8-Substituted analogues of 3-(3-cyclopentyloxy-4-methoxy-benzyl)-8- isopropyl-adenine: Highly potent and selective PDE4 inhibitors

Article abstract of DOI:10.1021/jm030603w

3-(3-Cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine V11294 (1) has been identified as a lead structure, which selectively inhibits human lung PDE4 (436 nM) and is also active in a number of in vitro and in vivo models of inflammation. Here we descri

Full text of DOI:10.1021/jm030603w

J. Med. Chem. 2005, 48, 1237-1243
1237
8-Substituted Analogues of 3-(3-Cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-
adenine: Highly Potent and Selective PDE4 Inhibitors
John W. F. Whitehead,* Gary P. Lee, Parviz Gharagozloo, Peter Hofer, Andre´ Gehrig, Peter Wintergerst,
Donald Smyth,† William McCoull,‡ Mohamed Hachicha, Aniket Patel, and Donald J. Kyle
Purdue Pharma LP, 6 Cedar Brook Drive, Cranbury, New Jersey 08512
Received December 5, 2003
3-(3-Cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine V11294 (1) has been identified as
a lead structure, which selectively inhibits human lung PDE4 (436 nM) and is also active in a
number of in vitro and in vivo models of inflammation. Here we describe the synthesis and
pharmacology of a series of highly potent 8-[(benzyloxy)methyl]-substituted analogues, with
potencies in the range 10-300 nM. In addition, several compounds showed interesting PDE4
subtype specificities, for example, the 3-thienyl derivative 5v, which showed 6-10 nM potency
at PDE4B, D3, and D5 and a 20- to 200-fold selectivity over A and D2, respectively.
Introduction
are expected to reach the market in 2005. The most
advanced of these are GlaxoSmithKline’s cilomilast
(Ariflo)16 and Byk Gulden’s roflumilast,17 currently in
phase 2/3 trials for asthma and chronic obstructive
pulmonary disease (COPD). Other PDE4 compounds
currently undergoing phase 2 trials include Celltech/
Schering-Plough’s D439618 and Almirall’s arofylline.19
1 has been identified as a lead structure, which
selectively inhibits human lung hPDE4 (Ki ) 436 nM)
and is also active in a number of in vitro and in vivo
models of inflammation.20-22 In addition, it is orally
active at doses that are not emetogenic,22 a side effect
that has proved dose limiting for some earlier PDE4
inhibitors. Considerable SAR studies around the purine
ring skeleton particularly at N-3, C-8, and the amino
group had led to the identification of 1 as a lead
structure, in particular the “rolipram-like” disubstituted
catechol moiety at N-3.21 During the synthesis of V11689
(2),21,23 the 8-hydroxy human metabolite of 1, the
8-benzyloxy analogue 5a was also prepared21 and was
found to be 3 times more potent than its parent in the
mixed PDE4 assay and over 30 times as potent in
PDE4B and 4D 3/5 assays. These interesting results
coincided with a publication, which described a series
of highly potent PDE4 inhibitors.24 These compounds,
hydroxamic acid analogues of rolipram, were suggested
to bind at the divalent metal-ion binding pocket of the
PDE4 enzyme active site.24 We speculated that the
increased potency of 5a could be due to binding of the
electron-rich benzylic moiety at this binding site. This
paper describes the synthesis and pharmacology of a
range of 8-benzyloxy analogues of 5a, a hitherto unex-
plored region of the purine skeleton.
Asthma is a chronic inflammatory disease character-
ized by reversible narrowing and inflammation of the
airways, accompanied by hyper-reactivity of the respira-
tory tract to external stimuli. The development of highly
effective drugs is urgently needed, because the mortality
and morbidity due to asthma are increasing despite
extensive treatment.1- 3
The central role of cyclic nucleotides, cyclic adenosine
monophosphate (cAMP) and cyclic guanosine mono-
phosphate (cGMP), in regulating the function of airway
smooth muscle, inflammatory cells, and immune cells
is well established.4-8 Cyclic nucleotide phosphodi-
esterases (PDEs) play a key role in the metabolism of
cAMP and cGMP. PDEs have been characterized into
11 distinct families of isozymes that differ in their
selectivity and specificity for the hydrolysis of cAMP and
cGMP to their inactive 5′-nucleotide products.9,10 The
PDE4 subtype consisting of four gene products (PDE4A
to PDE4D) is characterized by selective high-affinity
hydrolysis of cAMP. It is predominantly found within
airway smooth muscle and proinflammatory cells, mak-
ing it an attractive target for novel antiasthma and
antiinflammatory therapy.11-14 Inhibition of this en-
zyme results in increased cellular levels of cAMP, which
contribute to both the relaxation of airway smooth
muscle and the prevention of proinflammatory cell
activation and recruitment to the lungs and airways.
The prototype PDE4 inhibitor is rolipram, originally
developed as an antidepressant.15 This compound is
however highly emetic and therefore precluded from use
as a human therapeutic. First generation derivatives
also suffered from this side effect and in general had
low oral bioavailability. A new generation of highly
potent and orally available PDE4 inhibitors are looking
more promising in late stage clinical trials for both
asthma and chronic obstructive pulmonary disease and
Chemistry
2-(6-Amino-9H-purin-8-yl)-2-propanol 3 and 1-(6-
amino-9H-purin-8-yl)ethanol 6 were synthesized ac-
cording to the literature method25 starting from 6-chlo-
ropurine. These adenines 3 and 6 were then reacted
with 3-cyclopentyloxy-4-methoxy-benzyl chloride21 in
acetonitrile under reflux to give the 3-alkylated products
4 and 7. Carrying out this reaction in the absence of
base, ensures selective 3-alkylation with none of the
* To whom correspondence should be addressed. Phone: 609-409-
5166. Fax: 609-409-6930. E-mail: john.whitehead@pharma.com.
† Currently at OSI Pharmaceuticals, Watlington Road, Oxford, OX4
6LT, United Kingdom.
‡Currently at AstraZeneca, Alderley Park, Macclesfield, Cheshire,
SK10 4TG, United Kingdom.
10.1021/jm030603w CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/27/2005

1238 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Whitehead et al.
Scheme 1a
inflammatory cells in man and are therefore of more
relevance to the asthmatic disease state (Table 1).29 The
compounds were then tested against human PDE3 and
PDE5 enzymes from human blood platelets. With the
exception of 8b and 8c, which showed low-micromolar
PDE5 activity of 1.44 and 4.02 µM, respectively, the
remaining compounds were inactive with both PDE3
and PDE5 enzymes at 30 µM concentration.
Results and Discussion
With the exception of the carboxylic acid 5s (Ki ) 5230
nM), all the gem-dimethyl analogues 5a-v were more
potent at PDE4 (Ki ) 9.7-191 nM) than 1 (Ki ) 436
nM). The most potent compound was the methyl ester
5q (Ki ) 9.3 nM), a hydrogen bond acceptor, which is
47-fold more potent than the parent 1.
The carboxylic acid 5s is the most weakly active
compound in this series at all PDE4 subtype enzymes.
This cannot be due to steric requirements as the
similarly sized carboxamide 5r (Ki ) 25 nM) is one of
the most potent analogues. Rather, it must be due either
to the polarity of the carboxylate or to the entropically
unfavorable desolvation of the carboxylate anion re-
quired before binding can occur. The dimethyl series
5a-v were 10- to 20-fold more potent than their
monomethyl analogues 8a-c at all PDE4 subtype
enzymes. This may be due either to extra lipophilic
binding of the former or to the dimethyl analogue having
a greater conformational preference for the desired
bioactive conformation. A number of trends were seen
in the SAR of the dimethyl series 5a-v. At the 4-posi-
tion most substituents are well-tolerated. They showed
high potency at PDE4B, D3, and D5 subtypes (5-30
nM) and approximately 10-fold lower potency at PDE4A
and D2 subtypes (100-600 nM). The 3,4-dimethoxy 5i
and 3,5-dimethoxy 5j analogues are of interest in that
at PDE4A, B, D3, and D5 the 3,5-dimethoxy compound
is approximately twice as potent as the 3,4-dimethoxy
derivative. However at the D2 subtype, the 3,5-analogue
is 8 times as potent as the 3,4-analogue. Similarly, the
2-thienyl 5u and 3-thienyl 5v derivatives showed simi-
lar potencies at PDE4A, D3, and D5, but at PDE4B, the
3-thienyl 5v was 5 times more potent than the 2-thienyl
5u. However at PDE4D2, the potencies were reversed
with the 2-thienyl 5u being 6.5 times more potent than
the 3-thienyl 5v.
The 4-cyano derivative 5m shows an interesting
gradation of tolerance for the PDE4 subtype enzymes
being very potent at PDE4D5 (Ki ) 19.4 nM) and D3
(Ki ) 33.4 nM) but only weakly active against PDE4A
(Ki ) 1190 nM) and D2 (Ki ) 1840 nM). The trend is
D5 > D3 > B > A > D2. These observations taken
together could be used in the design of third generation
subtype selective PDE4 inhibitors which could lead to
increased tissue and cell selectivity, to decreased side
effects, and improved therapeutic index.33-35 Recent
literature supports this hypothesis; for instance, dual
PDE4A/B inhibition has been found to significantly
correlate with inhibition of LPS-stimulated TNF-R
secretion and with T-cell proliferation in peripheral
blood monocytes, while no correlation was found for
PDE4D inhibition. The PDE4C36 subtype is not gener-
ally found in proinflammatory cells, but it is highly
expressed in the CNS37 where PDE4 inhibitors are
a Reagents and conditions: (a) 3-cyclopentyloxy-4-methoxyben-
zyl chloride, DMF, 100 °C; (b) KH, THF, 50 °C f 0 °C, then benzyl
or aryl chloride, 0 °C f 25 °C; (c) NH4OH, EtOH, reflux, 18 h; (d)
NaOH, EtOH, room temperature, 4 h; (e) NaH, DMF, 50 °C f 0
°C, then benzyl chloride, 0 °C f 25 °C, 5 h.
9-alkylated regioisomer being formed as has been
observed in the literature.26
The hydroxy-alkyl side chain was then alkylated with
a range of benzyl and heteroaryl bromides and chlorides
to give the O-benzylated derivatives 5a-v and 8a-c
(Scheme 1). O-Alkylation was found to be accompanied
by O,N-dialkylation. The optimum conditions to mini-
mize this were NaH in DMF for the alkylation of 7 and
KH in THF for alkylations of the more sterically
hindered 4. The reactions were monitored by TLC and
stopped when the O,N-dialkylated product began to
appear. Yields were typically in the range 10-40%. The
carboxamide 5r and carboxylic acid 5s derivatives were
synthesized from the methyl ester 5q by hydrolysis
using aqueous ammonia and sodium hydroxide, respec-
tively. The carboxylic acid 5s was isolated as its sodium
salt (Scheme 1).
In Vitro Pharmacology
The two series of compounds 5a-v and 8a-c were
tested first against human PDE4 enzymes from human
lung tissue. They were then tested in human PDE4A,
B, D2, D3, and D5 subtypes, which had been cloned and
expressed in a baculovirus system.28 The hPDE4AL and
hPDE4BL are the long form of the cloned enzymes. The
PDE4C enzyme is an unstable clone, and so we were
not able to test our compounds in this enzyme subtype.
The PDE4C enzyme is mainly found in testis, skeletal
muscle, the CNS, and human fetal lung, while the
PDE4A, B, and D subtypes are expressed in many

8-Substituted Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1239
Table 1. PDE4 Subtype Enzyme Potencies (nM)
PDE4 (mix)
hPDE4AL
mean ( SEM
(n)
hPDE4BL
mean ( SEM
(n)
hPDE4D2
mean ( SEM
(n)
hPDE4D3
mean ( SEM
(n)
hPDE4D5
mean ( SEM
(n)
mean ( SEM
compd
R2 (or het)
(n)
rolipram
CI-930
zaprinast
1
7940 ( 1840 (6)
>50000 (6)
1470 ( 820 (5) 8460 ( 1260 (7) 382 ( 42 (5)
375 ( 80 (4)
>50000 (6)
>50000 (5)
>50000 (3)
>50000 (5)
>50000 (5)
>50000 (5)
>50000 (4)
>50000 (4)
8760 ( 3300 (5)
141 ( 17 (3)
4.1 ( 0.6 (3)
5.1 ( 0.6 (3)
5.1 ( 0.9 (3)
8.1 ( 5.5 (3)
7.6 ( 3.5 (3)
4.8 ( 1.8 (3)
6.8 ( 3.2 (3)
8.4 ( 4.6 (3)
7.8 ( 1.8 (3)
7.9 ( 1.2 (3)
13.3 ( 0.7 (3)
3.7 ( 1.4 (3)
19.4 ( 9.6(3)
6.6 ( 2.7 (3)
4.3 ( 1.4 (3)
7.2 ( 1.1 (3)
5.5 ( 1.7 (3)
4.9 ( 1.7 (3)
436 ( 23 (3)
191 ( 21 (3)
38 ( 2.1 (3)
30.3 ( 3.2 (3)
22.6 ( 2.2 (3)
19.7 ( 2.0 (3)
25.3 ( 2.6 (3)
45.3 ( 7.4 (3)
60.0 ( 6.4 (3)
3900 ( 1290 (3)
179 ( 65 (4)
137 ( 25.1 (3)
175 ( 32 (3)
113 ( 27 (3)
206 ( 26 (3)
126 ( 43 (3)
271 ( 76 (3)
208 ( 21 (3)
270 ( 42.1 (3) 2890 ( 805 (5)
172 ( 72.9 (3)
5.7 ( 0.7 (3)
7.1 ( 3.7 (3)
6.0 ( 3.3 (3)
5.6 ( 2.9 (3)
6.6 ( 3.0 (3)
5.8 ( 0.9 (3)
11.0 ( 6.1 (3)
7.4 ( 2.9 (3)
15.0 ( 4.5 (3)
9.0 ( 1.8 (3)
13.0 ( 1.6 (3)
6.6 ( 1.9 (3)
33.4 ( 8.0(3)
10.6 ( 4.8 (3)
5.0 ( 0.6 (3)
6.5 ( 3.6 (3)
3.0 ( 0.1 (3)
8.0 ( 2.1 (3)
5a
H
8.9 ( 3.0 (3)
11.0 ( 3.8 (3)
13.0 ( 4.8 (3)
10.3 ( 3.1(3)
9.8 ( 3.9 (3)
11.0 ( 2.0 (3)
18.9 ( 1.9 (3)
11.6 ( 3.4 (3)
413 ( 81 (5)
255 ( 53.5 (3)
296 ( 39 (3)
347 ( 52 (3)
347 ( 50 (3)
205 ( 34 (3)
427 ( 45 (3)
369 ( 74 (3)
5b
2-OMe
3-OMe
4-OMe
4-Me
5c
5d
5e
5f
4-Cl
5g
2-F
5h
3-F
5i
3,4-OMe
3,5-OMe
3,4-F
91.7 ( 23.2 (3) 405 ( 8(3)
42.8 ( 19.3 (4) 2640 ( 530 (3)
5j
44.0 ( 2.1(3)
49.0 ( 1.5 (3)
3,4-OCH2CH2O 35.0 ( 8.3 (3)
225 ( 29 (3)
244 ( 47.0 (3)
147 ( 27 (3)
1190 ( 170 (3)
166 ( 22 (3)
175 ( 41 (3)
415 ( 95 (3)
103 ( 13 (3)
155 ( 34 (3)
>50000 (3)
21.9 ( 8.1 (3)
17.3 ( 2.8 (3)
21.4 ( 11.3 (3) 218 ( 41 (3)
71.3 ( 18.2 (3) 1840 ( 290 (3)
9.7 ( 5.8 (3)
5.8 ( 1.3 (3)
26.6 ( 10.0 (3) 782 ( 225 (5)
5.2 ( 2.1 (3)
7.6 ( 3.2 (3)
>50000 (3)
33 ( 13 (3)
46.9 ( 20.0 (3) 412 ( 100 (4)
9.5 ( 3.1 (3)
340 ( 84 (3)
459 ( 63 (3)
5k
5l
5m
5n
4-CN
3,4-Cl
58.0 ( 2.0 (3)
26.0 ( 3.0 (3)
15.0 ( 3.0 (3)
27.0 ( 3.5 (3)
9.3 ( 1.0 (3)
25.0 ( 2.5 (3)
5230 ( 60 (3)
23.5 ( 1.0 (3)
355 ( 30 (3)
255 ( 8 (3)
5o
4-t-Bu
4-OCF3
4-CO2Me
4-CONH2
4-CO2H
4-CF3
2-thienyl
3-thienyl
H
5p
5q
246 ( 68 (3)
312 ( 32 (3)
>50000 (3)
5r
5s
4300 ( 2420 (3) >50000 (3)
5t
309 ( 34 (3)
580 ( 192 (4)
9.9 ( 4.7 (3)
7.0 ( 1.3 (3)
9.0 ( 4.7 (3)
10.3 ( 4.3 (3)
6.3 ( 1.5 (3)
6.6 ( 3.6 (3)
590 ( 107 (3)
322 ( 83 (3)
5u
53.3 ( 11.7 (3) 236 ( 65 (4)
83.3 ( 12.7 (3) 223 ( 72 (3)
5v
2680 ( 870 (3)
8a
1210 ( 140 (3) 15200 ( 9900 (3) 533 ( 164(3)
4450 ( 1560 (4) 640 ( 150 (4)
8b
3-OMe
3-F
1470 ( 170 (3) 5200 ( 930 (3)
204 ( 69 (3) 3390 ( 1350 (3)
430 ( 40 (3)
269 ( 95 (4)
5050 ( 620 (3)
4300 ( 580 (3)
493 ( 133 (4)
114.7 ( 24.3 (3) 231 ( 112 (3)
8c
on a Bruker DPX400 or AMX500 spectrometer. The spectra
were referenced to residual protonated solvent residues as an
internal standard, and chemical shifts are quoted in parts per
million (ppm) downfield from tetramethylsilane in the solvent
indicated in parentheses. Values are quoted to 0.01 ppm.
Signal multiplicities are described as s (singlet), d (doublet), t
(triplet), and q (quartet) with J being reported to the nearest
0.1 Hz. The symbol br indicates that the peak was broad. Low-
resolution mass spectra (MS) were recorded on a Micromass
Platform LC system with electrospray (ES) mode of ionization.
Intensities were recorded as percentages of the largest signal,
and only major peaks were recorded, as the mass to charge
(m/z) ratio.
believed to promote many of their side effects such as
emesis. Third generation inhibitors could target mini-
mizing PDE4C inhibition as a drug design strategy.
Interestingly, cilomilast (Ariflo)16 currently in clinical
phase trials shows 10-fold selectivity for PDE4D versus
other PDE4 subtypes.
Conclusions
The synthesis of the 8-benzyloxy analogues of 1 has
led to the discovery of highly potent and selective PDE4
inhibitors that are 10- to 20-fold more potent than the
parent compound. The 4-methylester derivative 5q was
the most potent compound in the series showing single
digit nanomolar potency at the B, D3, and D5 subtypes.
In addition, a number of compounds in particular the
4-cyano 5m, the 2- and 3-thienyl 5u and 5v, and the
3,4- and 3,5-dimethoxy 5i and 5j analogues showed
interesting PDE4 subtype specificities which could be
further exploited to design third generation PDE4
inhibitors with reduced side effect liability.
Preparation of 2-(6-Amino-9H-purin-8-yl)-2-propanol
(3). 2-(6-Amino-9H-purin-8-yl)-2-propanol 3 was prepared
25
according to the literature method.
Preparation of 2-{6-Amino-3-[3-(cyclopentyloxy)-4-
methoxybenzyl]-3H-purin-8-yl}-2-propanol (4). 2-(6-Amino-
9H-purin-8-yl)-2-propanol 3 (1.0 g, 5.18 mmol) was dissolved
in dry DMF (25 mL) with heating and stirring (45-50 °C)
under nitrogen. To the clear solution was added freshly
prepared 3-cyclopentyloxy-4-methoxy-benzyl chloride21 (1.3 g,
5.4 mmol), and the resulting mixture was heated to 100 °C
with stirring for 4 h. The cooled solution was evaporated to
dryness in vacuo, the residue was partitioned between satu-
rated sodium bicarbonate solution (400 mL) and dichlo-
romethane (400 mL), the organic phase was separated and
dried (MgSO4), and the solvent was evaporated to dryness in
vacuo. The residue was chromatographed over flash silica,
eluting with dichloromethane/methanol (8:1) to give a yellow
gum. Trituration with ice-cold acetone (50 mL) gave 4 as a
white solid (0.63 g, 31%): mp ) 207.2-209 °C. TLC (SiO2, CH2-
Cl2/MeOH, 8:1) Rf ) 0.22; detection, UV and Dragendorff’s
reagent. δH {400 MHz, DMSO-d6}: 1.50 (6H, s), 1.55-1.96 (8H,
m), 3.66 (3H, s), 4.66 (1H, m), 4.75 (1H, s,), 5.36 (2H, s), 6.86
(1H, d, J ) 8.3 Hz), 7.04 (1H, dd, J ) 8.2, 1.8 Hz), 7.24 (1H,
d, J ) 1.8 Hz), 7.73 (1H, bs), 8.46 (1H, s).
Experimental Section
Anhydrous diethyl ether (ether) and tetrahydrofuran (THF)
were obtained by distillation from sodium hydride/benzophen-
one ketyl under argon immediately prior to use. All other
reagents were used as obtained from commercial sources or
purified by standard methods. N,N-Dimethylformamide is
abbreviated to DMF and ethyl acetate to EtOAc. All reactions
were performed under an inert atmosphere unless otherwise
stated. Thin-layer chromatography was performed on glass
plates precoated with Merck silica gel 60 F254 0.2 mm, which
were visualized by quenching of UV fluorescence (λmax 254 nM)
and by staining with iodine vapor or Dragendorff’s reagent.
Rf values are quoted to the nearest 0.05. Flash chromatography
was performed on YMC 60A I-400/230 mesh silica. Melting
points (mp) were determined using an Electrothermal IA9200
apparatus and are uncorrected. 1H NMR spectra were recorded
Preparation of 1-(6-Amino-9H-purin-8-yl) Ethanol (6).
1-(6-Chloro-9H-purin-8-yl) ethanol (prepared from 6-chloro-9-
tetrahydropyran-2-yl-purine27 using n-butyllithium, in tet-

1240 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Whitehead et al.
rahydrofuran at -78 °C, followed by treatment with acetal-
dehyde, using the literature method25) (5.7 g, 28.7 mmol) was
dissolved in concentrated ammonia F ) 0.88 (30 mL), and the
solution was heated to 150 °C with stirring in a sealed tube
for 48 h. The cooled mixture was evaporated to dryness in
vacuo. The residue was chromatographed over flash silica
eluting with dichloromethane/methanol (5:1) to give 6 as a
white solid (4.27 g, 83%): mp 240.1-247 °C. TLC, SiO2 (CH2-
Cl2/MeOH, 5:1) Rf ) 0.41; detection, UV. δH {400 MHz, DMSO-
d6}: 1.32 (3H, d, J ) 6.6 Hz), 4.87 (1H, q, J ) 6.6 Hz), 5.8
(1H, bs), 7.05 (2H, bs), 8.11 (1H, s), 12.5-13.0 (1H, bs).
Preparation of 1-{6-Amino-3-[3-(cyclopentyloxy)-4-
methoxybenzyl]-3H-purin-8-yl}ethanol (7). 3-Cyclopentyl-
oxy-4-methoxybenzyl chloride21 (5.91 g, 24.55 mmol) and 1-(6-
amino-9H-purin-8-yl) ethanol 6 (4.0 g, 22.3 mmol) were heated
together in dry DMF (35 mL) with stirring at 100 °C under
nitrogen for 4 h. The solvent was evaporated to dryness in
vacuo, and the residue was partitioned between CH2Cl2/MeOH
(5:1) (400 mL) and saturated sodium bicarbonate solution (400
mL). The organic phase was separated and dried (MgSO4), and
the solvent was evaporated in vacuo to leave a yellow gum.
Flash chromatography (SiO2, CH2Cl2/MeOH, 12:1) gave a
yellow foam which was triturated with cold acetone (0 °C) to
give 7 (2.64 g, 31%) as a white solid: mp ) 199-202 °C. MH+
) 384.05 (100%). TLC (SiO2, CH2Cl2/MeOH, 12:1) Rf ) 0.25;
detection, UV and Dragendorff’s reagent. δH {400 MHz,
DMSO-d6}: 1.42 (2H, d, J ) 6.5 Hz), 1.48-1.70 (6H, m), 1.77-
1.88 (2H, m), 3.67 (3H, s), 4.67 (1H, m), 4.73 (1H, d, J ) 4.6
Hz), 4.79 (1H, m), 6.85 (1H, d, J ) 8.3 Hz), 6.99 (1H, dd, J )
8.2, 1.6 Hz), 7.24 (1H, d, J ) 1.6 Hz), 7.70 (2H, bs), 8.43 (1H,
s). Anal. (C20H25N5O5‚0.3H2O) C, H, N.
General Procedure for the Preparation of Fumarate
Salts. Fumaric acid (1.05 equiv) was dissolved in the minimum
quantity of anhydrous ether/methanol (10:1). This solution was
added dropwise to the adenines 5a-v or 8a-c (1 equiv) in a
minimum volume of ether, immediately forming a precipitate.
Filtration and washing with cold anhydrous ether (3 × 2 mL)
gave the desired salt as a crystalline solid. Evaporation of the
solvent and trituration with cold ether gave a second crop of
the salt.
Preparation of 8-(1-Benzyloxy-1-methyl-ethyl)-3-(3-
cyclopentyloxy-4-methoxy-benzyl)-3H-purin-6-ylamine
Hydrochloride (5a). Using the general procedure b, we
obtained the 3H-purin-6-ylamine 5a (205 mg, 35% yield) as a
white solid. It was dissolved in ether (40 mL) and treated with
hydrogen chloride 1 M in ether (0.5 mL, 0.5 mmol). The
mixture was filtered, washed with ether (20 mL), and dried
in vacuo to give the hydrochloride salt as a white solid: mp )
203 °C. δH {500 MHz, DMSO-d6}: 1.53-1.69 (2H, m), 1.74 (6H,
s), 1.80-1.97 (6H, m), 3.70 (3H, s), 4.48 (2H, s), 4.73 (1H, m),
5.45 (2H, s), 6.91 (1H, d, J ) 8.3 Hz), 7.11 (1H, dd, J ) 8.3,
2.0 Hz), 7.20 (1H, d, J ) 2.0 Hz), 7.24-7.37 (5H, m), 8.99 (1H,
s), 9.34 (1H, bs), 14.04 (2H, bs). Anal. (C28H33N5O3‚HCl) C, H,
N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(2-methoxy-benzyloxy)-1-methyl-ethyl]-3H-pu-
rin-6-ylamine (5b). Using the general procedure b, we
obtained the 3H-purin-6-ylamine 5b (10% yield): mp ) 202.3-
202.5 °C. δH {400 MHz, CDCl3}: 1.49-1.92 (14H, m), 3.81 (3H,
s), 3.86 (3H, s), 4.65-4.68 (1H, m), 4.81 (2H, s), 5.41 (2H, s),
6.78-6.95 (4H, m), 7.06 (1H, s), 7.20-7.32 (2H, m), 7.97 (1H,
s). Anal. (C29H35N5O3‚1.2H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(3-methoxy-benzyloxy)-1-methyl-ethyl]-3H-pu-
rin-6-ylamine Hemifumarate Hemihydrate (5c). Using
the general procedure b, we obtained the 3H-purin-6-ylamine
5c (10% yield). The fumarate salt was formed using the general
procedure above: mp ) 171.1-171.3 °C. δH {500 MHz,
CDCl3}: 1.54-1.59 (2H, m), 1.73-1.86 (12H, m), 3.77 (3H, s),
3.82 (3H, s), 4.44 (2H, s), 4.66-4.68 (1H, m), 5.45 (2H, s), 6.63
(1H, dd, J ) 8.0, 2.0 Hz), 6.81 (1H, d, J ) 8.0 Hz), 6.94-7.01
(5H, m), 7.17 (1H, t, J ) 8.0 Hz), 8.06 (1H, s). Anal.
(C29H35N5O4‚0.5C4H4O4‚0.5H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-methoxy-benzyloxy)-1-methyl-ethyl]-3H-pu-
rin-6-ylamine Hemifumarate (5d). Using the general pro-
cedure b, we obtained the 3H-purin-6-ylamine 5d (40% yield).
It was converted to the fumarate salt using the general
procedure above: mp ) 199.8-200.2 ° C. δH {500 MHz, DMSO-
d6}: 1.48-1.55 (2H, m), 1.63-1.68 (4H, m), 1.71 (6H, s), 1.77-
1.85 (2H, s), 3.70 (3H, s), 3.74 (3H, s), 4.17 (2H, s), 4.65-4.70
(1H, m), 5.45 (2H, s), 6.63 (1H, s), 6.83 (2H, d, J ) 8 Hz), 6.86
(2H, d, J ) 8.5 Hz), 7.10 (1H, dd, J ) 8.15, 1.5 Hz), 7.18 (2H,
d, J ) 8 Hz), 7.25 (1H, d, J ) 1.5 Hz), 7.80-7.98 (2H, brs),
8.55 (1H, s). Anal. C29H35N5O4‚0.5C4H4O4) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-methyl-benzyloxy)-1-methyl-ethyl]-3H-purin-
6-ylamine Hemifumarate (5e). Using the general procedure
b, we obtained the 3H-purin-6-ylamine 5e (35% yield). This
was converted to the fumarate salt using the general procedure
above: mp ) 196.9-197.2 °C. δH {500 MHz, CDCl3}: 1.55-
1.57 (2H, m), 1.73-1.86 (12H, m), 2.28 (3H, s), 3.83 (3H, s),
4.42 (2H, s), 4.65-4.68 (1H, m), 5.46 (2H, s), 6.81 (1H, d, J )
8.0 Hz), 6.94-6.98 (3H, m), 7.08 (2H, d, J ) 8.0 Hz), 7.29 (2H,
J ) 8.0 Hz), 8.06 (1H, s), 10.74 (1H, brs). Anal. (C29H35N5O3‚
0.5C4H4O4) C, H, N.
Preparation of 8-{1-[(4-Chlorobenzyl)oxy]-1-methyl-
ethyl}-3-[3-cyclopentyloxy)-4-methoxybenzyl]-3H-purin-
6-amine Hemifumarate Hydrate (5f). Using the general
procedure b, we obtained the 3H-purin-6-ylamine 5f (10%
yield). It was converted to the fumarate salt using the general
procedure above: mp ) 170.8-171.3 °C. δH {500 MHz,
CDCl3}: 1.56-1.58 (2H, m), 1.73-1.88 (12H, m), 3.83 (3H, s),
4.43 (2H, s), 4.66-4.69 (1H, m), 5.45 (2H, s), 6.81 (1H, d, J )
8.0 Hz), 6.92-6.96 (4H, m), 7.23 (2H, d, J ) 8.5 Hz), 7.34 (2H,
General Procedure B for the Alkylation of 4. Potassium
hydride (2.2 equiv, 35 wt % dispersion in mineral oil) under a
constant flow of argon was transferred to a flame dried round-
bottom flask. The potassium hydride was washed with pentane
(2 mL, dried over NaH). Compound 4 (1 equiv, 1 mmol), then
THF (15 mL) was added, and the solution was heated to 50
°C for 1 h. The resulting bright yellow solution was cooled to
0 °C, and the substituted benzyl halide (1.2 equiv) in THF (5
mL) was added dropwise over 20 min. The reaction was
maintained at 0 °C for 1 h and then allowed to warm to room
temperature. The reaction was stirred for 5 h at room
temperature or until visualization of the bis-benzylated mater-
ial by TLC. The reaction was recooled to 0 ° C, and water (10
mL) was added dropwise. EtOAc (10 mL) was added, and the
layers were separated. The aqueous layer was extracted with
EtOAc (2 × 10 mL), and the combined organics were dried
(MgSO4), filtered, and evaporated under reduced pressure. The
residue was triturated with cold ether (10 mL) to give
recovered starting material 4 (40-70%). Evaporation of the
ether filtrate under reduced pressure gave the crude product.
Purification by flash column chromatography [SiO2, EtOAc
(500 mL), and then EtOAc/MeOH/NH3, 60:10:1 (460 mL)] gave
the product (6-35%). This was converted to its crystalline
fumarate salt in most cases.
General Procedure E for the Alkylation of 7. Sodium
hydride (1.2 equiv, 60% dispersion in mineral oil) under a
constant flow of argon was transferred to a flame-dried round-
bottom flask. The sodium hydride was washed with pentane
(2 mL, dried over NaH). DMF (40 mL) and then compound 7
(1 mmol, 1.0 equiv) were added, and the solution was heated
to 50 °C for 30 min. The resulting bright yellow solution was
cooled to 0 °C, and the substituted benzyl halide (1.2 equiv)
in DMF (3 mL) was added dropwise. The reaction was
maintained at 0 °C for 1 h and then allowed to warm to room
temperature. The reaction was recooled to 0 °C, and water (10
mL) was added dropwise. EtOAc (20 mL) was added, and the
layers were separated. The organic layer was washed with
brine (3 × 10 mL), dried (MgSO4), filtered, and evaporated
under reduced pressure to give the crude product. Flash
chromatography (SiO2, EtOAc/MeOH/NH3, 100:10:1 to 40:10:
1; gradient elution) gave the product (3-35%) which if not
crystalline was converted to its fumarate salt.

8-Substituted Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1241
d, J ) 8.5 Hz), 8.08 (1H, s). Anal. (C28H32ClN5O3‚0.5C4H4O4‚
procedure b, we obtained the 3H-purin-6ylamine 5l (13%
yield). This was converted to the fumarate salt using the
general procedure above: mp ) 164.1-164.5 °C. δH {400 MHz,
CDCl3}: 1.53-1.58 (2H, m), 1.71-1.88 (12H, m), 3.82 (3H, s),
4.31 (2H, s), 4.64-4.65 (1H, m), 5.46 (2H, s), 5.87 (2H, s), 6.69
(1H, d, J ) 8.0 Hz), 6.81 (2H, m), 6.90-7.01 (4H, m), 8.02 (1H,
s). Anal. (C29H33N5O5‚0.5C4H4O4‚0.3H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-cyano-benzyloxy)-1-methyl-ethyl]-3H-purin-
6-ylamine (5m). Using the general procedure b, we obtained
the 3H-purin-6-yl-amine 5m (10% yield). This was converted
to the fumarate salt using the general procedure above: mp
) 170 °C. δH {400 MHz, CDCl3}: 1.55-1.62 (2H, m), 1.73 (6H,
s), 1.78-1.90 (8H, m), 3.83 (3H, s), 4.65-4.73 (1H, m), 4.91
(2H, s), 5.42 (2H, s), 6.80-6.85 (2H, m), 6.92-7.00 (2H, m),
7.50-7.59 (4H, m), 8.08 (1H, s), 11.26 (1H, brs).
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(3,4-dichloro-benzyloxy)-1-methyl-ethyl]-3H-pu-
rin-6-ylamine Hemifumarate Hemihydrate (5n). Using
the general procedure b, we obtained the 3H-purin-6ylamine
5n (29% yield). This was converted to the fumarate salt using
the general procedure above: mp ) 215 °C. δH {400 MHz,
CDCl3}: 1.32-1.42 (2H, m), 1.56-1.67 (8H, m), 3.62 (3H, s),
4.16 (2H, s), 4.49 (1H, m), 5.30 (2H, s), 6.60 (1H, d, J ) 8.3
Hz), 6.63 (1H, s), 6.78 (1H, dd, J ) 8.3, 2.0 Hz), 6.85 (1H, d, J
) 2.0 Hz), 7.02 (1H, dd, J ) 8.25, 1.9 Hz), 7.15 (1H, d, J ) 8.2
Hz), 7.37 (1H, d, J ) 1.83 Hz), 7.95 (1H, s). Anal. (C28H31-
Cl2N5O3‚0.5C4H4O4‚0.5H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-tert-butyl-benzyloxy)-1-methyl-ethyl]-3H-pu-
rin-6-ylamine Hemifumarate Hydrate (5o). Using the
general procedure b, we obtained the 3H-purin-6ylamine 5o
(29% yield). It was converted to the fumarate salt using the
general procedure above: mp ) 167 °C. δH {400 MHz,
CDCl3}: 1.23 (9H, s), 1.50 (2H, m), 1.70-1.78 (6H, m), 1.79
(6H, s), 3.77 (3H, s), 4.38 (2H, s), 4.63 (1H, m), 5.43 (2H, s),
6.76 (1H, d, J ) 8.2 Hz), 6.79 (1H, s), 6.97 (1H, dd, J ) 8.2,
2.0 Hz), 7.01 (1H, d, J ) 2.0 Hz), 7.23-7.28 (4H, m), 7.99 (1H,
s). Anal. (C32H41N5O3‚0.5C4H4O4‚H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-trifluoromethoxy-benzyloxy)-1-methyl-ethyl]-
3H-purin-6-ylamine Hemifumarate (5p). Using the general
procedure b, we obtained the 3H-purin-6ylamine 5p (19%
yield). It was converted to the fumarate salt using the general
procedure above: mp ) 210 °C. δH {400 MHz, CDCl3}: 1.33-
1.37 (8H, m), 2.15 (6H, s), 3.36 (3H, s), 3.95 (2H, s), 4.24 (1H,
brs), 5.06 (2H, s), 6.31 (1H, s), 6.36-6.38 (1H, s), 5.59-5.61
(1H, s), 6.67-6.69 (2H, m), 6.96-6.98 (1H, s), 7.79 (1H, s).
Anal. (C29H32F3N5O4‚0.5C4H4O4) C, H, N.
Preparation of 4-{2-[6-Amino-3-(3-cyclopentyloxy-4-
methoxy-benzyl)-3H-purin-8-yl]-2-methyl-propoxy}-ben-
zoic Acid Methyl Ester Hemifumarate Hemihydrate
(5q). Using the general procedure b, we obtained the 3H-purin-
6ylamine 5q (36% yield). It was converted to the fumarate salt
using the general method above: mp ) 188.5-189 °C. δH {400
MHz, DMSO-d6}: 1.45-1.51 (2H, m), 1.56-1.65 (4H, m), 1.69
(6H, s), 1.72-1.79 (2H, m), 3.65 (3H, s), 3.82 (3H, s), 4.32 (2H,
s), 4.63 (1H, m), 5.38 (2H, s), 6.60 (1H, s), 6.75 (1H, d, J ) 8.3
Hz), 7.02 (1H, dd, J ) 8.3, 1.7 Hz), 7.19 (1H, d, J ) 1.7 Hz),
7.40 (2H, d, J ) 8.2 Hz), 7.85 (2H, d J ) 8.2 Hz), 7.90-7.95
(2H, brs), 8.50 (1H, s), 13.15 (1H, bs). Anal. (C30H35N5O5‚
0.5C4H4O4‚0.5H2O) C, H, N.
Preparation of 4-{2-[6-Amino-3-(3-cyclopentyloxy-4-
methoxy-benzyl)-3H-purin-8-yl]-2-methyl-propoxy}-ben-
zamide Fumarate Hemihydrate (5r). The methyl ester 5q
(200 mg, 0.367 mmol) was dissolved in ethanol (5 mL).
Concentrated ammonia F ) 0.88 (2 mL) was added, and the
mixture was heated to 100 °C in a sealed tube for 18 h. The
cooled reaction mixture was evaporated to dryness in vacuo,
and the residue was chromatographed over flash silica (SiO2,
CH2Cl2/MeOH, 20:1) to give the amide 5r (151 mg, 75%) as a
white solid: mp ) 167.3-168.2 °C; Rf ) 0.07; detection, UV
and Dragendorff’s reagent. δH {400 MHz, DMSO-d6}: 1.45-
1.52 (2H, m), 1.57-1.67 (4H, m), 1.70 (6H, s), 1.72-1.80 (2H,
H2O) C, N. H analysis was 0.41% lower than theory.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(2-fluoro-benzyloxy)-1-methyl-ethyl]-3H-purin-
6-ylamine (5g). 2-{6-Amino-3-[3-(cyclopentyloxy)-4-methoxy-
benzyl]-3H-purin-8-yl}-2-propanol 4 (400 mg, 1.006 mmol) was
alkylated with 2-fluorobenzyl chloride (0.145 mL, 1.2 mmol)
using the general method b to give a colorless gum (120.9 mg,
0.239 mmol) after flash column chromatography (SiO2, CH2-
Cl2/MeOH, 20:1): Rf ) 0.15; detection, UV and Dragendorff’s
reagent. This was dissolved in ether (20 mL); fumaric acid
(27.8 mg, 0.239 mmol) in methanol(0.5 mL) was added, and
the mixture was allowed to crystallize slowly. The mixture was
filtered to give 5g (103.5 mg, 17%) as a white solid: mp )
202.5-203 °C. δH {400 MHz, DMSO-d6}: 1.47-1.52 (2H, m),
1.58-1.60 (4H, m), 1.70 (6H, s), 1.74-1.79 (2H, m), 3.66 (3H,
s), 4.30 (2H, s), 4.65-4.63 (1H, m), 5.40 (2H, s), 6.60 (1H, s,),
6.80 (1H, d, J ) 8.3 Hz), 7.06-7.14 (3H, m), 7.29 (1H, d, J )
7.5 Hz), 7.22-7.29 (1H, m), 7.42 (1H, t, J ) 6.7 Hz), 7.85-
8.00 (2H, bd), 8.51 (1H, s). Anal. (C28H32FN5O3‚0.5C4H4O4‚
0.25H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(3-fluoro-benzyloxy)-1-methyl-ethyl]-3H-purin-
6-ylamine Hemifumarate Hemihydrate (5h). 2-{6-Amino-
3-[3-(cyclopentyloxy)-4-methoxybenzyl]-3H-purin-8-yl}-2-
propanol 4 (400 mg, 1.006 mmol) was alkylated with 3-fluoro-
benzyl chloride (0.145 mL, 1.2 mmol) using method b to give
a white solid (126.9 mg, 0.25 mmol) after flash column
chromatography (SiO2, CH2Cl2/MeOH, 25:1): Rf ) 0.22; detec-
tion, UV and Dragendorff’s reagent. This was dissolved in
ether (15 mL); fumaric acid (29.1 mg, 0.25 mmol) in methanol
(0.4 mL) was added, and the resulting mixture was allowed
to crystallize slowly with cooling to 0 °C. The mixture was
filtered and dried in vacuo at 50 °C to give 5 h (102.1 mg,
16%) as a white solid: mp ) 197.5-198 °C. δH {400 MHz,
DMSO-d6}: 1.45-1.65 (6H, m), 1.69 (6H, s), 1.75-1.85 (2H,
m), 3.69 (1H, s), 4.27 (2H, s), 4.62-4.66 (1H, m), 5.39 (2H, s),
6.61 (1H, s), 6.79 (1H, d, J ) 8.3 Hz), 7.00-7.16 (4H, m), 7.22
(1H, d, J ) 1.67 Hz), 7.30 (1H, m), 7.75-7.95 (2H, m), 8.51
(1H, s). Anal. (C28H32FN5O3‚0.5C4H4O4) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(3,4-dimethoxy-benzyloxy)-1-methyl-ethyl]-3H-
purin-6-ylamine Fumarate Hemihydrate (5i). Using the
general procedure b, we obtained the 3H-purin-6ylamine 5i
(11% yield). This was converted to the fumarate salt using the
general method above: mp ) 160.2-160.8 °C. δH (400 MHz,
CDCl3): 1.58-1.59 (2H, m), 1.71 (6H, s), 1.78-1.88 (6H, m),
3.82 (3H, s), 3.83 (3H, s), 3.84 (3H, s), 4.66-4.70 (1H, m), 4.79
(2H, s), 5.40 (2H, s), 6.73-7.00 (8H, m), 8.10 (1H, s), 10.70
(1H, brs). Anal. (C30H37N5O5‚C4H4O4‚0.5H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(3,5-dimethoxy-benzyloxy)-1-methyl-ethyl]-3H-
purin-6-ylamine Fumarate (5j). Using the general proce-
dure b, we obtained the 3H-purin-6ylamine 5j (21% yield). This
was converted to the fumarate salt using the general procedure
above: mp ) 165.3-165.8 °C. δH {400 MHz, CDCl3}: 1.52-
1.59 (2H, m), 1.73-1.85 (12H, m), 3.73 (6H, s), 3.91 (3H, s),
4.38 (2H, s), 4.64-4.68 (1H, m), 5.43 (2H, s), 6.28 (1H, t, J )
2.5 Hz), 6.57 (2H, d, J ) 2.5 Hz), 6.79 (1H, d, J ) 8.0 Hz),
6.93-6.99 (4H, m), 8.03 (1H, s). Anal. (C30H37N5O6‚C4H4O4)
H, N. C analysis was 0.42% higher than theory.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(3,4-difluoro-benzyloxy)-1-methyl-ethyl]-3H-pu-
rin-6-ylamine Fumarate (5k). Using the general procedure
b, we obtained the3H-purin-6ylamine 5k (21% yield). This was
converted to the fumarate salt using the general procedure
above: mp ) 169.1-169.4 °C. δH {400 MHz, CDCl3 (1% DMSO-
d6)}: 1.38-1.42 (2H, m), 1.63-1.75 (12H, m), 3.66 (3H, s), 4.20
(2H, s), 4.51-4.54 (1H, m), 5.32 (2H, s), 6.64-6.66 (3H, m),
6.81-6.99 (4H, m), 7.14-7.19 (1H, m), 8.10 (1H, s). Anal.
(C28H31F2N5O3‚C4H4O4) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(3,4-methylenedioxy-benzyloxy)-1-methyl-ethyl]-
3H-purin-6-ylamine Hemifumarate (5l). Using the general

1242 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Whitehead et al.
s), 3.66 (3H, s), 4.31 (2H, s), 4.61-4.65 (1H, m), 5.40 (2H,s),
6.60 (2H, s), 6.79 (1H, d, J ) 8.3 Hz), 7.05(1H, dd, J ) 8.3, 1.8
Hz), 7.18 (1H, d, J ) 1.8 Hz), 7.32 (2H, d, J ) 8.1 Hz), 7.75
(2H, d, J ) 8.1 Hz), 8.45 (1H, s). Anal. (C29H34N6O4‚C4H4O4‚
0.5H2O) C, H, N.
6.69 (2H, s), 6.91 (1H, m), 7.09 (2H, m), 7.19 (1H, s), 7.37-
7.48 (2H, m), 8.84 (1H, bs), 8.98 (2H, bs), 13.85 (1H, bs).
HRMS: [M + 1]+ calcd for C26H32N5O3S, 494.2226; found,
494.2232. Anal. (C26H31N5O3‚0.5C4H4O4) C, H, N.
Preparation of 8-(1-Benzyloxy-ethyl)-3-(3-cyclopentyl-
oxy-4-methoxy-benzyl)-3H-purin-6-ylamine (8a). Using
the general procedure e, we obtained the 3H-purin-6ylamine
8a (16% yield): Rf ) 0.6 (EtOAc/MeOH/NH3, 10:1:1), mp )
210 °C. δH {400 MHz, CDCl3}: 1.56-1.61(2H, m), 1.78-1.91
(6H, m), 1.63 (3H, d, J ) 6.5), 3.84 (3H, s), 4.68-4.71 (1H, m),
4.85 (2H, d, J ) 15.5 Hz), 5.11 (1H, q, J ) 6.5 Hz), 5.44 (2H,
brs), 6.83 (1H, d, J ) 8 Hz), 6.94 (1H, dd, J ) 8, 2 Hz), 7.04
(1H, d, J ) 2 Hz), 7.24-7.41 (5H, m), 8.03 (1H, s). MS (ES)+
m/z: 496 (MNa+, 36%), 474 (MH+, 100%). Anal. (C27H31N5O3‚
0.25H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-methoxy-benzyloxy)-ethyl]-3H-purin-6-
ylamine (8b). Using the general procedure e, we isolated the
3H-purin-6ylamine 8b (5% yield): Rf ) 0.2 (EtOAc/MeOH/
NH3, 20:1:1), mp ) 204 °C. δH {400 MHz, CDCl3}: 1.56-1.67,
1.76-1.92 (11H, m), 3.80 (3H, s), 3.84 (3H, s), 4.68-4.72 (1H,
m), 4.77 (2H, brs), 5.09 (1H, q, J ) 6.5 Hz), 5.45 (2H, brs),
6.83-6.89 (4H, m), 6.95 (1H, dd, J ) 8, 2 Hz), 7.06 (1H, d, J
) 2 Hz), 7.30 (1H, bs), 8.00 (1H, brs). MS (ES+) m/z: 526
(MNa+, 28%), 504 (MH+, 100%). Anal. (C28H33N5O4‚0.25H2O)
C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-fluoro-benzyloxy)-ethyl]-3H-purin-6-
ylamine (8c). Using the general procedure e, we isolated the
3H-purin-6-ylamine 8c (3% yield): Rf ) 0.2 (EtOAc/MeOH/
NH3, 20:1:1), mp ) 217 °C. δH {400 MHz, CDCl3}: 1.60-1.69,
1.80-1.92 (8H, m), 1.72 (3H, d, J ) 7 Hz), 3.85 (3H, s), 4.69-
4.73 (1H, m), 4.84 (2H, brs), 5.16 (1H, q, J ) 7 Hz), 5.44 (2H,
brs), 6.86 (1H, d, J ) 8 Hz), 6.94 (1H, dd, J ) 8, 2 Hz), 6.99-
7.03 (3H, m), 7.36-7.53 (2H, m), 8.18 (1H, s). MS (ES+) m/z:
514 (MNa+, 41%), 492 (MH+, 100%). Anal. (C27H30FN5O3‚
0.25H2O) C, H. N analysis was 0.42% lower than theory.
PDE Binding Assay. The PDE4 enzyme was isolated from
cadaver human lung (8 h post-mortem); PDE3 and PDE5 were
isolated from freshly collected human platelets. The PDE4
isoform assays were obtained using recombinant hPDE4AL,
hPDE4BL, hPDE4D2, hPDE4D3, and hPDE4D5 from the
baculovirus system. They were obtained from Professor Conti
at Stanford University Medical Center. Assays were conducted
using [3H] cAMP SPA kit 40 µCi for PDE3 and PDE4; for the
PDE5 assay, [3H] cGMP SPA kit 40 µCi was used, both from
Amersham Biosciences Corp.31,32 In each assay, substrate
concentration was 200 nM, near the Km values for the enzymes
under the conditions of the assays.
Preparation of 4-{2-[6-Amino-3-(3-cyclopentyloxy-4-
methoxy-benzyl)-3H-purin-8-yl]-2-methyl-propoxy}-ben-
zoic Acid Sodium Salt Trihydrate (5s). The methyl ester
5q (200 mg, 0.367 mmol) was dissolved in ethanol (5 mL).
Sodium hydroxide (58.6 mg, 1.47 mmol) in water (0.2 mL) was
added, and the mixture was stirred at room temperature for
5 h, after which time a white precipitate formed. The mixture
was filtered, washed with cold ethanol (2 mL), and dried in
vacuo to give the sodium salt 5s (125 mg, 60%) as a white
solid: mp ) 317.8-319.6 °C. δH {400 MHz, DMSO-d6}: 1.46-
1.53 (2H, m), 1.55 (6H, s), 1.57-1.67 (4H, m), 1.78-1.85 (2H,
m), 3.66 (3H, s), 4.65-4.69 (1H, m), 5.47 (2H, m), 6.88 (1H, d,
J ) 8.3 Hz), 7.07 (1H, d, J ) 8.3 Hz), 7.17 (1H, s), 7.48 (2H, d,
J ) 8.1 Hz), 7.88 (2H, d, J ) 8.1 Hz), 9.05 (1H, s). Anal.
(C29H32N5O5Na‚3H2O) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1-(4-trifluoromethyl-benzyloxy)-1-methyl-ethyl]-
3H-purin-6-ylamine Hemifumarate (5t). Using the general
procedure b, we obtained the 3H-purin-6ylamine 5t (22%
yield). It was converted to the fumarate salt using the
procedure given: mp ) 196 °C. δH {500 MHz, CDCl3}: 1.25
(6H, s), 1.50-1.91 (8H, m), 3.80 (3H, s), 4.43 (2H, s), 4.68 (1H,
m), 5.47 (2H, s), 6.76 (1H, d, J ) 8.3 Hz), 6.96 (1H, dd, J )
8.3, 2 Hz), 7.02 (1H, d, J ) 2 Hz), 7.45-7.55 (4H, m), 7.95
(1H, s). Anal. (C29H32F3N5O3‚0.5C4H4O4) C, H, N.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1,1-dimethyl-2-(thiophen-2-yloxy)-ethyl]-3H-purin-
6-ylamine (5u). Potassium hydride 35% dispersion in mineral
oil (301.9 mg, 2.64 mmol) was washed with dry pentane (3 ×
10 mL) and suspended in dry THF (60 mL) under argon. 2-{6-
Amino-3-[3-(cyclopentyloxy)-4-methoxybenzyl]-3H-purin-8-yl}-
2-propanol 4 (500 mg, 1.26 mmol) was added, and the resulting
suspension was heated to 45-50 °C for 1 h. The mixture was
cooled to -10 °C; 2-chloromethylthiophene (183.8 mg, 1.39
mmol) in dry THF (20 mL) was added, and the mixture was
allowed to warm to room temperature with stirring for 48 h.
The mixture was quenched with brine (250 mL) and extracted
with EtOAc (2 × 250 mL); the organic phase was dried
(MgSO4), and the solvent was evaporated to dryness in vacuo
to leave a yellow gum. Flash chromatography(SiO2, CH2Cl2/
MeOH, 20:1) gave a yellow gum (67.9 mg, 0.137 mmol), which
was dissolved in ether/methanol (10:1, 2 mL), and fumaric acid
(16 mg, 0.137 mmol) in methanol (0.4 mL) was added. The
mixture was cooled in ice for 1 h and filtered to give 5u as a
white solid (35 mg, 6%): mp ) 162-165.9 °C. TLC (SiO2, CH2-
Cl2/MeOH, 20:1) Rf ) 0.21; detection, UV and Dragendorff’s
reagent. δH {400 MHz, DMSO-d6}: 1.48-1.53 (2H, m), 1.58
(4H, m), 1.67 (6H, s), 1.75-1.79 (2H, m), 3.60 (3H, s), 4.40 (2H,
s), 4.65 (2H, s), 5.41 (2H, s), 6.55 (2H, s), 6.83-6.86 (2H, m),
6.90 (1H, dd, J ) 8.4, 3.48 Hz), 7.09 (1H, dd, J ) 8.2, 1.4 Hz),
7.23 (1H, d, J ) 1.4 Hz), 7.75-7.95 (2H, bd), 8.51 (1H, s). Anal.
(C26H31N5O3S‚C4H4O4‚ 0.5 H2O) C, H, N.
Acknowledgment. We are very grateful to Profes-
sor Marco Conti at the Stanford University Medical
Center Department of Gynecology and Obstetrics for the
supply of the cloned human PDE4 subtype enzymes.
Supporting Information Available: Elemental analysis
data of compounds 5a-v, 6, and 8a-c. This material is
available free of charge via the Internet at http://pubs.acs.org.
Preparation of 3-(3-Cyclopentyloxy-4-methoxy-ben-
zyl)-8-[1,1-dimethyl-3-(thiophen-3-yloxy)-ethyl]-3H-purin-
6-ylamine Hemifumarate (5v). 2-{6-Amino-3-[3-(cyclopen-
tyloxy)-4-methoxybenzyl]-3H-purin-8-yl}-2-propanol 4 (500 mg,
1.26 mmol) was alkylated with 3-chloromethylthiophene (183.8
mg, 1.39 mmol) using the general method b to give a white
solid (22 mg, 44.56 µmol) after flash column chromatography
(CH2Cl2/MeOH, 25:1) and trituration with EtOAc (20 mL). TLC
(SiO2, CH2Cl2/MeOH, 25:1) Rf ) 0.24; detection, UV and
Dragendorff’s reagent. The solid was dissolved in methanol
(1 mL); fumaric acid (5.2 mg, 44.56 µmol) in methanol (1 mL)
was added, and the mixture was evaporated to dryness in
vacuo. The residue was dissolved in hot EtOAc (10 mL) and
allowed to crystallize slowly at 0 °C. The mixture was filtered
to give 5v as a white solid: mp ) 191-193 ° C. δH{400 MHz,
DMSO-d6}: 1.49-1.57 (2H, m), 1.62-1.69 (4H, m), 1.71 (6H,
s), 1.79-1.88 (2H, m), 3.70 (3H, s), 4.42 (1H, m), 5.48 (2H, s),
References
(1) Flemming, D. H.; Crombie, D. L. Prevalence of asthma and hay
fever in England and Wales. Br. Med. J. 1987, 294, 279-83.
(2) Sly, R. M. Increases in death from asthma. Ann. Allergy 1984,
53, 2-25.
(3) Barnes, P. J. Asthma deaths a continuing problem. Advanced
Medicine; Sheppard, M., Ed.; Bailliere Tindall: London, 1988;
pp 53-61.
(4) Beavo, J. A. Multiple isozymes of cyclic nucleotide phosphodi-
esterase. In Advances in Second Messenger and Phosphorylation
Research; Greengard, P., Robinson, G. A., Eds.; Raven Press:
New York, 1988; Vol. 22, pp 1-38.
(5) Beavo, J. A.; Reifsnyder, D. H. Primary sequence of cyclic
nucleotide phosphodiesterase isozymes and the design of selec-
tive inhibitors. Trends Pharmacol. Sci. 1990, 11, 150-155.
(6) Torphy, T. J.; Hay, D. W. P. Biochemical regulation of airway
smooth muscle tone: An overview. In Airway Smooth Muscle:
Modulation of Receptors and Response; CRC Press: Boca Raton,
FL, 1990; pp 39-68.

8-Substituted Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1243
(7) Kramer, G. M. The adenylate cyclase-cAMP-protein kinase
pathway and regulation of immune response. Immunol. Today
1988, 9, 222-229.
(25) Leonard, N. J.; Bryant, J. D. Regioselective electrophilic reac-
tions on substituted purines. Predominant intermediacy of 6-
or 8-purinyl carbanions. J. Org. Chem. 1979, 44 (25), 4612-4617.
(26) Leonard, N. J.; Fujii, T. The synthesis of 7-substituted adenines
through the use of a blocking group at the 3-position. Site of
alkylation of 7-substituted adenines. J. Am. Chem. Soc. 1963,
85, 3719.
(27) Press, J. B.; Falotico, R.; Hajos, Z. G.; Sawyers, R. A.; Kanojia,
R. M.; Williams, L.; Haertlein, B.; Kauffman, J. A.; Lakas-Weiss,
C.; Salata, J. Synthesis and structure-activity relationship of
6-substituted purine derivatives as novel selective positive
inotropes. J. Med. Chem. 1992, 35 (24), 4509-4515.
(28) Pollok, B. A.; Fuog, E. D.; Robbins, M. D.; Fisher, D. A.; Umland,
J. P.; Pillar, J. S.; Cheng, J. B. Baculovirus expression of human
phosphodiesterase (PDE) IV enzymes: Comparison with rPDE-
IV produced in mammalian and bacterial expression systems.
Presented at the Baculovirus and Insect Cell Expression Sym-
posium, Pinehurst, NC, March 26-30, 1995.
(29) Spina, D. The potential of PDE4 inhibitors in asthma or COPD.
Curr. Opin. Invest. Drugs 2000, 1 (2), 204-213.
(30) Xu, R. X.; Hassal, A. M.; Vanderwall, D.; Lambert, H. M.;
Holmes, W. D.; Luther, M. A.; Rocque, W. J.; Milburn, M. V.;
Zhao, Y.; Ke, H.; Nolte, R. T. Atomic structure of PDE4. Insights
into phosphodiesterase mechanism and specificity. Science 2000,
288, 1822-25.
(31) Weishaar, R. E.; Burrows, S. D.; Kobylarz, D. C.; Quade, M. M.;
Evans, D. B. Multiple molecular forms of cyclic nucleotide
phosphodiesterase in cardiac and smooth muscle and in plate-
lets. Isolation, characterization, and effects of various reference
phosphodiesterase inhibitors and cardiotonic agents. Biochem.
Pharmacol. 1986, 35, 787-800.
(32) Weishaar, R. E.; Kobylarz-Singer, D. C.; Keiser, J.; Haleen, S.
J.; Major, T. C.; Rapundalo, S.; Peterson, J. T.; Panek, R.
Subclass of cyclic GMP-specific phosphodiesterase and their role
in regulating the effects of atrial natriuretic factor. Hypertension
1990, 15, 528-40.
(33) Houslay, M. D.; Sullivan, M.; Bolger, G. B. The multienzyme
PDE4 cyclic adenosine monophosphate-specific phosphodi-
esterase family: Intracellular targeting, regulation, and selective
inhibition by compounds exerting anti-inflammatory and anti-
depressant actions. Adv. Pharmacol. 1998, 44, 225-342.
(34) Bolger, G.; Michaeli, T.; Martins, T.; Souness, J. E.; Rao, S.
Proposal for pharmacologically distinct conformers of PDE4
cyclic AMP phosphodiesterases. Cell. Signalling 1997, 9 (3-4),
227-36.
(35) Muller, T.; Engel, P.; Fozard, J. R. Subtypes of the type 4 cAMP
phosphodiesterases: Structure, regulation and selective inhibi-
tion. TIPS 1996, 17, 294-298.
(36) Obernolte, R.; Ratzliff, J.; Baecker, P. A. Multiple splice variants
of phosphodiesterase PDE4C cloned from human lung and testis.
Biochim. Biophys. Acta 1997, 1353, 287-297.
(8) Torphy, T. J.; Undem, B. J. Phosphodiesterase inhibitors: New
opportunities for the treatment of asthma. Thorax 1991, 12, 19-
27.
(9) Torphy, T. J. Phosphodiesterase enzymes: Molecular targets for
novel anti-asthma agents. Am. J. Respir. Crit. Care Med. 1998,
157, 351-370.
(10) Soderling, S. H.; Beavo, J. A. Regulation of cAMP and cGMP
signalling: New phosphodiesterases and new functions. Curr.
Opin. Cell Biol. 2000, 12, 174-179.
(11) Torphy, T. J.; Livi, G. P.; Christensen, S. B. Novel phosphodi-
esterase inhibitors for the therapy of asthma. Drug News
Perspect. 1993, 6, 537-545.
(12) Palfreyman, M. N. Phosphodiesterase inhibitors as antiasth-
matic agents. In Annual Reports in Medicinal Chemistry; Bristol,
J. A., Ed.; Academic Press: New York, 1994; Vol. 29, pp 185-
194.
(13) Norman, P. PDE4 Inhibitors 1998. Expert Opin. Ther. Pat. 1998,
8 (7), 771-784.
(14) Teixeira, M. M.; Gristwood, R. W.; Cooper, N.; Hallewell, P. G.
Phosphodiesterase (PDE)4 Inhibitors: Anti-inflammatory drugs
of the future? TIPS 1997, 18 (5), 164-170.
(15) Hebenstreit, G. F.; Fellerer, K.; Fichte, K.; Fischer, G.; Geifer,
N.; Meya, U.; Sastre-y-Hernandez, M.; Schany, W.; Schratzer,
M.; Soukop, W. Rolipram in major depressive disorder. Results
of a double blind comparative study with imipramine. Pharma-
copsychiatry 1989, 22 (4), 156-160.
(16) Barnette, M. S.; Christensen, S. B.; Essayan, D. M. SB207499
(Ariflo), a potent and selective second-generation phosphodi-
esterase 4 inhibitor: in vitro anti-inflammatory actions. J.
Pharmacol. Exp. Ther. 1988, 284, 420-426.
(17) IDDB Drug Report for roflumilast (BY-217).
(18) IDDB Drug Report for D-4396 (Sch-351591).
(19) LAS-31025. Clin. Trials Monitor 1997, 69, 25-26.
(20) Cavalla, D.; Gale, D. A case history of successful virtual research.
Drug News Perspect. 1997, 10, 470-476.
(21) Cavalla, D.; Chasin, M.; Hofer, P.; Gehrig, A.; Wintergerst, P.
Preparation of purine derivatives having phosphodiesterase IV
inhibiting activity. WO 9931102A1, 1999; 44 pp.
(22) Gale, D. D.; Hofer, P.; Spina, D.; Seeds, E. A.; Banner, K. H.;
Harrison, S.; Douglas, G.; Matsumoto, T.; Page, C. P.; Wong, R.
H.; Jordan, S.; Smith, F.; Banik, N.; Halushka, P. V.; Cavalla,
D.; Rotshteyn, Y.; Kyle, D. J.; Burch, R. M.; Chasin, M.
Pharmacology of a new cyclic nucleotide phosphodiesterase type
4 inhibitor, V11294. Pulm. Pharmacol. Ther. 2003, 16, 97-104.
(23) Subrahmanyam, V.; Renwick, A. B.; Walters, D. G.; Price, R. J.;
Tonelli, A. P.; Lake, B. G. Metabolism of a novel phosphodi-
esterase-IV inhibitor (V11294) by human hepatic cytochrome
P450 forms. Xenobiotica 2002, 32 (6), 521-534.
(24) Kleinman, E. F.; Campbel, E.; Giordano, L. A.; Cohan, V. L.;
Jenkinson, T. H.; Cheng, J. B.; Shirley, J. T.; Pettipher, R. E.;
Salter, E. D.; Hibbs, T. A.; DiCapua, F. M.; Bordner, J. Striking
effects of hydroxamic acid substitution on the phosphodiesterase
type 4 (PDE4) and TNFR inhibitory activity of two series of
rolipram analogues; implications for a new active site model of
PDE4. J. Med. Chem. 1998, 41, 266-270.
(37) Engels, P.; Adbel’ Al, S.; Hulley, P. Brain distribution of four
rat homologues of the Drosophila dunce cAMP phosphodi-
esterase. J. Neurosci. Res. 1995, 41, 169-78.
JM030603W