Adenosine kinase inhibitors. 2. Synthesis, enzyme inhibition, and antiseizure activity of diaryltubercidin analogues

Article abstract of DOI:10.1021/jm0000259

In the preceding article (Ugarkar et al. J. Med. Chem. 2000, 43) we reported that analogues of tubercidin are potent adenosine kinase (AK) inhibitors with antiseizure activity in the rat maximum electroshock (MES) model. Despite the discovery of several h

Full text of DOI:10.1021/jm0000259

2894
J . Med. Chem. 2000, 43, 2894-2905
Ad en osin e Kin a se In h ibitor s. 2. Syn th esis, En zym e In h ibition , a n d An tiseizu r e
Activity of Dia r yltu ber cid in An a logu es
Bheemarao G. Ugarkar,* Angelo J . Castellino, J ay M. DaRe, J oseph J . Kopcho, J ames B. Wiesner,
J uergen M. Schanzer, and Mark D. Erion
Metabasis Therapeutics Inc., 9390 Towne Centre Drive, San Diego, California 92121
Received J anuary 21, 2000
In the preceding article (Ugarkar et al. J . Med. Chem. 2000, 43) we reported that analogues
of tubercidin are potent adenosine kinase (AK) inhibitors with antiseizure activity in the rat
maximum electroshock (MES) model. Despite the discovery of several highly potent AK
inhibitors (AKIs), e.g., 5′-amino-5′-deoxy- 5-iodotubercidin (1c) (IC50 ) 0.0006 µM), no
compounds were identified that exhibited a safety, efficacy, and side effect profile suitable for
further development. In this article, we demonstrate that substitution of the tubercidin molecule
with aromatic rings at the N4- and the C5-positions not only retains AKI potency but also
improves in vivo activity. Synthesis of such compounds entailed transformation of 4-arylamino-
5-iodotubercidin analogues to their corresponding 5-aryl derivatives via the Suzuki reaction.
Alternatively, 4-N-arylamino-5-arylpyrrolo[2,3-d]pyrimidine bases were constructed and then
glycosylated with appropriately protected R-ribofuranosyl chlorides using a phase-transfer
catalyst. Several compounds exhibited potent activity in the rat MES seizure assay with ED50s
e 2.0 mg/kg, ip, and showed relatively mild side effects.
Ch a r t 1
In tr od u ction
In our continuing effort to develop adenosine regulat-
ing agents (ARAs) as an alternative to adenosine or its
receptor agonists, we have pursued inhibition of the
cytosolic enzyme adenosine kinase (AK).1 Inhibition of
AK was hypothesized to increase intracellular adenosine
levels which, following transport out of the cell, would
stimulate nearby adenosine receptors and induce a
protective pharmacological response. We have demon-
strated that adenosine kinase inhibitors (AKIs) exhibit
anticonvulsive effects against MES-induced seizures in
rats. Moreover, the effects are reversed by the nonspe-
cific adenosine receptor antagonist theophylline, sug-
gesting that the pharmacological effect is mediated by
an adenosine receptor.1 Based on the potent AKI activi-
ties reported for 5-iodotubercidin (1a , IC50 ) 0.026 µM)
and 5′-deoxy-5-iodotubercidin (1b, IC50 ) 0.009 µM)
(Chart 1) a number of tubercidin analogues were
prepared to study the SAR of AK inhibition.1 Our results
revealed that a halogen (I or Br) at the C5-position and
an NH2, Cl, or SCH3 at the C4-position of tubercidin
yield molecules that potently inhibit AK (IC50 e 0.1 µM).
The enzyme was found to accommodate both hydropho-
bic and hydrophilic substituents at the C5′-position,
with the 5′-amino-linked tubercidin analogues exhibit-
ing the highest potencies (1c, IC50 ) 0.0006 µM; 1d , IC50
) 0.0002 µM). The above compounds, however, were not
considered ideal development candidates for various
reasons. For example, 1a is suspected to be a cytotoxic
agent due to its 5′-phosphorylation by intracellular
kinases,2 whereas 1b is reported to produce side effects
such as sedation, hypothermia, and muscle flaccidity3
at doses similar to those required for inhibition of MES
seizure in rats.1 Furthermore, compounds 1c,d , though
very potent in the enzyme inhibition assay, exhibited
weak activity in the rat MES seizure assay.1 The weak
in vivo potency of these compounds was believed to be
due to their poor brain penetration or poor cell penetra-
tion. Consequently, we sought to make structural
changes that included large hydrophobic substituents
on the C4-NH2 and at the C5-position of the tubercidin
molecule in order to enhance the AK specificity and
selectivity, as well as enhance their brain/cell penetra-
tion.
In this report, we disclose the discovery that tuber-
cidin analogues with an aromatic amine at the C4-
position and an aromatic ring at the C5-position exhibit
potent AK inhibition and potent antiseizure activities.
Several of these compounds were found to exhibit
reduced side effects when compared with adenosine
receptor agonists. The synthesis, AK inhibition, anti-
seizure activity, and side effects of these potent AKIs
are described in detail.
Ch em istr y
Two separate strategies were employed for the syn-
thesis of AKIs shown in Table 1. The first strategy used
2′,3′-O-isopropylidene-protected 4-chloro-5-iodopyrrolo-
[2,3-d]pyrimidine nucleosides1 as the key starting ma-
* To whom correspondence should be addressed. Phone: 858-622-
5535. Fax: 858-622-5526. E-mail: ugarkar@mbasis.com.
10.1021/jm0000259 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/07/2000

Adenosine Kinase Inhibitors. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2895
Sch em e 1a
Sch em e 2a
a
(a) HC(OEt)3 or H3CC(OEt)3/p-TSA; (b) DMF/ArNH2; (c) H2O,
a
(a) 70% TFA; (b) ArB(OH)2/Pd(Ph3P)4; (c) H2/Pd-C.
reflux; (d) NBS/DMF.
terials, which were condensed with a number of anilines
and amines. Then the C5-aromatic ring was introduced
via the Suzuki reaction4 to generate the target molecules
after acid catalyzed removal of the protecting group. The
second strategy entailed construction of a fully substi-
tuted pyrrolopyrimidine base followed by glycosylation
and deprotection.
Condensation of 2′,3′-O-isopropylidene-protected nu-
cleosides 2a 1 and 2b1 with amines, including various
anilines, benzylamine, and cyclohexylamine, provided
the corresponding 4-N-substituted-amino-5-iodopyrrol-
opyrimidine nucleoside intermediates 3a -j, as shown
in Scheme 1. These intermediates were subjected to
acid-catalyzed deprotection to furnish 1a and 4a -i in
60-70% overall yields.
The key step in synthesizing the desired molecules
5a -p was the arylation of 5-iodo nucleoside intermedi-
ates 1b and 4a -i via the Suzuki reaction.4 For example,
reaction of 1b5 with phenylboronic acid in the presence
of Pd(PPh3)4 gave 5a in 65% yield. In general, unpro-
tected nucleosides gave lower yields of the desired
products presumably due to partial formation of 2′,3′-
O-cyclic borates (e.g., 6), whereas isopropylidene-
protected nucleosides 3g,j gave >85% yield of the
Suzuki products.
Attempts to condense 2a with p-cyanoaniline and
heteroarylamines, such as 2-, 3-, and 4-aminopyridines,
2-aminothiazole, 2-aminoimidazole, and 4-amino-1,2,4-
triazole, resulted in either no reaction or decomposition
of the starting material. This was attributed to poor
nucleophilicity of the amines. Therefore, when a strong
base such as t-BuOK was used for activating the amine
function, 2a readily condensed with p-cyanoaniline
giving the desired intermediate 3i in 82% yield. Unfor-
tunately, under the similar conditions only one het-
eroarylamine, 3-aminopyridine, reacted to give the
desired intermediate 3j in 79% yield.
formed 4-arylamino-5-arylpyrrolo[2,3-d]pyrimidine bases
followed by deprotection using 70% TFA. The hetero-
cycles 10a -e were synthesized by a procedure reported
by Taylor et al.,6 for the synthesis of 4-amino-5-phe-
nylpyrrolo[2,3-d]pyrimidine. For example, 2-amino-3-
cyano-4-phenylpyrrole (7)7 was converted to 3-cyano-2-
ethoxymethylenimino-4-phenylpyrrole (8a ),6 which was
further condensed with aniline in boiling DMF to give
a mixture of partially aromatized intermediate 9a and
the desired product 10a (Scheme 2). Complete conver-
sion of 9a to 10a was accomplished by diluting the
reaction mixture with water and refluxing for an ad-
ditional 4-8 h. This procedure of making pyrrolopyri-
midine bases was found also to be useful in the
synthesis of C2-methyl-substituted heterocycle 10e by
replacing triethyl orthoformate with triethyl orthoac-
etate in the first step. It should be noted here that the
efforts to isolate pure 9a or 9b for characterization were
unsuccessful due to their slow but spontaneous rear-
rangement to form the fully aromatized products during
purification by chromatography or crystallization.
This method of synthesizing pyrrolo[2,3-d]pyrimidines
was found to be effective for anilines with halogens and
electron-donating substituents. In contrast, anilines
with electron-withdrawing groups such as CN, COOEt,
and NO2, as well as heteroarylamines failed to react
with 8a .
Glycosylation of the heterocycles was accomplished
using appropriately protected R-ribofuranosyl chloride
as shown in Scheme 3. Sodium salt-mediated glycosy-
lation8 of 10a with 5-deoxy-2,3-O-isopropylidene-R-D-
ribofuranosyl chloride (12a )1 resulted in a poor yield of
the desired N7-glycosylated product 13a (30-35%) along
with two major byproducts, which are tentatively as-
signed as the N1-â-nucleoside 14 and the N7-R-nucleo-
side 15. Similarly, glycosylation using 50% NaOH/
methylene chloride and the phase-transfer catalyst
tris[2-(methoxyethoxy)ethyl]amine9 (TDA-1) gave 13a
(30-35%) along with 14 (12-20%) and 15 (10-15%).
The second strategy employed for the preparation of
the target compounds involved glycosylation of pre-

2896 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ugarkar et al.
Sch em e 3a
a
(a) HMPT/CCl4/toluene, -10 °C; (b) HMPT/CCl4/THF, -76 °C; (c) KOH/toluene/TDA-1; (d) 70% TFA; (e) H2, Pd/C.
During optimization of this procedure other phase-
transfer catalysts, such as benzyltrimethylammonium
bromide and benzyltributylammonium bromide, and
solvents, such as acetonitrile, 1,2-dichloroethane, 1,4-
dioxane, and THF, were examined. These experiments
gave less desirable product mixtures. However, a much
improved product profile resulted when the reaction was
carried out in toluene using 2 equiv of powdered KOH,
1 equiv of TDA-1, and 2 full equiv of 12a . Under these
conditions 13a was obtained in 45-50% yield. Moreover,
the formation of the N1-nucleoside was minimized, and
the R-nucleoside byproduct was eliminated. The higher
yield of the desired nucleoside using a phase-transfer
catalyst is attributed to better dissolution of the anion
of the heterocycle which facilitates efficient condensa-
tion the reaction.
The R/â ratio of chloro sugars was established to be
1
g95/5 based on the H NMR signal integration of the
anomeric proton of the respective isomers. When a
solution of 12a prepared in toluene was employed for
the glycosylation of 10a , the yield of 13a increased to
65%. The yield of 14 decreased to 8% and that of 15
was negligible.
The structural assignments of the glycosylation prod-
ucts were made by 1H NMR and UV spectroscopies. The
anomeric proton of 13a is a doublet at 6.3 ppm with a
coupling constant of 2.9 Hz which is characteristic of
other 2′,3′-O-isopropylidene-protected â-nucleosides of
pyrrolo[2,3-d]pyrimidine bases.1 Acid-catalyzed depro-
tection of 13a gave a product which was identical by
TLC, melting point, and 1H NMR to 5b prepared via
the Suzuki reaction. The structure of N1-glycosylated
product 14, however, was established based on earlier
studies10 which reported that the UV spectra of N1-
glycosylated pyrrolopyrimidine nucleosides have an
absorption maximum at a higher wavelength compared
to their N7-isomer. This trend was observed with
compounds 13a and 14 which showed λmax values at 297
and 304 nm, respectively. Thus, the major product was
assigned the N7-glycosyl structure 13a and the minor
The moderate yield (e50%) of 13a even with 2 full
equiv of the sugar was attributed to the short half-life
of the chloro sugar 12a (t1/2 e 3 h in THF) which may
be even shorter in the strongly basic reaction conditions.
Therefore, attempts were directed toward finding a
suitable solvent and conditions that would improve the
formation and stability of the R-chloro sugars. Since
toluene was found to be the solvent of choice for the
TDA-1-mediated glycosylation, chlorination of 11a was
attempted in toluene under a variety of reaction condi-
tions. Careful investigation revealed that the chlorina-
tion proceeded well in toluene at a warmer temperature
(-10 °C) vs THF (-78 °C). Furthermore, washing the
chlorination mixture with ice-cold brine and drying over
MgSO4 increased the half-life of 12a to >48 h at e4 °C.
1
product, the N1-glycosyl structure 14. Although the H
NMR spectra of the two isomers are very similar, the
chemical shifts for C2-H signals of 13a (8.40 ppm) and
14 (8.66 ppm) are slightly different. Also, when sub-
jected to trifluoroacetic acid-catalyzed deprotection 13a
gave 5b, whereas 14 underwent deglycosylation giving
10a as the only isolable product. Several attempts to

Adenosine Kinase Inhibitors. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2897
Sch em e 4
Sch em e 5a
a
(a) Acetoxyisobutyryl bromide; (b) Pd(OH)2/C, 10%, EtOAc, H2;
successfully deprotect 14 under mild acidic conditions
failed. Such acid instability of N1-glycosylated pyrrolo-
pyrimidine nucleosides is well-known.11 The R-nucleo-
side 15 obtained in the sodium salt glycosylation method
was assigned by 1H NMR spectrum based on the
downfield chemical shift and the large coupling constant
for the anomeric proton signal (6.7 ppm, J g 7 Hz).
The potent AKI activity exhibited by 5′-amino-5′-
deoxy nucleosides 1c,d prompted the synthesis of the
5′-amino nucleoside 16. Since earlier efforts had failed
to convert the 5′-OH of 1a to the corresponding 5′-amino
derivative 1c via a 5′-O-tosylate or Mitsunobu reaction,
the 5-azido-R-chloro sugar 12c was coupled to 10a to
give 13h . Deprotection followed by hydrogenation of 13h
gave the desired product 16 in an overall 80% yield. It
is important to note that, unlike 12a ,b, 12c could not
be generated in toluene due to the insolubility of the
starting material 11c in toluene at the reaction tem-
perature (-10 °C). Instead, it was prepared in THF by
the original procedure.1
With a view to evaluating the role of various OH
groups of the ribofuranosyl moiety on AKI activity, 2′-
deoxy nucleoside 19, 2′,5′-dideoxy nucleoside 22, and
3′,5′-dideoxy nucleoside 23 were prepared. Synthesis of
compound 19 was accomplished as shown in Scheme 4.
Glycosylation of 10a with 2-deoxy-3,5-di-O-toluoyl-R-D-
pentofuranosyl chloride (17)12 using the sodium salt
procedure8 gave 18 which was deprotected using sodium
methoxide in methanol to furnish 19 in an overall 51%
yield. Interestingly, in this glycosylation experiment the
formation of both N7-R- and N1-glycosylated byproducts
was negligible.
The structural assignments of 18 were made using
1H NMR spectroscopy. The signal for the anomeric
proton of 18 appears at 6.60 ppm as a doublet of
doublets (J 1 ) 6.01 Hz and J 2 ) 8.25 Hz) which is
consistent with the data reported for similar com-
pounds.9,13 Similarly, the signal for the anomeric proton
of the deprotected nucleoside 19 also appears as a sharp
doublet of doublets at 6.64 ppm which is characteristic
of 2′-deoxy nucleosides.13 The UV spectrum of 19 shows
the characteristic λmax at 298 nm (ꢀ 18900) at pH 7,13
thus confirming regiochemistry of the glycosylation.
The synthesis of the dideoxy nucleosides 22 and 23
was accomplished by reacting 5b with acetoxyisobutyryl
bromide14 in moist acetonitrile to give 20 (19%) and 21
(63%) which were separated by flash chromatography
(Scheme 5). The compounds were characterized by
comparing the chemical shifts and splitting patterns for
the anomeric protons with those reported for the known
2′-O-acetyl-3′-â-bromoadenosine and 3′-O-acetyl-2′-â-
bromoadenosine derivatives.14,15 Hydrogenation and
subsequent deprotection of 20 and 21 gave 22 and 23,
(c) MeOH/NaOMe.
Ta ble 1. AK Inhibitor SAR
a
AK IC50
compd
R
R′
A
B
C
D
Z
(µM)
4a
4b
4c16
4e
4f
4g
4h
4i
Ph
I
I
I
I
I
I
I
I
H
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
H
H
H
H
H
H
H
0.1
0.8
0.55
0.775
0.2
10
1.2
0.12
1.25
Ph-CH2
4-F-Ph
4-MeO-Ph
4-HO-Ph
cyclohexyl
4-CN-Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
OH OH OH
OH OH OH
4j
Ph
H
Ph
Ph-CH2
4-F-Ph
4-MeO-Ph Ph
4-HO-Ph Ph
cyclohexyl Ph
Ph
4-F-Ph
Ph
5a
5b
5c
5d 16
5e
5f
5g
5h
5i
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
H
H
H
H
H
H
H
0.32
0.0005
0.03
0.0015
0.006
0.001
0.25
Ph
OH OH OH
0.0008
0.026
0.0012
0.0027
0.014
0.001
0.0036
0.009
0.025
0.0023
0.0015
0.047
0.001
0.0015
0.0063
0.3
4-F-Ph
4-Cl-Ph
4-Cl-Ph
Ph
4-MeO-Ph
2-furanyl
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
H
H
H
H
H
H
H
H
H
H
H
H
5j
5k
5l
4-Cl-Ph
4-CN-Ph
4-CN-Ph
Ph
5m
5n
5o
5p
5q
5r
4-MeO-Ph 2-furanyl
3-pyridyl
4-Cl-Ph
4-Me-Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
5s
5t
Ph
Ph
Ph
Ph
Ph
Ph
Br OH OH
5u
16
19
22
23
H
H
H
H
H
OH OH N3
OH OH NH2
H
H
OH OH
OH
H
H
H
0.100
0.044
OH
a
Enzyme inhibition assays were performed on human recom-
binant AK enzyme. IC50 values are results of a single experiment.
respectively. These products exhibited characteristically
different NMR signal patterns which enabled definitive
structural assignments. For example, the anomeric
proton of 22 appears as a doublet of doublets at 6.63
ppm (J 1 ) 5.5 Hz and J 2 ) 5.8 Hz) confirming that it is
a 2′-deoxy nucleoside, whereas the anomeric proton of
23 appears as a sharp doublet at 6.0 ppm (J ) 2.2 Hz)
which is characteristic of a â-nucleoside with a C2′-OH.
Resu lts
The compounds were evaluated as inhibitors of the
recombinant human AK (Table 1). The IC50 values were

2898 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ugarkar et al.
determined as described previously.1 The results reflect
the importance of aromatic rings at the C4- and C5-
positions and also of the 2′- and 3′- OH groups of the
sugar component. Substituting the iodo group at the C5-
position of 1b with a phenyl ring resulted in a weaker
AKI (5a , IC50 ) 0.32 µM).1 Also a moderate loss of AKI
activity was observed when the C4-NH2 of 1b was
replaced with C6H5NH (4a , IC50 ) 0.1 µM). Remarkably,
inhibitory potency was enhanced when the above two
substitutions were both present (5b, IC50 ) 0.0005 µM).
As reported earlier, 5b undergoes oxidative metabolism
in vivo to form the corresponding p-OH compound 5f
which is eliminated as the glucoronate.16 This in vivo
hydroxylation of the aromatic ring was avoided by
substituting the p-position with F. Accordingly, we
prepared 5b analogues with CN, CH3, and OCH3 in the
para-position of the aniline ring and F or Cl in the para-
position of both the phenyl rings. These compounds,
however, were relatively less potent than 5b in the
enzyme inhibition assay.
Ta ble 2. Antiseizure Activity in Rat MES Seizure Model
% inhib ED50 % inhib ED50
compd 5 mg/kg, ipa (mg/kg, ip)b compd 5 mg/kg, ipa (mg/kg, ip)b
5b
5d
5e
5f
5h
5i
82
73
93
35
37
100
83
0
1.1
1.9
1.0
5.8
5l
87
100
50
62
50
0
1.1
0.7
5.0
7.1
5m
5o
5q
5r
5t
5u
16
>5a
g5a
1.1
1.7
>5a
g5a
>5a
5j
5k
50
0
>5a
a
Screened at 1 h after administration of 5 mg/kg, ip (N ) 8).
b
N ) 8/dose.
A moderate loss in AKI activity was observed when
the C4-aniline of 5b was replaced with 3-pyridylamine
(5p , IC50 ) 0.025 µM), whereas a much smaller loss
occurred when the C5-phenyl ring of 5b or 5e was
replaced with a furan ring (5n , IC50 ) 0.0036 µM; 5o,
IC50 ) 0.009 µM). Moderate loss in potency was also
observed when aniline in 5b was replaced with benzy-
lamine (5c, IC50 ) 0.03 µM), but a much greater loss in
potency resulted for the corresponding C4-N-cyclohexy-
lamino analogue 5g (IC50 ) 0.25 µM).
5-Halogen-substituted tubercidin analogues 1a ,b ,
with OH and H in their respective C5′-positions, showed
similar AKI potencies (1a , IC50 ) 0.026 µM; 1b, IC50
)
F igu r e 1. Effect of AKIs (5l,m ) on mean arterial blood
pressure (MAP) and heart rate at 10 mg/kg, ip. Data are means
( SEM (n ) 4).
0.009 µM), whereas the C5′-NH2 analogues 1c,d showed
a considerable increase in their ability to inhibit AK
(IC50 < 0.001 µM). However, such enhancement in AKI
potency was not observed with diaryl AKIs when the
C5′-OH of 5h was replaced with C5′-NH2 group to give
16 (IC50 ) 0.0063 µM).
General behavioral effects observed following admin-
istration of the AKIs were qualitatively similar to those
previously noted with other AKIs.1 These include de-
creased locomotor activity, hypothermia, and muscular
flaccidity. These side effects in rats were generally mild
at or below the anticonvulsant ED50s and are less
pronounced than those observed with equi-effective
doses of adenosine receptor agonists.18 Also, in contrast
to adenosine receptor agonists, AKIs (5l,m ) did not
cause a decrease in blood pressure or heart rate at doses
10-fold higher than the ED50 for inhibition of MES
seizures (Figure 1).
In contrast to the C5′-OH, both the C2′- and C3′-OH
groups are important for potent AKI activity as evi-
denced by the significant loss in the inhibitory potency
of the 2′-deoxy compounds 19 (IC50 ) 0.3 µM) and 22
(IC50 ) 0.1 µM) and the 3′-deoxy compound 23 (IC50
)
0.044 µM). The presence of a methyl group at the C2-
position of adenosine is reported to have a deleterious
effect on AK substrate efficiency, whereas a Br at the
8-position was well-tolerated.17 The corresponding nu-
cleosides 5s,t followed these trends with IC50s 0.047 and
0.001 µM, respectively.
Discu ssion
An tiseizu r e Activity. Compounds with IC50 < 0.05
µM were screened for anticonvulsant activity in the rat
MES seizure model. Ten AKIs showed potent activity
(Table 2). Compound 5b and several of its para-
substituted analogues (compounds 5d ,e,f,i,j,l,m ,o,q)
inhibited MES seizures with ED50s in the range of 0.7-
7.1 mg/kg. Some of these compounds were shown also
to exhibit potent activity with oral administration in
rats.16,18 Compounds 5h ,t,u and 16 which inhibited the
enzyme with e0.01 µM were found to be weak in the
MES seizure model; information on the metabolism or
pharmacokinetic characteristics is not currently avail-
able but will be needed to explain their in vivo potencies.
The present study evaluated the SAR of tubercidin
analogues with large hydrophobic substituents at the
C4- and C5-positions. The results suggest the possible
existence of one or more hydrophobic binding pockets
in the enzyme active site. The potent AKI activity
exhibited by 1a -d may therefore be attributed to a
hydrophobic interaction between the halogen atom
(I or Br) and a hydrophobic pocket. Replacement of the
C5-halogen atom, however, with a methyl1 or a phenyl
group failed to retain the AKI activity. Whereas,
attaching two phenyl rings to a tubercidin molecule
resulted in highly potent AKIs (e.g., 5b, IC50 ) 0.0005
µM). Replacement of the phenyl group on the C4-amino

Adenosine Kinase Inhibitors. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2899
with cyclohexyl or benzyl led to a significant loss in
potency. These results suggest that the aryl group forms
a distinct interaction with the AK binding site. Cur-
rently, we are evaluating the interactions of various
AKIs with the active site using 3D X-ray crystal
structure of the human AK-adenosine complex which
was recently solved at high resolution.19
hypotensive or bradycardic effects in rats. This hemo-
dynamic neutrality contrasts markedly with the severe
effects of adenosine receptor agonists, which exert
profound effects on blood pressure and heart rate. For
example, the adenosine receptor agonist cyclopentylad-
enosine reduced blood pressure and heart rate to one-
third of its baseline levels at a dose equalling the ED50
for inhibition of MES seizures,18 and other adenosine
receptor agonists have been shown to have similar
hemodynamic effects.20,21 The improved side effect
profile of diaryl AKIs compared with the agonists is a
significant finding and may be attributed to a relatively
site- and event-specific increase of adenosine levels
within brain tissue undergoing epileptiform activity.18
It is also notable that certain of these compounds
exemplified by 5b result in less overt toxicity, seen as
much higher LD50 in rats compared with the non-aryl
AKI 1a .18 Thus, the discovery of these new AKIs
provides an opportunity for clinical development of
selected compounds that offer this novel therapeutic
approach.
The potency of non-aryl AKIs is highly dependent on
the substituent at the C5′-position. In contrast, the
potency of diaryl AKIs is not affected by the nature of
the substituent at the C5′-position suggesting that the
aryl groups provide a significant proportion of the
binding energy. Binding through aromatic hydrophobic
interactions may change the orientation of these AKIs
relative to that of the natural substrate adenosine or
non-aryl AKIs. Under these circumstances the 5′-
position of diaryl AKIs may be placed in a position that
fails to interact significantly with the active site.
Alternatively, it is conceivable that non-aryl- and aryl-
substituted AKIs bind to different sites. For example,
the non-diaryl AKIs 1a -d could bind to the active site
of the enzyme, whereas the diaryl AKIs could bind to
the ATP site or to yet another site which may affect the
protein conformation and/or the conformation of the
active site.
Con clu sion
The previous manuscript showed that a combination
of groups such as a Br or I at the C5-position and a NH2,
Cl, and SCH3 at the C4-position was important for
potent AK inhibition, whereas the results in this report
suggest that two aryl rings, one on the C4-amine
function and another at the C5-position of tubercidin,
enable potent AKI activity in vitro and in vivo. In
contrast to the first series, an NH2 group at the C5′-
position did not increase the inhibitory potency of diaryl
AKIs. The results also emphasize that the C2′-OH and
C3′-OH are essential for potent AKI activity. Finally,
diaryltubercidin-based AKIs are potent anticonvulsant
agents which exhibit relatively mild side effects com-
pared to adenosine receptor agonists at equi-effective
doses and therefore represent a potential strategy to
harness the neuroprotective properties of adenosine
receptor activation.
The decreased inhibitory potency of the 2′-deoxy
compounds 19 and 22 and the 3′-deoxy compound 23
suggest that hydrogen-bonding interactions by C2′- and
C3′-OH groups are important for enzyme inhibition. The
poor AKI activity of 19 (IC50 ) 0.3 µM) also indicates
that the presence of a C5′-OH group does not compen-
sate for the lack of C2′-OH, reiterating the relatively
insignificant role of the C5′-OH or C5′-NH2 groups of
diaryl AKIs in binding to the enzyme. These observa-
tions parallel the previous observation made with
adenosine which is a better substrate than its 2′-deoxy
and 3′-deoxy derivatives.17
Although 5b showed potent anticonvulsant effect
(ED50 ) 1.1 mg/kg) when administered ip, it was less
potent orally (5.5 mg/kg, po), due to poor oral bioavail-
ability (<20%).16 In addition, pharmacokinetic studies
indicated that 5b had a relatively short half-life (∼1.1
h) in dogs and high hepatic clearance in rats, dogs, and
monkeys.16 The short half-life of 5b was determined to
be due to its rapid metabolism to the corresponding
p-hydroxy compound 5f which was cleared as the
glucoronate. Consequently, the p-fluoro-substituted com-
pound 5d was shown to have a longer half-life in dogs
(t1/2 ) 4.2 h).16 Other para-substituted AKIs showed
potent anticonvulsant activity (e.g., 5e,i,l-m ) and are
expected to have longer half-lives due to increased
stability toward oxidative metabolism. Some of the
potent inhibitors such as 5f,h ,k ,t,u and 16 exhibited
significantly reduced anticonvulsant potency; it could
be speculated that this may be due to alteration in the
pharmacokinetic characteristics responsible for distri-
bution to the site of action or metabolism resulting in a
less potent AKIs or both.
Exp er im en ta l Section
1H and 13C NMR spectra were obtained using a Varian
Gemini-200 spectrophotometer at 200 MHz. NOE experiments
were conducted on a Bruker AM-500 spectrophotometer at 500
MHz by NuMega Resonance Labs, Inc., San Diego, CA. The
chemical shifts are expressed in δ units with respect to
tetramethylsilane (δ 0.00) as an internal standard.The ultra
violet absorption spectra were recorded on Perkin-Elmer UV/
VIS spectrometer, Lambda 2, and the λmax are cm-1 units.
Melting points were determined using a Thomas-Hoover
capillary melting point apparatus and are uncorrected. Thin-
layer chromatography was performed on silica gel, GHLF 250-
µm plates. Silica gel, 230-400 mesh (E. Merck), was used for
the column chromatography. Elemental analyses were deter-
mined by Robertson Microlit Laboratories, Madison, NJ .
4-N-P h en yla m in o-5-iod o-7-(5-d eoxy-2,3-O-isop r op yl-
id en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (3a ). A
mixture of 2a 1 (2.5 g, 5.73 mmol), aniline (1.4 g, 15 mmol) and
sodium acetate (2.5 g, 18 mmol) taken in ethanol (25 mL) and
heated to reflux for 24 h. The reaction mixture was concen-
trated under reduced pressure. The residue was dissolved in
ethyl acetate and washed with 0.5 N HCl solution (25 mL).
The organic layer was dried (MgSO4), evaporated and the
residue was purified by chromatography on a silica gel column
(20% ethyl acetate in hexanes) to give 3a as a colorless glassy
solid (2.2 g, 78.5%): 1H NMR (DMSO-d6) δ 1.25 (d, 3H), 1.35
and 1.55 (2s, 6H), 4.17 (m, 1H), 4.7 (m, 1H), 5.35 (m, 1H),
Of the diaryl AKIs tested, the two compounds with
the most potent anticonvulsant activity, 5l,m , showed
little or no effect on blood pressure or heart rate at doses
well above those required for inhibition of MES seizures.
These results parallel the previous finding18 that an-
other active diaryl AKI, compound 5b, was devoid of

2900 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ugarkar et al.
6.2 (d, J ) 3.7 Hz, 1H), 7.4 (m, 5H), 7.85 (s, 1H), 8.27 (s, 1H,
5.3 (m, 1H), 6.21 (d, J ) 3.2 Hz, 1H, 1′-CH), 7.8 (d, 2H), 7.9
(d, 2H), 7.91 (s, 1H), 8.45 (s, 1H), 8.75 (br s, 1H, exchangeable
with D2O).
4-N-(3-P yr id yl)a m in o-5-iod o-7-(5-d eoxy-2,3-O-isop r o-
pyliden e-â-D-r ibofu r an osyl)pyr r olo[2,3-d]pyr im idin e (3j).
Condensation of 3-aminopyridine with 2a 1 by the procedure
described for 3i gave 3j as a glassy solid in 82% yield: 1H NMR
(DMSO-d6) δ, 1.28 (d, 3H), 1.32 and 1.58 (2s, 6H), 4.17 (m,
1H), 4.75 (m, 1H), 5.28 (m, 1H), 6.21 (d, J ) 3.5 Hz, 1H, 1′-
CH), 7.35-8.9 (m, 6H).
exchangeable with D2O), 8.38 (s, 1H).
4-N-Ben zyla m in o-5-iod o-7-(5-d eoxy-2,3-O-isop r op yl-
id en e-â-D-r ib ofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (3b ).
Condensation of benzylamine with 2a by the procedure
described for 3a gave 3b as a glassy solid in 83% yield: 1H
NMR (DMSO-d6) δ 1.23 (d, 3H), 1.36 and 1.6 (2s, 6H), 4.2 (m,
1H), 4.65 (m, 1H), 4.8 (d, 2H), 5.32 (m, 1H), 6.18 (d, J ) 3.5
Hz, 1H, 1′-CH), 7.22 (m, 7H), 8.2 (s, 1H).
4-N-(4-F lu or op h en yl)a m in o-5-iod o-7-(5-d eoxy-2,3-O-
isop r op ylid en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im i-
d in e (3c). Condensation of 4-fluoroaniline with 2a by the
procedure described for 3a gave 3c as a glassy solid in 77%
4-N-P h en ylam in o-5-iodo-7-(5-deoxy-â-D-r ibofu r an osyl)-
p yr r olo[2,3-d ]p yr im id in e (4a ). A solution of 3a (500 mg, 1
mmol) in 70% TFA (10 mL) was stirred at room temperature
for 45 min and concentrated under high vacuum. The residue
was coevaporated with water (2 × 10 mL) and ethanol (10 mL),
and the resulting semisolid was stirred with aqueous NaHCO3
for 10 min. The solid was collected by filtration, washed with
water, dried and crystallized from boiling ethanol to give 4a
as needles (358 mg, 78%): mp 230-233 °C; 1H NMR (DMSO-
d6) δ 1.3 (d, 3H), 3.8-4.0 and 4.45 (m, 3H), 5.18 (d, 1H,
exchangeable with D2O), 5.4 (d, 1H, exchangeable with D2O),
6.14 (d, J ) 5.4 Hz, 1H, 1′-CH), 7.47 (m, 6H), 8.28 (s, 1H,
exchangeable with D2O), 8.4 (s, 1H). Anal. (C17H17IN4O3) C,
H, N.
4-N-Ben zyla m in o-5-iod o-7-(5-d eoxy-â-D-r ibofu r a n osyl)-
p yr r olo[2,3-d ]p yr im id in e (4b). Compound 3b was subjected
to deprotection by the procedure described for 4a to give 4b
as a crystalline solid in 63% yield: mp 198-199 °C; 1H NMR
(DMSO-d6) δ 1.28 (d, 3H), 3.9 (m, 1H), 4.4 (m, 2H), 4.8 (d, 2H),
5.1 (d, 1H, exchangeable with D2O) 5.33 (d, 1H, exchangeable
with D2O), 6.0 (d, J ) 5.5 Hz, 1H, 1′-CH), 6.8-7.4 (m, 6H),
7.65 (s, 1H), 8.18 (s, 1H). Anal. (C18H19IN4O3) C, H, N.
4-N-(4-F lu or op h en yl)a m in o-5-iod o-7-(5-d eoxy-â-D-r ibo-
fu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (4c). Compound 3c
was subjected to deprotection by the procedure described for
4a to give 4c as a crystalline solid in 69% yield: mp 190-192
°C; 1H NMR (DMSO-d6) δ 1.29 (d, 3H), 3.86 (m, 1H), 4.42 (m,
2H), 5.2 and 5.43 (2d, 2H, exchangeable with D2O), 6.15 (d, J
) 5.6 Hz, 1H, 1′-CH), 7.35-7.9 (m, 5H), 8.35 (s, 1H, exchange-
able with D2O), 8.38 (s, 1H). Anal. (C17H16FIN4 O3) C, H, N.
4-N-(4-Ch lor op h en yl)a m in o-5-iod o-7-(5-d eoxy-â-D-r ibo-
fu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (4d ). Compound 3d
was subjected to deprotection by the procedure described for
4a to give 4d as a crystalline solid in 75% yield: mp 201-202
°C; 1H NMR (DMSO-d6) δ 1.3 (d, 3H), 3.9 (m, 1H), 4.42 (m,
2H), 5.15 and 5.4 (2d, 2H, exchangeable with D2O), 6.1 (d, J
) 5.8 Hz, 1H, 1′-CH), 7.4-7.9 (m, 5H), 8.32 (s, 1H, exchange-
able with D2O), 8.4 (s, 1H). Anal. (C17H16ClIN4O3‚0.33H2O) C,
H, N.
1
yield: H NMR (DMSO-d6) δ 1.25 (d, 3H), 1.32 and 1.53 (2s,
6H), 4.2 (m, 1H), 4.7 (m, 1H), 5.3 (m, 1H), 6.2 (d, J ) 3.3 Hz,
1H, 1′-CH), 7.5 (m, 4H), 7.85 (s, 1H), 8.27 (s, 1H, exchangeable
with D2O), 8.39 (s, 1H).
4-N-(4-Ch lor op h en yl)a m in o-5-iod o-7-(5-d eoxy-2,3-O-
isop r op ylid en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im i-
d in e (3d ). Condensation of 4-chloroaniline with 2a by the
procedure described for 3a gave 3d as a glassy solid in 65%
1
yield: H NMR (DMSO-d6) δ 1.24 (d, 3H), 1.33 and 1.56 (2s,
6H), 4.3 (m, 1H), 4.7 (m, 1H), 5.4 (m, 1H), 6.2 (d, J ) 3.5 Hz,
1H, 1′-CH), 7.5 (m, 4H), 7.8 (s, 1H), 8.2 (s, 1H, exchangeable
with D2O), 8.4 (s, 1H).
4-N-(4-Meth oxyp h en yl)a m in o-5-iod o-7-(5-d eoxy-2,3-O-
isop r op ylid en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im i-
d in e (3e). Condensation of 4-methoxyaniline with 2a by the
procedure described for 3a gave 3e as a glassy solid in 65%
1
yield: H NMR (DMSO-d6) δ 1.26 (d, 3H), 1.32 and 1.53 (2s,
6H), 3.77 (s, 3H), 4.18 (m, 1H), 4.75 (m, 1H), 5.3 (m, 1H), 6.19
(d, J ) 3.06 Hz, 1H, 1′-CH), 6.97 (d, 2H), 7.6 (d, 2H), 7.81 (s,
1H), 8.1 (s, 1H, exchangeable with D2O), 8.31 (s, 1H).
4-N-(4-Hyd r oxyp h en yl)a m in o-5-iod o-7-(5-d eoxy-2,3-O-
isop r op ylid en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im i-
d in e (3f). Condensation of 4-aminophenol with 2a 1 by the
procedure described for 3a gave 3f as a glassy solid in 62%
yield: 1H NMR (CDCl3) δ 1.3 (d, 3H), 1.31 and 1.6 (2s, 6H),
4.17 (m, 1H), 4.7 (m, 1H), 5.31 (m, 1H), 6.2 (d, J ) 3.3 Hz, 1H,
1′-CH), 6.8 (d, 2H), 7.5 (d, 2H), 7.8 (s, 1H), 8.0 (s, 1H,
exchangeable with D2O), 8.3 (s, 1H), 9.3 (s, 1H, exchangeable
with D2O).
4-N-Cycloh exyla m in o-5-iod o-7-(5-d eoxy-2,3-O-isop r o-
pyliden e-â-D-r ibofu r an osyl)pyr r olo[2,3-d]pyr im idin e (3g).
Condensation of cyclohexylamine with 2a 1 by the procedure
described for 3a gave 3g as a glassy solid in 88% yield: 1H
NMR (DMSO-d6) δ 1.65 (m, 19H), 4.17 (m, 2H), 4.7 (m, 1H),
5.35 (m, 1H), 6.15 (d, J ) 3.5 Hz, 1H, 1′-CH), 6.22 (d, 1H,
exchangeable with D2O), 7.7 (s, 1H), 8.22 (s, 1H).
4-N-(4-Meth oxyp h en yl)a m in o-5-iod o-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (4e). Compound 3e
was subjected to deprotection by the procedure described for
4a to give 4e as microplates in 70% yield: mp 190-192 °C;
1H NMR (DMSO-d6) δ 1.3 (d, 3H), 3.8 (s, 3H), 3.95 (m, 1H),
4.42 (m, 2H), 5.15 (d, 1H, exchangeable with D2O), 5.38 (d,
1H, exchangeable with D2O), 6.1 (d, J ) 5.7 Hz, 1H, 1′-CH),
7.0 and 7.65 (2d, 4H), 7.8 (s, 1H), 8.1 (s, 1H, exchangeable with
D2O), 8.3 (s, 1H). Anal. (C18H19IN4O4) C, H, N.
4-P h en yla m in o-5-iod o-7-(5-O-ter t-bu tyld im eth ylsilyl-
2,3-O-isop r op ylid en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p y-
r im id in e (3h ). Condensation of aniline with 2b by the
procedure described for 3a gave 3h as a glassy solid in 68%
yield: 1H NMR (CDCl3) δ 0.55 (m, 15H), 1.4 and 1.66 (2s, 6H),
3.85 (m, 2H), 4.35 (m, 1H), 5.0 (m, 1H), 5.2 (m, 1H), 6.33 (d, J
) 3.1 Hz, 1H, 1′-CH),7.4 (m, 5H), 7.85 (s, 1H), 8.27 (s, 1H,
exchangeable with D2O), 8.38 (s, 1H).
4-N-(4-Hyd r oxyp h en yl)a m in o-5-iod o-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (4f). Compound 3f
was subjected to deprotection by the procedure described for
4a to give 4f as a crystalline solid in 73% yield: mp 233-235
4-N-(Cya n op h en yl)a m in o-5-iod o-7-(5-d eoxy-2,3-O-iso-
p r op ylid e n e -â-D-r ib ofu r a n osyl)p yr r olo[2,3-d ]p yr im i-
d in e (3i). To a solution of 4-cyanoaniline (450 mg, 4 mmol) in
dry DMF (15 mL) was added a 1 M solution of t-BuOK in tert-
butyl alcohol (4 mL) over a period of 10 min. The resulting
dark red solution was cooled in an ice bath, treated with 2a
(800 mg, 1.8 mmol) in portions, and stirred at room temper-
ature for 1 h. The reaction mixture was concentrated under
high vacuum and the residue was partitioned between brine
and ethyl acetate. The organic layer was dried (MgSO4) and
evaporated and the residue was purified by chromatography
on a silica gel column (5% methanol in methylene chloride) to
furnish a solid which was crystallized from ethanol to give 3i
1
°C; H NMR (DMSO-d6) δ 1.35 (d, 3H), 3.9 (m, 1H), 4.45 (m,
2H), 5.15 (d, 1H, exchangeable with D2O), 5.4 (d, 1H, exchange-
able with D2O), 6.08 (d, J ) 5.5 Hz, 1H, 1′-CH), 6.8 (d, 2H),
7.5 (d, 2H), 7.78 (s, 1H, 6-CH), 8.0 (s, 1H, exchangeable with
D2O), 8.29 (s, 1H), 9.3 (s, 1H, exchangeable with D2O). Anal.
(C17H17IN4O4) C, H, N.
4-N-Cycloh exyla m in o-5-iod o-7-(5-d eoxy-â-D-r ib ofu r a -
n osyl)p yr r olo[2,3-d ]p yr im id in e (4g). Compound 3g was
subjected to deprotection by the procedure described for 4a to
give 4g as crystalline solid in 68% yield: mp 161-164 °C; 1H
NMR (DMSO-d6) δ 1.65 (m, 13H), 3.65 (m, 4H), 5.1 (1d, 1H,
exchangeable with D2O), 5.35 (1d, 1H, exchangeable with D2O),
1
(760 mg, 82%): mp 110-112 °C; H NMR (DMSO-d6) δ 1.28
(d, 3H), 1.3 and 1.55 (2s, 6H), 4.2 (m, 1H, 4′-CH), 4.71 (m, 1H),

Adenosine Kinase Inhibitors. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2901
6.0 (d, J ) 5.5 Hz, 1H, 1′-CH), 6.2 (d, 1H, exchangeable with
D2O), 7.62 (s, 1H), 8.2 (s, 1H). Anal. (C17H23IN4O3‚0.5H2O) C,
H, N.
(d, 1H, exchangeable with D2O), 6.17(d, J ) 5.6 Hz, 1H, 1′-
CH), 7.42 (m, 10H), 8.41 (s, 1H). Anal. (C24H24N4O4) C, H, N.
4-N-(4-Hyd r oxyp h en yl)a m in o-5-p h en yl-7-(5-d eoxy-â-D-
r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5f). Compound 3f
was reacted with phenylboronic acid by the procedure de-
scribed for 5a to give 4-N-(4-hydroxyphenyl)amino-5-phenyl-
7-(5-deoxy-2,3-O-isopropylidene-â-D-ribofuranosyl)pyrrolo[2,3-
d]pyrimidine as a glassy solid: 1H NMR (DMSO-d6) δ 1.3 (d,
3H), 1.32 and 1.57 (2s, 6H), 4.19 (m, 1H), 4.75 (m, 1H), 5.38
(m, 1H), 6.28 (d, J ) 3.1 Hz, 1H, 1′-CH), 7.2 (m, 11H), 8.35 (s,
2-CH), 9.21 (s, 1H, exchangeable with D2O). This intermediate
was subjected to TFA catalyzed deprotection as described for
4a to give 5f as a crystalline solid in an overall 55% from 2a :
188-189 °C; 1H NMR (DMSO-d6) δ 1.35 (d, 3H), 4.25 (m, 3H),
5.1 (d, 1H, exchangeable with D2O), 5.4 (d, 1H, exchangeable
with D2O), 6.18 (d, J ) 6.0 Hz, 1H, 1′-CH), 7.2 (m, 11H), 8.31
(s, 1H), 9.2 (s, 1H, exchangeable with D2O). Anal. (C23H22N4O4)
C, H, N.
4-N-(Cya n oyp h en yl)a m in o-5-iod o-7-(5-d eoxy-â-D-r ibo-
fu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (4h ). Compound 3i
was subjected to deprotection by the procedure described for
4a to give 4h as a crystalline solid in 65% yield: mp 259-261
°C; 1H NMR (DMSO-d6) δ 1.32 (d, 3H), 3.95 (m, 2H), 4.50 (m,
1H), 5.18 (d, 1H, exchangeable with D2O), 5.4 (d, 1H, exchange-
able with D2O), 6.11 (d, J ) 5.8 Hz, 1H, 1′-CH), 6.8 (d, 2H),
7.8-8.0 (m, 5H), 8.5 (s, 1H), 8.75 (s, 1H, exchangeable with
D2O).
4-N-P h en yla m in o-5-iod o-7-â-D-r ib ofu r a n osylp yr r olo-
[2,3-d ]p yr im id in e (4i). Compound 3h was subjected to
deprotection by the procedure described for 4a to give 4i as
needles in 62% yield: mp 224-225 °C; 1H NMR (DMSO-d6) δ
3.62 (m, 2H), 3.95 (m, 3H), 5.1-5.5 (m, 3H, exchangeable with
D2O), 6.12 (d, J ) 5.8 H, 1H, 1′-CH), 7.05-7.9 (m, 6H), 8.28
(br s, 1H, exchangeable with D2O), 8.39 (s, 1H). Anal. (C17H17
IN4O4) C, H, N.
-
4-N-Cycloh exylam in o-5-ph en yl-7-(5-deoxy-â-D-r ibofu r a-
n osyl)p yr r olo[2,3-d ]p yr im id in e (5g). Compound 3g was
reacted with phenylboronic acid by the procedure described
for 5a to give 4-N-cyclohexylamino-5-phenyl-7-(5-deoxy-2,3-O-
isopropylidene-â-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine as a
glassy solid in 85% yield: 1H NMR (DMSO-d6) δ 1.27 (d, 3H),
1.31 and 1.6 (2s, 6H), 1.51 (m, 10H), 4.12 (m, 2H), 4.75 (m,
1H), 5.15 (d, 1H, exchangeable with D2O), 5.35 (m, 1H), 6.23
(d, J ) 3.4 Hz, 1H, 1′-CH), 7.55 (m, 6H), 8.28 (s, 1H). This
intermediate was subjected to TFA catalyzed deprotection by
the procedure described for 4a to give 5g as needles in 68%
yield: mp 156-158 °C; 1H NMR (DMSO-d6) δ 1.3 (d, 3H), 1.55
(m, 10H), 4.23 (m, 4H), 5.23 (m, 3H, exchangeable with D2O),
6.1 (d, J ) 6.5 Hz, 1H, 1′-CH), 7.5 (m, 6H), 8.22 (s, 1H). Anal.
(C23H28N4O3‚0.5H2O) C, H, N.
4-N-P h en ylam in o-5-ph en yl-7-â-D-r ibofu r an osylpyr r olo-
[2,3-d ]p yr im id in e (5h ). Compound 4h was reacted with
phenylboronic acid by the procedure described for 5a to give
5h as microplates in 58% yield: mp 228-229 °C; 1H NMR
(DMSO-d6) δ 3.58 (m, 2H), 4.25 (m, 3H), 5.25 (m, 3H,
exchangeable with D2O), 6.19 (d, J ) 6.2 Hz, 1H, 1′-CH), 7.35
(m, 11H), 7.7 (s, 1H), 8.4 (s, 1H). Anal. (C23H22N4O4) C, H, N.
4-N-P h en yla m in o-7-â-D-r ibofu r a n osylp yr r olo[2,3-d ]p y-
r im id in e (4j). Compound 4i (460 mg, 1 mmol) was dissolved
in methanol (50 mL) and purged with argon. To the solution
was added 10% Pd/C (50 mg) and subjected to hydrogenation
under 20 psi of H2 for 12 h. The reaction mixture was filtered
through a Celite pad and the filtrate was evaporated to
dryness. The residue was crystallized from ethanol to obtain
4j as microcrystals (250 mg, 75%): mp 145-146 °C; 1H NMR
(DMSO-d6) δ 3.68 (m, 2H), 4.0 (m, 3H), 5.1-5.5 (m, 3H,
exchangeable with D2O), 6.2 (d, J ) 5.6 H, 1H, 1′-CH), 6.95-
7.9 (m, 7H), 7.9 (br s, 1H, exchangeable with D2O), 8.39 (s,
1H). Anal. (C17H18N4O4) C, H, N.
4-Am in o-5-ph en yl-7-(5-deoxy-â-D-r ibofu r an osyl)pyr r olo-
[2,3-d ]p yr im id in e (5a ). To a solution of 4-amino-5-iodo-7-(5-
deoxy-â-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine1 (1b; 376 mg,
1 mmol), and tetrakis(triphenylphosphine)palladium (115 mg,
0.1 mmol) in diglyme (25 mL) were added a saturated solution
of Na2CO3 (4 mL) and a solution of phenylboronic acid (488
mg, 4 mmol) in ethanol (7 mL). The resulting heterogeneous
mixture was heated at 100 °C for 4 h. The reaction mixture
was cooled, filtered through a Celite pad and the Celite pad
was washed with ethyl acetate. The filtrate was evaporated
under reduced pressure and the residue was purified by
chromatography on a silica gel column (10% methanol in
methylene chloride) to give 5a as solid microplates (255 mg,
78%): mp 106-109 °C; 1H NMR (DMSO-d6) δ 1.29 (d, 3H),
3.95 (m, 2H), 4.5 (m, 1H), 5.1 (d, 1H, exchangeable with D2O),
5.4 (d, 1H, exchangeable with D2O), 6.15 (d, J ) 6.2 Hz, 1H,
1′-CH), 5.8-6.3 (br s, 2H, exchangeable with D2O), 7.55 (m,
6H), 8.18 (s, 1H). Anal. (C17H18N4O3‚H2O) C, H, N.
4-N-(4-F lu or op h en yl)a m in o-5-(4-flu or op h en yl)-7-(5-
d eoxy-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5i).
Compound 4c was reacted with 4-fluorophenylboronic acid by
the procedure described for 5a to give 5i as a crystalline solid
1
in 62% yield: mp 204-205 °C; H NMR (DMSO-d6) δ 1.3 (d,
3H), 3.9 (m, 2H), 4.5 (m, 1H), 5.15 (d, 1H, exchangeable with
D2O), 5.4 (d, 1H, exchangeable with D2O), 6.2 (d, J ) 5.7 Hz,
1H, 1′-CH), 7.15 (t, 2H), 7.35 (t, 2H), 7.65 (m, 6H), 8.4 (s, 1H).
Anal. (C23H20F2N4O3) C, H, N.
4-N-P h en yla m in o-5-(4-ch lor op h en yl)-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5j). Compound 4a
was reacted with 4-chlorophenylboronic acid by the procedure
described for 5a to give 5j as a crystalline solid in 58% yield:
4-N-P h en yla m in o-5-p h en yl-7-(5-d eoxy-â-D-r ibofu r a n o-
syl)p yr r olo[2,3-d ]p yr im id in e (5b). Compound 4a was re-
acted with phenylboronic acid by the procedure described for
5a to give 5b as a crystalline solid in 70% yield: 207-208 °C;
1H NMR (DMSO-d6) δ 1.3 (d, 3H), 3.93 (m, 2H), 5.03 (m, 1H),
5.1 (d, 1H, exchangeable with D2O), 5.45 (d, 1H, exchangeable
with D2O), 6.18 (d, J ) 6.9 Hz, 1H, 1′-CH), 6.95-7.7 (m, 6H),
8.42 (s, 1H). Anal. (C23H22N4O3) C, H, N.
1
mp 235-236 °C; H NMR (DMSO-d6) δ 1.32 (d, 3H), 4.22 (m,
3H), 5.17 (d, 1H, exchangeable with D2O), 5.4 (d, 1H, exchange-
able with D2O), 6.17 (d, J ) 5.6 Hz, 1H, 1′-CH), 7.35 (m, 11H),
8.3 (s, 1H). Anal. (C23H21ClN4O3) C, H, N.
4-N-(4-Ch lor op h en yl)a m in o-5-(4-ch lor op h en yl)-7-(5-
d eoxy-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5k ).
Compound 4d was reacted with 4-chlorophenylboronic acid by
the procedure described for 5a to give 5k as needles in 50%
yield: mp 199-201 °C; 1H NMR (DMSO-d6) δ 1.31 (d, 3H),
4.22 (m, 3H), 5.15 (d, 1H, exchangeable with D2O), 5.4 (d, 1H,
exchangeable with D2O), 6.18 (d, J ) 5.9 Hz, 1H, 1′-CH), 7.55
(m, 9H), 8.0 (br s, 1H, exchangeable with D2O), 8.4 (s, 1H).
Anal. (C23H20Cl2N4O3) C, H, N.
4-N-Ben zyla m in o-5-p h en yl-7-(5-d eoxy-â-D-r ibofu r a n o-
syl)p yr r olo[2,3-d ]p yr im id in e (5c). Compound 4b was re-
acted with phenylboronic acid by the procedure described for
5a to give 5c as crystalline solid in 63% yield: mp 139-141
1
°C; H NMR (DMSO-d6) δ 1.25 (d, 3H), 3.93 (m, 2H), 4.5 (m,
1H), 4.71 (d, 2H), 5.1 (d, 1H, exchangeable with D2O), 5.4 (d,
1H, exchangeable with D2O), 6.07 (d, J ) 6.7 Hz, 1H, 1′-CH),
5.95 (t, 1H, exchangeable with D2O), 6.12 (d, 1H, J ) 6.3 Hz,
1′-CH), 7.2-7.6 (m, 11H), 8.22 (s, 1H). Anal. (C24H24N4O3) C,
H, N.
4-N-(4-Cya n op h en yl)a m in o-5-p h en yl-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5l). Compound 4h
was reacted with phenylboronic acid by the procedure de-
scribed for 5a to give 5l as a crystalline solid in 52% yield:
4-N-(4-Meth oxyp h en yl)a m in o-5-p h en yl-7-(5-d eoxy-â-D-
r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5e). Compound
4e was reacted with phenylboronic acid by the procedure
described for 5a to give 5e as needles in 59% yield:162-165
1
mp 194-196 °C; H NMR (DMSO-d6) δ 1.35 (d, 3H), 4.55 (m,
1
°C; H NMR (DMSO-d6) δ 1.31 (d, 3H), 3.72 (s, 3H), 3.95 (m,
1H), 4.95 (m, 2H), 5.2 (d, 1H, exchangeable with D2O), 5.4 (d,
1H), 4.49 (m, 2H), 5.12 (d, 1H, exchangeable with D2O), 5.4
1H, exchangeable with D2O), 6.2 (d, J ) 6.1 Hz, 1H, 1′-CH),

2902 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ugarkar et al.
7.4-7.9 (m, 9H), 8.3 (br s, 1H, exchangeable with D2O), 8.51
4-N-(4-F lu or op h en yl)a m in o-5-p h en ylp yr r olo[2,3-d ]p y-
r im id in e (10c). Condensation of 4-fluoroaniline with 8a by
the procedure described for 10a gave 10c as a light pink solid
in 66% yield: mp 231-233 °C; 1H NMR (DMSO-d6) δ 7.0-7.7
(m, 11H), 8.35 (s, 1H), 12.1 (br s, 1H, exchangeable with D2O);
13C NMR (DMSO-d6) δ 102.62, 116.55, 116.94, 117.00, 123.19,
123.29, 123.34, 128.48, 130.31, 130.50, 135.52, 137.51, 137.58,
152.545, 153.18, 155.16, 158.90, 161.65.
4-N-(4-Meth ylp h en yl)a m in o-5-p h en ylp yr r olo[2,3-d ]p y-
r im id in e (10d ). Condensation of p-toluidine with 8a by the
procedure described for 10a gave 10d as an off-white solid in
68% yield: mp 180-182 °C; 1H NMR (DMSO-d6) δ 2.2 (s, 3H),
7.35 (m, 11H), 8.33 (s, 1H), 11.2 (br s, 1H, exchangeable with
D2O); 13C NMR (DMSO-d6) δ 24.82, 101.10, 113.10, 117.84,
120.73, 121.57, 126.08, 127.67, 128.15, 134.26, 138.60, 148.99,
150.55, 154.49.
(s, 1H). Anal. (C24H21N5O3) C, H, N.
4-N-(4-Cya n op h en yl)a m in o-5-(4-m eth oxyp h en yl)-7-(5-
d eoxy-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5m ).
Compound 4h was reacted with 4-methoxyphenylboronic acid
by the procedure described for 5a to give 5m as a crystalline
solid in 50% yield: mp 207-210 °C; 1H NMR (DMSO-d6) δ
1.35 (d, 3H), 3.85 (s, 3H), 4.55 (m, 1H), 4.95 (m, 2H), 5.18 (d,
1H, exchangeable with D2O), 5.4 (d, 1H, exchangeable with
D2O), 6.2 (d, J ) 5.9 Hz, 1H, 1′-CH), 7.1-7.8 (m, 9H), 8.2 (br
s, 1H, exchangeable with D2O), 8.5 (s, 1H). Anal. (C25H23N5O4)
C, H, N.
4-N-P h en ylam in o-5-(2-fu r an yl)-7-(5-deoxy-â-D-r ibofu r a-
n osyl)p yr r olo[2,3-d ]p yr im id in e (5n ). Compound 4a was
reacted with furan-2-boronic acid by the procedure described
for 5a to give 5n as needles in 53% yield: mp 214-216 °C; 1H
NMR (DMSO-d6) δ 1.32 (d, 3H), 3.95(m, 2H), 4.55 (m, 1H),
5.18 (d, 1H, exchangeable with D2O), 5.42 (d, 1H, exchangeable
with D2O), 6.19 (d, J ) 6.0 Hz, 1H, 1′-CH), 6.8 (dd, 1H), 7.46
(m, 5H), 8.0 (s, 1H), 8.05 (2d, 2H), 8.41 (s, 1H), 9.0 (s, 1H,
exchangeable with D2O). Anal. (C21H20N4O4) C, H, N.
4-N-(4-Meth oxyp h en yl)a m in o-5-(2-fu r a n yl)-7-(5-d eoxy-
â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5o). Com-
pound 4e was reacted with furan-2boronic acid by the proce-
dure described for 5a to give 5o as a crystalline solid in 59%
yield: mp 222-224 °C; 1H NMR (DMSO-d6) δ 1.34 (d, 3H),
3.79 (s, 3H), 3.97 (m, 1H), 4.47 (m, 2H), 5.18 (d, 1H, exchange-
able with D2O), 5.42 (d, 1H, exchangeable with D2O), 6.15 (d,
J ) 5.7 Hz, 1H, 1′-CH), 6.7 (dd, 1H), 6.95 (d, 1H), 8.0 (d, 1H),
7.4 (m, 5H), 8.35 (s, 1H), 8.8 (s, 1H, exchangeable with D2O).
Anal. (C22H22N4O5) C, H, N.
2-Meth yl-4-N-p h en yla m in o-5-p h en ylp yr r olo[2,3-d ]p y-
r im id in e (10e). A mixture of 2-amino-4-phenylpyrrole-3-
carbonitrile (9.15 g, 0.05 mol), triethyl orthoacetate (25 mL,
135 mmol) and p-toluenesulfonic acid (75 mg) in dry THF (100
mL) was heated to reflux for 30 min. Volatiles were removed
under reduced pressure and the residue kept under high
vacuum to give 2-ethoxymethylmethyleneimino-4-phenylpyr-
role-3-carbonitrile (8b) as a dark solid: 1H NMR (DMSO-d6)
δ 1.32 (t, 3H), 4.26 (q, 2H), 7.06 (d, 1H), 7.2-7.7 (m, 6H), 11.43
(br.d, 1H, exchangeable with D2O). This intermediate was
condensed with aniline (7.5 g, 0.16 mol) by the procedure
described for 10a to give 10e as an off-white solid (6.5 g,
1
43%): mp >240 °C; H NMR (DMSO-d6) δ 2.54 (s, 3H), 6.9-
7.7 (m, 11H), 11.89 (br.s, 1H, exchangeable with D2O); 13C
NMR (DMSO-d6) δ 24.52, 97.82, 113.98, 118.05, 119.73, 120.
95, 125.88, 127.62, 127.67, 128.00, 134.06, 138.76, 151.43,
152.25, 158.47.
4-N-(3-P yr idyl)am in o-5-ph en yl-7-(5-deoxy-â-D-r ibofu r a-
n osyl)p yr r olo[2,3-d ]p yr im id in e (5p ). Compound 3j was
reacted with phenylboronic acid by the procedure described
for 5a to give 4-N-(3-pyridyl)amino-5-phenyl-7-(2,3-O-isopro-
pylidene-5-deoxy-â-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine in-
termediate as a glassy solid in 88% yield: 1H NMR (DMSO-
d6) δ 1.3 (d, 3H), 1.35 and 1.58 (2s, 6H), 4.31 (m, 1H), 4.78 (m,
1H), 5.40 (m, 1H), 6.31 (d, J ) 3.4 Hz, 1H, 1′-CH), 8.0 (m, 11H).
This material was subjected to TFA catalyzed deprotection to
give 5p as crystalline microplates in 70% yield: mp 211-212
6-Br om o-4-N-p h en yla m in o-5-p h en ylp yr r olo[2,3-d ]p y-
r im id in e (10f). To a stirred solution of 10a (10.0 g, 35 mmol)
in dry DMF (200 mL) was added N-bromosuccinimide (6.7 g,
41 mmol) in portions over 10 min. After 30 min the solid was
collected by filtration and washed with DMF (10 mL). The
combined filtrate and washings were evaporated under re-
duced pressure, and the residue stirred with water (50 mL) to
give a second crop, which was collected by filtration and
washed with water. The two crops were combined, stirred in
hot ethanol (75 mL) for 10 min and cooled to room tempera-
ture. The resulting pale yellow solid was collected by filtration,
washed with cold ethanol and dried under vacuum to give 10f
as an off-white solid (12.27 g, 96%): mp 245-249 °C; 1H NMR
(DMSO-d6) δ 6.9-7.7 (m, 10H) 8.33 (s, 1H), 12.9 (br s, 1H,
exchangeable with D2O); 13C NMR (DMSO-d6) δ 102.58,
107.03, 113.93, 119.41, 122.64, 128.30, 128.88, 129.15, 120.37,
132.79, 139.16, 140.90, 151.38, 152.38.
1
°C; H NMR (DMSO-d6) δ 1.3 (d, 3H), 3.95 (m, 1H), 4.52 (m,
2H), 5.12 (d, 1H, exchangeable with D2O), 5.4 (d, 1H, exchange-
able with D2O), 6.2 (d, J ) 5.9 Hz, 1H, 1′-CH), 7.25-8.7 (m,
11H). Anal. (C22H21N5O5‚0.25H2O) C, H, N.
4-N-P h en yla m in o-5-p h en ylp yr r olo[2,3-d ]p yr im id in e
(10a ). A mixture of 2-amino-4-phenylpyrrole-3-carbonitrile
(18.3 g, 0.1 mol),7 triethyl orthoformate (50 mL, 0.3 mol) and
p-toluenesulfonic acid (75 mg) in dry THF (100 mL) was heated
to reflux for 30 min. The solvent was evaporated under reduced
pressure and the residue kept under high vacuum to give
2-ethoxymethyleneimino-4-phenylpyrrole-3-carbonitrile (8a )6
as a dark solid: 1H NMR (DMSO-d6) δ 1.25-1.4 (t, 3H), 4.3
(q, 2H), 7.37 (m, 6H), 8.38 (s, 1H), 11.8 (br d, 1H, exchangeable
with D2O). A mixture of this intermediate and aniline (15 g,
0.16 mol) was dissolved in dry DMF (100 mL) and refluxed
for 45 min. To the reaction mixture water (150 mL) was added
carefully through the condenser and refluxed for 8 h. Upon
cooling, a solid formed in the reaction mixture which was
collected by filtration, washed with water and dried in air. The
crude material was decolorized and crystallized from ethyl
acetate to give 10a as needles (18.5 g, 65%): mp 240-243 °C;
1H NMR (DMSO-d6) δ 7.3 (m, 12H), 8.36 (s, 1H), 11.5 (br s,
1H, exchangeable with D2O); 13C NMR (DMSO-d6) δ 100.10,
114.15, 118.44, 120.58, 121.23, 125.97, 127.67, 128.00, 133.86,
138.50, 149.96, 150.46, 152.49.
5-Deoxy-2,3-O-isop r op ylid en e-r-D-r ibofu r a n osyl Ch lo-
r id e (12a ). A 100-mL three-neck flask was charged with a
solution of 5-deoxy-2,3-O-isopropylidene-D-ribofuranose (11a 1;
5.3 g, 30.5 mmol), carbon tetrachloride (3.4 mL, 35.3 mmol)
and toluene (60 mL). The contents were cooled to ca. -15 °C
in a dry ice acetone bath. A 85% solution of hexamethylphos-
phorus triamide (6.6 mL, 34.7 mmol) was added dropwise
maintaining the internal temperature between -10 and -5
°C over a period of 20 min. After stirring the reaction mixture
for an additional 20 min at ca. -5 °C, the pale yellow solution
was transferred to a 125 mL separatory funnel, washed with
ice-cold brine (100 mL) and the organic layer was dried
(MgSO4). A small aliquot was pulled out and evaporated to
1
make a sample for H NMR spectrum: 1H NMR (CDCl3) δ 1.29
(d, 3H), 1.31 and 1.65 (2s, 6H), 4.37 (m, 1H), 4.80 (m, 2H) and
6.15 (d, J ) 3.8 Hz, 1H, 1-CH). The presence of the â-chloro
isomer was determined by the singlet at 6.1 ppm which is
consistent with our earlier observation for the â-chloro sugar.1
The approximate ratio of the R/â-chloro sugars was determined
by comparison of signal integrations for the anomeric protons
and found to be 95:5. This material was used immediately in
the glycosylation experiment.
4-N-(4-Ch lor op h en yl)a m in o-5-p h en ylp yr r olo[2,3-d ]p y-
r im id in e (10b). Condensation of 4-chloroaniline with 8a by
the procedure described for 10a gave 10b as off-white solid in
1
62% yield: mp 248-250 °C; H NMR (DMSO-d6) δ 7.0-7.75
(m, 11H), 8.38 (s, 1H), 11.5 (br s, 1H, exchangeable with D2O);
13C NMR (DMSO-d6) δ 101.82, 117.05, 117.44, 117.80, 123.33,
123.45, 123.58, 128.55, 129.61, 129.89, 135.44, 137.68, 138.01,
151.14, 152.38, 156.06, 159.10, 162.35.
5-O-ter t-Bu tyld im eth ylsilyl-2,3-O-isop r op ylid en e-r-D-
r ibofu r a n osyl Ch lor id e (12b). Chlorination of 5-O-tert-

Adenosine Kinase Inhibitors. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2903
butyldimethylsilyl-2,3-O-isopropylidene-R-D-ribofuranose (11b)
1′-CH), 7.25 (m, 12H), 8.52 (s, 1H). This intermediate was
subjected to TFA-catalyzed deprotection followed by crystal-
lization from boiling ethanol to give 5h in 58% yield which
was identical by TLC, melting point, and 1H NMR to the one
obtained from 4h via Suzuki reaction.
1
was carried out by the procedure described for 12a . H NMR
of this material was identical to the one obtained by Wilcox’s
procedure.18 The presence of â-chloro isomer was confirmed
by the singlet at 6.15 ppm, which is consistent with our earlier
observation.1 The approximate ratio of the R/â-chloro sugars
was determined by comparison of signal integrations for the
anomeric protons and found to be 85:15. This mixture of chloro
sugars was used immediately in the following glycosylation
experiment.
4-N-P h en yla m in o-5-p h en yl-7-(5-d eoxy-2,3-O-isop r op yl-
id en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (13a ).
Glycosyla tion of 10a by th e Sod iu m Sa lt Meth od . To an
ice-cold solution of 10a (2.86 g, 10 mmol) in dry acetonitrile
(50 mL) was added NaH (80% dispersion in oil, 0.33 g, 11
mmol) in small portions, and stirred for 30 min. A solution of
12a (prepared from 6.1 g of 11a , 20 mmol) prepared by the
original procedure1 was cannulated into the reaction mixture
and stirred overnight at room temperature. The reaction was
concentrated under reduced pressure to give a residue, which
was stirred with ethyl acetate (50 mL) and filtered. The filtrate
was evaporated, and the crude product was purified by
chromatography on silica gel (25% ethyl acetate in hexanes)
to provide 13a as a glassy product (1.5 g, 35%): 1H NMR
(DMSO-d6) δ 1.28 (d, 3H), 1.3 and 1.6 (2s, 6H), 4.22 (m, 1H),
4.8 (m, 1H), 5.4 (m, 1H), 6.3 (d, J ) 2.9 Hz, 1H, 1′-CH), 7.42
(m, 12H), 8.4 (s, 1H); UV (methanol) nm-1 λmax 290 (ꢀ 18300),
λmin 258 (ꢀ 2300).
4-N-(4-Ch lor op h en yl)a m in o-5-p h en yl-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5q). Glycosylation
of 10b with 12a by the TDA-1 method gave 13b in 63% yield
as a glassy solid: 1H NMR (CDCl3) δ 1.4 (d, 3H), 1.38 and 1.6
(2s, 6H), 4.28 (m, 1H), 4.65 (m, 1H), 5.3 (m, 1H), 6.25 (d, J )
3.4 Hz, 1H, 1′-CH), 6.9-7.55 (m, 12H), 8.5 (s, 1H). TFA-
catalyzed deprotection of this intermediate gave a semisolid
that was crystallized from boiling ethanol to furnish 5q as a
crystalline solid in 68% yield: mp 176-178 °C; 1H NMR
(DMSO-d6) δ 1.35 (d, 3H), 4.25 (m, 3H), 5.17 (d, 1H, exchange-
able with D2O), 5.4 (d, 1H, exchangeable with D2O), 6.2 (d, J
) 5.7 Hz, 1H, 1′-CH), 7.25-7.8 (m, 10H), 8.41 (s, 1H). Anal.
(C23H21ClN4O3) C, H, N.
4-N-(4-Met h ylp h en yl)a m in o-5-p h en yl-7-(5-d eoxy-â-D-
r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5r ). Glycosyla-
tion of 10d with 12a by the TDA-1 method gave 13d in 68%
yield as a glassy solid: 1H NMR (DMSO-d6) δ 1.3 (d, 3H), 1.33
and 1.56 (2s, 6H), 2.26 (s, 3H), 4.2 (m, 1H), 4.75 (m, 1H), 5.40
(m, 1H), 6.29 (d, J ) 3.0 Hz, 1H, 1′-CH), 7.35 (m, 11H), 8.41
(s, 1H). This intermediate was subjected to TFA-catalyzed
deprotection and the resulting crude product crystallized from
ethanol to give 5r as needles in 68% yield: mp 180-182 °C;
1H NMR (DMSO-d6) δ 1.31 (d, 3H), 2.29 (s, 3H), 3.91 (m, 1H),
4.55 (m, 2H), 5.15 (d, 1H, exchangeable with D2O), 5.4 (d, 1H,
exchangeable with D2O), 6.17 (d, J ) 5.8 Hz, 1H, 1′-CH), 7.35
(m, 11H), 8.4 (s, 1H). Anal. (C24H24N4O3) C, H, N.
2-Meth yl-4-N-p h en yla m in o-5-p h en yl-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5s). Glycosylation
of 10e with 12a by the TDA-1 method gave 13e as a glassy
solid in 58% yield: 1H NMR (CDCl3) δ 1.29 (d, 3H), 1.38 and
1.68 (2s, 6H), 2.56 (s, 3H,), 4.2 (m, 1H), 4.75 (m, 1H), 5.40 (m,
1H), 6.29 (d, J ) 3.0 Hz, 1H, 1′-CH), 6.9-7.7 (m, 11H). This
intermediate was subjected to deprotection by the procedure
described for 4a and the resulting crude product crystallized
from ethanol to give 5s as needles in 58% yield: 194-196 °C;
1H NMR (DMSO-d6) δ 1.32 (d, 3H), 2.57 (s, 3H), 3.94 (m, 2H),
4.53 (m, 1H), 5.15 (d, 1H, exchangeable with D2O), 5.37 (d,
1H, exchangeable with D2O), 6.19 (d, J ) 5.4 Hz, 1H, 1′-CH),
7.25 (m, 12H). Anal. (C24H24N4O3) C, H, N.
Further elution of the column provided 14 (850 mg, 20%)
which was isolated as a yellow glassy solid: 1H NMR (DMSO-
d6) δ 1.26 (d, 3H), 1.32 (s, 3H), 1.54 (s, 3H), 4.32 (m, 1H), 4.90
(m, 1H), 5.55 (m, 1H), 6.41 (s, 1H, J ) 5.7 Hz 1′-CH), 7.4 (m,
12H), 8.66 (s, 1H); UV (methanol) nm-1 λmax 304 (ꢀ 17600),
λmin 275 (ꢀ 2100).
Continued elution provided 15 (635, 15%) as a glassy solid:
1H NMR (DMSO-d6) δ 1.28 (d, 3H), 1.15 (s, 3H), 1.25 (s, 3H),
4.55 (m, 1H), 4.76 (m, 1H), 6.70 (d, J ) 7.4 Hz, 1H), 7.42 (m,
12H), 8.4 (s, 1H); UV (methanol) nm-1
λmax 292 (ꢀ 18500), λmin
256 (ꢀ 1900).
4-N-P h en yla m in o-5-p h en yl-7-(5-d eoxy-2,3-O-isop r op yl-
id en e-â-D-r ibofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (13a ).
Glycosylation of 10a by P h ase-Tr an sfer Catalysis Meth od
(TDA-1 Meth od ). To a well-stirred mixture of 10a (2.86 g,
10 mmol), powdered KOH (1.2 g, 20 mmol) and TDA-1 (3.3
mL, 10 mmol) in toluene (40 mL) was added the chloro sugar
12a (prepared from 11a , 3.5 g, 20 mmol). After stirring the
reaction mixture at room temperature overnight, the dark
solution was transferred to a separatory funnel and washed
successively with water, 0.5 N HCl solution (10 mL) and again
with water. The organic layer was separated and concentrated
under reduced pressure and the residue was purified by
chromatography on a silica gel column (20% ethyl acetate in
hexane) to provide 13a as a glassy solid (2.9 g, 65%). Further
elution provided 14 (320 mg, 6.7%).
6-Br om o-4-N-p h en yla m in o-5-p h en yl-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5t). The hetero-
cycle 10f was coupled to 12a by the TDA-1 method gave 13f
in 62% yield as a glassy solid: 1H NMR (CDCl3) δ 1.32 (d, 3H),
1.35 and 1.57 (2s, 6H), 4.20 (m, 1H), 4.91 (m, 1H), 5.71 (m,
1H), 6.3 (d, J ) 2.5 Hz, 1H, 1′-CH), 7.35 (m, 12H), 8.48 (s,
1H). This intermediate was subjected to deprotection by the
procedure described for 4a to give 5t as a crystalline solid in
72% yield: mp 168-169 °C; 1H NMR (DMSO-d6) δ 1.34 (d,
3H), 3.9 (m, 1H), 4.21 (m, 1H), 5.1-5.4 (m, 3H), 6.06 (d, J )
4.48 Hz, 1H, 1′-CH), 7.3 (m, 12H), 8.45 (s, 1H). Anal. (C23H21
-
4-N-P h en yla m in o-5-p h en yl-7-(5-d eoxy-â-D-r ibofu r a n o-
syl)p yr r olo[2,3-d ]p yr im id in e (5b). Deprotection of 13a by
the procedure described for 4a gave 5b which was identical
BrN4O3) C, H, N.
4-N-P h en yla m in o-5-p h en yl-7-(5-a zid o-5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5u ). Glycosylation
of 10a using 5-azido-5-deoxy-2,3-O-isopropylidene-R-D-ribo-
furanosyl chloride, 12c,1 by the TDA-1 method gave 13h in
58% yield as a glassy solid: 1H NMR (DMSO-d6) δ 1.33 and
1.55 (2s, 6H), 3.67 (d, 2H), 4.27 (m, 1H), 5.00 (m, 1H), 5.40
(m, 1H), 6.29 (d, J ) 2.8 Hz, 1H, 1′-CH), 7.05-7.9 (m, 11H),
8.31 (s, 1H, exchangeable with D2O), 8.38 (s, 1H). This material
was deprotected by the procedure described for 4a to give 5u
as needles in 58% yield: mp 108-109 °C dec;1H NMR (DMSO-
d6) δ 3.65 (m, 2H), 4.10 (m, 1H), 4.57 (m, 2H), 5.35 (d, 1H,
exchangeable with D2O), 5.6 (d, 1H, exchangeable with D2O),
6.29 (d, J ) 5.86 Hz, 1H, 1′-CH), 7.35 (m, 12H), 8.42 (s, 1H).
Anal. (C23H21N7O3) C, H, N.
1
by TLC, melting point, and H NMR to the one prepared from
4a via Suzuki reaction.
4-N-(4-F lu or op h en yl)a m in o-5-p h en yl-7-(5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (5d ). Glycosylation
of 10c with 12a by the TDA-1 method gave 13c in 66% yield
1
as a glassy solid: H NMR (DMSO-d6) δ 1.28 (d, 3H), 1.3 and
1.6 (2s, 6H), 4.21 (m, 1H), 4.8 (m, 1H), 5.4 (m, 1H), 6.3 (d, J )
3.2 Hz, 1H), 7.43 (m, 11H), 8.4 (s, 1H). TFA-catalyzed depro-
tection of 13c by the procedure described for 4a followed by
crystallization from boiling ethanol gave 5d in 68% yield which
was identical by TLC, melting point, and 1H NMR to the one
obtained from 4c via Suzuki reaction.
4-N-P h en ylam in o-5-ph en yl-7-â-D-r ibofu r an osylpyr r olo-
[2,3-d ]p yr im id in e (5h ). Glycosylation of 10a with 12b using
the TDA-1 method gave 13g in 62% yield as a foam: 1H NMR
(CDCl3) δ 0.55 (m, 15H), 1.4 and 1.66 (2s, 6H), 3.85 (m, 2H),
4.35 (m, 1H), 5.0 (m, 1H), 5.2 (m, 1H), 6.43 (d, J ) 3.1 Hz, 1H,
4-N-P h en yla m in o-5-p h en yl-7-(5-a m in o-5-d eoxy-â-D-r i-
bofu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (16). A solution of
5u (24 mg, 0.05 mmol) in ethanol (5 mL) was purged with N2,
treated with 10% Pd on C (25 mg) and hydrogenated under 1

2904 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ugarkar et al.
atm of H2. The catalyst was removed by filtration through a
Celite pad and the pad was washed with ethanol. The
combined filtrate and washings were filtered through a 0.45-
µm membrane. The filtrate was evaporated to dryness and the
residue dried under high vacuum at 60 °C to give 16 as an
amorphous solid (18 mg, 80%): mp 150-153 °C; 1H NMR
(DMSO-d6) δ 2.9-3.1 (m, 2H), 3.1-3.8 (br s, 2H, exchangeable
with D2O), 3.9-4.22 and 4.5-4.65 (3m, 3H), 5.1-5.6 (2 br s,
2H, exchangeable with D2O), 6.2 (d, 1H, J ) 6.1 Hz, 1′-CH),
6.95-7.7 (m, 12H), 8.42 (s, 1H). Anal. (C23H23N5O3‚1.2H2O)
C, H, N.
under 50 psi of H2 on a Parr apparatus. After 72 h the catalyst
was removed by filtration and washed with methanol. The
combined filtrate and washings were evaporated and the
residue was purified by chromatography over silica gel (25%
ethyl acetate in hexanes) to give 4-N-phenylamino-5-phenyl-
7-(3-O-acetoxy-2,5-dideoxy-â-D-ribofuranosyl)pyrrolo[2,3-d]py-
rimidine as a glassy solid (55 mg, 75%). This material was
subjected to deprotection according to the procedure described
for 18 to give 22 as off-white needles from aqueous ethanol
1
(34 mg, 68%): mp 91-92 °C; H NMR (DMSO-d6) δ 1.25 (d,
3H), 2.25 and 2.7 (2m, 2H), 3.9 (m, 1H), 4.16 (m, 1H), 5.32 (d,
1H, exchangeable with D2O), 6.63 (dd, J 1 ) 5.5 Hz and J 2
)
4-N-P h en ylam in o-5-ph en yl-7-(2-deoxy-â-D-er yth r open to-
fu r a n osyl)p yr r olo[2,3-d ]p yr im id in e (19). Sodium hydride
(300 mg, 7.0 mmoL, 60% dispersion in mineral oil) was rinsed
with hexane under N2 atmosphere. Dry acetonitrile (70 mL)
was introduced into the flask and the flask was immersed in
an ice bath. Powdered 10a (1.75 g, 6.1 mmol) was added in
three equal portions with constant stirring. After the addition
was complete, the cooling bath was removed and stirring was
continued until the evolution of H2 gas ceased. A solution of
2-deoxy-3,5-di-O-toluoyl-R-D-erythropentofuranosyl chloride12
(17; 2.37 g, 6.1 mmol) in acetonitrile (10 mL) was added over
a 5 min period. TLC (silica gel, 2:1 hexanes-EtOAc) after 1.5
h indicated complete consumption of the chloro sugar. The
reaction mixture was filtered to remove the unreacted 10a .The
filtrate was evaporated, and the crude product was purified
by chromatography over silica gel (25% EtOAc in hexanes) to
give pure 4-N-phenylamino-5-phenyl-7-(3,5-di-O-toluoyl-2-
deoxy-â-D-erythropentofuranosyl)pyrrolo[2,3-d]pyrimidine (18)
as a glassy solid (2.55 g, 65%): 1H NMR (DMSO-d6) δ 2.35
and 2.45 (2s, 6H), 2.77 and 3.13 (2m, 2H), 4.6 (m, 3H), 5.8 (m,
5.8 Hz, 1H, 1′-CH), 6.95-7.7 (m, 12H), 8.49 (s, 1H). Anal.
(C23H22N4O2‚0.6H2O) C, H, N.
4-N-P h en ylam in o-5-ph en yl-7-(3,5-dideoxy-â-D-r ibofu r a-
n osyl)p yr r olo[2,3-d ]p yr im id in e (23). By the procedure
described for 22, 21 was subjected to hydrogenation and the
resulting intermediate 4-N-phenylamino-5-phenyl-7-(2-O-
acetyl-3,5-dideoxy-â-D-ribofuranosyl)pyrrolo[2,3-d]pyrimi-
dine was subjected to base-catalyzed deprotection to give 23
as needles from aqueous ethanol: yield 57%; mp 80-83 °C;
1H NMR (DMSO-d6) δ 1.30 (d, 3H), 2.03 (m, 2H), 4.36 (m, 1H),
4.53 (m, 1H), 5.60 (d, 1H, exchangeable with D2O), 6.18 (d,
1H, J ) 2.2 Hz, 1′-CH), 6.95-7.7 (m, 12H), 8.39 (s, 1H). Anal.
(C23H22N4O2) C, H, N.
En zym e Assa y. AK activity was measured in a radio-
chemical assay similar to the procedure of Yamada et al.,23
with minor modifications as described previously.1 The results
are shown in Table 1.
MES Seizu r e Assa y. Rat MES seizure activity was deter-
mined by the procedure described previously,1 and the results
are shown in Table 2.
1H), 6.83 (dd, J ) 6.01 Hz and J 2 ) 8.25 Hz, 1H, 1′-CH),
1
Hem od yn a m ic Stu d ies. The hemodynamic effects of 5l,m
6.95-8.05 (m, 19H), 8.4 (s, 1H). This material was dissolved
in methanol (70 mL) to which a freshly prepared solution of
sodium methoxide (4 mL, 2 M solution in methanol) was
added. The mixture was stirred at room temperature for 1 h
before glacial acetic acid was added gradually to adjust the
pH to ∼4. The off-white solid formed was collected by filtration,
washed with small volumes of methanol (2 × 5 mL) and
crystallized from boiling ethanol to give 19 as a crystalline
solid (1.28 g, 79%): mp 89-96 °C; 1H NMR (DMSO-d6) δ 2.25
and 2.57 (2m, 2H), 3.55 (m, 3H), 4.37 (m, 1H), 5.01 (t, 1H,
exchangeable with D2O), 5.29 (d, 1H, exchangeable with D2O),
6.65 (dd, J 1 ) 7.1 Hz and J 2 ) 9.35 Hz, 1H, 1′-CH), 6.95-7.7
(m, 12H), 8.38 (s, 1H). Anal. (C23H22N4O3) C, H, N.
were determined by the protocol described earlier.18
Ack n ow led gm en t. The authors acknowledge Dr.
Paul VanPoelje, Dr. David Dumas, and Patrick McCur-
ley for their help in setting up AK inhibition assays.
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