Synthesis and cytostatic activity of novel 6-(Difluoromethyl)purine bases and nucleosides

Article abstract of DOI:10.1055/s-2006-942365

An expeditious synthesis of modified purine bases and nucleosides bearing a difluoromethyl group in position 6 is described starting from the corresponding 6-(hydroxymethyl)purine derivatives. Their oxidation using Dess-Martin reagent gave 6-formylpurines

Full text of DOI:10.1055/s-2006-942365

1848
PAPER
Synthesis and Cytostatic Activity of Novel 6-(Difluoromethyl)purine Bases and
Nucleosides
N
ovel 6-(Diflu
e
oromethyl)
t
purine
e
Bases and N
r
ucleoside
s
Šilhár, Radek Pohl, Ivan Votruba, Michal Hocek*
Centre for New Antivirals and Antineoplastics, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Re-
public, Flemingovo nam. 2, 16610 Prague 6, Czech Republic
Fax +420(2)20183559; E-mail: hocek@uochb.cas.cz
Received 7 December 2005; revised 30 January 2006
methyl and difluoromethyl groups the corresponding or-
Abstract: An expeditious synthesis of modified purine bases and
ganometallics are hardly accessible. Therefore, purine
derivatives containing these groups must be prepared by
functional group transformations of other types of 6-
nucleosides bearing a difluoromethyl group in position 6 is de-
scribed starting from the corresponding 6-(hydroxymethyl)purine
derivatives. Their oxidation using Dess–Martin reagent gave 6-
formylpurines that were converted to the title compounds by reac- [(substituted)methyl]purines. Protected 6-methylpurines
tion with Deoxofluor. 6-(Difluoromethyl)purine ribonucleoside ex-
erted significant cytostatic effect against leukemia cell lines.
were reported to be deprotonated by NaHMDS at the
methyl group. Quenching of the anion with NFSI gave 6-
Key words: purines, nucleobases, nucleosides, fluorination, anti-
neoplastic activity
(fluoromethyl)purines in moderate yields [trace amounts
of 6-(difluoromethyl)purines were formed as by-prod-
ucts]. We have recently reported8 a facile synthesis of 6-
(fluoromethyl)purines via direct deoxyfluorination or by
Several types of purines bearing C-substituents in position multistep functional group transformation starting from
6 are biologically active (Figure 1). 6-Aryl and 6- now easily available 6-(hydroxymethyl)purines. Appar-
hetarylpurine,1 6-trifluoromethylpurine,2 as well as 6-(hy- ently, the hydroxymethyl group is a suitable precursor for
droxymethyl)purine3 ribonucleosides display significant many other transformations.
cytostatic activity and some 6-hetarylpurine ribonucleo-
sides exert4 also potent antiviral activity against HCV. 6-
Methylpurine and its ribonucleoside are highly cytotoxic5
and its liberation by purine nucleoside phosphorylases
from its non-toxic deoxyribonucleoside was proposed as
a novel principle in the gene therapy of cancer.6 Fluoro-
methyl and difluoromethyl groups are isosteric to methyl
groups and fluorine is often used as surrogate of oxygen
in biologically active compounds. Hydrophobic nucleo-
base surrogates, including fluorinated and trifluoromethy-
lated (het)arenes, are7 very promising tools in chemical
biology (e.g. extension of the genetic alphabet etc.). The
biological activities and other applications of the above-
mentioned important classes of compounds imply that
further logical members of the series, 6-(fluoromethyl)pu-
rines and 6-(difluoromethyl)purines, are of great interest
both in medicinal chemistry and chemical biology. Sur-
prisingly, these simple compounds have not been reported
until very recently when we8 and others9 have published a
synthesis of 6-(fluoromethyl)purines and the correspond-
ing ribonucleoside was found to possess significant cyto-
static effect.8 Here we report the synthesis and biological
activity of 6-(difluoromethyl)purine bases and nucleo-
sides as the last missing members of this interesting class
of compounds.
R
R = aryl
Me
N
N
N
known
cytostatics
CF3
CH2F
CH2OH
N
HO
O
CHF2 - hitherto unknown,
this study
OH OH
Figure 1 Biologically active substituted 6-methylpurine nucleosi-
des
Our strategy for the preparation of the desired 6-(difluo-
romethyl)purines was to perform a deoxofluorination of
the corresponding 6-formylpurines. 6-Formylpurines
were reported11 several times in the past but their synthe-
ses based on multistep or direct oxidations of 6-methylpu-
rines were rather low-yielding procedures with limited
applicability. We have tried to prepare these key interme-
diates by oxidation of 6-(hydroxymethyl)purines 1a–d
readily available by cross-coupling of 6-halopurines with
(acetoxymethyl)zinc iodide3 followed by chemoselective
deacetylation.8 The oxidation was performed using Dess–
Martin periodinane [1,1,1-tris(acetyloxy)-1,1-dihydro-
1,2-benziodoxol-3-(1H)-one] which is a general reagent
for oxidation of alcohols to aldehydes.12 Thus the protect-
ed 6-(hydroxymethyl)purine bases and nucleosides 1a–d
were treated with Dess–Martin reagent at ambient temper-
ature to give quantitative conversion to 6-formylpurines
2a–d. These compounds occur in the form of the alde-
hydes 2 in absence of water but in presence of even traces
of water (e.g. NMR measurement in DMSO-d6) they oc-
Purines bearing C-substituents in position 6 are generally
prepared by cross-coupling reactions10 of 6-halopurines
with organometallics. However, in the case of fluoro-
SYNTHESIS 2006, No. 11, pp 1848–1852
x
x.
x
x
.
2
0
0
6
Advanced online publication: 05.05.2006
DOI: 10.1055/s-2006-942365; Art ID: T16705SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Novel 6-(Difluoromethyl)purine Bases and Nucleosides
1849
cur as mixtures of aldehydes 2 and hydrates 3. Analytical- kemia HL60 cells (ATCC CCL 240), human cervix carci-
ly pure hydrate 3a was obtained by crystallization of 2a noma HeLa S3 cells (ATCC CCL 2.2), and human T
from acetone–water mixture. For the synthetic purposes lymphoblastoid CCRF-CEM cell line (ATCC CCL 119).
the aldehydes 2 were directly used in the next step after The results are summarized in Table 2. 6-(Difluorometh-
column chromatography and drying.
yl)purine ribonucleoside (4f) exerted significant cytostat-
ic effect (IC50 in micromolar range) against leukemia cell
lines HL-60 and CCRF-CEM but virtually no effect
against solid tumor (HeLa S3) while the deoxyribonucle-
oside as well as the purine bases were inactive in all these
cell lines.
The deoxyfluorinations were performed using Deoxofluor
{[bis(2-methoxyethyl)amino]sulfur trifluoride}13 that was
also previously found to be the best reagent for deoxy-
fluorinations of 6-(hydroxymethyl)purines.8 Thus the
formylpurines 2 were treated with Deoxofluor at –20 °C
and the reaction mixture was then stirred at ambient tem- The 6-substituted purine nucleosides could be considered
perature for 10–16 hours until the consumption of the as transition state analogues for adenosine deaminase
aldehydes 2. The desired protected 6-(difluoro- (ADA), an important catabolic enzyme. In previous stud-
methyl)purine bases and nucleosides 4a–d were easily ies, 6-(hydroxymethyl)purine ribonucleoside was found3
isolated in acceptable yields of ca. 50% (Table 1) as the to be a moderate inhibitor of ADA while the fluoromethyl
only products besides tarry, chromatographically immo- derivative8 was inactive. Adenosine deaminase (ADA) in-
bile mixture of side-products of decomposition and/or po- hibition was studied16 on calf intestinal adenosine amino-
lymerization of starting compounds.
hydrolase (EC 3.5.4.4.). Both riboside 4f and
deoxyriboside 4g exerted only very low inhibitory effect
to ADA.
Standard deprotection of 6-(difluoromethyl)purine inter-
mediates 4b–d was used to prepare the final free 6-(di-
fluoromethyl)purine base and nucleosides 4e–g It could be concluded, that the activity of 6-(difluoro-
(Scheme 1). The THP protective group in position 9 of methyl)purine derivatives is comparable to that of 6-(fluo-
purine is easily cleavable under mild acidic conditions. romethyl)purines8 and 6-(trifluoromethyl)purines;2 their
Thus compound 4b was deprotected using Dowex 50X8 ribonucleosides are active against leukemia cell lines
(H+ form)14 in ethanol to afford free base 4e in 74% of while other derivatives are inactive. In addition, they do
yield. The ester groups in 4c,d were cleaved by sodium not significantly inhibit ADA.
methoxide in methanol at room temperature15 to give free
nucleosides 4f,g in about 83–90% of yield (Table 1).
Table 2 Cytostatic and ADA Inhibitory Activity of Title Com-
pounds
Table 1 Yields of the Particular Steps in the Synthesis
Compound
IC50 (mmol/L)a
Entry
Starting
compound
Reagent
Product yield (%)
HL60
CCRF-CEM
NA
ADA
4a
4e
4f
NAb
NA
1
2
1a
1b
1c
1d
2a
2b
2c
2d
4b
4c
4d
Dess–Martin
Dess–Martin
Dess–Martin
Dess–Martin
Deoxofluor
Deoxofluor
Deoxofluor
Deoxofluor
Dowex (H+)
MeONa
2a (93)a
2b (95)
2c (98)
2d (93)
4a (49)
4b (52)
4c (48)
4d (49)
4e (74)
4f (83)
4g (90)
NA
NA
NA
1.05 0.001
NA
1.02 0.001
NA
31.5 2.1
18.0 0.9
3
4g
4
a Values are means of four experiments, standard deviation is given
5
in parentheses.
b NA = not active (inhibition of cell growth at 10 mM was lower than
6
30%).
7
In conclusion, 6-(difluoromethyl)purine bases and nucleo-
sides can be prepared by this expeditious methodology in
reasonable yields from 6-(hydroxymethyl)purines. They
complement the series of cytostatic 6-methylpurine deriv-
atives.
8
9
10
11
MeONa
NMR spectra were recorded on Bruker Avance 400 (1H NMR at
400 MHz, 13C NMR at 100.6 MHz) or Bruker Avance 500 (500
a Isolated in the form of a hydrate 3a after crystallization from water–
acetone.
1
MHz for H NMR and 125.8 MHz for 13C NMR) spectrometers.
Chemical shifts (in ppm, d scale) were referenced to TMS as inter-
nal standard. Complete assignment of all NMR signals was per-
formed using a combination of H,H-COSY, H,C-HSQC and H,C-
HMBC experiments. Melting points were determined on a Kofler
block and are uncorrected. Optical rotations were measured at 25 °C
on a Autopol IV (Rudolph Research Analytical) polarimeter and
[a]D values are given in 10–1 deg cm2 g–1. Mass spectra were mea-
Title 6-(difluoromethyl)purine bases and nucleosides
4a,e,f,g were screened for biological activity. In vitro cy-
tostatic activity tests (inhibition of cell growth) were per-
formed using the following cell cultures: mouse leukemia
L1210 cells (ATCC CCL 219), human promyelocytic leu-
Synthesis 2006, No. 11, 1848–1852 © Thieme Stuttgart · New York

1850
P. Šilhár et al.
PAPER
O
HO
N
OH
OH
O
O
Cl
1. AcOCH2ZnI, Pd(PPh3)4
2. aq NH3, ZnCl2, EtOH
I
OAc
OAc
N
N
AcO
N
N
N
N
N
N
N
N
+
refs 3 and 8
CH2Cl2
r.t., 2 h
92–98 %
N
N
N
N
N
R
R
R
R
1a–d
2a–d
3a–d
in presence of
H2O
In compounds 1–4:
TolO
TolO
(MeOCH2CH2)2NSF3 (3.5 equiv)
CH2Cl2
O
O
R = a Bn,
b THP
c
d
–20 °C to r.t., 16 h
48–52 %
OTol
TolO
83%
OTol
[H+]
MeONa/
MeOH
CHF2
90%
N
N
74%
N
HO
HO
O
O
N
e H
R
g
f
HO
OH
OH
4a–d
4e–g
THP = tetrahydropyran-2-yl; Tol = 4-methylbenzoyl
Scheme 1 Synthesis of 6-(difluoromethyl)purine bases and nucleosides
sured on a ZAB-EQ (VG Analytical) spectrometer using FAB (ion-
ization by Xe, accelerating voltage 8 kV, glycerol matrix).
Cytostatic activity tests were performed as described in the litera-
ture.1a ADA inhibition assay was performed by standard technique
given in the literature.16
IR (CHCl3): 3372, 2951, 2861, 1602, 1496, 1407, 1334, 1087, 1045,
959 cm–1.
1H NMR (500 MHz, CDCl3): d = 1.67–1.89, 2.02–2.24 [m, 6 H,
CH2(THP)], 3.81 [td, J = 2.6, 11.7 Hz, 1 H, bCH2O(THP)], 4.22 [ddt,
J = 1.8, 4.3, 11.7 Hz, 1 H, aCH2O(THP)], 5.88 [dd, J = 2.5, 10.4 Hz,
1 H, CHO(THP)], 8.53 (s, 1 H, H-8), 9.19 (s, 1 H, H-2), 10.50 (s, 1 H,
CHO).
13C NMR (125.8 MHz, CDCl3): d = 22.6, 24.8, 31.9 [CH2(THP)], 68.9
[CH2O(THP)], 82.4 [CHO(THP)], 131.5 (C-5), 146.4 (CH-8), 146.8 (C-
6), 152.7 (CH-2), 153.7 (C-4), 191.6 (CHO).
Preparation of 6-Formylpurines (2); General Procedure
Dess–Martin periodinane (510 mg, 1.2 mmol) was added to a solu-
tion of 6-(hydroxymethyl)purine 1 (1 mmol) in CH2Cl2 (15 mL).
After consumption of starting material (2 h), the reaction mixture
was quenched with sat. aq NaHCO3 (15 mL) and extracted with
CHCl3 (4 × 20 mL). The collected organic layers were dried over
MgSO4, evaporated and chromatographed on a column of silica gel
(hexanes–acetone, 3:1–2:1).
MS (FAB): m/z (%) = 233 (5) [MH+], 149 (44), 85 (52).
HRMS (FAB): m/z calcd for C11H13N4O2: 233.1033; found:
233.1042.
9-Benzyl-6-formylpurine (2a)
Yield: 93%. Crystallization from acetone–H2O afforded an analyti-
cally pure hydrate 3a; mp 119–120 °C.
6-Formyl-9-(2,3,5-tri-O-toluoyl-b-D-ribofuranosyl)purine (2c)
Yield: 98%.
IR (CHCl3): 1726, 1612, 1591, 1495, 1408, 1331, 1267, 1180, 1093,
1020, 840 cm–1.
3a
IR (KBr): 3381, 3087, 2935, 1605, 1590, 1510, 1405, 1341, 1211,
1H NMR (500 MHz, CDCl3): d = 2.38, 2.42, 2.43 [3 × s, 9 H, 3 ×
CH3(Tol)], 4.67 (dd, J5¢b,4¢ = 4.0 Hz, Jgem = 12.3 Hz, 1 H, H-5¢b), 4.85
(td, J4¢,5¢ = 3.1, 4.0 Hz, J4¢,3¢ = 4.6 Hz, 1 H, H-4¢), 4.93 (dd, J5¢a,4¢ = 3.1
Hz, Jgem = 12.3 Hz, 1 H, H-5¢a), 6.21 (dd, J3¢,4¢ = 4.6 Hz, J3¢,2¢ = 5.8
Hz, 1 H, H-3¢), 6.41 (t, J2¢,1¢ = 5.4 Hz, J2¢,3¢ = 5.8 Hz, 1 H, H-2¢), 6.52
(d, J1¢,2¢ = 5.4 Hz, 1 H, H-1¢), 7.16, 7.24, 7.25 [3 × m, 6 H, 3 ×
2 H(m-Tol)], 7.80, 7.93, 7.97 [3 × m, 6 H, 3 × 2 H(o-Tol)], 8.46 (s, 1 H,
H-8), 9.05 (s, 1 H, H-2), 10.45 (s, 1 H, CHO).
13C NMR (125.8 MHz, CDCl3): d = 21.7, 21.8 [CH3(Tol)], 63.2 (CH2-
5¢), 71.4 (CH-3¢), 73.7 (CH-2¢), 81.3 (CH-4¢), 87.2 (CH-1¢), 125.5,
125.9, 126.4 [C(i-Tol)], 129.3, 129.3, 129.4 [CH(m-Tol)], 129.73,
129.87 [CH(o-Tol)], 131.97 (C-5), 144.4, 144.7, 144.8 [C(p-Tol)], 147.1
(CH-8), 147.2 (C-6), 152.9 (CH-2), 154.2 (C-4), 165.2, 165.4, 166.1
(CO), 191.3 (CHO).
1099, 1043 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 5.53 (s, 2 H, CH2Ph), 6.27 (t,
J = 7.8 Hz, 1 H, CH), 6.55 (d, J = 7.8 Hz, 2 H, 2 × OH), 7.26–7.37
(m, 5 H, Ph), 8.74 (s, 1 H, H-8), 8.92 (s, 1 H, H-2).
13C NMR (100.6 MHz, DMSO-d6): d = 46.7 (CH2Ph), 87.0 (CH),
127.8, 128.1, 128.9 (CHPh), 129.6 (C-5), 136.7 (Ci-Ph), 146.7 (CH-
8), 151.9 (C-4), 151.9 (CH-2), 158.7 (C-6).
MS (FAB): m/z (%) = 257 (10) [MH+], 239 (5), 149 (6), 91 (43).
HRMS (FAB): m/z calcd for C13H12N4O2: 257.1039; found:
257.1028.
Anal. Calcd for C13H12N4O2: C, 60.93; H, 4.72; N, 21.86. Found: C,
60.76; H, 4.71; N, 21.58.
MS (FAB): m/z (%) = 635 (1) [MH+], 487 (42), 369 (5), 149 (7), 119
(100).
6-Formyl-9-(tetrahydropyran-2-yl)purine (2b)
Yield: 95%.
HRMS (APCI): m/z calcd for C35H31N4O8: 635.2142; found:
635.2175.
Synthesis 2006, No. 11, 1848–1852 © Thieme Stuttgart · New York

PAPER
Novel 6-(Difluoromethyl)purine Bases and Nucleosides
1851
9-(2-Deoxy-3,5-di-O-toluoyl-b-D-erythro-pentofuranosyl)-6-
formylpurine (2d)
HRMS (FAB): m/z calcd for C11H13F2N4O: 255.1052; found:
255.1061.
Yield: 93%.
6-(Difluoromethyl)-9-(2,3,5-tri-O-toluoyl-b-D-ribofurano-
syl)purine (4c)
Yield: 48%; white foam.
IR (CHCl3): 1719, 1612, 1589, 1492, 1406, 1326, 1269, 1179, 1102,
1020, 939, 841 cm–1.
1H NMR (400 MHz, CDCl3): d = 2.40, 2.45 [2 × s, 6 H, 2 × CH3(Tol)],
2.91 (ddd, J2¢b,3¢ = 2.3 Hz, J2¢b,1¢ = 5.9 Hz, Jgem = 14.4 Hz, 1 H, H-2¢b),
3.21 (ddd, J2¢a,3¢ = 6.4 Hz, J2¢a,1¢ = 8.2 Hz, Jgem = 14.4 Hz, 1 H, H-2¢a),
4.64–4.71 (m, 2 H, H-5¢b, H-4¢), 4.82 (m, 1 H, H-5¢a), 5.86 (dt,
IR (CCl4): 3039, 1732, 1612, 1600, 1499, 1411, 1331, 1266, 1114,
1068, 1020 cm–1.
1H NMR (500 MHz, CDCl3): d = 2.38, 2.42, 2.43 [3 × s, 9 H, 3 ×
CH3(Tol)], 4.67 (dd, J5¢b,4¢ = 4.1 Hz, Jgem = 12.4 Hz, 1 H, H-5¢b), 4.86
(td, J4¢,5¢ = 3.1, 4.1 Hz, J4¢,3¢ = 4.6 Hz, 1 H, H-4¢), 4.92 (dd, J5¢a,4¢ = 3.1
Hz, Jgem = 12.4 Hz, 1 H, H-5¢a), 6.20 (dd, J3¢,4¢ = 4.6 Hz, J3¢,2¢ = 5.8
Hz, 1 H, H-3¢), 6.40 (t, J2¢,1¢ = 5.4 Hz, J2¢,3¢ = 5.8 Hz, 1 H, H-2¢), 6.50
(d, J1¢,2¢ = 5.4 Hz, 1 H, H-1¢), 7.01 (t, JH,F = 53.8 Hz, 1 H, CHF2),
7.16, 7.23, 7.25 [3 × m, 6 H, 3 × H(m-Tol)], 7.80, 7.92, 7.98 [3 × m, 6
H, 3 × H(o-Tol)], 8.36 (s, 1 H, H-8), 8.92 (s, 1 H, H-2).
13C NMR (100.6 MHz, CDCl3): d = 21.7, 21.8 [CH3(Tol)], 63.2 (CH2-
5¢), 71.3 (CH-3¢), 73.6 (CH-2¢), 81.2 (CH-4¢), 87.1 (CH-1¢), 111.8
(t, JC,F = 242 Hz, CHF2), 125.5, 125.9, 126.5 [C(i-Tol)], 129.25,
129.30, 129.37 [CH(m-Tol)], 129.7, 129.9 [CH(o-Tol)], 131.5 (C-5),
144.4, 144.7, 144.8 [C(p-Tol)], 145.3 (CH-8), 150.1 (t, JC,F = 26 Hz,
C-6), 152.5 (CH-2), 152.7 (C-4), 165.2, 165.4, 166.2 [CO(Tol)].
J3¢,4¢ = 2.3 Hz, J3¢,2¢ = 2.3, 6.4 Hz, 1 H, H-3¢), 6.65 (dd, J1¢,2¢ = 5.9, 8.2
Hz, 1 H, H-1¢), 7.21, 7.30 [2 × m, 4 H, 2 × H(m-Tol)], 7.87, 7.98 [2 ×
m, 4 H, 2 × H(o-Tol)], 8.48 (s, 1 H, H-8), 9.12 (s, 1 H, H-2), 10.46 (s,
1 H, CHO).
13C NMR (100.6 MHz, CDCl3): d = 21.7, 21.8 [CH3(Tol)], 37.9 (CH2-
2¢), 63.7 (CH2-5¢), 75.0 (CH-3¢), 83.4 (CH-4¢), 85.2 (CH-1¢), 126.3,
126.5 [C(i-Tol)], 129.31, 129.33 [CH(m-Tol)], 129.6, 129.8 [CH(o-Tol)],
132.0 (C-5), 144.3, 144.7 [C(p-Tol)], 146.8 (CH-8), 146.9 (C-6),
152.7 (CH-2), 154.0 (C-4), 165.9, 166.1 [CO(Tol)], 191.4 (CHO).
MS (FAB): m/z (%) = 501 (1) [MH+], 353 (2), 149 (40), 119 (82).
HRMS (APCI): m/z calcd for C27H25N4O6: 501.1774; found:
501.1786.
MS (FAB): m/z (%) = 657 (0.5) [MH+], 487 (16), 119 (100).
Deoxyfluorinations with Deoxofluor; General Procedure
A 6-formylpurine 2 (0.5 mmol) was dissolved in CH2Cl2 (10 mL)
under an Ar atmosphere and cooled to –20 °C. Deoxofluor (0.32
mL, 1.75 mmol) was then added dropwise through septum and the
reaction mixture was allowed to warm to r.t. and stirred for 10–16
h. Then the reaction was quenched with 5% NaHCO3 and extracted
with CHCl3. Organic phase was dried over MgSO4, concentrated
and chromatographed on silica gel (hexanes–EtOAc, 2:1–1:1).
HRMS (APCI): m/z calcd for C35H31F2N4O7: 657.2161; found:
657.2145.
9-(2-Deoxy-3,5-di-O-toluoyl-b-D-erythro-pentofuranosyl)-6-(di-
fluoromethyl)purine (4d)
Yield: 49%; white foam.
IR (CCl4): 3040, 1727, 1613, 1600, 1496, 1409, 1329, 1267, 1209,
1178,1101, 1068, 1021 cm–1.
9-Benzyl-6-(difluoromethyl)purine (4a)
Yield: 49%; white crystals; mp 78–79 °C.
1H NMR (500 MHz, CDCl3): d = 2.40, 2.45 [2 × s, 6 H, 2 × CH3(Tol)],
2.90 (ddd, J2¢b,3¢ = 2.2 Hz, J2¢b,1¢ = 5.9 Hz, Jgem = 14.2 Hz, 1 H, H-2¢b),
3.19 (ddd, J2¢a,3¢ = 6.4 Hz, J2¢a,1¢ = 8.2 Hz, Jgem = 14.2 Hz, 1 H, H-2¢a),
4.65–4.70 (m, 2 H, H-5¢b, H-4¢), 4.80 (m, 1 H, H-5¢a), 5.85 (dt,
IR (CCl4): 3070, 3036, 1598, 1501, 1408, 1330, 1209, 1109, 1066
cm–1.
1H NMR (400 MHz, CDCl3): d = 5.50 (s, 2 H, CH2Ph), 7.05 (t,
JH,F = 53.9 Hz, 1 H, CHF2), 7.31–7.42 (m, 5 H, Ph), 8.20 (s, 1 H, H-
8), 9.10 (t, JH,F = 0.7 Hz, 1 H, H-2).
J3¢,4¢ = 2.2 Hz, J3¢,2¢ = 2.2, 6.4 Hz, 1 H, H-3¢), 6.63 (dd, J1¢,2¢ = 5.9, 8.2
Hz, 1 H, H-1¢), 7.02 (t, JH,F = 53.9 Hz, 1 H CHF2), 7.21, 7.30 [2 ×
m, 4 H, 2 × H(m-Tol)], 7.87, 7.98 [2 × m, 4 H, 2 × H(o-Tol)], 8.38 (s, 1
H, H-8), 8.98 (s, 1 H, H-2).
13C NMR (100.6 MHz, CDCl3): d = 47.6 (CH2Ph), 112.1 (t, JC,F
=
242 Hz, CHF2), 127.9, 128.9, 129.3 (CHPh), 130.6 (C-5), 134.5 (Ci-
Ph), 146.4 (CH-8), 149.8 (t, JC,F = 26 Hz, C-6), 152.5 (CH-2), 153.2
(C-4).
13C NMR (125.8 MHz, CDCl3): d = 21.7, 21.8 [CH3(Tol)], 37.9 (CH2-
2¢), 63.8 (CH2-5¢), 75.0 (CH-3¢), 83.4 (CH-4¢), 85.2 (CH-1¢), 111.9
(t, JC,F = 242 Hz, CHF2), 126.3, 126.5 [C(i-Tol)], 129.3, 129.3
[CH(m-Tol)], 129.6, 129.8 [CH(o-Tol)], 131.5 (C-5), 144.3, 144.7
[C(p-Tol)], 144.9 (CH-8), 150.0 (t, JC,F = 26 Hz, C-6), 152.3 (CH-2),
152.5 (C-4), 165.9, 166.1 [CO(Tol)].
19F NMR (188.2 MHz, CDCl3): d = –118.41 (d, JF,H = 53.9 Hz).
MS (FAB): m/z (%) = 523 (1) [MH+], 353 (5), 170 (16), 119 (75).
MS (FAB): m/z (%) = 261 (32) [MH+], 242 (3), 171 (4), 91 (100).
HRMS (FAB): m/z calcd for C13H11F2N4: 261.0952; found:
261.0946.
Anal. Calcd for C13H10F2N4: C, 60.00; H, 3.87; F, 14.60; N, 21.53.
Found: C, 59.80; H, 3.83; F, 14.31; N, 21.19.
HRMS (FAB): m/z calcd for C27H25F2N4O5: 523.1793; found:
6-(Difluoromethyl)-9-(tetrahydropyran-2-yl)purine (4b)
523.1803.
Yield: 52%; white foam.
6-(Difluoromethyl)-9H-purine (4e)
IR (CCl4): 2950, 2858, 1598, 1592, 1498, 1410, 1329, 1210, 1109,
1088, 1066 cm–1.
6-(Difluoromethyl)-9-(tetrahydropyran-2-yl)purine (4b; 144 mg,
0.566 mmol) was dissolved in EtOH (20 mL) and Dowex 50 WX8
(H+ form; ca. 30 mg) was added. This suspension was stirred for 1.5
h at 75 °C. After a complete deprotection of starting material, the re-
action mixture was filtered and Dowex was washed with NH3–
EtOH. Volatiles were evaporated in vacuo. Crude product was chro-
matographed on silica gel (hexanes–EtOAc, 1:2–0:1) and crystal-
lized from EtOAc–hexanes to afford a white solid (71 mg, 74%; mp
244–245 °C).
1H NMR (500 MHz, CDCl3): d = 1.66–1.88, 2.04–2.22 [m, 6 H,
CH2(THP)], 3.81 [td, J = 2.6, 11.8 Hz, 1 H, bCH2O(THP)], 4.21 [ddt,
J = 1.8, 4.4, 11.8 Hz, 1 H, aCH2O(THP)], 5.86 [dd, J = 2.5, 10.5 Hz,
1 H, CHO(THP)], 7.05 (t, JH,F = 53.9 Hz, 1 H, CHF2), 8.43 (s, 1 H, H-
8), 9.06 (s, 1 H, H-2).
13C NMR (125.8 MHz, CDCl3): d = 22.6, 24.8, 31.9 (CH2THP),
68.9 (CH2OTHP), 82.3 (CHOTHP), 112.0 (t, JC,F = 242 Hz, CHF2),
130.9 (C-5), 144.5 (CH-8), 149.7 (d, JC,F = 26 Hz, C-6), 152.25
(CH-2), 152.34 (C-4).
IR (KBr): 3081, 1621, 1607, 1479, 1382, 1325, 1229, 1103, 1075,
1041 cm–1.
MS (FAB): m/z (%) = 255 (11) [MH+], 171 (100), 152 (9), 85 (64).
Synthesis 2006, No. 11, 1848–1852 © Thieme Stuttgart · New York

1852
P. Šilhár et al.
PAPER
1H NMR (400 MHz, CDCl3): d = 7.32 (t, JH,F = 53.6 Hz, 1 H, CHF2),
8.78 (s, 1 H, H-8), 9.03 (s, 1 H, H-2).
Hz, CHF2), 131.0 (C-5), 147.1 (CH-8), 148.7 (t, JC,F = 25 Hz, C-6),
151.9 (CH-2), 152.7 (C-4).
13C NMR (100.6 MHz, CDCl3): d = 112.6 (t, JC,F = 239 Hz, CHF2),
126.5 (C-5), 146.5 (t, JC,F = 26 Hz, C-6), 148.3 (CH-8), 151.8 (CH-
2), 157.4 (C-4).
19F NMR (188.2 MHz, DMSO-d6): d = –118.78 (d, JF,H = 53.3 Hz).
MS (FAB): m/z (%) = 287 (18) [MH+], 171 (63), 152 (5), 117 (10).
HRMS (FAB): m/z calcd for C11H13F2N4O3: 287.0956; found:
287.0950.
MS (FAB): m/z (%) = 171 (100) [MH+], 152 (12), 134 (17).
HRMS (FAB): m/z calcd for C6H5F2N4: 171.0482; found: 171.0477.
Anal. Calcd for C11H12F2N4O3: C, 46.16; H, 4.23; F, 13.27; N,
19.57. Found: C, 46.00; H, 4.30; F, 13.23; N, 19.56.
6-(Difluoromethyl)-9-(b-D-ribofuranosyl)purine (4f)
A methanolic solution of MeONa (c = 1 M; 0.1 mL, 0.1 mmol) was
added to a solution of 6-(difluoromethyl)purine ribonucleoside 4c
(230 mg, 0.35 mmol) in MeOH (20 mL). After a complete deprotec-
tion of starting material (16 h, monitoring by TLC), the reaction
mixture was adsorbed on silica gel and chromatographed (EtOAc–
MeOH, 1:0–9:1) to give a white solid (88 mg, 83%); crystallization
Acknowledgment
This work is a part of the research project Z4 055 0506. It was sup-
ported by the Grant Agency of the Czech Republic (grant No. 203/
03/0035), by the ‘Centre of New Antivirals and Antineoplastics’
(1M0508), by the Program of Targeted Projects of Academy of Sci-
ences of the Czech Republic (1QS400550501) and by Sumitomo
Chemical, Inc. (Osaka, Japan).
20
from MeOH–EtOAc–heptane; mp 181–182 °C; [a]D –30.5 (c =
2.75, MeOH).
IR (KBr): 3401, 3327, 1607, 1590, 1499, 1405, 1332, 1212, 1118,
1089, 1048 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 3.59 (ddd, J5¢b,4¢ = 4.0 Hz,
J5¢b,OH = 5.7 Hz, Jgem = 12.1 Hz, 1 H, H-5¢b), 3.71 (dt, J5¢a,4¢ = 4.0 Hz,
J5¢a,OH = 5.3 Hz, Jgem = 12.1 Hz, 1 H, H-5¢a), 4.00 (q, J4¢,3¢ = 3.8 Hz,
References
(1) (a) Hocek, M.; Holý, A.; Votruba, I.; Dvořáková, H. J. Med.
Chem. 2000, 43, 1817. (b) Hocek, M.; Holý, A.; Votruba, I.;
Dvořáková, H. Collect. Czech. Chem. Commun. 2001, 66,
483.
J
4¢,5¢ = 4.0 Hz, 1 H, H-4¢), 4.21 (br q, J3¢,4¢ = 3.8 Hz, J3¢,2¢ = 4.9 Hz,
J3¢,OH = 5.2 Hz, 1 H, H-3¢), 4.62 (q, J2¢,3¢ = 4.9 Hz, J2¢,1¢ = 5.4 Hz,
J2¢,OH = 5.7 Hz, 1 H, H-3¢), 5.11 (t, JOH,5¢ = 5.3, 5.7 Hz, 1 H, OH-5¢),
5.28 (d, JOH,3¢ = 5.2 Hz, 1 H, OH-3¢), 5.60 (d, JOH,2¢ = 5.7 Hz, 1 H,
OH-2¢), 6.10 (d, J1¢,2¢ = 5.4 Hz, 1 H, H-1¢), 7.36 (t, JH,F = 53.3 Hz, 1
H, CHF2), 9.01 (s, 1 H, H-8), 9.09 (s, 1 H, H-2).
13C NMR (100.6 MHz, DMSO-d6): d = 61.3 (CH2-5¢), 70.4 (CH-3¢),
74.1 (CH2-2¢), 85.9 (CH-4¢), 88.1 (CH-1¢), 112.3 (t, JC,F = 240 Hz,
CHF2), 131.0 (C-5), 147.1 (CH-8), 148.9 (t, JC,F = 25 Hz, C-6),
152.1 (CH-2), 153.0 (C-4).
(2) Hocková, D.; Hocek, M.; Dvořáková, H.; Votruba, I.
Tetrahedron 1999, 55, 11109.
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2004, 6, 3225. (b) Šilhár, P.; Pohl, R.; Votruba, I.; Hocek,
M. Collect. Czech. Chem. Commun. 2005, 70, 1669.
(4) Hocek, M.; Nauš, P.; Pohl, R.; Votruba, I.; Furman, P. A.;
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W. R.; Wells, A. H.; Gillespie, G. Y.; Sorscher, E. J. Hum.
Gene Ther. 1997, 8, 1637.
19F NMR (188.2 MHz, DMSO-d6): d = –118.80 (t, JF,H = 53.3 Hz).
MS (FAB): m/z (%) = 303 (30) [MH+], 171 (100), 152 (16).
HRMS (FAB): m/z calcd for C11H13F2N4O4: 303.0905; found:
(7) (a) Henry, A. A.; Olsen, A. G.; Matsuda, S.; Yu, C.;
Geierstanger, B. H.; Romesberg, F. E. J. Am. Chem. Soc.
2004, 126, 6923. (b) Lai, J. S.; Kool, E. T. Chem. Eur. J.
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Chem. Eur. J. 2005, 11, 2125.
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J. A.; Secrist, J. A. III Nucleosides, Nucleotides Nucleic
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303.0908.
Anal. Calcd for C11H12F2N4O4: C, 43.71; H, 4.00; F, 12.57; N,
18.54. Found: C, 43.68; H, 3.95; F, 12.78; N, 18.19.
9-(2-Deoxy-b-D-erythro-pentofuranosyl)-6-(difluoromethyl)pu-
rine (4g)
Prepared from 6-(difluoromethyl)purine 2¢-deoxynucleoside 4d
(150 mg, 0.287 mmol) by the procedure described for 4f. Yield: 74
mg (90%); white solid; crystallization from MeOH–EtOAc–hep-
tane; mp 122–123 °C; [a]D20 +28.0 (c = 2.54, MeOH).
IR (KBr): 3343, 3264, 1596, 1501, 1339, 1326, 1208, 1101, 1061,
1029 cm–1.
1H NMR (500 MHz, DMSO-d6): d = 2.39 (ddd, J2¢b,3¢ = 3.9 Hz,
J2¢b,1¢ = 6.4 Hz, Jgem = 13.4 Hz, 1 H, H-2¢b), 2.80 (ddd, J2¢a,3¢ = 6.0
(11) (a) Giner-Sorolla, A.; Zimmerman, I.; Bendich, A. J. Am.
Chem. Soc. 1959, 81, 2515. (b) Giner-Sorolla, A. Chem.
Ber. 1968, 101, 611. (c) Tanji, K.; Satoh, R.; Higashino, T.
Chem. Pharm. Bull. 1992, 40, 227.
(12) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
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H. S. J. Org. Chem. 1999, 64, 7048.
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60, 1386.
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1705.
Hz, J2¢a,1¢ = 6.9 Hz, Jgem = 13.4 Hz, 1 H, H-2¢a), 3.54 (ddd, J5¢b,4¢
=
4.7 Hz, J5¢b,OH = 5.5 Hz, Jgem = 11.8 Hz, 1 H, H-5¢b), 3.63 (dt,
J
J
5¢a,4¢ = 5.1 Hz, J5¢a,OH = 5.5 Hz, Jgem = 11.8 Hz, 1 H, H-5¢a), 3.90 (td,
4¢,3¢ = 3.2 Hz, J4¢,5¢ = 4.7, 5.1 Hz, 1 H, H-4¢), 4.46 (dq, J3¢,4¢ = 3.2 Hz,
J3¢,OH = 4.1 Hz, J3¢,2¢ = 3.9, 6.0 Hz, 1 H, H-3¢), 4.98 (t, JOH,5¢ = 5.5 Hz,
1 H, OH-5¢), 5.39 (d, JOH,3¢ = 4.1 Hz, 1 H, OH-3¢), 6.52 (t, J1¢,2¢ = 6.4,
6.9 Hz, 1 H, H-1¢), 7.35 (t, JH,F = 53.3 Hz, 1 H, CHF2), 8.96 (s, 1 H,
H-8), 9.08 (s, 1 H, H-2).
13C NMR (125.8 MHz, DMSO-d6): d = 39.5 (CH2-2¢), 61.6 (CH2-
5¢), 70.6 (CH-3¢), 84.1 (CH-1¢), 88.28 (CH-4¢), 112.3 (t, JC,F = 240
(16) Agarwal, R. P.; Parks, R. E. Jr. In Methods in Enzymology,
Vol. 51; Hoffe, P. A.; Jones, M. E., Eds.; Academic Press:
New York, 1978, 502.
Synthesis 2006, No. 11, 1848–1852 © Thieme Stuttgart · New York