNTHESIS AND BIOLOGICAL ACTIVITY OF 2-FLUORO ADENINE AND 6-METHYL PURINE NUCLEOSIDE ANALOGS
AS PRODRUGS FOR SUICIDE GENE THERAPY OF CANCER, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 881-885, DOI:
10.1081/NCN-200059237
To link to this article: http://dx.doi.org/10.1081/NCN-200059237
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):881–885, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059237
SYNTHESIS AND BIOLOGICAL ACTIVITY OF 2-FLUORO ADENINE AND
6-METHYL PURINE NUCLEOSIDE ANALOGS AS PRODRUGS FOR
SUICIDE GENE THERAPY OF CANCER
A. V. Silamkoti, P. W. Allan, A. E. A. Hassan, A. T. Fowler, E. J. Sorscher,
5
W. B. Parker, and J. A. Secrist III
Southern Research Institute, Birmingham,
Alabama, USA
5
A novel series of 6-methylpurine nucleoside derivatives with substitutions at 5’-position have been
synthesised. These compounds bear a 5’-heterocycle such as triazole or a imidazole with a two carbon
chain, and an ether, thio ether or amine. To extend the SAR study of 2-fluoroadenine and 6-methyl
purine nucleosides, their corresponding aÀlinker nucleosides with L-xylose and L-lyxose were also
synthesized. All of these compounds have been evaluated for their substrate activity with E. coli PNP.
INTRODUCTION
Suicide gene therapy is a novel approach among those explored for selectively
killing tumor cells without harming normal cells. A strategy has been developed
earlier based on the selective expression of E. coli purine nucleoside phosphorylase
(E. coli PNP) in tumor cells, making these cells sensitive to an otherwise nontoxic or
weakly toxic agent. E. coli PNP selectively cleaves certain weakly toxic purine
nucleosides, such as certain 6-methylpurine and 2-fluoroadenine nucleosides to
their toxic purine or purine analogs.[1]
Several analogs of 6-methylpurine and 2-fluoroadenine nucleosides have been
prepared earlier for their substrate activity to E. coli PNP. Recently, we have been
examining a variety of mutant enzymes of E. coli PNP. These have included
mutation of Met 64 and His 4. Thus, our synthetic efforts have focused on finding
prodrugs specifically tailored to these mutant enzymes, thereby providing
additional specificity to the cleavage process. To that end, we have recently
synthesized 6-methyl-9-[5-O-[2-(1H-1,2,4-triazol-l-yl)ethyl]-b-D-ribofuranosyl]-9H (4)
This investigation was supported by the National Cancer Institute, National Cooperative Drug Discovery
Grant (U19CA67763). We thank Dr. J.M. Riordan, Mr. Mark Richardson, Mrs. J.C. Bearden, and Mrs. S.
Campbell for spectral and HPLC analyses. Ms. Stephanie Brannon for help in preparation of this poster.
Address correspondence to J. A. Secrist III, Southern Research Institute, Birmingham, AL 35255-5305, USA;
E-mail: secrist@sri.org
881
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882
A. V. Silamkoti et al.
9-[[5-deoxy-5-[2-(1H-imidazol-4-yl)ethyl]-thio]-b-D-ribofuranosyl]-6-methyl-9H-purine
(10), 2-fluoro-9-a-L-xylofuranosyl-9-H-purin-6-amine (14), 9-a-L-lyxofuranosyl-6-
methyl-9-H-purine (16), 2-fluoro-9-a-L-lyxofuranosyl-9-H-purine-6-amine (17) and
9-(2-deoxy-a-L-lyxofuranosyl)-2-fluoro-9-H-purine-6-amine (18). Compounds 4, 7,
and 10 are designed to provide 5-membered ring replacements for the imidazole
that is listd in various His4 mutations. The other compounds focus on Met64
mutations. All of these newly synthesized compounds were evaluated for their
substrate activity to E. coli PNP and several mutant forms of E. coli PNP. The
detailed synthesis of these compounds is presented.
CHEMISTRY
The target compound 9H-purine-9-[5-O-[2-(1H-1,2,4-triazol-1-yl)ethyl]-b-D-ribo-
furanosyl]-6-methyl (4) was synthesized as depicted in Scheme 1. Inosine was
converted to 2 following the usual method.[2] The two carbon ether linkage was
generated by reacting 2 with ethyl bromoacetate under basic conditions to yield the
corresponding ester, 9H-purine-9-[5-O-[ethyl-2-acetate]-2,3-isopropylidene-b-D-ribo-
furanosyl]-6-methyl. The ester functional group was reduced to alcohol with LiBH4
in the presence of MeOH to afford the hydroxyl compound 9H-purine-9-[5-O-
[ethanol-2-yl]-2,3-isopropylidene-b-D-ribofuranosyl]-6-methyl. This hydroxyl group
was then converted to the mesityl derivate 9H-purine-9-[5-O-[2-(O-methanesul-
phonyl)-ethyl]-2,3-isopropylidene-b-D-ribofuranosyl]-6-methyl (3). 3 was coupled
with triazole under basic conditions to give 9H-purine-9-[5-O-[2-(1H-1,2,4-triazol-1-
yl)ethyl]-2,3-isopropylidene-b-D-ribofuranosyl]-6-methyl this on acidic deprotection
of isopropylidene group afforded 4.
The synthesis of 9H-purine-9-[[5-deoxy-5-[2-(1H-imidazol-4-yl)ethyl]-thio]-b-D-
ribofuranosyl]-6-methyl (7) is outlined in Scheme 2. 2 was converted to the thio
ester 9H-purine-9-[[5-deoxy-5-(acetyl)-thio]-b-D-ribofuranosyl]-6-methyl by reacting
with thiol acetic acid, under Mitsunobu conditions. Followed by the condensation
with 4-(2-methanesulphonylethyl)-N-(t-butyloxycarbonyl)-1H-imidazole (6) in
SCHEME 1 Reagents and conditions: a) acetone, HC(OEt)3, TsOH, RT, 4 hrs; b) Ac2O-pyridine, 40°C, 2 hrs;
c) POCl3, DMF, 1,2-Dichloroethane, reflux, 3 hrs; d) MeZnCl, Tetrakis, THF, RT—reflux, 2 hrs; e) MeONa-MeOH,
RT, 1 hr; f) NaNH2, 1,4-dioxane, BrCH2CO2Et, reflux, 2 hrs; g) LiBH4, Et2O-MeOH, 0°C, 5–10 mins;
h) MsCl,TEA, DCM, 0°C, 1 hr; i) NaH, Triazole, 1,4-dioxane, reflux, 2 hrs; j) 1N HCl, THF, RT, overnight.

A. V. Silamkoti et al.
883
SCHEME 2 Reagents and conditions: a) Boc2O, MeOH, TEA, RT, overnight; b) MsCl, TEA DCM, RT, 1 hr;
c) CH3COSH, Ph3P, DEAD, overnight; d) NaOMe, comp-4, MeOH, 0°C-RT, overnight; e)1N HCL, THF,
RT, overnight.
MeOH in the presence of NaOMe to give 9H-purine-9-[[5-deoxy-5-[2-(1H-imidazol-
4-yl)ethyl]-thio]-2,3-isopropylidene-b-D-ribofuranosyl]-6-methyl. This on hydrolysis of
isopropylidene group afforded the target molecule 7.
Preparation of 9H-purine-9-[[5-deoxy-5-[2-(1H-imidazol-4-yl)ethyl]-amino]-b-D-
ribofuranosyl]-6-methyl (10) was accomplished by following the steps as described
in Scheme 3. The mesityl analogue of 2 was condensed with N-1-bis[t-
butoxycarbonyl]-histamine (9), in DMF and NaH as base, the resulting compound
9H-purine-9-[[5-deoxy-5-[2-(1H-imidazol-4-yl)ethyl]-amino-5-N-(t-butyloxycarbonyl)]-
b-D-ribofuranosyl]-6-methyl was deprotected to afford 10.
Scheme 4 describes the synthetic pathway of the target molecule 9-H-purin-6-
amine-2-fluoro-9-a-L-xylofuranosyl (14). The conversion of L-xylose to 1-chloro-
2,3,5-tri-O-benzyl-L-xylofuranoside,[4,5] (11), was accomplished by the usual
method. The silylated derivative of 2-fluoroadenine (13) was coupled with 11
under neutral condition, using powdered molecular sieves, in 1,2-dichloroethane at
reflux temperature. The resulting nucleoside 9-H-purin-6-amine-2-fluoro-9-a-L-2,3,5-
tri-O-benzyl-xylofuranosyl was deblocked with BCl3 to give 14.
9-H-Purine-9-a-L-lyxofuranosyl-6-methyl (16), 9-H-purin-6-amine-2-fluoro-9-a-L-
lyxofuranosyl (17) and 9-H-purin-6-amine-9-(2-deoxy-a-L-lyxofuranosyl)-2-fluoro
(18) were synthesized following the steps illustrated in Scheme 5. L-lyxose was
converted to 1-O-acetyl-2,3,5-tri-O-benzoyl-L-lyxofuranose (12),[6] following the
reported procedure. 12 was coupled with the respective silylated derivatives of 13
& 15 using SnCl4 as the coupling agent. Saponification of the resulting nucleosides
led to compounds 17 & 16, respectively. The 2’-hydroxy of 17 was deoxygenated
following the reported procedures[7] with some modifications to afford 18.
SCHEME 3 Reagents and conditions: a) Boc2O, MeOH, TEA, RT, overnight; b) MsCl, TEA, DCM, RT, 1 hr;
c) Comp-9, NaH, DMF, 0°C–100°C, 2 hrs; d) 1N HCl, THF, RT, overnight.

884
A. V. Silamkoti et al.
SCHEME 4 Reagents and conditions: a) N,O-bis(trimethylsilyl)acetamide, AcCN, reflux one hour; b) Comp-
11 molecular sieves, 1,2-dichloroethane, reflux; c) BCl3 DCM, 0°C-RT, overnight.
1
The structures of all the target molecules were confirmed by MS, H-NMR,
HPLC, and CHN analysis.
RESULTS
Compounds 4, 7, and 10 were incubated with crude extracts (100 mg/mL) of
E. coli that were transfected with E. coli PNP or the various E. coli PNP mutants
(H5A, H5I, H5L, H5M, H5V, and M64V). Compound 10 was not cleaved by any
of the extracts. Compound 7 was readily cleaved by non-transfected cells and its
cleavage has not enhanced in any of the bacterial extracts that had been transfected.
These results indicated that 7 was a good substrate for an enzyme other than PNP
that was endogenously expressed in the bacterium. Compound 4 was not cleaved
by purified E. coli PNP or the M64V mutant. However, it was cleaved at a rate of
3 nmoles/mg/hr in non-transfected cells. Greater cleavage was detected in E. coli
that had been transfected with H5A (9 nmoles/mg-hr), H5I (15 nmoles/mg-hr), H5L
SCHEME 5 Reagents and conditions: a) HMDS, (NH4)2 SO4, reflux, 31 h; b) Comp-12 SnCl4, AcCN, À10°C
to RT, 3–5 h; c) 0.5 M KOH, EtOH, RT, 5 h; d) HMDS, TMSCl, 1,2-dichloroethane, reflux, 2 h; e) NaOMe,
MeOH, 5°C, 2 h; f) TIPDSCl2, pyridine, RT, overnight; g) ImC(S)Im, DMAP, AcCN, 80°C, overnight; h) TTMSS,
ButOOBut, toluene, reflux, 2 h; i) Et4NFxH2O, AcCN, RT, 1 h.

A. V. Silamkoti et al.
885
TABLE 1 E. coli PNP and M64V Incubated with 100 mM of Each Compound Shown
Compound
E. coli PNP
M64V
M64V/WT
MeP-dR#
528,000
8,000
<1
218
2,600
593,000
244,000
4
10,000
32,000
1
30
>4
50
12
17
14
16
18
The rate of cleavage was determined by HPLC separation of base from nucleoside. Each number is
the average of at least 2 determinations and is represented in nmol/mg/hr.
(21 nmoles/mg-hr), and H5V (12 nmoles/mg-hr). Since these values were sig-
nificantly above the rate of cleavage in non-transfected cells, these enzymes had
increased ability to cleave this compound over wild-type enzyme, although we
believe that the rate cleavage is too low to be useful in a gene therapy approach for
the treatment of cancer. F-araA, which is a poor substrate for E. coli PNP, is cleaved
at a rate of 1000 nmoles/mg/hr in cells transfected with E. coli PNP (Table 1).
Since 9-H-purin-6-amine-2-fluoro-9-a-L-lyxofuranosyl (17) was a good substrate
for the M64V mutant, we made other analogs with similar structure to explore the
SAR of this enzyme (14, 16, 18).
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