Regioselective metalation of 6-methylpurines: Synthesis of fluoromethyl purines and related nucleosides for suicide gene therapy of cancer

Article abstract of DOI:10.1080/15257770903091938

Metalation of 6-methyl-9-(tetrahydro-2H-pyran-2-yl)purine (10) with lithiating agents of varying basicities such as n-BuLi and LiHMDS in THF at -78C resulted in metalation at both of the 6-CH3 moiety and the 8-CH position, irrespective of the molar equiva

Full text of DOI:10.1080/15257770903091938

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Regioselective Metalation of
6-Methylpurines: Synthesis of
Fluoromethyl Purines and Related
Nucleosides for Suicide Gene Therapy of
Cancer
Abdalla E. A. Hassan a , William B. Parker a , Paula W. Allan a & John
A. Secrist III a
a Southern Research Institute, Drug Discovery Division, Birmingham,
Alabama, USA
Published online: 29 Jul 2009.
To cite this article: Abdalla E. A. Hassan , William B. Parker , Paula W. Allan & John A. Secrist III
(2009) Regioselective Metalation of 6-Methylpurines: Synthesis of Fluoromethyl Purines and Related
Nucleosides for Suicide Gene Therapy of Cancer, Nucleosides, Nucleotides and Nucleic Acids, 28:5-7,
642-656, DOI: 10.1080/15257770903091938
To link to this article: http://dx.doi.org/10.1080/15257770903091938
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Nucleosides, Nucleotides and Nucleic Acids, 28:642–656, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903091938
REGIOSELECTIVE METALATION OF 6-METHYLPURINES: SYNTHESIS
OF FLUOROMETHYL PURINES AND RELATED NUCLEOSIDES FOR
SUICIDE GENE THERAPY OF CANCER
Abdalla E. A. Hassan, William B. Parker, Paula W. Allan, and
John A. Secrist III
Southern Research Institute, Drug Discovery Division, Birmingham, Alabama, USA
Metalation of 6-methyl-9-(tetrahydro-2H-pyran-2-yl)purine (10) with lithiating agents of vary-
2
ing basicities such as n-BuLi and LiHMDS in THF at −78◦C resulted in metalation at both of
the 6-CH3 moiety and the 8-CH position, irrespective of the molar equivalence of the base. On the
other hand, a regioselective metalation at the 6-CH3 moiety of 10 was observed with NaHMDS or
KHMDS, under similar conditions. Treatment of the potassium salts of 10 and of the protected ri-
boside derivative 6-methyl-9-(β-D-2,3,5-tri-O-tert-butyldimethylsilylribofuranosyl)purine (22) with
N-fluorobenzenesulfonamide (NFSI) at −78◦C gave the corresponding 6-fluoromethylpurine deriva-
tives 11 and 23, respectively, in good yields. Deprotection of 11 and 23 under standard conditions
gave 6-fluoromethylpurine (6-FMeP, 3) and 6-fluoromethyl-9-(β-d-ribofuranosyl)purine (6-FMePR,
4), respectively, in high yield. Both 3 and 4 demonstrated cytotoxic activity against CCRF-CEM
cells in culture. 6-FMePR is a good substrate for E. coli purine nucleoside phosphorylase (E.
coli PNP) with a comparable substrate activity to that of the parent nucleoside, 6-methyl-9-(β-
d-ribofuranosyl)purine (6-MePR, 21). The cytotoxic activity of 6-FMeP along with the substrate
activity of 6-FMePR with E. coli PNP meet the fundamental requirements for using 6-FMeP as a
potential toxin in PNP/prodrug based cancer gene therapy.
Keywords Metalation; electrophilic fluorination; purine nucleoside phosphorylase;
suicide gene therapy of cancer
INTRODUCTION
We have developed a cancer gene therapy strategy that is based on
the activation of a non-toxic purine nucleoside (prodrug) to a highly
Received 6 March 2009; accepted 29 May 2009.
Dedicated to Dr. Morris J. Robins on his 70th birthday.
This investigation was supported by a National Cooperative Drug Discovery Grant (CA67763) from
the National Cancer Institute. We thank J. M. Riordan, M. D. Richardson and J. C. Bearden of the
Molecular Spectroscopy Section of Southern Research Institute for analytical and spectral data and S.
Campbell for HPLC analyses.
Address correspondence to John A. Secrist III, Southern Research Institute, P.O. Box 55305,
Birmingham, AL, 35255-5305. E-mail: secrist@sri.org
642

Regioselective Metalation of 6-Methylpurines
643
CHART 1
toxic purine analog by a non-human gene, E. coli purine nucleoside
phosphorylase (E. coli PNP), which can be selectively expressed in tumor
cells.[1–6] E. coli PNP differs from human PNP in its ability to accept not
only 6-oxopurine nucleosides, but also 6-aminopurine and certain other
6-substituted purine nucleoside analogs as substrates.[7,8] This property
has been used to cleave relatively non-toxic purine nucleoside analogs to
very toxic purine analogs, which readily diffuse across cell membranes and
have high bystander activity.[1,3] The toxic adenine analogs of most interest
to date are 6-methylpurine (6-MeP, 1) and 2-fluoroadenine (2)[4] (Chart
1); however, we still continue to search for the optimal toxin/prodrug
combination that would have the desired biological properties. The small
size and powerful electron-withdrawing properties of fluorine dramatically
affect the physical (e.g., lipophilicity), chemical, and biological properties
of organic compounds. Substitution of hydrogen atoms for fluorine at
the nucleobase[9–17] and at the sugar moiety[18–20] of nucleosides have
produced several anticancer and antiviral drugs as well as other molecules
that are undergoing clinical investigation. Herein, we report on selective
metalation at the 6-CH3 moiety of 6-methylpurine derivative 10 and its
application for the synthesis of 6-FMeP (3) as a potential toxin for appli-
cation in PNP-based cancer gene therapy (Chart 1). We have also applied
the methodology to a nucleoside precursor to prepare 6-FMePR (4). A
preliminary account of this research has previously been published.[21] In
addition, another very useful approach to 6-fluoromethylpurine derivatives
has appeared.[22]
RESULTS AND DISCUSSION
Chemistry
During the course of the synthesis of 5ꢁ-alkoxy derivatives of 6-methyl-
9-(β-d-ribofuranosyl)purine as potential prodrugs for 6-MeP, we have ob-
served under basic conditions a C-alkylation at the 6-methyl along with
the desired O-alkylation at the 5ꢁ-hydroxyl. The precursor for the alky-

644
A. E. A. Hassan et al.
SCHEME 1 a) Ac2O, pyridine, 4 hours, room temperature., 95%; b) CH3ZnBr, Pd (PPh3)4, THF, 1
hour, 55◦C, then MeOH/NH3, 4 hours, room temperature, 77%; c) t-BuOK, CH3I, THF, 30 minutes,
0◦C, 87% for 8 and 7% for 9.
lation reaction, 6-methyl-9-(2,3-O-isopropylidene-β-D-ribofuranosyl)purine
(7),[23] was synthesized in good yield (Scheme 1) following our reported
procedure.[24] Treatment of 7 with t-BuOK (1.2 equiv.) in THF in the
presence of MeI gave the O,C-bisalkylated derivative 9 in 7% yield along
with the desired 5ꢁ-O-methyl product 8 in 87% yield (Scheme 1). When
the above reaction was carried out with the stronger base NaH (1.5 equiv.)
under similar conditions, the amount of 9 was increased at the expense of
the monoalkylated product 8 (9:8, 1.1:1). These results led us to examine
the feasibility of the synthesis of 6-fluoromethylpurines utilizing a selective
metalation-electrophilic fluorination of the 6-CH3 moiety of 6-methylpurine
derivatives.
We first attempted deprotonation of 6-methylpurine derivative 10[25]
using t-BuOK in THF and quenching with N -fluorobenzenesulfonimide
(NFSI) at −40◦C or at −78◦C. At both temperatures a complex mixture
was obtained and none of the desired monofluorinated product 11 was
isolated (Scheme 2). This result was attributed to the higher acidity of the
fluoromethyl protons as compared to the starting material. This conclusion
was supported by conducting the reaction in the presence of electrophiles
with similar electronic characteristics under similar conditions. Treatment
of 10 with NaHMDS in the presence of diphenyl disulfide at −78◦C gave
6-[bis(phenylthio)methyl] derivative 12 in 78% yield as a sole product and
neither the 8-phenylthio or 6-phenylthiomethyl derivatives were isolated.
Similar results were also obtained upon treatment of 10 with NaHMDS at
−78◦C and quenching with TsCl to give a mixture of 6-chloromethylpurine
derivative 13 and 6-trichloromethyl derivative 14 in good yields. To our
surprise, lithiation[26] of 10 with 5 equivalents of n-BuLi or LDA in THF
at −78◦C in the presence of I2 gave 8-iodo-6-methylpurine derivative 15 in
82% yield based on recovered starting material.

Regioselective Metalation of 6-Methylpurines
645
SCHEME 2 a) NaHMDS, (PhS)2, THF, 30 minutes, −78◦C—room temperature, 78%; b) LDA, THF, I2,
1 hour, −78◦C, 48% c) NaHMDS, TsCl, THF, 20 minutes, −78◦C, 41% for 13 and 8% for 14; d) KHMDS,
NFSI, THF, −80◦C, 57%. e) 1N HCl, THF, 72 hours, room temperature, 95%.
These results led us to examine the role of the basicity, the counter
cation, and the molar equivalents of the base on the regioselectivity of
the metalation of 10 in the presence of CH3I as electrophile (Scheme 3).
Lithiation of 10 with n-BuLi (5 equiv.) in THF at −78◦C, and quenching
with CH3I resulted in alkylation at the 6-CH3 and the C-8 positions to
give the 6-ethyl-8-methyl purine derivative 17 in 75% isolated yield (Table
1, entry 1). The use of LDA (1 equiv.) under similar conditions gave the
C-8 alkylated product 6,8-dimethylpurine 16[27] as the major product along
with a mixture of the starting material 10, 6-ethyl-8-methylpurine 17, and

646
A. E. A. Hassan et al.
SCHEME 3 a) Lithiating agent Table 1, CH3I, 30 minutes, −78◦C; b) NaHMDS or KHMDS, CH3I, 30
minutes, −78◦C.
6-ethylpurine 19 as a minor product (Table 1, entry 2). Increasing the
molar equivalence of LDA (2 equiv.) increased the yield of the bisalkylation
product 17 at the expense of the C-8 alkylation product 16 (Table 1,
entry 3). Similar results were also obtained with Et2NLi (Table 1, entries
4, 5), with additional equivalents resulting in significant formation of the
trialkylated product 18. When the relatively weak base LiHMDS was used, a
moderately regioselective lithiation at the 6-CH3 moiety was observed (Table
1, entry 7). These results demonstrate that lithiation of 6-methylpurine
derivative 10 with the strong bases n-BuLi, LDA, or LiNEt2 is poorly selective
regardless of the molar equivalence, while the weaker base LiHMDS induces
a selective metalation at the 6-CH3 moiety. We next examined metalation
of 10 with bases NaHMDS and KHMDS under the same conditions as
used for the lithium salt. With NaHMDS metalation at the 6-CH3 occurred
regioselectively to give a mixture of 6-ethylpurine derivative 19 and 6-
isopropylpurine derivative 20[28,29] in a ratio of (19:20, 10.5:1; Table 1, entry
7). No 8-methylpurine was formed, as confirmed from the HPLC analysis of
the crude product. Similar regioselective metalation at the 6-CH3 moiety
was obtained with KHMDS (Table 1, entry 8). These results as presented in
Table 1 correlate the metalation regioselectivity with the nature of the metal
ion as well as strength and steric aspects of the base.
We next attempted the metalation of 10 using KHMDS (5 equiv.) in
THF at −78◦C, followed by treatment with NSFI (1.1 equiv.). A complex
mixture containing the desired product 11 was obtained, as judged by

Regioselective Metalation of 6-Methylpurines
647
TABLE 1 Effect of the base on the regioselectivity of the metallation of 10
Ratiob
Basea (molar
Entry
equivalents)
10
16
17
18
19
20
1
2
3
4
5
6
7
8
n-Bu Li (5 eq.)
LDA (1 eq.)
LDA (2 eq.)
LiNEt2 (1.2 eq.)
LiNEt2 (5 eq.)
LiHMDS (5 eq.)
NaHMDS (5 eq.)
KHMDS (5 eq.)
0.5
22.6
2.3
51.6
—
2.0
0.04
0.02
2.9
61.5
33.0
23.5
16.6
—
83.8
8.6
48.6
3.5
31.3
14.0
—
2.3
—
—
3.6
3.7
3.1
18.6
12.7
54.1
89.4
92.1
—
—
—
—
—
11.9
8.5
5.5
—
36.5
10.6
—
0.45
0.35
—
—
a
Metallation reactions were performed in THF −78◦C for 30 minutes at and quenched with CH3I
(10 equiv.).
bThe ratio of the products was determined by the% area of the HPLC chromatograms after a flash
silica gel column chromatography, not normalized to 100%.
mass spectral analysis of the crude product. After examination of several
ratios of KHMDS and NSFI as well as variations in the reaction time of
the fluorination step, we found that the use of two equivalents of both
KHMDS and NSFI followed by quenching the fluorination reaction in less
than 5 minutes at −78◦C, produced the desired 6-FMeP derivative 11 in
1
58% yield. The H-NMR spectrum of 11 shows the 6-CH2F signals as two
sets of dd with the characteristic large fluorine-proton coupling constant;
J F,H = 46.7 Hz. Applying the same sequence of metalation-fluorination
(KHMDS/NSFI/THF/−78◦C) reactions to the ribonucleoside derivative 22
produced the corresponding 6-fluoromethylpurine derivative 23 in 48%
yield (Scheme 4). Deprotection of 11 and 23 under conventional conditions
gave 6-FMeP (3) and 6-FMePR (4) in high yields.
SCHEME 4 a) KHMDS, NFSI, THF, −80◦C, 47%; b) Et4NF·xH2O, CH3CN, 2 hours, room temperature,
95%.

648
A. E. A. Hassan et al.
TABLE 2 Cleavage of 6-FMePR (4) and 6-MePR (21) by E. coli PNP
Entry
Compound
Mean SD (nmoles/mg/hr)a
1
2
3
6-FMePR
6-MePR
Adenosine
66, 000
98, 000
398, 000
aCompounds (100 µM) were incubated with E. coli PNP and the substrates and products were
separated by HPLC as described.[6] Number is the average of at least two determinations.
Biology
The newly synthesized compounds, 3 and 4 were evaluated for their
cytotoxic activity against CCRF-CEM cells as described previously.[4] Cells
were incubated with various concentrations of compound for 72 hours, and
the concentration of 6-FMeP (3) that inhibited cell growth by 50% (IC50)
was 20 µM (N = 2), which was greater than the IC50 of 6-methylpurine (1.2
µM).[4] The ribonucleoside derivative 4 also showed potent cytotoxic activity
with an IC50 of 0.07 µM, which was similar to that of 6-MePR (0.05 µM).
These results suggested that 6-FMeP and 6-FMePR were substrates for the
activating enzymes (adenine phosphoribosyltransferase and adenosine ki-
nase, respectively) and that 6-FMeP nucleotides formed from these analogs
were toxic to human cells, as are the MeP nucleotides. 6-FMeP-R was also a
good substrate for E. coli PNP. The specific activity of 66,000 nmoles/mg-hr
was similar to 98,000 nmoles of 6-MeP-R cleaved/mg-hr (Table 2), which
indicated that the fluorine atom did not affect enzyme activity.
CONCLUSIONS
We have demonstrated that a regioselective metalation at the 6-CH3 of
6-methylpurine derivative 10 is achieved with bases NaHMDS and KHMDS,
while less selective metalation at both C-8 and at the alkyl side chain(s) is
observed with various lithium bases. We have utilized this observation for
the synthesis of the novel purine derivative 6-FMeP, 3 and its ribonucleoside
derivative 6-FMePR, 4 via metalation-fluorination. 6-FMeP (3) was found
to have moderate cytotoxic activity against CCRF-CEM cells in vitro, and
its ribonucleoside derivative 4 was found to have a good substrate activity
with E. coli PNP. These biological properties suggest that 6-FMeP could be
considered as a toxin in the PNP-based gene therapy of cancer, and that a
6-FMeP-containing nucleoside suitably blocked to prevent phosphorylation
could be a suitable prodrug cleavable to 6-FMeP.
EXPERIMENTAL
Melting points were determined on a Mel-temp apparatus and are
1
uncorrected. H NMR and 13C NMR spectra were recorded on a Nicolet

Regioselective Metalation of 6-Methylpurines
649
NT 300NB spectrometer (Madison, WI, USA) operating at 300.635 MHz
(1H) or 75.6 MHz (13C). Chemical shifts were expressed in parts per
million from tetramethylsilane. The hydrogen-decoupled 13C NMR was
assigned by comparison of the J CH values obtained from hydrogen-coupled
13C NMR spectra, and when necessary, selective hydrogen decoupling was
performed in order to confirm the assignments. The NOE experiments
were conducted in degassed CDCl3. To minimize the effects of magnetic
perturbations with the sample nonspinning, eight FIDs were recorded with
the decoupler set to a desired frequency and eight FID’s were recorded
with the decoupler off-resonance. Ultraviolet absorption spectra were de-
termined on Perkin-Elmer Lambda 9 spectrometer (Norwalk, CT, USA) by
dissolving each compound in methanol or water and diluting 10-fold with
0.1 N HCl, pH 7 buffer, and 0.1 N NaOH. Mass spectra were recorded
on a Varian/MAT 311A double-focusing mass spectrometer (Palo Alto,
CA, USA) in the fast atom bombardment (FAB) mode (glycerol matrix).
HPLC analysis were carried out on a Hewlett-Packard 1100 series liquid
chromatograph (Wilmington, DE, USA) with a Phenomenex Sphereclone
5 µ ODS (1) column (4.6 mm × 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 (Newark, DE, USA) precoated
(250 µm) silica gel (GF) plates. Compounds 5[10] and 10[11] have been
previously fully characterized. The 6-fluoromethylpurine derivatives 3 and 4
have full characterization data that agrees with the literature.[22] The various
other 6- and 8-substituted purines are characterized with spectral data alone.
6-Chloro-9-(5-O-acetyl-2,3-O-isopropylidene-β-
D-ribofuranosyl)purine (6)
Acetic anhydride (90 µL, 0.92 mmol) was added to a solution of 5[10]
(150 mg, 0.46 mmol) in dry pyridine (5 mL) at 0◦C. The mixture was
stirred for 4 hours at room temperature, EtOH (1 mL) and the solvents
were evaporated in vacuo. Water work up and flash silica gel column
chromatography (eluate: 30% EtOAc in hexanes) gave (160 mg, 95%) of
1
6 as a white solid: MS m/z 369 (M+1)+, H NMR (CDCl3) δ 8.79 (1H, s,
H-2), 8.25 (1H, s, H-8), 6.20 (1H, d, H-1ꢁ, J = 2.2 Hz), 5.43 (1H, dd, H-2ꢁ, J
= 2.2, J = 6.4 Hz), 5.03 (1H, dd, H-3ꢁ, J = 3.5, J = 6.4 Hz), 4.54 (1H, ddd,
H-4ꢁ, J 3 ,4 = 3.5, J 4 ,5a = 4.2, J 4 ,5b = 5.9 Hz), 4.36 (1H, dd, H5ꢁa, J 4 ,5a
=
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
4.2, J 5 a,5 b = 12.1 Hz), 4.24 (1H, dd, H5ꢁb, J 4 ,4b = 5.9, J 5 a,5 b = 12.1 Hz),
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
1.99 (3H, s, Ac), 1.65 (3H, s, CMe2), 1.41 (3H, s, CMe2).
6-Methyl-9-(2,3-O-isopropylidene-β-D-ribofuranosyl)purine
(7)[23,24]
Pd (PPh3)4 (46 mg, 0.04 mmol) in THF (1 mL) was added to a solution
of CH3ZnBr (0.23 mL, 0.95 mmol) in THF (4 mL) at room temperature.

650
A. E. A. Hassan et al.
Compound 6 (0.14 g, 0.38 mmol) in THF (1 mL) was added and the mixture
was stirred for 1 hour at 55◦C. The solvent was evaporated in vacuo and
the residue was partitioned between EtOAc and H2O. The organic phase
ꢁ
was dried (MgSO4) and evaporated. The residue was dissolved in MeOH
saturated with NH3 (5 mL) and stirred for 4 hours at room temperature.
The solvent was evaporated and the residue was purified by silica gel
chromatography (eluate; 1% MeOH in CHCl3) to give (89 mg, 77%) of 7 as
a white solid; HPLC 100% (RT 5.297, 0.01 M NH4H2PO4: MeOH 60: 40);
MS m/z 307.1 (M+1)+; UV λmax (pH 1) 264.2, λmax (pH 7) 260.3, λmax (pH
13) 260.5 nm; 1H NMR (CDCl3) δ 8.84 (1H, s, H-2), 8.09 (1H, s, H-8), 5.92
(1H, d, H-1ꢁ, J = 4.8 Hz), 5.86 (1H, dd, 5ꢁ-OH, J = 2.0, J = 11.7 Hz), 5.23
(1H, br dd, H-2ꢁ), 5.13 (1H, br dd, H-3ꢁ), 4.56 (1H, br d, H-4ꢁ), 3.99 (1H,
m, H-5ꢁa), 3.82 (1H, m, H-5ꢁb), 2.88 (3H, s, 6-CH3), 1.66 (3H, s, CMe2),
1.39 (3H, s, CMe2); Anal. Calcd. for C14H18N4O4·: C 54.89, H 5.92, N 18.29;
Found: C 54.52, H 5.96, N 18.41.
6-Methyl-9-(2,3-O-isopropylidene-5-O-methyl-β-
D-ribofuranosyl)purine (8) and 6-ethyl-9-(2,3-O-isopropylidene-
5-O-methyl-β-D-ribofuranosyl)purine (9)
A 1M solution of t-BuOK in THF (0.22 mL, 0.26 mmol) was added to a
solution of 7 (66 mg, 0.22 mmol) in THF (2 mL) at 0◦C and the mixture was
stirred for 5 minutes. CH3I (50 µL, 0.52 mmol) was added and the mixture
was stirred for 30 minutes at 0◦C. The volatiles were evaporated and the
residue was purified by a flash silica gel chromatography (eluate: 40%-20%
hexanes in EtOAc) to give (64 mg, 87%) of 8 as a colorless syrup and (5 mg,
7%) of 9 as a colorless syrup: Spectroscopic parameters for 8: MS m/z 321
(M+1)+; UV λmax (pH 1) 264.0, λmax (pH 7) 260.2, λmax (pH 13) 260.5 nm;
1H NMR (CDCl3) δ. 8.87 (1H, s, H-2), 8.23 (1H, s, H-8), 6.28 (1H, d, H-1ꢁ,
J = 2.5 Hz), 5.27 (1H, dd, H-2ꢁ, J = 2.4, J = 6.2 Hz), 4.98 (1H, dd, H-3ꢁ,
J = 2.3, J = 6.2 Hz), 4.52 (1H, m, H-4ꢁ), 3.64 (1H, dd, H-5ꢁa, J = 3.2, J =
10.4 Hz), 3.57 (1H, dd, H-5ꢁb, J = 4.2, J = 10.4 Hz), 3.33 (3H, s, 5ꢁ-O-CH3),
2.87 (3H, s, 6-CH3), 1.65 (3H, s, CMe2), 1.40 (3H, s, CMe2); spectroscopic
parameters for 9: MS m/z 335 (M+1)+; UV λmax (pH 1) 265.4, λmax (pH 7)
1
260.8, λmax (pH 13) 261.3 nm; H NMR (CDCl3) δ. 8.91 (1H, s, H-2), 8.28
(1H, s, H-8), 6.28 (1H, d, H-1ꢁ, J = 2.4 Hz), 5.27 (1H, dd, H-2ꢁ, J = 2.5, J =
6.3 Hz), 4.98 (1H, dd, H-3ꢁ, J = 2.3, J = 6.2 Hz), 4.51 (1H, m, H-4ꢁ), 3.64
(1H, dd, H-5ꢁa, J = 3.3, J = 10.4 Hz), 3.57 (1H, dd, H-5ꢁb, J = 4.2, J = 10.4
Hz), 3.34 (3H, s, 5ꢁ-O-CH3), 3.23 (2H, q, 6-CH2CH3, J = 7.6 Hz), 1.65 (3H,
s, CMe2), 1.44 (3H, t, 6-CH2CH3, J = 7.6 Hz), 1.40 (3H, s, CMe2).
6-[Bis(phenylthio)methyl]-9-(tetrahydro-2H-pyran-2-yl)purine (12)
1M solution of NaHMDS in THF (1.9 mL) was added dropwise to a
solution of 10 (100 mg, 0.46 mmol) in THF (3 mL) at −78◦C and the

Regioselective Metalation of 6-Methylpurines
651
mixture was stirred for 5 minutes. A solution of diphenyl disulfide (0.23
g, 2.3 mmol) in THF (3 mL) was added and the mixture was further stirred
for 30 minutes at room temperature. NH4Cl solution (1M, 3 mL) was added
and the solvents were evaporated under reduced pressure. The residue was
partitioned between CHCl3 and H2O, the organic phase was dried over
(MgSO4), evaporated, and purified by short flash silica gel column (eluate;
1% MeOH in CHCl3) to (155 mg, 78%) of 12 as a pale yellow solid: MS
1
m/z 435 (M+1)+, H NMR (CDCl3) δ 8.91 (1H, s, H-2), 8.22 (1H, s, H-8),
7.95–7.20 (10H, m, 6-CHSPh2), 6.21 (1H, s, 6-CHSPh2), 5.77 (1H, dd, H-1ꢁ, J
= 2.6, J = 9.9 Hz), 4.18 (1H, m, H-5ꢁa), 3.79 (1H, m, H-5ꢁb), 2.12–1.68 (6H,
m, H-2ꢁa,b, H-3ꢁa,b, and H-4ꢁa,b).
6-Fluoromethyl-9-(tetrahydro-2H-pyran-2-yl)purine (11)[21,22]
A pre-cooled solution of KHMDS (1.17 g, 5.85 mmol) in THF (20 mL)
was added dropwise to a solution of 10 (1.06 g, 4.87 mmol) in THF (20
mL) over 5 minutes at −80◦C. The mixture was stirred for 30 minutes, and
then treated with a cold solution of NFSI (1.9 g, 5.85 mmol) in THF (5 mL)
at −80◦C. The mixture was stirred for 5 minutes at the same temperature,
then quenched with NH4Cl (1N , 10 mL). The mixture was partitioned
between EtOAc and H2O, the organic phase was dried over (MgSO4), and
evaporated. The residue was purified by a flash silica gel column (eluate 1%
MeOH in CHCl3) to give (0.513 g, 57.8%, based on a recovered 0.24 g, 23%
of 10) of 11 as a pale yellow solid: MS m/z 237.1 (M+1)+, UV λmax (pH 1)
261.1, λmax (pH 7) 265.6, λmax (pH 13) 274.9 nm; 1H NMR (CDCl3) δ 9.00
(1H, s, H-2, 1J C,H = 206.3 Hz), 8.34 (1H, s, H-8, 1J C,H = 212.3 Hz), 5.97 (1H,
dd, 6-CH2aF, J = 12.6, J F,H = 46.7 Hz), 5.87 (1H, dd, 6-CH2bF, J = 12.6, J F,H
= 46.7 Hz), 5.83 (1H, dd, H-1ꢁ, J = 2.2, J = 5.1 Hz), 4.20 (1H, m, H-5ꢁa),
3.81 (1H, m, H-5ꢁb), 2.15–1.69 (6H, m, H-2ꢁa,b, H-3ꢁa,b, and H-4ꢁa,b).
6-Fluoromethylpurine (3)[21,22]
1 N HCl (4 mL) was added to a solution of 11 (0.2 g, 0.85 mmol) in THF
(5 mL) and the mixture was stirred for 72 hours at room temperature. The
volatiles were evaporated in vacuo and the concentrated aqueous solution
was applied to Dowex 50 W (H+) column. The column was washed with
H2O until no UV absorbing fractions were observed, then eluted with
2.5% NH4OH. The collected fractions were evaporated and the residue was
purified by flash silica gel column (eluate; 15% MeOH in CHCl3) to give
(123 mg, 95%) of 3 as a pale yellow solid: m.p. 204–206◦C; MS m/z 153. 1
1
(M+1)+, H NMR (DMSO-d6) δ 13.65 (1H, br s, 9-NH), 8.93 (1H, s, H-2),
8.68 (1H, s, H-8), 5.83 (2H, d, 6-CH2F, J = 46.6 Hz); 13C NMR (DMSO-d6)
δ 156.13 (C-4), 151.63 (C-2), 150.81 (C-6), 146.64 (C-8), 127.07 (C-5), 81.33

652
A. E. A. Hassan et al.
(C-6, J = 165.9 Hz); Anal. Calcd. for C6H5N4F, C 47.37, H 3.31, N 36.53;
found C 47.25, H 3.42, N 36.32.
6-Chloromethyl-9-(tetrahydro-2H-pyran-2-yl)purine (13) and
6-trichloromethyl-9-(tetrahydro-2H-pyran-2-yl)purine (14)
1 M solution of NaHMDS in THF (0.45 mL) was added dropwise to a
solution of 10 (0.1 g, 0.45 mmol) in THF (3 mL) at −78◦C and the mixture
was stirred for 0.5 hours at −78◦C. A pre-cooled solution of TsCl (0.35
g, 1.84 mmol) in THF (2 mL) was added at −80◦C and the mixture was
stirred for 20 minutes at the same temperature. A 1 N solution of NH4Cl
(4 mL) was added and the mixture was warmed to room temperature,
partitioned between EtOAc and H2O, and the organic phase was dried
over (MgSO4) and evaporated. The residue was purified by a flash silica
gel column (eluate: 40% EtOAc in hexanes) to give (45 mg, 41%) of 13 as a
pale yellow syrup and (11 mg, 8%) of 14 as a pale yellow syrup: spectroscopic
parameters for 13; UV λmax (pH 1) 267.1, λmax (pH 7) 268.6, λmax (pH 13)
1
268.3; H NMR (CDCl3) δ 8.99 (1H, s, H-2), 8.34 (1H, s, H-8), 5.82 (1H,
dd, H-1ꢁ, J = 2.6, J = 10.0 Hz), 5.07 (1H, d, 6-CH2aCl, J = 11.7 Hz), 5.02
(1H, d, 6-CH2bCl, J = 11.7 Hz), 4.20 (1H, m, H-5ꢁa), 3.80 (1H, m, H-5ꢁb),
2.19–1.68 (6H, m, H-2ꢁa,b, H-3ꢁa,b, and H-4ꢁa,b); spectroscopic parameters for
14: H NMR (CDCl3) δ 9.06 (1H, s, H-2), 8.40 (1H, s, H-8), 5.84 (1H, dd, H-1ꢁ,
J = 2.5, J = 10.1 Hz), 4.20 (1H, m, H-5ꢁa), 3.80 (1H, m, H-5ꢁb), 2.208–1.68
(6H, m, H-2ꢁa,b, H-3ꢁa,b, and H-4ꢁa,b).
8-Iodo-6-methyl-9(tetrahydro-2H-pyran-2-yl)purine (15)
LDA (0.5M, 4.2 mL) was added dropwise to a solution of 10 (90 mg,
0.41 mmol) in THF (3 mL) at −78◦C and the mixture was stirred for 30
minutes. I2 (0.5 4 g, 2.1 mmol) in THF (1 mL) was and the mixture was
stirred for 1 hour at −78◦C. 10% Aqueous Na2S2O3 (10 mL) was added
and the mixture was stirred for 30 minutes at room temperature. EtOAc
was added to the mixture and the organic phase was separated, dried over
MgSO4 and evaporated. The residue was purified by a flash silica gel column
(eluate: 3% MeOH in CHCl3) to give (41 mg, 48%) of 10 and (60 mg, 43%)
of 15 as a white solid: UV λmax (pH 1) 282.9 and 217.2, λmax (pH 7) 271.5,
λmax (pH 13) 271.7; 1H NMR (CDCl3) δ 8.77 (1H, s, H-2,1J C,H = 204.8 Hz,),
5.65 (1H, dd, H-1ꢁ, J = 2.4, J = 11.4 Hz), 4.23 (1H, m, H-5ꢁa), 3.75 (1H,
m, H-5ꢁb), 3.16 (1H, m, H-2ꢁ), 2.82 (3H, s, 6-CH3), 2.18–1.61 (5H, m, H-2ꢁb,
H-3ꢁa,b, and H-4ꢁa,b).

Regioselective Metalation of 6-Methylpurines
653
6,8-Dimethyl-9(tetrahydro-2H-pyran-2-yl)purine (16)
LDA (2M, 0.21 mL) was added dropwise to a solution of 10 (92 mg,
0.42 mmol) in THF (3 mL) at −78◦C. The mixture was stirred for 30
minutes, and then MeI (0.25 mL, 4.2 mmol) was added and the mixture
was stirred for further 1 hour at −78◦C. A solution of NH4Cl (1N, 3 mL) was
added and the whole was partitioned between EtOAc and H2O. The organic
phase was separated, dried over (MgSO4) and evaporated under reduced
pressure. The residue was purified by a flash silica gel column (eluate: 3%
MeOH in CHCl3) to give 69 mg as a mixture of 10 and 15: HPLC [eluate;
H2O:CH3CN, 20 minutes linear gradient from 10–90%; 10 (22.59%, RT =
12.49 minutes); 16 (61.47%, RT = 14.77 minutes)]. An analytical sample
of 16 was obtained by preparative TLC (eluate 1.5% MeOH in CHCl3): MS
m/z 233 (M+1)+; 1H NMR (CDCl3) δ 8.77 (1H, s, H-2), 5.79 (1H, dd, H-1ꢁ,
J = 2.5, J = 5.9 Hz), 4.21 (1H, m, H-5ꢁa), 3.75 (1H, m, H-5ꢁb), 2.81 (3H,
s, 6-CH3), 2.79 (3H, s, 8-CH3), 2.48 (1H, m, H-2ꢁa), 2.08 (1H, m, H-2ꢁb),
1.92–1.66 (6H, m, H-3ꢁa,b, and H-4ꢁa,b).
6-Ethyl-8-methyl-9-(tetrahydro-2H-pyran-2-yl)purine (17)
A 1.6 M solution of n-BuLi in hexanes (1.07 mL, 1.72 mmol) was added
to a solution of 10 (75 mg, 0.34 mmol) in THF (5 mL) at −78◦C. The
mixture was stirred for 30 minutes under −70◦C, then CH3I (0.2 mL) was
added and the mixture was stirred for 30 minutes. A solution of NH4Cl (1N,
3 mL) was added and the mixture was partitioned between EtOAc and H2O.
The organic phase was separated, dried over (MgSO4) and evaporated. The
residue was purified by a flash silica gel column (eluate: 3% MeOH in
CHCl3) to give (62 mg, 75%) of 17 as a pale yellow syrup: MS m/z 247.2
(M+1)+, 1H NMR (CDCl3) δ 8.81 (1H, s, H-2), 5.80 (1H, dd, H-1ꢁ, J = 2.4, J
= 8.9 Hz), 4.20 (1H, m, H-5ꢁa), 3.75 (1H, m, H-5ꢁb), 3.18 (2H, q, 6-CH2CH3,
J = 7.5 Hz),.2.78 (3H, s, 8-CH3), 2.49 (1H, m, H-2ꢁa), 2.08–1.63 (5H, m,
H-2ꢁb, H-3ꢁa,b, and H-4ꢁa,b), 1.41 (3H, t, 6-CH2CH3, J = 7.5 Hz).
6,8-Diethyl-9-(tetrahydro-2H-pyran-2-yl)purine (18)
MS m/z 262.2 (M+1)+, 1H NMR (CDCl3) δ 8.81 (1H, s, H-2), 5.73 (1H,
dd, H-1ꢁ, J = 2.3, J = 11.2 Hz), 4.21 (1H, m, H-5ꢁa), 3.73 (1H, m, H-5ꢁb),
3.19 (2H, q, 6-CH2CH3, J = 7.7 Hz),.3.11 (2H, q, 8-CH2CH3, J = 7.6 Hz),
2.65 (1H, m, H-2ꢁa), 2.08–1.62 (5H, m, H-2ꢁb, H-3ꢁa,b, and H-4ꢁa,b), 1.47 (3H,
t, 6-CH2CH3, J = 7.5 Hz), 1.47 (3H, t, 8-CH2CH3, J = 7.6 Hz).

654
A. E. A. Hassan et al.
6-Ethyl-9-(tetrahydro-2H-pyran-2-yl)purine (19) and
6-isopropyl-9-(tetrahydro-2H-pyran-2-yl)purine (20)[13]
A solution of KHMDS (0.35 g. 1.65 mmol) in THF (3 mL) was added
dropwise to a solution of 10 (72 mg, 0.33 mmol) in THF (3 mL) at
−78◦C. Upon the addition of the base, an orange red colored mixture
developed. The mixture was stirred for 15 minutes at −78◦C, and then
quenched with MeI (0.2 mL, 3.3 mmol). The mixture was stirred for 5
minutes, 1 N NH4Cl (3 mL) was added, and then was partitioned between
EtOAc and H2O. The organic phase was separated, dried over (MgSO4) and
evaporated. The residue was purified by a flash silica gel column (eluate: 3%
MeOH in CHCl3) to give 72 mg as a mixture of 19 and 20: HPLC [eluate;
H2O:CH3CN, 20 minutes linear gradient from 10–90%; 19 (89.4%, RT =
14.07 minutes); 20 (8.5%, RT = 15.08 minutes)]. Spectroscopic parameters
1
for 19: MS m/z 233 (M+1)+, H NMR (CDCl3) δ 8.89 (1H, s, H-2), 8.24
(1H, s, H-8), 5.79 (1H, dd, H-1ꢁ, J = 3.7, J = 5.9 Hz), 4.19 (1H, m,
H-5ꢁa), 3.80 (1H, m, H-5ꢁb), 3.24 (1H, q, 6-CH2CH3, J = 7.5 Hz),.2.13–1.69
(6H, m, H-2ꢁa,b, H-3ꢁa,b, and H-4ꢁa,b), 1.45 (3H, t, 6-CH2CH3, J = 7.5 Hz).
Spectroscopic parameters for 20: 1H NMR (CDCl3) δ 8.92 (1H, s, H-2), 8.24
(1H, s, H-8), 5.80 (1H, m, H-1ꢁ), 4.19 (1H, m, H-5ꢁa), 3.82 (1H, m, H-5ꢁb),
3.78 (1H, m, 6-CHMe2), 2.14–1.68 (6H, m, H-2ꢁb, H-3ꢁa,b, and H-4ꢁa,b), 1.45
(6H, d, 6-CHMe2, J = 7.1 Hz).
6-Fluoromethyl-9-(2,3,5-tri-O-tert-butyldimethylsilyl-β-
D-ribofuranosyl)purine (23)
A 1 M solution of NaHMDS in THF (4.4 mL) was added dropwise to
a solution of 22 (2.23 g, 3.66 mmol) in THF (25 mL) at −78◦C and the
mixture was stirred for 25 minutes. A pre-cooled solution of NFSI (1.43
g, 4.4 mmol) in THF (15 mL) was added at −80◦C and the mixture was
stirred for 5 minutes at the same temperature. A solution of NH4Cl (1N,
10 mL) was added and the mixture was warmed to room temperature,
partitioned between EtOAc and H2O, and the organic phase was dried over
(MgSO4), and evaporated. The residue was purified by a flash silica gel
column (eluate: 10% EtOAc in hexanes) to give (1.1 g, 47.9%) of 21 as a
white glassy solid: MS m/z 628 (M+1)+; UV λmax (pH 1) 276.1, 216.5, λmax
1
(pH 7) 268.0, λmax (pH 13) 268.0; H NMR (CDCl3) δ 8.99 (1H, s, H-2),
8.50 (1H, s, H-8), 6.15 (1H, d, H-1ꢁ, J = 5.2 Hz), 5.91 (1H, dd, 6-CH2aF, J a,b
= 12.6, J H,F = 46.6 Hz), 5.89 (1H, dd, 6-CH2bF, J a,b = 12.6, J H,F = 46.6 Hz),
4.65 (1H, dd, H-2ꢁ, J 1 ,2 = 5.2, J 2 ,3 = 4.3 Hz), 4.32 (1H, dd, H-3ꢁ, J 1 ,2
=
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
5.2, J 3 ,4 = 3.6 Hz), 4.16 (1H, ddd, H-4ꢁ, J 3 ,4 = 3.6, J 4 ,5a = 2.6, J 4 ,5b = 3.7
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
Hz), 4.03 (1H, dd, H5ꢁa, J 4 ,5a = 2.6, J 5 a,5 b = 11.7 Hz), 3.81 (1H, dd, H5ꢁb,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
J 4 ,4b = 2.6, J 5 a,5 b = 11.7 Hz), 0.97–0.78 (27 H, m, tert-BuSiMe2), 0.15- -0.25

Regioselective Metalation of 6-Methylpurines
655
(18H, 6s, tert-BuSiMe2); Anal. Calcd. for C29H25N4O4Si3F; C 55.55, H 8.84;
N 8.94, found C 55.38, H 8.65, N 8.97.
6-Fluoromethyl-9-(β-D-ribofuranosyl)purine (4)
Solid Et4NF·x H2O (1.2 g, 7.97 mmol) was added to a solution of 23
(1, 1.59 mmol) in CH3CN (10 mL) at room temperature. The mixture was
stirred for 2 hours and the solvent was evaporated under reduced pressure.
The residue was purified by a flash silica gel column (eluate; 10% EtOH in
CHCl3) to give (430 mg, 95%) of 4 as a white solid: m.p. 185–187◦C; MS m/z
285 (M+1)+; UV λmax (pH 1) 268.3, λmax (pH 7) 264.2, λmax (pH 13).264.7;
1H NMR (DMSO-d6) δ 8.99 (1H, s, H-2, 1J C,H = 206.3 Hz), 8.91 (1H, s, H-8,
1J C,H = 215.9 Hz), 6.07 (1H, d, H-1ꢁ, J = 5.5 Hz), 5.86 (2H, d, 6-CH2a,bF, J H,F
= 46.6 Hz), 5.58 (1H, d, 2ꢁ-OH, J = 5.9 Hz), 5.28 (1H, d, 3ꢁ-OH, J = 4.9 Hz),
5.12 (1H, t, 5ꢁ-OH, J = 5.3 Hz), 4.64 (1H, ddd, H-2ꢁ, J 1 ,2 = 5.5, J 2 ,3 = 5,
ꢁ
ꢁ
ꢁ
ꢁ
J 2 ,2 OH = 5.9 Hz), 4.20 (1H, q, H-3ꢁ, J 2 ,3 = 5.0, J 3 ,4 = 3.6, J 3 ,3 OH = 4.9 Hz),
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
3.99 (1H, q, H-4ꢁ, J 3 ,4 = 3.6, J 4 ,5 a, = 4.1, J 4 ,5 a = 4.0 Hz), 3.70 (1H, ddd,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
H-5ꢁa, J 4 ,5 a = 4.0, J 5 a, 5 -OH = 5.3, J 5 a,5 b = 11.9 Hz), 3.59 (1H, ddd, H-5ꢁb,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
J 4 ,5 b = 4.1, J 5 b, 5 -OH = 5.8, J 5 a,5 b = 11.9 Hz), 13C NMR (DMSO-d6) δ 153.42
(C-6), 151.88 (C-2), 151.56 (C-4), 145.55 (C-8), 131.75 (C-5), 87.72 (C-1ꢁ),
85.72 (C-4ꢁ), 80.52 (1J CF = 167.0 Hz), 73.78 (C-2ꢁ), 70.24 (C-3ꢁ), 61.18 (C-5ꢁ);
Anal. Calcd. for C11H13N4O4F, C 46.48, H 4.61, N 19.71; found C 46.38, H
4.62, N 19.66.
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
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