Novel and selective calcitonin-inducing agents

Article abstract of DOI:10.1021/jm990558l

A series of xanthine sulfonamides is presented as a class of calcitonin (CT) inducers - a potentially new method for treating diseases associated with postmenopausal bone loss such as osteoporosis. We have found that certain di-n-butylxanthine sulfonamides 4 upregulate CT transcription in a CT-luciferase reporter gene assay (CT-luci) and increase the production and release of CT in a CT secretion/RIA assay (CTS). In addition, these compounds do not have potent PDE4 inhibitory activity as do the related xanthine methylene ketones such as denbufyllene (2). One compound in particular (9) shows a transcription activation ratio (TAR) of 2.1 in CT-luci, a CTS increase of 3.6-fold, and a PDE4 (phosphodiesterase type IV) IC₅₀ = 4.1 μM. In addition, this compound showed a statistically significant 47% trabecular bone protection in ovariectomized-induced osteopenia (OVX) rats as determined by assay when administered for 4 weeks at 30 mg/kg/day, i.p. by quantitative computed tomography (QCT). When administered p.o., compound 9 shows 50% trabecular bone protection when administered for 3 weeks at 50 mg/kg/day, i.p. This compared with salmon CT which shows 62% trabecular bone protection when administered at 50 IU/kg/day for 4 weeks.

Full text of DOI:10.1021/jm990558l

J . Med. Chem. 2000, 43, 1223-1233
1223
Novel a n d Selective Ca lciton in -In d u cin g Agen ts
Adam M. Gilbert,*,† Stephen Caltabiano,‡ Denise Roberts,§ Sam F. W. Sum,† Gerardo D. Francisco,† Kitae Lim,†
Magda Asselin,† J ohn W. Ellingboe,† Yogendra Kharode,‡ Anna Cannistraci,‡ Rita Francis,‡
Mark TrailSmith,‡ and David Gralnick‡
Chemical Sciences, Wyeth-Ayerst Research, 401 North Middletown Road, Pearl River, New York 10965-1299, Bone Metabolism
and Osteoporosis Research, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, Pennsylvania 19087-4588, and
OSI Pharmaceuticals, 106 Charles Lindbergh Boulevard, Uniondale, New York 11553-3632
Received November 8, 1999
A series of xanthine sulfonamides is presented as a class of calcitonin (CT) inducers - a
potentially new method for treating diseases associated with postmenopausal bone loss such
as osteoporosis. We have found that certain di-n-butylxanthine sulfonamides 4 upregulate CT
transcription in a CT-luciferase reporter gene assay (CT-luci) and increase the production and
release of CT in a CT secretion/RIA assay (CTS). In addition, these compounds do not have
potent PDE4 inhibitory activity as do the related xanthine methylene ketones such as
denbufyllene (2). One compound in particular (9) shows a transcription activation ratio (TAR)
of 2.1 in CT-luci, a CTS increase of 3.6-fold, and a PDE4 (phosphodiesterase type IV) IC50
)
4.1 µM. In addition, this compound showed a statistically significant 47% trabecular bone
protection in ovariectomized-induced osteopenia (OVX) rats as determined by assay when
administered for 4 weeks at 30 mg/kg/day, i.p. by quantitative computed tomography (QCT).
When administered p.o., compound 9 shows 50% trabecular bone protection when administered
for 3 weeks at 50 mg/kg/day, i.p. This compared with salmon CT which shows 62% trabecular
bone protection when administered at 50 IU/kg/day for 4 weeks.
In tr od u ction
approved for clinical use. However, the stimulation of
endogenous calcitonin synthesis and release by inducer
compounds would be expected to result in a similar
therapeutic effect as the administration of nasal s-CT.
To our knowledge, there have been no citings in the
literature to a CT inducer. Related to this idea, one
series of small organic molecules (1) has recently been
claimed to act as CT mimetics (Chart 1).8
Calcitonin (CT) is a 32-amino acid polypeptide hor-
mone secreted by the parafollicular or C cells of the
thyroid gland in response to elevated blood levels of
calcium. This hormone decreases blood calcium (hypo-
calcemic activity) primarily by inhibiting bone resorp-
tion through plasma membrane-associated receptors on
the osteoclast. High-turnover bone loss, as seen with
hypercalcemia of malignancy, estrogen withdrawal fol-
lowing the onset of menopause, and certain antiinflam-
matory or arthritis therapies, has recently been shown
to be preventable by the administration of salmon
calcitonin (s-CT).1 As recently demonstrated for post-
menopausal osteoporosis, s-CT treatment leads not only
to a maintenance of bone mass and total body calcium
but also to a decrease in the incidence of hip and
vertebral fractures.2 Thus, it is apparent that calcitonin
is an appropriate therapeutic for the prevention and
treatment of osteoporosis by virtue of its hypocalcemic
activity.
Although s-CT has demonstrated efficacy in the
prevention of high-turnover bone loss, a limitation for
its widespread use is the lack of oral bioavailability
necessitating administration by parental (intramuscu-
lar), intrapulmonary,3 or, most commonly, intranasal
routes.4 References to oral delivery systems for s-CT
have appeared in the patent literature: stable formula-
tions,5 polymer conjugation,6 and delivery agent-medi-
ated oral absorption,7 but currently none have been
High-throughput screening of our compound library
identified a potential CT-inducer compound, denbufyl-
lene (2), which was found to stimulate CT transcription
as assessed by the measurement of luciferase activity
from a CT promotor/luciferase receptor construct (CT-
luci) as well as CT secretion and release in a CT
secretion/RIA assay (CTS). However, we were aware
that 2 displays polypharmacy, most notably phosphodi-
esterase type IV (PDE4) inhibition.9 Moreover, xanthine
PDE4 inhibitors such as 2 possess emetic side effects
which prevent their clinical use due to their affinity for
a hypothesized high-affinity rolipram (3) binding site
in addition to their affinity for the catalytic site of
PDE4.10 There has been a suggested correlation between
PDE4 inhibitory activity and bone-sparing activity in
the literature: 2 has been reported to reduce bone loss
in ovariectomy-induced osteopenic (OVX) rats;11 how-
ever it is not clear whether the bone-sparing activity of
2 is due to its ability to increase CT transcription/CT
secretion or whether it was due to its PDE4 inhibitory
activity.
In this paper, we disclose a series of xanthine sul-
fonamides 4 that induce the expression and release of
endogenous calcitonin without the potent PDE4 inhibi-
tory activity of denbufylline (2). In addition, some of
these compounds were found to have bone-sparing
* To whom correspondence should be addressed. Phone: (914) 732-
4865. Fax: (914) 732-5561. E-mail: gilbera@war.wyeth.com.
† Wyeth-Ayerst Research, NY.
‡ Wyeth-Ayerst Research, PA.
§ OSI Pharmaceuticals.
10.1021/jm990558l CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/25/2000

1224 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
Ch a r t 1
Sch em e 1
activity in OVX rats which could lead to their use as a
new class of antiosteoporotic agents.
measure the ability of a compound to increase cellular
levels and secretion of human CT, and (3) a U937 cell
PDE4 enzyme assay. In this assay, compounds were
first tested for PDE4 activity at 30 µM; a percent
rolipram PDE4 activity was determined with no rolip-
ram inhibition set to 0% and full rolipram inhibition
set at 100% (PDE4 30). Compounds of further interest
were tested in this assay at multiple concentrations so
that a proper IC50 could be determined.
Compounds which displayed a transcription activa-
tion ratio (TAR) equal to or greater than 2.0 at 30 µM,
a CTS increase of equal to or greater than 2.0-fold at
30 µM, and a PDE4 IC50 g 1.0 µM were advanced to
OVX studies to measure bone-sparing activity which
was determined after 4 weeks of dosing using quantita-
tive computed tomography (QCT) assessment of femoral
bone mineral density.14,15
Ch em istr y
Xanthine sulfonamides 7-21, 23-29, and 37-41 are
prepared by sulfonating N(1),N(3)-di-n-butylxanthines
512 and 6 with various sulfonyl chlorides using either
(a) K2CO3 in acetone at 23 °C or (b) i-Pr2NEt in CH2Cl2
at 23 °C. Sulfonamides 31-36 were prepared by selec-
tively sulfonating N(3)-n-butylxanthine 3013 at N(7)
with 3,4-dimethoxybenzenesulfonyl chloride in CH2Cl2
at 23 °C, followed by alkylation at N(3) with alkyl
bromides using K2CO3 in DMF at 23 °C (Scheme 1).
P h a r m a cology
Newly synthesized compounds were evaluated in vitro
using (1) a CT-luci reporter gene assay (CT-luci) to
measure the ability of a compound to increase tran-
scription of a luciferase receptor gene linked to the
human CT gene, (2) a CT secretion/RIA assay (CTS) to
Resu lts a n d Discu ssion
The N(1),N(3)-di-n-butylxanthine sulfonamides in
Table 1 are representative of those compounds which

Calcitonin-Inducing Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1225
Ta ble 1. In Vitro Profile of N(1),N(3)-Dibutylxanthine Sulfonamides

1226 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Ta ble 1 (Continued)
Gilbert et al.
a
b
Structures of compounds confirmed by 1H NMR, IR, and MS. Analytical results are within (0.4% of the theoretical value. Calcitonin
luciferase assay (CT-luci) transcription activation ratio (TAR): transcription of a luciferease receptor gene linked to the human CT promotor
gene expressed as a ratio of luciferase activity in the presence of test compound (at 30 µM) compared to the luciferase activity in the
untreated control. c Calcitonin secretion assay (CTS): stimulation of CT secretion as expressed as a ratio of secretion activity in the
d
presence of test compound compared to the secretion activity in the untreated control. PDE4 30 assay: percent rolipram activity of the
PDE4 assay at 30 µM concentration of test compound. e PDE4 assay: concentration at which 50% of the activity of the PDE4 assay is
inhibited by test compound. n: number of experiments.
showed increased CT-luci/CTS values at 30 µM in
conjunction with having reduced PDE4 inhibitory activ-
ity compared to that of denbufylline (2). Practically all
of these compounds have CT-luci results similar to the
known PDE4 inhibitors denbufylline (2) and rolipram
(3) as well as elevated CTS values. Other replacements
for the sulfonamide moiety in this series - alkyl, ester,
amide - produced molecules which fail to increase CT-
luci/CTS and possessed moderate to potent PDE4
inhibitory activity.
Me compound 14 has excellent CT-luci and good CTS
data while still having moderate PDE4 acivity (IC50
)
1.3 µM). Dramatic increases in CTS activity are seen
with the 2,5-disubstituted phenyl compounds 15 and 16
(15 CTS: 6.6, 16 CTS: 6.6) although additional PDE4
inhibition is seen as well (15 PDE4 30: 92%, 16 PDE4
30: 86%).
4-Substituted phenyl sulfonamides give compounds
with the desired in vitro profile - good CT-luci and CTS
activity accompanied by weak to moderate PDE4 inhibi-
tion. All of the following 4-substituted aryl sulfon-
amides: the 4-OMe analogue 17, the 3,4-dimethoxy
analogue 19, the 4-On-Bu analogue 20, the 4-OCF3
analogue 21, the 4-NHOH compound 21, and the 4-t-
Bu analogue 28 possess CT-luci TARs from 1.8 to 2.5,
accompanied by CTS values from 4.6 to 5.6. All com-
pounds were moderate to weak PDE4 inhibitors, par-
ticularly compound 21 which shows no PDE4 inhibitory
activity. Combining ortho and para substitution leads
to potent compounds as well. The 2,4,6-trimethyl ana-
logue 26 possesses good CT-luci and CTS activity (CT-
luci: 1.9, CTS: 4.0) and moderate PDE4 inhibition
(IC50: 6.0 µM). The 2,4,5-trisubstituted compound 29
also possesses an excellent activity profile (CT-luci: 2.5,
CTS: 5.3, PDE4 IC50 ) 23.3 µM) consistent with
properties of an ortho- and a para-substituted sulfon-
amide.
The sulfonamide substituent was varied in order to
optimize the in vitro profile of the compounds in Table
1. Although the methyl sulfonamide 7 possesses good
CT-luci activity (TAR: 1.9) as well as good activity in
the CTS assay (CTS: 4.7), it also shows relatively potent
PDE4 inhibitory activity when tested at 30 µM (PDE4
30: 84%). This was surprising considering that this
compound had a relatively weak PDE4 IC50 (IC50: 25.2
µM). Longer alkyl chains (8: n-Pr, 9: (3-Cl)-n-Pr, 10:
n-Bu, and 11: n-octyl) maintained CT-luci and CTS
activity while showing reduced PDE4 inhibition. Sul-
fonamide 9 showed slightly more PDE4 inhibitory
activity than the other alkyl analogues when tested in
the PDE4 30 assay but only had an IC50 of 4.1 µM.
Aryl substituents on the sulfonamide give active
compounds as well. The simple phenyl compound 12
possesses good CT-luci and CTS activity, with very little
PDE4 inhibitory activity. Ortho substituents on the
phenyl ring can lead to increased CT-luci and CTS
activity but often at the expense of adding more PDE4
inhibition. For instance the o-F compound 13 maintains
the good CT-luci activity of 12 (CT-luci: 2.0) but lacks
much of its CTS activity (CTS: 1.7). However, the o-CO2-
In addition, other kinds of aromatic sulfonamides
possess our desired in vitro activity profile. The 1-naph-
thyl compound 24 has an in vitro profile very similar
to that of the Ph compound 12. The isosteric thiophene
sulfonamide 25 also shows a good in vitro profile
although its CTS activity is lower than that of 12.

Calcitonin-Inducing Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1227
Ta ble 2. In Vitro Profile of 3-Substituted 3,4-Dimethoxybenzene Sulfonamides
a
Structures of compounds confirmed by 1H NMR, IR, and MS. Analytical results are within (0.4% of the theoretical value unless
b
otherwise noted. Calcitonin luciferase assay (CT-luci) transcription activation ratio (TAR): transcription of a luciferase receptor gene
linked to the human CT promotor gene expressed as a ratio of luciferase activity in the presence of test compound (at 30 µM) compared
to the luciferase activity in the untreated control. c Calcitonin secretion assay (CTS): stimulation of CT secretion as expressed as a ratio
of secretion activity in the presence of test compound compared to the secretion activity in the untreated control. e PDE4 30 assay: percent
rolipram activity of the PDE4 assay at 30 µM concentration of test compound. e PDE4 assay: concentration at which 50% of the activity
of the PDE4 assay is inhibited by test compound. n: number of experiments.
Finally benzyl sulfonamide 26 shows excellent CT-luci
and CTS activity with a moderate PDE4 IC50 of 3.2 µM.
The CT-inducer activity of all of the aryl sulfonamides
without substantial PDE4 inhibition is noteworthy since
aryl sulfonamides (particularly 3,4-dimethoxyphenyl
sulfonamides) have previously been published as having
potent PDE4 inhibitory activity.16
values (1.5-1.8) and moderate CTS values (2.2-4.1).
However, all of these compounds have substantially
more PDE4 inhibitory activity than the corresponding
C(6) hydrogen compounds (see Table 1).
Several compounds from Table 1 with good CT-luci/
CTS values and weak to moderate PDE4 activity were
moved forward to OVX testing where bone-sparing
activity was determined by QCT measurements after 4
weeks of compound administration at 30 mg/kg/day
(Table 4). The 3-Cl-n-propyl sulfonamide 9 gave a
statistically significant 47% bone mineral density (BMD)
protection of trabecular bone when administered i.p.,
where 0% represents the BMD of the OVX rats and
100% is the BMD of sham-operated rats. This result
compares favorably to both s-CT, the marketed form of
CT, which gives a 62% bone-sparing effect when ad-
ministered i.p. at 50 IU/kg and to denbufyllene (2)
which gives a 49% bone-sparing effect at 30 mg/kg, i.p.
Compound 9 was also tested on p.o. administration at
50 mg/kg and gave a statistically significant 50% BMD
protection after 3 weeks of dosing. It is important to note
that 9 has a PDE4 IC50 of 4.1 µM; thus it is doubtful
that the bone-sparing activity could be due to PDE4
inhibition. In addition, further in vivo pharmacological
characterization showed increases in both thyroidal
calcitonin mRNA level and plasma calcitonin levels,
consistent with induction of endogenous calcitonin (data
not shown).
The observed CT-luci/CTS activity coupled with mini-
mal to moderate PDE4 inhibition is due to a combina-
tion of the sulfonamide moiety in addition to the
substituent at the N(3) position of the xanthine as
shown in Table 2. While compound 19 (N(3): n-Bu)
displays the desired in vitro profile, 31 (N(3): H) and
32 (N(3): n-octyl) have reduced PDE4 activity but at
the expense of CT-luci and CTS. Compounds 33 and 34
display excellent CT-luci activity (33 CT-luci: 2.8, 34
CT-luci: 2.9) but lack any CTS activity. In addition, both
of these compounds containing branched alkyl chains
at N(3) possess PDE4 inhibitory activity that ap-
proaches that of rolipram. Compounds 35 (N(3): allyl)
and 36 (N(3): 3-methylbutyl) show both excellent CT-
luci and CTS activity but have substantial PDE4
activity. The branching of the alkyl chains at N(3)
leading to more PDE4 inhibition is consistent with
known xanthine PDE4 SAR.17
The hydrogen at C(6) in several xanthine sulfon-
amides was replaced with a Me group in an attempt to
increase the CT-luci/CTS activity of these compounds.
The results of this replacement are presented in Table
3. Compounds 37-41 all possess moderate CT-luci
Other xanthine sulfonamides selected for OVX studies
were phenyl sulfonamides. Compounds 10, 18, 19, 24,

1228 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
Ta ble 3. In Vitro Profile of N(1),N(3)-Di-n-butyl-6-methylxanthine Sulfonamides
a
b
Structures of compounds confirmed by 1H NMR, IR, and MS. Analytical results are within (0.4% of the theoretical value. Calcitonin
luciferase assay (CT-luci) transcription activation ratio (TAR): transcription of a luciferase receptor gene linked to the human CT promotor
gene expressed as a ratio of luciferase activity in the presence of test compound (at 30 µM) compared to the luciferase activity in the
untreated control. c Calcitonin secretion assay (CTS): stimulation of CT secretion as expressed as a ratio of secretion activity in the
d
presence of test compound compared to the secretion activity in the untreated control. PDE4 30 assay: percent rolipram activity of the
PDE4 assay at 30 µM concentration of test compound. e PDE4 assay: concentration at which 50% of the activity of the PDE4 assay is
inhibited by test compound. n: number of experiments.
Ta ble 4. Ovariectomized-Induced Osteopenia Studies of Selected Compounds in Rats
Method of
Administration
Duration of
Assay (weeks)
Percent Protection
Compound
na
(trabecular BMD, 30 mg/kg/dayb
Salmon CT
2
9
9
10
18
19
24
26
27
29
i.p.
i.p.
i.p.
p.o.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
8
7
7
8
7
8
8
8
8
8
8
4
4
4
3
4
4
4
4
4
4
4
62 ( 5c*
49 ( 9*
47 (8*
50 ( 10d*
23 ( 7
30 ( 7
35 ( 8
37 ( 9
25 ( 8
50 ( 7*
52 ( 9*
a
b
d
Number of animals tested. Trabecular bone mineral density measured by QCT. c 50 IU/kg. 50 mg/kg/day. *p < 0.05: results are
statistically significant.
and 26 all showed some bone protection activity at 30
mg/kg/day, i.p., but none of the results were statistically
significant nor were these compounds as protective as
9. Compounds 27 and 29 however gave substantial
BMD protection (27: 50%, 29: 52%) at 30 mg/kg/day,
i.p. These compounds also have PDE4 IC50s of 6.0 and
23.3 µM, respectively. It should be noted that all rats
during the OVX studies did not suffer any species-
specific PDE4 inhibitory effects (rats: apnea) when
treated with any of the xanthine sulfonamides, an
observation that correlates with the weak to moderate
PDE4 inhibitory activity of these compounds.
activity. Many of these compounds have CT-luci TARs
of over 2.0 at 30 µM and CTS values of over 3.0 at 30
µM as well as moderate to weak PDE4 IC50s (g2.0 µM).
Several of these molecules were put into OVX rats and
were shown to be bone sparing. Compounds 9, 27, and
29 were shown to have statistically significant bone-
sparing activity with 9 having oral bone-sparing activity
at 50 mg/kg. All of these observations are consistent
with compound 9 and possibly several of the xanthine
sulfonamides presented above acting as CT inducers,
which represents a potentially novel method of treating
diseases of bone loss.
Con clu sion s
Exp er im en ta l Section
We have demonstrated that a series of xanthine
sulfonamides 4 can increase CT transcription secretion
in vitro with only moderate to weak PDE4 inhibitory
Melting points were determined on a Thomas-Hoover Mel-
temp apparatus and are uncorrected. The proton nuclear
magnetic resonance (1H NMR) spectra were recorded at 300

Calcitonin-Inducing Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1229
MHz on a Bruker DPX-300 spectrometer using tetramethyl-
silane (δ 0.0) as an internal standard. Infrared (IR) spectra
were obtained as KBr pellets. Combustion analyses were
obtained using a Perkin-Elmer series II 2400 CHNS/O ana-
lyzer. Mass spectra were obtained using a Micromass platform
electrospray ionization quadrapole mass spectrometer. Flash
chromatography was performed using EM Science 230-400
mesh silica gel. Thin-layer chromatography (TLC) was per-
formed in Analtech silica gel GHLF 250-µm prescored plates.
(s, 1H); IR (KBr, cm-1) 1710, 1668; MS 405 (MH)+. Anal.
C
19H24N4O4S: C, H, N.
1,3-Dib u t yl-7-(2-flu or ob en zen esu lfon yl)-3,7-d ih yd r o-
p u r in e-2,6-d ion e (13). Compound 13 was prepared according
to method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
(2-F)PhSO2Cl: yield 86%; mp 122-123 °C; 1H NMR (300 MHz,
CDCl3) δ 0.89 (t, J ) 7.5 Hz, 3H), 0.96 (t, J ) 7.5, 3H); 1.22-
1.43 (m, 4H), 1.45-1.54 (m, 2H), 1.72 (pent, J ) 7.5 Hz, 2H),
3.87 (t, J ) 7.5 Hz, 2H), 4.08 (t, J ) 7.4 Hz, 2H), 7.19 (t, J )
9.1 Hz, 1H), 7.44 (t, J ) 7.9 Hz, 1H), 7.68-7.78 (m, 1H), 8.37
(s, 1H), 8.44 (td, J ) 7.9, 1.7 Hz, 1H); IR (KBr, cm-1) 1713,
1665; MS 423 (MH)+. Anal. C19H23FN4O4S: C, H, N.
1,3-Dibu tyl-7-m eth a n esu lfon yl-3,7-d ih yd r op u r in e-2,6-
d ion e (7). Meth od A. To 500 mg (1.89 mmol) of 1,3-dibutyl-
3,7-dihydropurine-2,6-dione12 in 30 mL of acetone at 23 °C was
added 1.04 g (7.56 mmol) of K2CO3 followed by 0.15 mL (216
mg, 1.89 mmol) of MeSO2Cl. After stirring at 23 °C for 30 min,
the reaction mixture was filtered, and the filtrate collected and
evaporated to give a white solid. This solid was then triturated
with 30 mL of petroleum ether to give 500 mg (1.46 mmol, a
77% yield) of the title compound as a white crystalline solid:
2-(1,3-Dibu tyl-2,6-d ioxo-1,2,3,6-tetr a h yd r op u r in e-7-su l-
fon yl)ben zoic Acid Meth yl Ester (14). Compound 14 was
prepared according to method A using 1,3-dibutyl-3,7-dihy-
dropurine-2,6-dione and (2-CO2Me)PhSO2Cl: yield 60%; mp
1
106-109 °C; H NMR (300 MHz, CDCl3) δ 0.88-1.0 (m, 6H),
1.30-1.43 (m, 4H), 1.50-1.60 (m, 2H), 1.66-1.77 (m, 2H), 3.92
(t, J ) 7.6 Hz, 2H), 3.96 (s, 3H), 4.09 (t, J ) 7.6 Hz, 2H), 7.65-
1
mp 98-100 °C; H NMR (300 MHz, CDCl3) δ 0.93-0.99 (m,
7.81 (m, 3H), 8.32 (s, 1H), 8.86-8.94 (m, 1H); IR (KBr, cm-1
)
6H), 1.42-1.49 (m, 4H), 1.55-1.58 (m, 2H), 1.60-1.70 (m, 2H),
3.85 (s, 3H), 3.99-4.04 (m, 2H), 4.05-4.16 (t, 2H), 8.17 (s, 1H),
8.55; IR (KBr, cm-1) 1715, 1663, 1537, 1397, 1182; MS 343
(MH)+. Anal. C14H22N4O4S: C, H, N.
1711, 1668; MS 463 (MH)+. Anal. C21H26N4O6S: C, H, N.
1,3-Dibu tyl-7-(2,5-d im eth ylben zen esu lfon yl)-3,7-d ih y-
d r op u r in e-2,6-d ion e (15). Compound 15 was prepared ac-
cording to method A using 1,3-dibutyl-3,7-dihydropurine-2,6-
1,3-Dibu tyl-7-p r op a n esu lfon yl-3,7-d ih yd r op u r in e-2,6-
d ion e (8). Meth od B. To 412 mg (1.56 mmol) of 1,3-dibutyl-
3,7-dihydropurine-2,6-dione in 15 mL of CH2Cl2 at 23 °C were
added 0.41 mL (302 mg, 2.34 mmol) of i-Pr2NEt and 0.19 mL
(245 mg, 1.72 mmol) of n-PrSO2Cl. After stirring at 23 °C at
24 h, the reaction mixture as poured into 50 mL of brine and
extracted with 2 × 30 mL of EtOAc. The combined organics
were washed with 1 × 50 mL of H2O and 1 × 50 mL of brine,
dried over MgSO4, filtered and evaporated to a light yellow
oil. Flash chromatography on silica gel, eluting with CH2Cl2/
EtOAc (40/1), gave the title compound as an off-white solid.
Recrystallization from hot hexanes/EtOAc gave 156 mg (0.42
1
dione and (2,5-Me2)PhSO2Cl: yield 73%; mp 120-122 °C; H
NMR (300 MHz, CDCl3) δ 0.89 (t, J ) 7.3 Hz, 3H), 0.96 (t, J
) 7.4 Hz, 3H), 1.23-1.43 (m, 4H), 1.46-1.56 (m, 2H), 1.63-
1.78 (m, 2H), 2.46 (s, 6H), 3.88 (t, J ) 7.5 Hz, 2H), 4.08 (t, J
) 7.5 Hz, 2H), 7.17 (d, J ) 7.8 Hz, 1H), 7.37 (dd, J ) 1.9, 7.8
Hz, 1H), 8.32 (d, J ) 2.0 Hz, 1H), 8.35 (s, 1H); IR (KBr, cm-1
)
1712, 1666; MS 433 (MH)+. Anal. C21H28N4O4S: C, H, N.
1,3-Dibu tyl-7-(5-flu or o-2-m eth ylben zen esu lfon yl)-3,7-
d ih yd r op u r in e-2,6-d ion e (16). Compound 16 was prepared
according to method A using 1,3-dibutyl-3,7-dihydropurine-
2,6-dione and (5-F,2-Me)PhSO2Cl: yield 84%; mp 88-90 °C;
1H NMR (300 MHz, CDCl3) δ 0.90 (t, J ) 7.3 Hz, 3H), 0.96 (t,
J ) 7.4 Hz, 3H), 1.26-1.46 (m, 4H), 1.48-1.56 (m, 2H), 1.68-
1.74 (m, 2H), 2.48 (s, 3H), 3.88 (t, J ) 7.6 Hz, 2H), 4.08 (t, J
) 7.6 Hz, 2H), 7.26-7,33 (m, 2H), 8.24 (dd, J ) 2.2, 8.0 Hz,
1H), 8.35 (s, 1H); IR (KBr, cm-1) 1711, 1667; MS 437 (MH)+.
Anal. C20H25FN4O4S: C, H, N.
1
mmol, a 27% yield) of 8 as a white solid: mp 73-74 °C; H
NMR (300 MHz, CDCl3) δ 0.92-1.01 (m, 6H), 1.11 (t, J ) 7.4
Hz, 3H), 1.32-1.48 (m, 4H), 1.60-1.79 (m, 4H), 1.92 (hex, J
) 7.6 Hz, 2H), 3.96-4.06 (m, 4H), 4.12 (t, J ) 7.6 Hz, 2H),
8.15 (s, 1H); IR (KBr, cm-1) 1699, 1667, 1376; MS 371 (MH)+.
Anal. C16H26N4O4S: C, H, N.
1,3-Dibu tyl-7-(4-m eth oxyben zen esu lfon yl)-3,7-dih ydr o-
p u r in e-2,6-d ion e (17). Compound 17 was prepared according
to method A using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
(4-OMe)PhSO2Cl: yield 80%; mp 68-70 °C; 1H NMR (300
MHz, CDCl3) δ 0.91-0.99 (m, 6H), 1.38 (m, 4H), 1.53-1.75
(m, 4H), 3.89 (s, 3H), 3.94 (t, J ) 7.5 Hz, 2H), 4.06 (t, J ) 7.5
Hz, 2H), 7.03 (d, J ) 8.1 Hz, 2H), 8.24 (d, J ) 8.1 Hz, 2H),
8.27 (s, 1H); IR (KBr, cm-1) 1714, 1667, 1167; MS 435 (MH)+.
Anal. C20H26N4O5S: C, H, N.
1,3-Dib u t yl-7-(3-ch lor op r op a n e-1-su lfon yl)-3,7-d ih y-
d r op u r in e-2,6-d ion e (9). Compound 9 was prepared accord-
ing to method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione
1
and Cl(CH2)3SO2Cl: yield 90%; mp 71-73 °C; H NMR (300
MHz, CDCl3) δ 0.90-1.01 (m, 6H), 1.32-1.49 (m, 4H), 1.56-
1.80 (m, 4H), 2.30-2.42 (m, 4H), 3.69 (t, J ) 6.1 Hz, 2H), 4.01
(t, J ) 7.6 Hz, 2H), 4.12 (t, J ) 7.4 Hz, 2H), 4.23 (t, J ) 7.6
Hz, 2H), 8.15 (s, 1H); IR (KBr, cm-1) 1704, 1670, 1385; MS
406 (MH)+. Anal. C16H25ClN4O4S: C, H, N.
1,3-Dibu tyl-7-(3-n itr oben zen esu lfon yl)-3,7-d ih yd r op u -
r in e-2,6-d ion e (18). Compound 18 was prepared according
to method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
1,3-Dibu tyl-7-bu ta n esu lfon yl-3,7-d ih yd r op u r in e-2,6-d i-
on e (10). Compound 10 was prepared according to method A
using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and n-BuSO2-
Cl: yield 82%; mp 185-187 °C; 1H NMR (300 MHz, CDCl3) δ
0.93-0.99 (m, 9H), 1.39-1.88 (m, 11H), 3.98-4.09 (m, 6H),
4.10-4.19 (m, 4H), 8.14 (s, 1H); IR (KBr, cm-1) 1717, 1672,
1466, 1194; MS 385 (MH)+. Anal. C17H28N4O4S: C, H, N.
1
(3-NO2)PhSO2Cl: yield 79%; mp 178-179 °C; H NMR (300
MHz, CDCl3) δ 0.88-0.99 (m, 6H), 1.28-1.43 (m, 4H), 1.50-
1.62 (m, 2H), 1.62-1.75 (m, 2H), 3.93 (t, J ) 7.5 Hz, 2H), 4.10
(t, J ) 7.5 Hz, 2H), 7.86 (t, J ) 8.1 Hz, 1H), 8.34 (s, 1H), 8.56
(dd, J ) 2.0, 8.1 Hz, 1H), 8.77 (d, J ) 8.0 Hz, 1H), 9.01 (t, J )
2.0 Hz, 1H); IR (KBr, cm-1) 1717, 1661, 1538; MS 450 (MH)+.
Anal. C19H23N5O6S: C, H, N.
1,3-Dibu tyl-7-octa n esu lfon yl-3,7-d ih yd r op u r in e-2,6-d i-
on e (11). Compound 11 was prepared according to method B
using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and n-octylSO2-
Cl: yield 98%; mp 59-60 °C; 1H NMR (300 MHz, CDCl3) δ
0.89 (t, J ) 7.5 Hz, 3H), 0.92-1.00 (m, 6H), 1.20-1.32 (m, 8
H), 1.34-1.49 (m, 6H), 1.57-1.68 (m, 2H), 1.70-1.79 (m, 2H),
1.79-1.89 (m, 2H), 3.97-4.05 (m, 4H), 4.12 (t, J ) 7.5 Hz, 2H),
8.14 (s, 1H); IR (KBr, cm-1) 1708, 1667, 1373, 1178; MS 441
(MH)+. Anal. C21H36N4O4S: C, H, N.
1,3-Dib u t yl-7-(3,4-d im et h oxyb en zen esu lfon yl)-3,7-d i-
h yd r op u r in e-2,6-d ion e (19). Compound 19 was prepared
according to method B using 1,3-dibutyl-3,7-dihydropurine-
2,6-dione and (3,4-OMe)PhSO2Cl: yield 81%; mp 134-135 °C;
1H NMR (300 MHz, CDCl3) δ 0.88-0.99 (m, 6H), 1.30-1.43
(m, 4H), 1.50-1.62 (m, 2H), 1.63-1.76 (m, 2H), 3.91-4.00 (m,
2H), 3.95 (s, 3H), 3.99 (s, 3H), 4.09 (t, J ) 7.5 Hz, 2H), 6.98 (d,
J ) 8.7 Hz, 1H), 7.86 (dd, J ) 2.3, 8.7 Hz, 1H), 7.97 (d, J )
2.3 Hz, 1H), 8.25 (s, 1H); IR (KBr, cm-1) 1713, 1668, 1513,
1273; MS 465 (MH)+. Anal. C21H28N4O6S: C, H, N.
7-Ben zen esu lfon yl-1,3-d ibu tyl-3,7-d ih yd r op u r in e-2,6-
d ion e (12). Compound 12 was prepared according to method
A using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and PhSO2-
Cl: yield 61%; mp 144-146 °C; 1H NMR (300 MHz, CDCl3) δ
0.92 (t, J ) 7.5 Hz, 6H), 1.31-1.43 (m, 4H), 1.51-1.75 (m, 4H),
3.96 (t, J ) 7.5 Hz, 2H), 4.03 (t, J ) 7.5 Hz, 2H), 7.60 (t, J )
8.2 Hz, 2H), 7.68-7.76 (m, 1H), 8.29 (d, J ) 8.1 Hz, 2H), 8.29
7-(4-Bu toxyben zen esu lfon yl)-1,3-d ibu tyl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (20). Compound 20 was prepared according
to method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
1
(4-n-OBu)PhSO2Cl: yield 88%; mp 84-86 °C; H NMR (300

1230 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
MHz, CDCl3) δ 0.85-1.00 (m, 9H), 1.10-1.41 (m, 4H), 1.42-
1.53 (m, 2H), 1.53-1.61 (m, 2H), 1.61-1.75 (m, 2H), 1.75-
1.81 (m, 2H), 3.94 (t, J ) 7.6 Hz, 2H), 4.01-4.09 (m, 4H), 7.01
(d, J ) 8.1 Hz, 2H), 8.18 (d, J ) 8.1 Hz, 2H), 8.26 (s, 1H); IR
(s, 3H), 2.61 (s, 6H), 3.83 (t, J ) 7.4 Hz, 2H), 4.14 (t, J ) 7.4
Hz, 2H), 7.00 (s, 2H), 8.35 (d, J ) 2.1 Hz, 1H); IR (KBr, cm-1
)
1712, 1673, 1363; MS 447 (MH)+. Anal. C22H30N4O4S: C, H,
N.
(KBr, cm-1) 1718, 1668, 1169; MS 477 (MH)+. Anal. C23H32
N4O5S: C, H, N.
-
1,3-Dib u t yl-7-(4-ter t-b u t ylb en zen esu lfon yl)-3,7-d ih y-
d r op u r in e-2,6-d ion e (28). Compound 28 was synthesized
according to method B using 1,3-dibutyl-3,7-dihydropurine-
2,6-dione and 4-tert-butylbenzenesulfonyl chloride: yield 57%;
1,3-Dib u t yl-7-(4-t r iflu or om et h oxyb en zen esu lfon yl)-
3,7-d ih yd r op u r in e-2,6-d ion e (21). Compound 21 was syn-
thesized according to method B using 1,3-dibutyl-3,7-dihydro-
purine-2,6-dione and (4-OCF3)PhSO2Cl: yield 79%; mp 128-
129 °C; 1H NMR (300 MHz, CDCl3) δ 0.89-0.99 (m, 4H), 1.28-
1.43 (m, 4H), 1.52-1.63 (m, 2H), 1.63-1.78 (m, 2H), 3.94 (t, J
) 7.5 Hz, 2H), 4.07 (t, J ) 7.5 Hz, 2H), 7.40 (d, J ) 8.1 Hz,
2H), 8.28 (s, 1H), 8.37 (d, J ) 8.1 Hz, 2H); IR (KBr, cm-1) 1720,
1673, 1272, 1180, 1169; MS 489 (MH)+. Anal. C20H23F3N4O5S:
C, H, N.
1
mp 105-107 °C; H NMR (300 MHz, CDCl3) δ 0.90-0.97 (m,
6H), 1.36 (s, 9H), 1.35-1.42 (m, 4H), 1.64-1.74 (m, 4H), 3.73
(t, J ) 7.2 Hz, 2H), 4.06 (t, J ) 7.5 Hz, 2H), 7.60 (d, J ) 8.7
Hz, 2H), 8.18 (d, J ) 8.8 Hz, 2H), 8.28 (s, 1H); IR (KBr, cm-1
)
1714, 1671, 1183, 1121; MS 461 (MH)+. Anal. C23H32N4O4S:
C, H, N.
1,3-Dibu tyl-7-(4-ch lor o-2,5-d im eth ylben zen esu lfon yl)-
3,7-d ih yd r op u r in e-2,6-d ion e (29). Compound 29 was syn-
thesized according to method A using 1,3-dibutyl-3,7-dihydro-
purine-2,6-dione and 4-chloro-2,5-dimethylbenzenesulfonyl
chloride: yield 77%; mp 138-140 °C; 1H NMR (300 MHz,
CDCl3) δ 0.90 (t, J ) 7.5 Hz, 3H), 0.95 (t, J ) 7.5 Hz, 3H),
1.23-1.46 (m, 4H), 1.50-1.59 (m, 2H), 1.63-1.72 (m, 2H), 2.44
(s, 3H), 2.46 (s, 3H), 3.88 (t, J ) 7.5 Hz, 2H), 4.07 (t, J ) 7.5
1,3-Dibu tyl-7-(4-n itr oben zen esu lfon yl)-3,7-d ih yd r op u -
r in e-2,4-d ion e (22). Compound 22 was synthesized according
to method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
1
(4-NO2)PhSO2Cl: yield 69%; mp 138-139 °C; H NMR (300
MHz, CDCl3) δ 0.89-0.98 (m, 6H), 1.29-1.42 (m, 3H), 1.52-
1.62 (m, 3H), 1.65-1.74 (m, 2H), 3.92 (t, J ) 7.5 Hz, 2H), 4.08
(t, J ) 7.5 Hz, 2H), 8.31 (s, 1H), 8.42 (d, J ) 8.1 Hz, 2H), 8.51
(d, J ) 8.1 Hz, 2H); IR (KBr, cm-1) 1717, 1671, 1178; MS 450
(MH)+. Anal. C19H23N5O6S: C, H, N.
Hz, 2H), 7.27 (s, 1H), 8.33 (s, 1H), 8.40 (s, 1H); IR (KBr, cm-1
)
1711, 1665, 1172; MS 467 (MH)+. Anal. C21H27N4O4S: C, H,
N.
1,3-Dib u t yl-7-(4-h yd r oxya m in ob en zen esu lfon yl)-3,7-
d ih yd r op u r in e-2,6-d ion e (23). To 4.75 g (10.57 mmol) of 22
in 100 mL EtOAc was added 1.0 g of 10% Pd/C. This
heterogeneous mixture was hydrogenated under 1 atm of H2
at 23 °C for 8 h (until H2 uptake ceased). The reaction mixture
was then filtered through Celite and evaporated to an off-white
foam. Recrystallization from hot hexanes/EtOAc gave 3.91 g
(8.98 mmol, an 88% yield) of 23 as a white solid: mp 148-
150 °C; 1H NMR (300 MHz, CDCl3) δ 0.88-0.98 (m, 6H), 1.35
(sept, J ) 8.0 Hz, 4H), 1.51-1.61 (m, 2H), 1.61-1.73 (m, 2H),
3.93 (t, J ) 7.5 Hz, 2H), 4.08 (t, J ) 7.5 Hz, 2H), 5.85 (s, 1H),
7.03 (d, J ) 8.1 Hz, 2H), 7.19 (brs, 1H), 8.11 (d, J ) 8.1 Hz,
2H), 8.26 (s, 1H); IR (KBr, cm-1) 1707, 1659, 1595, 1167; MS
420 (MH)+. Anal. C19H25N5O6S: C, H, N.
1,3-Dibu tyl-8-m eth yl-3,7-d ih yd r op u r in e-2,6-d ion e (30).
6-Amino-1,3-di-n-butyl-5-nitrosouracil12 (15.0 g, 52.8 mmol),
1.5 g 10% Pd/C and 150 mL DMF were hydrogenated at 40
psi in a Parr shaker for 4 h. After completion, the reaction
mixture was filtered through Celite, and evaporated to give
crude 5,6-diamino-1,3-di-n-butyluracil as a dark oil. To this
oil were added 20 mL of acetic acid, 40 mL of DMF, 16.1 g (84
mmol) of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
drochloride, and 177 mg (1.45 mmol) of DMAP, and the
resulting mixture was stirred overnight. The reaction mixture
was concentrated on a rotary evaporator, 100 mL of 2 N NaOH
was added and this mixture was refluxed for 4 h. After cooling
to 23 °C, the reaction mixture was adjusted to pH ∼ 7 with 6
N HCl which caused a precipitate to form. The solid was
collected to give 14.1 g (50.7 mmol, a 96% yield) of the title
1,3-Dibu tyl-7-(n a p h th a len e-1-su lfon yl)-3,7-d ih yd r op u -
r in e-2,6-d ion e (24). Compound 24 was synthesized according
to method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
1-naphthalenesulfonyl chloride: yield 80%; mp 135-136 °C;
1H NMR (300 MHz, CDCl3) δ 0.82-0.97 (m, 6H), 1.20-1.39
(m, 4H), 1.41-1.53 (m, 2H), 1.60-1.72 (m, 2H), 3.83 (t, J )
7.5 Hz, 2H), 4.02 (t, J ) 7.5 Hz, 2H), 7.57-7.78 (m, 3H), 7.98
(d, J ) 8.2 Hz, 1H), 8.22 (d, J ) 8.1 Hz, 1H), 8.47 (d, J ) 8.2
1
compound as a yellow solid: mp 144-146 °C; H NMR (300
MHz, CDCl3) δ 0.91-0.98 (m, 6H), 1.36-1.46 (m, 4H), 1.64-
1.70 (m, 2H), 1.71-1.80 (m, 2H), 2.58 (s, 3H), 4.06-4.14 (m,
4H), 13.10 (brs, 1H); IR (KBr, cm-1) 1701, 1650; MS 279 (MH)+.
3-Bu tyl-7-(3,4-dim eth oxyben zen esu lfon yl)-3,7-dih ydr o-
p u r in e-2,6-d ion e (31). To a suspension of 5.0 g (26.0 mmol)
of 3-N-butylxanthene13 in 50 mL CH2Cl2 were added 3.0 mL
(2.93 g, 38.0 mmol) of pyridine, 7.4 g (31.0 mmol) of 3,4-
dimethoxybenzenesulfonyl chloride and 3.5 g (28.65 mmol) of
DMAP. The suspension was stirred at 23 °C for 5 days. The
remaining precipitate was filtered and discarded. The resulting
solution was washed with 1 × 100 mL of brine, dried over
MgSO4, filtered and evaporated to give a white solid. This solid
was crystallized from hot ethyl acetate to give 4.0 g (9.8 mmol,
a 38% yield) of the title compound as a white solid: mp 195-
Hz, 1H), 8.55 (s, 1H), 8.97 (d, J ) 8.2 Hz, 1H); IR (KBr, cm-1
)
1712, 1664; MS 455 (MH)+. Anal. C23H26N4O4S: C, H, N.
1,3-Dibu tyl-7-(th ioph en e-2-su lfon yl)-3,7-dih ydr opu r in e-
2,6-d ion e (25). Compound 25 was synthesized according to
method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
1
2-thiophenesulfonyl chloride: yield 63%; mp 145-146 °C; H
NMR (300 MHz, CDCl3) δ 0.90-0.99 (m, 6H), 1.31-1.46 (m,
4H), 1.55-1.76 (m, 4H), 3.98 (t, J ) 7.4 Hz, 2H), 4.08 (t, J )
7.4 Hz, 2H), 7.19 (t, J ) 4.0 Hz, 1H), 7.83 (dd, J ) 1.4, 5.0 Hz,
1
198 °C; H NMR (300 MHz, DMSO-d6) δ 0.90 (t, J ) 7.5 Hz,
1H), 8.26 (s, 1H), 8.33 (dd, J ) 1.4, 4.0 Hz, 1H); IR (KBr, cm-1
)
3H), 1.23-1.33 (m, 2H), 1.53-1.63(m, 4H), 3.85 (s, 3H), 3.87
(s, 3H), 7.24 (d, J ) 8.6 Hz, 1H), 7.81 (d, J ) 2.2 Hz, 1H), 7.84
(dd, J ) 2.2, 8.6 Hz, 1H), 8.70 (s, 1H) 11.38 (s, 1H); IR (KBr,
cm-1) 1710, 1597, 1031; MS 409 (MH)+. Anal. C17H20N4O6S:
C, H, N.
1713, 1670, 1392, 1176; MS 411 (MH)+. Anal. C17H22N4O4S2:
C, H, N.
1,3-Dibu tyl-7-p h en ylm eth a n esu lfon yl-3,7-d ih yd r op u -
r in e-2,6-d ion e (26). Compound 26 was synthesized according
to method B using 1,3-dibutyl-3,7-dihydropurine-2,6-dione and
benzylsulfonyl chloride: yield 56%; mp 117-118 °C; 1H NMR
(300 MHz, CDCl3) δ 0.90-1.01 (m, 6H), 1.31-1.52 (m, 4H),
1.62-1.79 (m, 4H), 4.00-4.13 (m, 4H), 5.23 (s, 2H), 7.21-7.43
(m, 5H), 7.77 (s, 1H); IR (KBr, cm-1) 1705, 1672, 1438; MS
419 (MH)+. Anal. C20H26N4O4S: C, H, N.
1,3-Dibu tyl-7-(2,4,6-tr im eth ylben zen esu lfon yl)-3,7-d i-
h yd r op u r in e-2,6-d ion e (27). Compound 27 was synthesized
according to method B using 1,3-dibutyl-3,7-dihydropurine-
2,6-dione and 2,4,6-trimethylbenzenesulfonyl chloride: yield
90%; mp 125-127 °C; 1H NMR (300 MHz, CDCl3) δ 0.87 (t, J
) 7.2 Hz, 3H), 0.96 (t, J ) 7.2 Hz, 3H), 1.15-1.30 (m, 2H),
1.30-1.48 (m, 2H), 1.49-1.56 (m, 2H), 1.65-1.78 (m, 2H), 2.32
3-Bu tyl-7-(3,4-d im eth oxyben zen esu lfon yl)-1-octyl-3,7-
d ih yd r op u r in e-2,6-d ion e (32). To a suspension of 408 mg
(1.0 mmol) of 3-N-butyl-7-(3,4-dimethoxybenzenesulfonyl)-1H-
3,7-dihydropurine-2,6-dione in 4 mL of DMF was added 165
mg (1.2 mmol) K2CO3, followed by 212 mg (1.02 mmol) of
1-bromooctane. The reaction was stirred at 50 °C for 5 h and
then at 23 °C overnight. After pouring into 100 mL water, the
product was extracted with 2 × 50 mL of EtOAc. The combined
organics were dried over MgSO4, filtered and evaporated to
give a white solid. The solid was triturated with Et2O and
filtered to give 280 mg (0.54 mmol, a 54% yield) of the title
compound as a while solid: mp 110-112 °C; 1H NMR (300
MHz, CDCl3) δ 0.87 (t, J ) 7.4 Hz, 3H), 0.94 (t, J ) 7.5 Hz,

Calcitonin-Inducing Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1231
3H), 1.22-1.43 (m, 12H), 1.61-1.72 (m, 4H), 3.95 (s, 3H), 3.98
(s, 3H), 4.07 (t, J ) 7.5 Hz, 4H), 8.97 (d, J ) 8.7 Hz, 1H), 7.86
(dd, J ) 2.3, 8.6 Hz, 1H), 7.97 (d, J ) 2.2 Hz, 1H), 8.25 (s,
1H); IR (KBr, cm-1) 1716, 1667, 1272; MS 521 (MH)+. Anal.
C25H36N4O6S: C, H, N.
3-Bu t yl-1-cyclop r op ylm e t h yl-7-(3,4-d im e t h oxyb e n -
zen esu lfon yl)-3,7-d ih yd r op u r in e-2,6-d ion e (33). Com-
pound 33 was prepared according to the procedure for 32 using
bromomethylcyclopropane in place of 1-bromooctane: yield
82%; mp 139-141 °C; 1H NMR (300 MHz, CDCl3) δ 0.41-0.52
(m, 4H), 0.95 (t, J ) 7.5 Hz, 3H), 1.15-1.26 (m, 1H), 1.38 (sex,
J ) 7.4 Hz, 2H), 1.61-1.74 (m, 2H), 3.87 (d, J ) 7.1 Hz, 2H),
3.95 (d, 3H), 3.99 (s, 3H), 4.07 (t, J ) 7.5 Hz, 2H), 7.86 (d, J )
2.3, 8,6 Hz, 1H), 7.96 (d, J ) 2.3 Hz, 1H), 8.26 (s, 1H); IR (KBr,
cm-1) 1713, 1666, 1270; MS 463 (MH)+. Anal. C21H26N4O6S:
C, H, N.
3-Bu tyl-1-cyclobu tylm eth yl-7-(3,4-d im eth oxyben zen e-
su lfon yl)-3,7-d ih yd r op u r in e-2,6-d ion e (34). Compound 34
was prepared according to the procedure for 32 using bro-
momethylcyclobutane in place of 1-bromooctane: yield 51%;
mp 150-151 °C; 1H NMR (300 MHz, CDCl3) δ 0.93 (t, J ) 7.5
Hz, 3H), 1.38 (sex, J ) 7.4 Hz, 2H), 1.57-1.72 (m, 2H), 1.76-
1.85 (m, 4H), 1.91-2.00 (m, 2H), 2.60-2.72 (m, 1H), 3.95 (s,
3H), 3.99 (s, 3H), 4.06 (t, J ) 7.5 Hz, 2H), 6.96 (d, J ) 8.5 Hz,
1H), 7.86 (dd, J ) 2.2, 8.6 Hz, 1H), 7.97 (d, J ) 2.2 Hz, 1H),
8.25 (s, 1H); IR (KBr, cm-1) 1712, 1667, 1272; MS 477 (MH)+.
Anal. C22H28N4O6S: C, H, N.
2H), 1.32-1.49 (m, 4H), 1.60-1.69 (m, 2H), 3.06 (s, 3H), 3.77
(t, J ) 7.4 Hz, 2H), 4.01 (t, J ) 7.5 Hz, 2H), 7.57-7.71 (m,
3H), 7.97 (dd, J ) 2.1, 8.2 Hz, 1H), 8.19 (d, J ) 8.1 Hz, 1H),
8.23 (d, J ) 8.2 Hz, 1H), 8.76 (d, J ) 8.2 Hz, 1H); IR (KBr,
cm-1) 1711, 1672; MS 469 (MH)+. Anal. C24H25N4O4S: C, H,
N.
1,3-Dibu tyl-7-(3,4-d im eth oxyben zen esu lfon yl)-8-m eth -
yl-3,7-d ih yd r op u r in e-2,6-d ion e (40). Compound 40 was
prepared according to method A using 1,3-di-n-butyl-8-methyl-
3,7-dihydropurine-2,6-dione and (3,4-OMe)PhSO2Cl: yield
78%; mp 145-147 °C; 1H NMR (300 MHz, CDCl3) δ 0.89-0.97
(m, 6H), 1.29-1.43 (m, 4H), 1.51-1.72 (m, 4H), 2.83 (s, 3H),
3.95 (s, 3H), 3.98 (s, 3H), 3.97 (t, 2H, obscured by the δ 3.95
and 3.98 singlets), 4.03 (t, J ) 7.4 Hz, 3H); IR (KBr, cm-1
)
1707, 1659; MS 479 (MH)+. Anal. C22H30N4O6S: C, H, N.
7-Ben zen esu lfon yl-1,3-d ib u t yl-8-m et h yl-3,7-d ih yd r o-
p u r in e-2,6-d ion e (41). Compound 41 was prepared according
to method A using 1,3-di-n-butyl-8-methyl-3,7-dihydropurine-
2,6-dione and PhSO2Cl: yield 50%; mp 87-89 °C; 1H NMR
(300 MHz, CDCl3) δ 0.88-0.98 (m, 6H), 1.26-1.43 (m, 4H),
1.50-1.61 (m, 2H), 1.62-1.73 (m, 4H), 2.88 (s, 3H), 3.93 (t, J
) 7.5 Hz, 2H), 4.03 (t, J ) 7.3 Hz, 2H), 7.61 (t, J ) 8.4 Hz,
2H), 7.66-7.73 (m, 1H), 8.25 (d, J ) 8.2 Hz, 2H); IR (KBr,
cm-1) 1711, 1667; MS 419 (MH)+. Anal. C20H26N4O4S: C, H,
N.
P h a r m a cologica l Meth od s. 1. Ca lciton in /Lu cifer a se
Rep or ter Gen e Exp r ession Assa y/Ca lciton in Secr etion
Assa y. Assessment of the ability of compounds to stimulate
human calcitonin expression was performed using a reporter
cell line, designated C3 which has been previously described
in the patent literature.18 This human medullary thyrocarci-
noma cell line (TT; ATCC) was stably transfected with 3 kb
(from the translation start site) of the upstream elements of
the human calcitonin gene fused to a luciferase reporter
construct. For assay of transcriptional activation ability, C3
cells were plated at a density of 6500-7500 cells/well in a 96-
plate microtiter plate. Twenty-four to forty-eight hours later,
compounds were added to the wells in triplicate. Compounds
were tested at a concentration of 10 µg/mL in 0.5% DMSO.
Compounds that exhibited a transcription activation ratio
(TAR) equal to or greater than 1.5 (equivalent to a 50%
increase in transcription or greater) advanced to primary
followup (CA-FUP), in which compounds were rescreened at
four concentrations: 10, 2, 0.4, and 0.08 µg/mL. Controls were
distributed throughout the plate and included (1) unstimulated
cells for basal luciferase expression and (2) cells stimulated
with 1 mM 8-CPT-c-AMP (results in 2.1 ( 0.3-fold induction).
The plates were incubated for 12 h in a humidified CO2
incubator, washed and then lysed in luciferase assay buffer
as previously described.18 The luciferase activity was deter-
mined with a commercially available assay (Promega) by
measurement of the light signal in a luminometer.
TAR r a tio: Stimulation of calcitonin promoter-dependent
transcription is expressed as a ratio of luciferase activity (CT-
luci) in the presence of test compound compared to the CT-
luci activity in the untreated control.
Following stimulation of the C3 cells for the determination
of transcription activation, culture media was aspirated and
assayed for calcitonin levels in duplicate using a commercially
available radioimmunoassay (RIA; Nichols Institute Diagnos-
tics, kit #40-2125) according to the manufacturers instructions.
Secr etion r a tio: Stimulation of calcitonin secretion is
expressed as a ratio of secretion activity in the presence of
test compound compared to the secretion activity in the
untreated control.
3-Bu t yl-7-(3,4-d im et h oxyb en zen esu lfon yl)-1-(3-m et h -
ylbu t-2-en yl)-3,7-d ih yd r op u r in e-2,6-d ion e (35). Compound
35 was prepared according to the procedure for 32 using
1-bromo-3-methylbutene in place of 1-bromooctane: yield 71%;
1
mp 92-95 °C; H NMR (300 MHz, CDCl3) δ 0.94 (t, J ) 7.5
Hz, 3H), 1.25-1.41 (m, 2H), 1.54-1.59 (m, 4H), 1.68 (s, 3H),
1.71 (s, 3H), 3.95 (s, 3H), 3.98 (s, 3H), 4.06 (t, J ) 9 Hz, 2H),
4.57 (d, J ) 6 Hz, 1H), 5.20 (t, J ) 12 Hz, 1H), 6.95 (d, J ) 9
Hz, 1H), 7.82 (dd, J ) 2.1, 10.8 Hz, 1H), 8.05 (d, J ) 2.1 Hz,
1H) 8.24 (s, 1H); IR (KBr, cm-1) 1711, 1677; MS 477(MH)+.
Anal. C22H28N4O6S: C, H, N.
3-Bu t yl-7-(3,4-d im et h oxyb en zen esu lfon yl)-1-(3-m et h -
ylbu tyl)-3,7-d ih yd r op u r in e-2,6-d ion e (36). Compound 36
was prepared according to the procedure for 32 using 1-bromo-
3-methylbutane in place of 1-bromooctane: yield 57%; mp
1
134-135 °C; H NMR (300 MHz, DMSO-d6) δ 0.87-0.93 (m,
3H), 0.89 (d, J ) 7.3 Hz, 6H), 1.21-1.40 (m, 6H), 1.43-1.65
(m, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 3.92 (t, J ) 7.5 Hz, 2H),
7.24 (d, J ) 8.3 Hz, 1H), 7.81-7.87 (m, 2H), 8.73 (s, 1H); IR
(KBr, cm-1) 1712, 1668, 1273; MS 479 (MH)+. Anal. C22H30
N4O6S: C, H, N.
-
1,3-Dib u t yl-7-(3-ch lor op r op a n e-1-su lfon yl)-8-m et h yl-
3,7-d ih yd r op u r in e-2,6-d ion e (37). Compound 37 was pre-
pared according to method A using 1,3-di-n-butyl-8-methyl-
3,7-dihydropurine-2,6-dione and Cl(CH2)3SO2Cl: yield 71%; mp
1
76-78 °C; H NMR (300 MHz, CDCl3) δ 0.92-0.99 (m, 6H),
1.33-1.42 (m, 4H), 1.57-1.65 (m, 2H), 1.67-1.74 (m, 2H),
2.32-2.41 (m, 2H) 2.74 (s, 3H), 3.69 (t, J ) 6.2 Hz, 2H), 3.99
(t, J ) 7.6 Hz, 2H), 4.08 (t, J ) 7.5 Hz, 2H), 4.23 (t, J ) 7.5
Hz, 2H); IR (KBr, cm-1) 1705, 1522; MS 419 (MH)+. Anal.
C
17H27ClN4O4S: C, H, N.
1,3-Dibu tyl-8-m eth yl-7-(3-n itr oben zen esu lfon yl)-3,7-d i-
h yd r op u r in e-2,6-d ion e (38). Compound 38 was prepared
according to method A using 1,3-di-n-butyl-8-methyl-3,7-
dihydropurine-2,6-dione and (3-NO2)PhSO2Cl: yield 65%; mp
166-168 °C; 1H NMR (300 MHz, CDCl3) δ 0.88-0.97 (m, 6H),
1.21-1.43 (m, 4H), 1.48-1.77 (m, 4H), 2.92 (s, 3H), 3.90 (t, J
) 7.5 Hz, 2H), 4.04 (t, J ) 7.4 Hz, 2H), 7.84 (t, J ) 8.1, 1H),
8.55 (dd, J ) 2.0, 8.2 Hz, 1H), 8.71 (dd, J ) 2.0, 8.2 Hz, 1H),
8.95 (t, J ) 2.0 Hz, 1H); IR (KBr, cm-1) 1705, 1669; MS 464
(MH)+. Anal. C20H25N5O6S: C, H, N.
2. Ova r iectom y-In d u ced Osteop en ia in Ra ts. The abil-
ity of compounds to prevent ovariectomy-induced bone loss in
female rats was determined in mature Sprague-Dawley CD
rats, weighing 225-250, and the time of ovariectomy. Rats
were either bilaterally ovariectomized (OXV) or sham-operated
(SHAM) and allowed 4-5 days recovery before initiation of
dosing. Rats were housed in metal cages (3-5 rats/cage) with
access to food and water ad libitum on a 14/10 h day/night
cycle. The concentration of the drug stock was calculated to
1,3-Dibu tyl-8-m eth yl-7-(n a p h th a len e-1-su lfon yl)-3,7-d i-
h yd r op u r in e-2,6-d ion e (39). Compound 39 was prepared
according to method A using 1,3-di-n-butyl-8-methyl-3,7-
dihydropurine-2,6-dione and 1-naphthalenesulfonyl chloride:
1
yield 60%; mp 96-98 °C; H NMR (300 MHz, CDCl3) δ 0.84
(t, J ) 7.4 Hz, 3H), 0.93 (t, J ) 7.4 Hz, 3H), 1.15-1.22 (m,

1232 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
be delivered in a volume of 0.1 mL/100 g of body weight and
was administered either intraperitoneally (i.p.) or orally (p.o.).
The drug solution or a uniform suspension was prepared in
1% Tween 80 in normal saline and was administered daily
(five times a week) for 6 weeks.
manufacturer’s instructions. Additionally, 1 µM [3H]cAMP
assays were conducted in the absence and presence of 10 µM
of the selective PDE4 inhibitor rolipram. Fractions which
showed cAMP hydrolytic activity which was inhibitable by
rolipram were pooled.
A sequential double labeling of mineralized tissue with a
fluorochrome was employed to determine the osseous changes
(especially the bone formation) and the mineralization rates.
Each animal was administered either 90 or 15 mg/kg calcein
(Sigma Chemical Co.), subcutaneously approximately 2 days
and then 9 days prior to the termination of the study,
respectively.
When prepared in this manner, approximately 80% of total
cAMP PDE activity is eluted by buffer B containing 0.7 M
sodium acetate. This PDE activity, which consists of >90%
PDE4 (as evidenced by its susceptibility to inhibition by
rolipram), was stored in 30% ethylene glycol at 20 °C; 15 µL
of the PDE was typically required to obtain sufficient activity
for inhibition assays (approximately 25000 dpm). Enzyme
activity was determined at 37 °C. The amount of enzyme and
duration of assay were adjusted to ensure that less than 25%
of the substrate was consumed under these conditions. PDE
activities of U937 cell PDE4 preparations have been found to
be linear for at least 30 min. To test inhibition of PDE4, a
test compound was added to the reaction mixture, usually at
concentrations ranging from 0.001 to 10 µM. Inhibition by a
test compound was measured as a percent reduction of total
PDE activity and calculated as follows:
Four weeks after the initiation of treatment, each rat was
evaluated for total and trabecular density (TotD and TrabD)
of the proximal tibia. TotD and TrabD were measured in
anesthetized rats using a XCT-960M (Stratec Medizintechnik,
Pforzheim, Germany). Measurements were performed as fol-
lows: 10 min prior to scanning, animals were anesthetized
with an i.p. injection of 50 mg/kg ketamine (Bristol Labora-
tories, Syracuse, NY), xylazine (5 mg/kg), and 1 mg/kg acepro-
mazine (Aveco, Ft. Dodge, IA). The right hind limb was passed
through a polycarbonate tube with a diameter of 25 mm and
taped to an acrylic frame with the ankle joint at a 90° angle
and the knee joint at 180°. The polycarbonate tube was affixed
to a sliding platform that maintained it parallel to the aperture
of the XCT. The platform was adjusted so that the distal end
of the femur and the proximal end of the tibia would be in the
scanning field. A two-dimensional scout view was run for a
length of 10 mm and a line resolution of 0.2 mm. After the
scout view was displayed on the monitor, the proximal end of
the tibia was located. The CT scan was initiated 3.4 mm distal
from this point. The CT scan was 1 mm thick, had a voxel
(three-dimensional pixel) size of 0.140 mm, and consisted of
145 projections through the slice.
A
B
× 100 ) % inhibition of PDE4
where A is the PDE activity (mean dpm - background dpm)
in the presence of test compound and B is the total PDE4
activity (mean dpm - background dpm) in the absence of test
compound. Percent inhibition of PDE4 values were then
normalized to rolipram values where the rolipram percent
inhibition of PDE IV is set to 100%.
IC50s were estimated by linear regression analysis using the
percent inhibition data bracketing 50% inhibition.
The difference in bone mineral density percent of the
proximal tibia between a treatment group and the vehicle
group was analyzed using a one-way analysis of variance with
Dunnett’s test or other multiple comparison methods. Com-
pounds were designated active if p < 0.05 vs vehicle value.
Refer en ces
(1) Bilezikian, J . P. Current and Future Nonhormonal Approaches
to the Treatment of Osteoporosis. Int. J . Fertil. 1996, 41, 148-
155.
(2) Revilla, M.; Hernandez, E. R.; Villa, L. F.; Alvarez de Buergo,
M. Total and Regional Bone Mineral Content and Fracture Rate
in Postmenopausal Osteoporosis Treated with Salmon Calcito-
nin: A Prospective Study. Calcif. Tissue Int. 1995, 56, 181-185.
(3) Deftos, L. J .; Nolan, J . J .; Seely, B. L.; Clopton, P. L.; Cote, G.
J .; Whitham, C. L.; Florek, L. J .; Christensen, T. A.; Hill, M. R.
Intrapulmonary Drug Delivery of Salmon Calcitonin. Calcif.
Tissue Int. 1997, 61, 345-347.
(4) Plosker, G. L.; McTavish, D. M. Intranasal Salcatonin (Salmon
Calcitonin). Drugs Aging 1996, 8, 378-400.
(5) Baudys, M.; Kim, S. W. Stabilization and Oral Delivery of
Calcitonin. WO Patent 9800155.
(6) Sakuma, S.; Suzuki, N.; Kikuchi, H.; Hiwatari, K.-i.; Arikawa,
K.; Kishida, A.; Akashi, M. Absorption Enhancement of Orally
Administered Salmon Calcitonin by Polystyrene Nanoparticles
Having Poly(N-isopropylacrylamide) Branches on Their Sur-
faces. Int. J . Pharm. 1997, 158, 69-78.
3. In h ibition of P h osp h od iester a se IV (P DE4) Isola ted
fr om Hu m a n U937 Cells. The human leukemic cell line
(U937) was maintained in RPMI 1640 medium supplemented
with 10% heat-inactivated fetal calf serum and penicillin/
streptomycin (100 units/100 µg/mL) in a humidified 5% CO2
atmosphere at 37 °C. Starter cells were maintained in continu-
ous logarithmic growth by seeding them in 75 cm2 vented
tissue culture flasks at a concentration of 3 × 104 cells/mL
and passing them every 3-4 days when the cells reached
approximately 8 × 105 cells/mL. For experiments, 3-4 day
starter cells were seeded in 225 cm2 flasks at 1 × 105 cells/mL
and harvested 3-4 days later at approximately 6 × 105 cells/
mL.
For isolation of PDE4 enzyme activity, U937 cells were
activated with 10 µM dibutyryl cAMP for a period of 4 h (to
upregulate PDE4) and then harvested by centrifugation at
1200g for 10 min in 250-mL conical centrifuge tubes. The pellet
from each 200-mL flask was resuspended in 5 mL of buffer A
[10 mM Tris-HCl, 5 mM MgCl2, 4 mM EGTA, 5 mM 2-mer-
captoethanol, 1 µM leupeptin, 1 µM pepstatin A, and 5 µM
phenylmethanesulfonyl fluoride (PMSF) (pH 7.8)], and the
cells were lysed using three cycles of freezing (3 min in dry
ice/acetone) followed by thawing (warm water). The extract
was centrifuged for 20 min at 1200g to remove cell debris and
the supernatant was immediately loaded onto a 1.6 × 70 cm
DEAE-Sepharose CL-6B anion-exchange column equilibrated
with buffer A. The column was next washed with 2.5 column
volumes of buffer B [10 mM Tris-HCl, 5 mM 2-mercaptoetha-
nol, 0.1 µM leupeptin, 0.1 µM pepstatin A, and 0.1 µM PMSF
(pH 7.8)], and PDEs were eluted with a step gradient consist-
ing of 80 mL each of buffer B containing 0.4 or 0.7 M sodium
acetate (80 mL/h, 8 mL/fraction). To determine which families
of PDEs were present, fractions were assayed for hydrolytic
activity with 1 µM [3H]cAMP or 1 µM [3H]cGMP using a
commercially available assay (Amersham) according to the
(7) (a) Labroo, V. M.; Beigel, S. Synthetic Calcitonin Mimetics. U.S.
Patent 5698672. (b) Milstein, S. J .; Barantsevitch, E. N.; Sarubbi,
D. J .; Leone-Bay, A.; Paton, D. R. Oral Drug Delivery Composi-
tions and Methods. U.S. Patent 5766633.
(8) Baindur, N.; Labroo, V.; Stroop, S.; Beigel, S.; Martinez, T.;
Petrie, C.; Orme, M. W.; Mckernan, P. A.; Moore, E. E. Calcitonin
Mimetics. WO Patent 9837077.
(9) Nicholson, C. D.; J ackman, S. A.; Wilke, R. The Ability of
Denbufylline to Inhibit Cyclic Nucleotide Phosphodiesterase and
its Affinity for Adenosine Receptors and the Adenosine Re-
Uptake Site. Br. J . Pharmacol. 1989, 97, 889-897.
(10) Duplantier, A. J .; Biggers, M. S.; Chambers, R. J .; Cheng, J . B.;
Cooper, K.; Damon, D. B.; Eggler, J . F.; Kraus, K. G.; Marfat,
A.; Masamune, H.; Pillar, J . S.; Shirley, J . T.; Umland, J . P.;
Watson, J . W. Biarylcarboxylic Acids and Amides: Inhibition
of Phosphsdiesterase Type IV versus [3H]Rolipram Binding
Activity and Their Relationship to Emetic Behavior in the Ferret.
J . Med. Chem. 1996, 39, 120-125.
(11) Miyamoto, K.-I.; Waki, Y.; Horita, T.; Kasugai, S.; Ohya, K.
Reduction of Bone Loss by Denbufylline, an Inhibitor of Phos-
phodiesterase 4. Biochem. Pharmacol. 1997, 54, 613-661.
(12) Kim, H. O.; J i, X.-d. Melmon, N.; Olch, M. E.; Listikis, G.;
J acobson, K. A. Structure-Activity Relationships of 1,3-Dialkyl-
xanthine Derivatives at Rat A3 Adenosine Receptors. J . Med.
Chem. 1994, 37, 3373-3382.

Calcitonin-Inducing Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1233
(13) Doub, L.; Krolls, U.; Vandenbelt, J . M.; Fisher, Myron W.
N-Sulfanilyl-l-alkylcytosines. New Highly Active Class of “Soluble,”
Short-acting Sulfanilamides. J . Med. Chem. 1970, 13, 242-246.
(14) Hagiwara, S.; Yang, S. O.; Glucer, C. C.; Bendavid, E.; Genant,
H. K. Noninvasive Bone Mineral Density Measurement in the
Evaluation of Osteoporosis. Osteoporosis 1994, 20, 651-669.
(15) Genant, H. K.; Lang, T. F.; Engelke, K.; Fuerst, T.; Gluer, C.
C.; Majumdar, S.; J ergas, M. Advances in the Noninvasive
Assessment of Bone Density, Quality and Structure. Calcif. Tisue
Int. 1996, 59 (Suppl. 1), S10-S15.
Maxey, R.; Miotla, J .; Naylor, R. J .; Runcie, K. A.; Tuladhar, B.;
Warneck, J . B. H. Aryl Sulfonamides as Selective PDE4 Inhibi-
tors. Bioorg. Med. Chem. Lett. 1998, 8, 2635-40.
(17) Montana, J . G.; Cooper, N.; Dyke, H. J .; Gowers, L.; Gregory, J .
P.; Hellewell, P. G.; Miotla, J .; Morris, K.; Naylor, R.; Tuladhar,
B.; Warneck, J . B. H. PDE Inhibitors: New Xanthine Analogues.
Bioorg. Med. Chem. Lett. 1998, 8, 2925-2930.
(18) Foulkes, J . G.; Leichfried, F.; Pieler, C.; Stephenson, J . R.;
Franco, R. Method of Discovering Chemicals Capable of Func-
tions as Gene Expression Modulators. U.S. Patent 5,665,543.
(16) Montana, J . G.; Buckley, G. M.; Cooper, N.; Dyke, H. J .; Gowers,
L.; Gregory, J . P.; Hellewell, P. G.; Kendall, H. J .; Lowe, C.;
J M990558L