Identification of Adenine and Benzimidazole Nucleosides as Potent Human Concentrative Nucleoside Transporter 2 Inhibitors: Potential Treatment for Hyperuricemia and Gout

Article abstract of DOI:10.1021/acs.jmedchem.5b01884

To test the hypothesis that inhibitors of human concentrative nucleoside transporter 2 (hCNT2) suppress increases in serum urate levels derived from dietary purines, we previously identified adenosine derivative 1 as a potent hCNT2 inhibitor (IC₅₀ = 0.64 μM), but further study was hampered due to its poor solubility. Here we describe the results of subsequent research to identify more soluble and more potent hCNT2 inhibitors, leading to the discovery of the benzimidazole nucleoside 22, which is the most potent hCNT2 inhibitor (IC₅₀ = 0.062 μM) reported to date. Compound 22 significantly suppressed the increase in plasma uric acid levels after oral administration of purine nucleosides in rats. Because compound 22 was poorly absorbed orally in rats (F = 0.51%), its pharmacologic action was mostly limited to the gastrointestinal tract. These findings suggest that inhibition of hCNT2 in the gastrointestinal tract can be a promising approach for the treatment of hyperuricemia.

Full text of DOI:10.1021/acs.jmedchem.5b01884

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Article
Identification of Adenine and Benzimidazole Nucleosides
as Potent Human Concentrative Nucleoside Transporter 2
Inhibitors: Potential Treatment for Hyperuricemia and Gout
Kazuya Tatani, Masahiro Hiratochi, Norihiko Kikuchi, Yu Kuramochi, Shinjiro
Watanabe, Yuji Yamauchi, Fumiaki Itoh, Masayuki Isaji, and Satoshi Shuto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01884 • Publication Date (Web): 23 Mar 2016
Downloaded from http://pubs.acs.org on March 24, 2016
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Identification of Adenine and Benzimidazole
Nucleosides as Potent Human Concentrative
Nucleoside Transporter 2 Inhibitors: Potential
Treatment for Hyperuricemia and Gout
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Kazuya Tatani,*,†,‡ Masahiro Hiratochi,† Norihiko Kikuchi,†,# Yu Kuramochi,†,ǁ Shinjiro
Watanabe,† Yuji Yamauchi,† Fumiaki Itoh,† Masayuki Isaji,† and Satoshi Shuto‡,§
†Central Research Laboratories, Kissei Pharmaceutical Co., Ltd., 4365ꢀ1, Hotakakashiwabara,
Azumino, Nagano 399ꢀ8304, Japan
‡Faculty of Pharmaceutical Sciences and §Center for Research and Education on Drug
Discovery, Hokkaido University, Kitaꢀ12, Nishiꢀ6, KitaꢀKu, Sapporo 060ꢀ0812, Japan
Abstract
To test the hypothesis that inhibitors of human concentrative nucleoside transporter 2 (hCNT2)
suppress increases in serum urate levels derived from dietary purines, we previously identified
adenosine derivative 1 as a potent hCNT2 inhibitor (IC50 = 0.64 ꢁM), but further study was
hampered due to its poor solubility. Here we describe the results of subsequent research to
identify more soluble and more potent hCNT2 inhibitors, leading to the discovery of the
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benzimidazole nucleoside 22, which is the most potent hCNT2 inhibitor (IC50 = 0.062 µM)
reported to date. Compound 22 significantly suppressed the increase in plasma uric acid levels
after oral administration of purine nucleosides in rats. Because compound 22 was poorly
absorbed orally in rats (F = 0.51%), its pharmacologic action was mostly limited to the
gastrointestinal tract. These findings suggest that inhibition of hCNT2 in the gastrointestinal tract
can be a promising approach for the treatment of hyperuricemia.
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Introduction
Uric acid is the end product of purine metabolism in human beings because a gene encoding
uricase has undergone mutational silencing during hominoid evolution.1ꢀ3 Uricase is an enzyme
involved in the degradation of uric acid to the more soluble allantoin, and is active in a wide
range of organisms, from microorganism to mammals.4ꢀ8 The loss of uricase activity has led to
higher uric acid levels in human beings compared with most other mammals.8,9 Uric acid is a
weak acid with a pKa of 5.75 and is present predominantly in the form of urate at a physiologic
pH of 7.4.3,10,11 In extracellular fluid, uric acid exists mainly as a solution of monosodium urate,
which is a poorly soluble excretion product unlike allantoin, and thus has the potential to cause
characteristic problems in human beings.3,10
Hyperuricemia is a condition in which serum urate levels are abnormally high. The definition
of hyperuricemia, though currently arbitrary, is a serum urate concentration in excess of the
solubility limit at physiologic temperature and pH (~ 6.8 mg/dL).11ꢀ16 Persistent hyperuricemia is
widely accepted as the primary risk factor for urate deposition diseases, such as gouty arthritis,
gouty tophi, and renal damage.15,16 In addition, a number of reports suggest an association
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between hyperuricemia and pathologic conditions other than urate deposition disease.17ꢀ22 For
example, recent epidemiologic studies demonstrated that serum urate levels positively correlate
with the frequency of metabolic syndrome23ꢀ28 and the risk for developing hypertension.29ꢀ35
Therefore, treatment of hyperuricemia has become increasingly important.
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Serum urate levels are controlled through the balance among dietary purines, purine
catabolism, and urate excretion.36,37 Therefore, hyperuricemia can result from the excessive
intake of purines, the increase in purine catabolism, the decrease in urate excretion, or
combinations of them. Currently, two types of therapeutic drugs are available for the treatment of
hyperuricemia.36 Xanthine oxidase inhibitors and uricosuric agents reduce serum urate levels by
blocking the biosynthesis and the tubular reabsorption of urate, respectively. However, no
therapeutic drug focused on the third causative factor, dietary purine, has yet been developed.
Patients with hyperuricemia are advised to refrain from habitual intake of a purineꢀrich diet.
Hyperuricemia can be evoked in healthy subjects by ingesting yeast nucleic acid for several
days.38ꢀ40 On the other hand, recent epidemiologic studies report that high levels of meat and
seafood consumption are associated with high serum urate levels41 and an increased risk of gouty
arthritis.42 In contrast, a low purine diet lowers serum urate levels in patients with
hyperuricemia.43,44 Together, these findings suggest that orallyꢀingested purines increase serum
urate levels and thus suppressing their gastrointestinal absorption could be a promising approach
for treating hyperuricemia.
While dietary purines comprise various forms, such as DNA, RNA, nucleoproteins,
oligonucleotides, purine nucleotides, purine nucleosides, and purine bases, most of these forms
are unlikely to directly pass through the plasma membranes due to their high molecular weights
and/or highly hydrophilic nature. Thus, carrierꢀmediated transport is probably involved in the
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gastrointestinal absorption of purines. Nucleoside transporter proteins have been extensively
studied as potential carriers.45ꢀ50 Currently, they are roughly classified into two groups in
mammals. One group comprises three concentrative nucleoside transporters, CNT1, CNT2, and
CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively), which mediate unidirectional
sodiumꢀ and/or protonꢀdependent transport.51,52 Another group comprises four equilibrative
nucleoside transporters, ENT1, ENT2, ENT3, and ENT4 (SLC29A1, SLC29A2, SLC29A3, and
SLC29A4, respectively), which mediate bidirectional sodiumꢀ and protonꢀindependent transport
(facilitated diffusion).53 In previous reports, we estimated the likelihood that human
concentrative nucleoside transporter 2 (hCNT2) plays a central role in the gastrointestinal
absorption of purines on the basis of the expression profile and inherent substrate selectivity of
the seven transporters in the gastrointestinal tract:54,55 In human beings, (1) CNT1, CNT2, ENT1,
and ENT2 are recognized as the major players in the enterocyte.56ꢀ58 (2) CNT1 and CNT2 are
located on the apical side, while ENT1 and ENT2 are on the basolateral side.45,48,50,56ꢀ58 (3)
CNT2 is a purine nucleosideꢀpreferring transporter, whereas CNT1 is a practically pyrimidine
nucleosideꢀspecific transporter.50,51 Thus, we hypothesized that hCNT2 inhibitors would
suppress the elevation of serum urate levels derived from dietary purines.54,55 To test this
hypothesis, we searched for potent hCNT2 inhibitors, which led to the discovery of compound 1
(Figure 1).55 Further studies using compound 1 were hampered, however, due to its poor
solubility in aqueous medium. For example, the solubility of compound 1 in the Japanese
Pharmacopoeia second fluid for a dissolution test (JP2, pH 6.8)59 at 37 °C was 0.003 mg/mL,
indicating that 1 is practically insoluble. Hence, more soluble potent hCNT2 inhibitors are
required. In the present paper, we describe structureꢀactivity relationship studies leading to the
discovery of potent hCNT2 inhibitors with increased solubility, and their suppressive effect on
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an increase of plasma uric acid levels derived from orally administered purine nucleosides in
rats.
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Figure 1. A potent hCNT2 inhibitor discovered in our previous study.
Results and Discussion
Improving solubility by manipulating the biphenyl conformation.
To enhance the solubility of compound 1, we focused on its highly hydrophobic biphenyl
moiety, and synthesized various compounds with modifications in this region. All newly
synthesized final products were evaluated primarily for their inhibitory effects on sodiumꢀ
dependent inosine uptake in COSꢀ7 cells transiently expressing hCNT2 following the previously
described procedure.54,55
First, the terminal benzene ring was replaced with a pyridine ring (Table 1). The 3ꢀpyridyl
analog 3 exhibited potent hCNT2 inhibitory activity similar to that of compound 1, whereas the
2ꢀ and 4ꢀpyridyl analogs (compounds 2 and 4) resulted in somewhat reduced activity.
Table 1. Inhibitory Effects of Compounds 2ꢀ4 on hCNT2 and rat CNT2 (rCNT2) Activities
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IC50 (ꢁM)a
compd
R1
hCNT2
0.64 ± 0.19
1.5 ± 0.5
1.0 ± 0.2
2.7 ± 0.9
rCNT2
inactiveb
inactiveb
>100c
6
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phenyl
1
2
3
4
pyridinꢀ2ꢀyl
pyridinꢀ3ꢀyl
pyridinꢀ4ꢀyl
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inactiveb
aConcentration of each compound required to inhibit inosine uptake by 50%. Data are
expressed as the mean ± standard error of the mean (SEM) of at least three independent
experiments unless otherwise stated. bNo inhibition was observed at the maximum concentration
of 100 µM. c11% inhibition was observed at the maximum concentration of 100 µM.
We then introduced a polar functional group on the endoꢀbenzene ring (Table 2). Among the
compounds substituted at the 2ꢀposition, the 2ꢀhydroxy derivative 5 was tolerated without a
significant loss of inhibitory activity. While the inhibitory activity of the 2ꢀmethoxy derivative 6
was comparable to that of 5, introduction of larger alkoxy groups, including ethoxy, propoxy,
and isopropoxy groups, to the 2ꢀposition tended to reduce the potency depending on their
bulkiness (compounds 7ꢀ9). The 3ꢀsubstituted derivatives 10ꢀ14 similarly reduced the potency
depending on the bulkiness of the substituent introduced. Thus, irrespective of the substitution
position, increased lipophilicity of substituents correlated with decreased activity of the
compounds.
Table 2. Inhibitory Effects of Compounds 5ꢀ17 on hCNT2 and rCNT2 Activities
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IC50 (ꢁM)a
rCNT2
compd
R2
R3
hCNT2
7
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H
H
0.64 ± 0.19
1.2 ± 0.6
1.3 ± 0.2
2.3 ± 0.6
5.2 ± 1.5
10.4 ± 1.0
1.3 ± 0.1
1.1 ± 0.1
5.4 ± 1.3
inactiveb
9.3 ± 1.8
3.3 ± 0.8
5.5 ± 1.4
3.1 ± 0.3
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
inactiveb
>100c
1
5
OH
OMe
OEt
OPr
OPrꢀi
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H
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H
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OMe
OEt
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OCH2CO2Me
OCH2CONH2
OCH2CH2OH
H
H
>100d
aConcentration of each compound required to inhibit inosine uptake by 50%. Data represent
b
mean ± SEM of at least three independent experiments unless otherwise stated. No inhibition
was observed at the maximum concentration of 100 µM. cLess than 5% inhibition was observed
d
at the maximum concentration of 100 µM. 49% inhibition was observed at the maximum
concentration of 100 µM.
Although substitution with a small group at both the 2ꢀ and 3ꢀpositions of the endoꢀbenzene
ring did not reduce the activity, we thought that substitution at the 2ꢀposition would probably
improve the solubility more effectively than substitution at the 3ꢀposition because steric
repulsion between the 2ꢀsubstituent and the terminal benzene ring might make the planar
conformation of the biphenyl moiety unstable, and thus the biphenyl moiety would become
stabilized by assuming a twisted conformation to avoid the steric repulsion. Actually, recent
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studies suggested that disruption of molecular planarity is a good strategy for improving the
aqueous solubility of poorly soluble compounds.60,61 Therefore, we estimated the substituent
effects on the conformation of compound 1, especially that of the biphenyl moiety, using its 2ꢀ
and 3ꢀmethyl derivatives (A and B, respectively) as model compounds by performing theoretical
calculations using the density functional theory B3LYP/6ꢀ31G* method in Spartan'14. When
comparing the most stable conformers (Figure 2), the biphenyl moiety of 2ꢀmethylated A was
significantly more twisted (57.4°) than that of 3ꢀmethylated B (36.6°) and unmethylated 1
(37.3°).
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Figure 2. Simulation of substituent effects on the biphenyl structure
If the biphenyl moiety of the compound actually assumes a nonꢀplanar twisted conformation,
its crystallinity would decrease compared with the planar one to improve the solubility. To test
this hypothesis, we measured the solubility of compounds in JP2 at 37 °C using the 2ꢀsubstituted
derivatives 6ꢀ7 and their 3ꢀsubstituted congeners 11ꢀ12 (Table 3). As expected, each of the 2ꢀ
substituted compounds 6 and 7 had significantly improved solubility compared with the parent
compound 1. In contrast, despite introduction of the same substituents, the solubility of 3ꢀ
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substituted compounds 11 and 12 were unchanged compared to the parent compound 1. These
results correlated well with the simulation of the biphenyl conformations shown in Figure 2.
On the basis of the above encouraging results, other substituents at the 2ꢀposition were
investigated. Because alkoxy groups introduced at the 2ꢀposition tended to decrease hCNT2
inhibitory activity depending on their bulkiness and lipophilicity, we examined alkoxy groups
substituted with a polar functional group (Table 2). Although the size of the 2ꢀmethoxyꢀ2ꢀ
oxoethoxy and 2ꢀhydroxyethoxy groups was comparable to or greater than the propoxy group,
compounds 15 and 17 exhibited more potent hCNT2 inhibitory activity than the propoxy
derivative 8, but less potent than the ethoxy derivative 7. In contrast, the 2ꢀaminoꢀ2ꢀoxoethoxy
derivative 16 resulted in inhibitory activity equal to that of 8.
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Table 3. Substituent Effects on the Solubility in JP2
compd R2
R3
solubility (mg/mL)a
H
H
0.003
0.215
0.121
0.004
0.002
1
6
OMe
OEt
H
H
H
7
OMe
OEt
11
12
H
aSolubility in JP2 (pH 6.8) after incubation at 37 °C for 1 h.
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Attempt to improve inhibitory activity against rat CNT2 (rCNT2) in adenosine derivatives.
We next examined the inhibitory activity of the relatively potent hCNT2 inhibitors 3, 5, and 6
against rCNT2.54 Unexpectedly, both compounds 5 and 6 were inactive against rCNT2 up to the
maximum concentration of 100 ꢁM (Table 2), whereas 3 exhibited slight inhibitory activity
(Table 1). These findings might be due to differences in the amino acid sequence between
hCNT2 and rCNT2: hCNT2 is one amino acid shorter than rCNT2 and 83% identical to
rCNT2.57,58 Hence, we also evaluated all other compounds including parent compound 1, and
confirmed that most of these compounds were inactive, and only compounds 16 and 17 had weak
activity (Tables 1 and 2).
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Comparison of the 2ꢀethoxy derivative 7 with its hydroxylated analog 17 revealed that the
terminal hydroxy group is crucial for exerting rCNT2 inhibitory activity. Therefore, we paid
attention to the homologs of compound 17 and synthesized the 3ꢀhydroxypropoxy, 4ꢀ
hydroxybutoxy, and 5ꢀhydroxypentyloxy derivatives 18ꢀ20. As summarized in Table 4,
elongation of the ethylene linker in compound 17 enhanced the inhibitory activity against both
hCNT2 and rCNT2. Because the 4ꢀhydroxybutoxy derivative 19 exhibited the most potent
inhibitory activity against both hCNT2 and rCNT2 among the homologs, we selected this
compound for further evaluation and measured its solubility in JP2 at 37 °C. As a result,
compound 19 showed much higher solubility (0.190 mg/mL) than the parent compound 1 (Table
4), suggesting that the 4ꢀhydroxybutoxy group enhanced the twisted structure of the biphenyl
moiety to increase solubility, as mentioned above.
Table 4. in vitro Profiles of Compounds 17ꢀ20 and Improved Solubility of 19 in JP2
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IC50 (µM)a
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compd
R4
solubility (mg/mL)d
hCNT2
0.64 ± 0.19
3.1 ± 0.3
1.1 ± 0.1
0.94± 0.24
1.6 ± 0.1
rCNT2
inactiveb
>100c
H
0.003
NDe
1
O(CH2)2OH
O(CH2)3OH
O(CH2)4OH
O(CH2)5OH
17
18
19
20
45.1 ± 2.9
38 ± 13
60 ± 20
NDe
0.190
NDe
aConcentration of each compound required to inhibit inosine uptake by 50%. Data represent
b
mean ± SEM of at least three independent experiments unless otherwise stated. No inhibition
c
was observed at the maximum concentration of 100 ꢁM. 49% inhibition was observed at the
maximum concentration of 100 ꢁM. dSolubility in JP2 (pH 6.8) after incubation at 37 °C for 1 h.
eND, not determined.
Subsequently, compound 19 was evaluated by performing a purine load test in rats. Serum urate
levels in rats are quite low (0.5ꢀ1 mg/mL),8 in contrast to human beings due to the existence of
uricase activity, and therefore they were pretreated with the uricase inhibitor potassium oxonate
(100 mg/kg) to induce serum urate accumulation.64 Oral administration of a purine mixture (50
mg/kg), an equal mixture of adenosine, inosine, and guanosine, significantly elevated plasma
uric acid levels in the rats (Figure 3). On the other hand, the purine mixtureꢀinduced elevation of
plasma uric acid levels was suppressed when compound 19 was administered simultaneously at a
dose of 50 mg/kg. The suppressive ratio was calculated to be 42%, but unfortunately it was not
statistically significant. This may be due to insufficient inhibitory activity against rCNT2 (IC50 =
38 ꢁM).
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##
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Potassium Oxonate
Purine mixture
Compound 19 (mg/kg)
+
ꢀ
ꢀ
+
+
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+
+
50
Figure 3. Effect of compound 19 on the elevation of plasma uric acid levels derived from orally
administered purine mixture (an equal mixture of adenosine, guanosine, and inosine, 50 mg/kg)
in rats. Data are expressed as the mean ± SEM (n = 5). ##: p < 0.01 versus a group treated with
only potassium oxonate (100 mg/kg). NS, not significant.
Scaffold hopping from adenine to benzimidazole.
On the basis of the aboveꢀmentioned in vitro profiles, it was conceivable that the different
susceptibility between hCNT2 and rCNT2 observed in this study was a characteristic common to
compounds having the 8ꢀ(benzylamino)adenosine substructure. Therefore, we designed
compounds based on a scaffold hopping strategy to find compounds with enhanced inhibitory
activity against rCNT2.
Because the sugar moiety has important interactions with CNT2,65ꢀ67 and the substituent at the
8ꢀposition of adenosine was highly optimized through our study, we performed adenine moietyꢀ
hopping of compound 19 and designed the hypoxanthine riboside 21 and the benzimidazole
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riboside 22. As shown in Table 5, conversion to the inosine congener 21 not only reduced the
inhibitory activity against both hCNT2 and rCNT2, but also increased the susceptibility
difference. Replacement of adenine with the benzimidazole forming 22, however, considerably
enhanced the inhibitory activity against both hCNT2 and rCNT2. Thus, benzimidazole congener
22 was identified as the most potent hCNT2 inhibitor (IC50 = 0.062 µM) reported to date, which
was also a potent inhibitor against rCNT2 (IC50 = 1.5 µM).68 In addition, the solubility of
compound 22 in JP2 at 37 °C (0.120 mg/mL) was much higher than that of parent compound 1.
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19
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22
23
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Table 5. in vitro Profiles and Solubility Data of Compounds 19, 21, and 22
IC50 (µM)a
compd
solubility (mg/mL)c
hCNT2
0.94± 0.24
20.1 ± 2.0
rCNT2
38 ± 13
>1000b
1.5 ± 0.1
0.190
NDd
19
21
22
0.062 ± 0.017
0.120
aConcentration of each compound required to inhibit inosine uptake by 50%. Data represent
b
mean ± SEM of at least three independent experiments unless otherwise stated. 18% inhibition
c
was observed at the maximum concentration of 1000 ꢁM. Solubility in JP2 (pH 6.8) after
incubation at 37 °C for 1 h. dND, not determined.
Although the issue of the susceptibility difference remained, compound 22 had more than 25ꢀ
fold more potent inhibitory activity than 19 against rCNT2, and therefore was evaluated by
purine load test in the same manner as described above (Figure 4). Compound 22 significantly
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suppressed the increase in plasma uric acid levels derived from the purine mixture at a lower
dose of 1.0 mg/kg, which was probably due to enhanced in vitro activity. A higher dose of
compound 22 (10 mg/kg), however, resulted in little enhancement of the suppressive effect.
These results suggested that rCNT2 played a central role in the absorption of purine nucleosides
through the gastrointestinal tract, while pathways other than via rCNT2 might be involved in the
uptake of orallyꢀingested purine nucleosides, including their breakdown products, in rats.
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###
***
6
***
5
4
3
2
1
0
Potassium Oxonate
Purine mixture
Compound 22 (mg/kg)
+
ꢀ
ꢀ
+
+
ꢀ
+
+
0.1
+
+
1.0
+
+
10
Figure 4. Effects of compound 22 on the elevation of plasma uric acid levels derived from orally
administered purine mixture (50 mg/kg) in rats. Data are expressed as the mean ± SEM. (n = 5ꢀ
11). ###: p < 0.001 versus a group treated with only potassium oxonate (100 mg/kg). ***: p <
0.001 versus a group treated with potassium oxonate and purine mixture.
Finally, the oral absorption properties of compound 22 were investigated in rats (Table 6). The
maximum plasma concentration value (Cmax) of compound 22 (0.11 ꢁM) after oral
administration (10 mg/kg) was below oneꢀtenth of the IC50 value of 22 (1.5 ꢁM), and the oral
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bioavailability (F) was calculated to be 0.51%. These results suggested that compound 22 was
poorly absorbed orally, and that its action was mostly limited to the gastrointestinal tract.
Therefore, the pharmacologic effects of compound 22 in the purine load test were probably due
to the uptake inhibition of purine nucleosides via rCNT2 through the gastrointestinal tract.
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Table 6. Pharmacokinetic Parameters of Compound 22 in Rats after Intravenous (iv) and Oral
(po) Administrationsa
parameter
CL (mL/h/kg)
Vdss (mL/kg)
t1/2 (h)
ivb
394
117
1.7
5.0
ꢀ
poc
ꢀ
ꢀ
ꢀ
AUC0ꢀinf (ꢁM·h)
Cmax (ꢁM)
tmax (h)
ꢀ
0.11
0.83
0.25
0.51
ꢀ
AUC0ꢀx (ꢁM·h)
F (%)
ꢀ
ꢀ
b
a Data are expressed as the mean (n = 3). Compound 22 was dissolved in 50% (v/v) N,Nꢀ
dimethylacetamide in 5% glucose solution and administered intravenously at a dose of 1.0 mg/kg.
cCompound 22 was suspended in sodium carboxymethyl cellulose and administered orally at a
dose of 10 mg/kg.
Chemistry
The adenosine derivatives 2ꢀ4 were synthesized by nucleophilic aromatic substitution of
commercially available 8ꢀbromoadenosine with the amines 24, 28, and 30, which were prepared
from commercially available starting materials (Scheme 1). The amine 24 was prepared by
reducing 4ꢀ(pyridinꢀ2ꢀyl)benzonitrile 23 with LiAlH4. SuzukiꢀMiyaura coupling of 4ꢀ
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cyanophenylboronic acid 25 with 3ꢀbromopyridine and subsequent treatment with LiAlH4 gave
4ꢀ(pyridinꢀ3ꢀyl)benzylamine, which was purified after conversion to corresponding
trifluoroacetamide 27. Alkaline hydrolysis of compound 27 afforded amine 28. In a similar
fashion, amine 30 could be synthesized starting from compound 25 without the additional
protectionꢀdeprotection step for purification.
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Scheme 1. Synthesis of Compounds 2ꢀ4a
aReagents and conditions: (a) LiAlH4, THF, 60 °C; (b) 8ꢀbromoadenosine, iꢀPr2NEt, EtOH,
120 °C, in a screw tube; (c) 3ꢀbromopyridine or 4ꢀbromopyridine hydrochloride, Pd(PPh3)4, 2.0
M aq Na2CO3, MeCN, 80 °C; (d) CF3CO2Et, EtOH, rt; (e) 2.0 M aq NaOH, EtOH, 60 °C.
Adenosine derivatives 5ꢀ9 and 15ꢀ20 were synthesized starting from commercially available 4ꢀ
hydroxyꢀ3ꢀmethoxybenzonitrile 31 (Schemes 2ꢀ4). Compound 31 was converted to its triflate,
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followed by SuzukiꢀMiyaura coupling with phenylboronic acid to give the biphenyl 32.
Reduction of compound 32 with LiAlH4 and subsequent nucleophilic aromatic substitution with
8ꢀbromoadenosine furnished 6. In addition, methyl ether 32 was converted to the corresponding
benzyl ether 35 by stepwise treatment with BBr3 and BnBr before reduction with LiAlH4 to
afford amine 36. Condensation of compound 36 with 8ꢀbromoꢀ2ʹ,3ʹ,5ʹꢀtrisꢀOꢀ(tertꢀ
butyldimethylsilyl)adenosine, which was prepared according to the method described in the
literature69 with some modifications, and subsequent catalytic hydrogenation provided the
common intermediate 38. Treatment of compound 38 with NH4F yielded 5. On the other hand,
the hydroxyl group of compound 38 was alkylated with the appropriate alkyl halide followed by
treatment with NH4F to give 7ꢀ9 and 15. Reactions of the ester 15 with NH3 and NaBH4 afforded
the amide 16 and alcohol 17, respectively. Alkylation of compound 34 with the appropriate alkyl
bromide and subsequent reduction with LiAlH4 provided amines 46ꢀ48, which were then
subjected to nucleophilic aromatic substitution with 8ꢀbromoadenosine before removal of the
benzyl groups by catalytic hydrogenation to furnish 18ꢀ20.
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22
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Scheme 2. Synthesis of Compound 6a
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aReagents and conditions: (a) Tf2O, Et3N, DCM, rt; (b) PhB(OH)2, Pd(PPh3)4, Na2CO3, H2O,
DMF, 80 °C; (c) LiAlH4, THF, 60 °C; (d) 8ꢀbromoadenosine, iꢀPr2NEt, EtOH, 120 °C, in a
screw tube.
3
4
5
6
7
8
9
Scheme 3. Synthesis of Compounds 5, 7ꢀ9, and 15ꢀ17a
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18
19
20
21
22
23
24
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BnO
NH2
HO
CN
BnO
CN
BnO
N
N
a
b
c
d
NH2
N
N
H
32
N
O
TBSO
TBSO
34
35
36
OTBS
37
HO
R6O
R6O
NH2
N
NH2
N
NH2
N
N
N
N
N
N
e
g
f
N
H
N
H
O
N
H
N
N
N
N
O
O
R5O
TBSO
TBSO
HO
R5O
OR5
OTBS
HO
OH
38: R5 = TBS
5: R = H
39: R6 = Et
40: R6 = Pr
7: R6 = Et
8: R6 = Pr
9: R6 = i-Pr
15: R6 = CH2CO2Me
16: R6 = CH2CONH2
17: R6 = (CH2)2OH
f
41: R6 = i-Pr
42: R6 = CH2CO2Me
h
i
aReagents and conditions: (a) BBr3 (1.0 M in DCM), DCM, 30 °C; (b) BnBr, K2CO3, DMF, rt;
(c) LiAlH4, THF, 60 °C; (d) 8ꢀbromoꢀ2ʹ,3ʹ,5ʹꢀtrisꢀOꢀ(tertꢀbutyldimethylsilyl)adenosine69, iꢀ
Pr2NEt, EtOH, 120 °C, in a screw tube; (e) H2, 10% PdꢀC, AcOEt, rt; (f) NH4F, MeOH, reflux;
(g) R6I or R6Br, K2CO3, DMF, 35ꢀ50 °C; (h) NH3 (2.0 M in MeOH), rt; (i) NaBH4, EtOH, rt.
Scheme 4. Synthesis of Compounds 18ꢀ20a
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aReagents and conditions: (a) R7Br, K2CO3, DMF, 50 °C; (b) LiAlH4, THF, 60 °C; (c) 8ꢀ
bromoadenosine, iꢀPr2NEt, EtOH, 120 °C, in a screw tube; (d) H2, 10% PdꢀC, MeOH, 50 °C.
The adenosine derivatives 10ꢀ14 were synthesized starting from commercially available 4ꢀ
bromoꢀ2ꢀhydroxybenzonitrile 52 following a methodology similar to that described above
(Scheme 5). SuzukiꢀMiyaura coupling with phenylboronic acid after benzylation of compound
52 and subsequent reduction with LiAlH4 provided the amine 54. Condensation of compound 54
with 8ꢀbromoꢀ2ʹ,3ʹ,5ʹꢀtrisꢀOꢀ(tertꢀbutyldimethylsilyl)adenosine69 followed by catalytic
hydrogenation yielded the common intermediate 56. The silyl ethers of compound 56 were
cleaved by treatment with NH4F to give 10. On the other hand, the hydroxy group of compound
56 was alkylated with the appropriate alkyl iodide before treatment with NH4F to afford 11ꢀ14.
Scheme 5. Synthesis of Compounds 10ꢀ14a
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aReagents and conditions: (a) BnBr, K2CO3, DMF, rt; (b) PhB(OH)2, Pd(PPh3)4, Na2CO3, H2O,
DMF, 80 °C; (c) LiAlH4, THF, 60 °C; (d) 8ꢀbromoꢀ2ʹ,3ʹ,5ʹꢀtrisꢀOꢀ(tertꢀ
butyldimethylsilyl)adenosine69, iꢀPr2NEt, EtOH, 120 °C, in a screw tube; (e) H2, 10% PdꢀC,
AcOEt, rt; (f) NH4F, MeOH, reflux; (g) R9I, K2CO3, DMF, rtꢀ50 °C.
The inosine derivative 21 was synthesized by treating the adenosine derivatives 50 with
NaNO2 in aqueous acetic acid followed by catalytic hydrogenation to remove the benzyl group
(Scheme 6).
Scheme 6. Synthesis of Compound 21a
aReagents and conditions: (a) NaNO2, AcOH, H2O, rt; (b) H2, 10% PdꢀC, MeOH, 50 °C.
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The benzimidazole nucleoside 22 was synthesized starting from commercially available 2ꢀ
chloroꢀ1Hꢀbenzimidazole 62 (Scheme 7). Compound 62 was silylated with N,Oꢀ
7
8
9
bis(trimethylsilyl)acetamide, followed by glycosidation with 1,2,3,5ꢀtetraꢀOꢀacetylꢀβꢀDꢀ
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ribofuranose in the presence of TMSOTf to give 63. Treatment of compound 63 with NaOMe
provided 64, which was then subjected to a nucleophilic aromatic substitution reaction with 47
followed by catalytic hydrogenation to furnish 22.
Scheme 7. Synthesis of Compound 22a
aReagents and conditions: (a) N,Oꢀbis(trimethylsilyl)acetamide, MeCN, 80 °C then TMSOTf,
1,2,3,4ꢀtetraꢀOꢀacetylꢀβꢀDꢀribofuranose, rt; (b) tꢀBuOK, MeOH, rt; (c) 47, iꢀPr2NEt, EtOH, 120
°C, in a screw tube; (d) H2, 10% PdꢀC, MeOH, 50 °C.
Conclusion
We searched for potent hCNT2 inhibitors with increased solubility starting from compound 1, a
potent but poorly soluble inhibitor that we reported previously. Substituents at the 2ꢀposition of
the biphenylꢀ4ꢀyl substructure in compound 1 effectively ameliorated the solubility in JP2 at
37 °C. Introduction of terminallyꢀhydroxylated alkoxy groups was crucial for acquisition of the
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rCNT2 inhibitory activity, which led to the discovery of compound 19. Subsequent scaffold
hopping of compound 19 by adenine replacement yielded the benzimidazole nucleoside 22 as the
most potent hCNT2 inhibitor reported to date. Compound 22 exhibited potent inhibitory activity
against rCNT2 and significantly suppressed increases in plasma urate levels in a purine load test
using rats. Compound 22 had a poorly absorbable nature after oral administration in rats, and
thus its pharmacologic action is likely limited to the gastrointestinal tract. These findings suggest
that inhibition of hCNT2 in the gastrointestinal tract can be a promising approach for the
treatment of hyperuricemia.
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Experimental Section
General Methods. Reagents and solvents were purchased from commercial sources and used
without further purification. All moisture and air sensitive reactions were carried out in an ovenꢀ
dried flask under an argon atmosphere. Reaction conditions and yields were not optimized. All
melting points were determined on a Yanaco micro melting point apparatus (MPꢀJ3) and are
uncorrected. Reactions were monitored by thin layer chromatography (TLC) using Merck
Kieselgel 60 F254 plates or Fuji Silysia Chemical Ltd. Chromatorex NHꢀTLC plates. Flash
column chromatography was performed on Biotage prepacked columns or Yamazen HiꢀFlash
columns using an automated flash chromatography systems Biotage Isolera One (Biotage AB,
Uppsala, Sweden) or Wꢀprep 2XY (Yamazen Corporation, Osaka, Japan). 1H NMR spectra were
recorded on a Bruker AV400M spectrometer at 400.1 MHz. Chemical shifts (δ) are reported in
parts per million (ppm) units using tetramethylsilane as an internal standard. Data are presented
as follows; chemical shift, multiplicity (s, singlet; d, doublet; dd, double doublet; t, triplet; q,
quartet; m, multiplet; br, broad; br s, broad singlet), coupling constant and integration. 13C NMR
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spectra were recorded on a Bruker AV400M spectrometer at 100.6 MHz with complete proton
decoupling. Chemical shifts (δ) are reported in parts per million (ppm) with the solvent as the
internal reference (δ 77.16 in CDCl3 or δ 39.52 in DMSOꢀd6).70 High resolution mass spectra
(HRMS) were recorded on an Agilent Technologies 6520 AccurateꢀMass QꢀTOF instrument
using electrospray ionization (ESI, positive or negative ion mode). The purity of all tested
compounds was determined to be ≥95% by HPLCꢀUV analysis, which was performed on a
Shimadzu LCꢀVP series instrument (Shimadzu Corporation, Kyoto, Japan) with the following
parameters: analytical column, Inertsil ODSꢀ3, 4.6 x 250 mm, 5 ꢁm (GL Sciences Inc., Tokyo,
Japan); mobile phase, linear gradient of 10ꢀ90% MeOH in 0.1% (v/v) aq HCO2H for 45 min with
flow rate of 1.0 mL/min; column temperature, 40 °C; injection volume, 10 ꢁL; compound
concentration, 2 mM in DMSO; detection wavelength, 260 nm.
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8ꢀ[4ꢀ(Pyridinꢀ2ꢀyl)benzylamino]adenosine (2). A mixture of 24 (343 mg, 1.86 mmol), 8ꢀ
bromoadenosine (215 mg, 0.621 mmol), and iꢀPr2NEt (482 mg, 3.73 mmol) in EtOH (5.18 mL)
was heated in a screw tube at 120 °C with stirring for 90 h. The reaction mixture was allowed to
cool to room temperature and then concentrated under reduced pressure. The residue was
purified by flash column chromatography on NH silica gel (gradient; 8ꢀ15% MeOH in DCM)
followed by flash column chromatography on silica gel (gradient; 10ꢀ20% MeOH in DCM) to
1
give 2 (235 mg, 84%) as a yellow foam. H NMR (DMSOꢀd6) δ 3.53ꢀ3.73 (m, 2H), 3.96ꢀ4.05
(m, 1H), 4.09ꢀ4.17 (m, 1H), 4.55ꢀ4.81 (m, 3H), 5.18 (d, J = 4.0 Hz, 1H), 5.32 (d, J = 6.8 Hz,
1H), 5.92 (dd, J = 4.0, 6.3 Hz, 1H), 5.96 (d, J = 7.5 Hz, 1H), 6.54 (br s, 2H), 7.28ꢀ7.37 (m, 1H),
7.43ꢀ7.53 (m, 2H), 7.55ꢀ7.67 (m, 1H), 7.80ꢀ7.97 (m, 3H), 7.99ꢀ8.10 (m, 2H), 8.60ꢀ8.69 (m, 1H).
13C NMR (DMSOꢀd6) δ 45.1, 61.8, 71.0, 71.2, 85.9, 86.6, 117.1, 120.1, 122.5, 126.5, 127.5,
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137.3 (2C), 140.9, 148.7, 149.6, 149.9, 151.4, 152.5, 156.0. HRMS calcd for C22H24N7O4
(M+H)+ 450.1884, found 450.1886.
7
8
9
8ꢀ[4ꢀ(Pyridinꢀ3ꢀyl)benzylamino]adenosine (3). The title compound was synthesized from 28
(160 mg, 0.867 mmol) and 8ꢀbromoadenosine (100 mg, 0.289 mmol) according to the procedure
described for 2. The crude product was purified by flash column chromatography on NH silica
gel (gradient; 8ꢀ15% MeOH in DCM) followed by flash column chromatography on silica gel
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1
(gradient; 10ꢀ24% MeOH in DCM) to afford 3 (99.9 mg, 77%) as a beige amorphous solid. H
NMR (DMSOꢀd6) δ 3.55ꢀ3.74 (m, 2H), 3.96ꢀ4.06 (m, 1H), 4.10ꢀ4.20 (m, 1H), 4.54ꢀ4.82 (m, 3H),
5.16 (d, J = 4.0 Hz, 1H), 5.30 (d, J = 6.8 Hz, 1H), 5.85ꢀ6.00 (m, 2H), 6.53 (br s, 2H), 7.44ꢀ7.55
(m, 3H), 7.61 (t, J = 6.0 Hz, 1H), 7.64ꢀ7.73(m, 2H), 7.90 (s, 1H), 8.02ꢀ8.10 (m, 1H), 8.55 (dd, J
= 1.8, 4.8 Hz, 1H), 8.84ꢀ8.91 (m, 1H). 13C NMR (DMSOꢀd6) δ 45.0, 61.8, 71.0, 71.1, 85.9, 86.6,
117.0, 123.9, 126.8, 127.9, 134.0, 135.5 (2C), 140.0, 147.6, 148.3, 148.5, 149.9, 151.4, 152.4.
HRMS calcd for C22H24N7O4 (M+H)+ 450.1884, found 450.1879.
8ꢀ[4ꢀ(Pyridinꢀ4ꢀyl)benzylamino]adenosine (4). A mixture of 30 (227 mg, 1.23 mmol), 8ꢀ
bromoadenosine (142 mg, 0.410 mmol), and iꢀPr2NEt (318 mg, 2.46 mmol) in EtOH (4.10 mL)
was heated in a screw tube at 120 °C with stirring for 96 h. The reaction mixture was allowed to
cool to room temperature and then concentrated under reduced pressure. To the suspension of the
residue in DCM (20.0 mL) was added iꢀPr2NEt (159 mg, 1.23 mmol), and the resulting mixture
was stirred at room temperature for 20 h. The precipitate was collected by filtration, washed with
DCM, airꢀdried, and dried under reduced pressure to give an orange brown solid (256 mg). The
solid (252 mg) was suspended in EtOH (30.0 mL) and heated under reflux with stirring for 1 h
before being allowed to cool to room temperature. The precipitate was collected by filtration,
washed with EtOH, airꢀdried, and dried under reduced pressure to give 4 (93.0 mg, 50%) as a
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pale brown solid. H NMR (DMSOꢀd6) δ 3.54ꢀ3.71 (m, 2H), 3.96ꢀ4.06 (m, 1H), 4.10ꢀ4.20 (m,
1H), 4.52ꢀ4.84 (m, 3H), 5.16 (d, J = 4.0 Hz, 1H), 5.30 (d, J = 6.8 Hz, 1H), 5.90 (dd, J = 4.0, 6.0
Hz, 1H), 5.95 (d, J = 7.5 Hz, 1H), 6.52 (br s, 2H), 7.48ꢀ7.59 (m, 2H), 7.62 (t, J = 6.2 Hz, 1H),
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7.66ꢀ7.86 (m, 4H), 7.90 (s, 1H), 8.58ꢀ8.69 (m, 2H). C NMR (DMSOꢀd6) δ 45.0, 61.8, 71.0,
71.1, 85.8, 86.5, 117.0, 121.1, 126.7, 127.9, 135.6, 141.3, 146.8, 148.6, 149.9, 150.2, 151.3,
152.5. HRMS calcd for C22H24N7O4 (M+H)+ 450.1884, found 450.1884.
8ꢀ(2ꢀHydroxybiphenylꢀ4ꢀylmethylamino)adenosine (5). To a solution of 38 (367 mg, 0.455
mmol) in MeOH (9.09 mL) was added NH4F (1.01 g, 27.3 mmol), and the resulting mixture was
heated under reflux with stirring for 24 h. The reaction mixture was allowed to cool to room
temperature, diluted with H2O (9.09 mL), and then stirred for 1 h. The precipitate was collected
by filtration, washed with MeOH/H2O (1/1), airꢀdried, and then recrystallized from EtOH to give
1
5 (75.0 mg, 36%) as an offꢀwhite solid. mp 240ꢀ242 °C. H NMR (DMSOꢀd6) δ 3.56ꢀ3.75 (m,
2H), 3.96ꢀ4.08 (m, 1H), 4.10ꢀ4.19 (m, 1H), 4.46ꢀ4.63 (m, 2H), 4.72ꢀ4.82 (m, 1H), 5.17 (d, J =
4.3 Hz, 1H), 5.24 (d, J = 6.8 Hz, 1H), 5.85ꢀ5.93 (m, 1H), 5.95 (d, J = 7.3 Hz, 1H), 6.51 (br s,
2H), 6.84ꢀ6.91 (m, 1H), 6.92ꢀ6.99 (m, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.23ꢀ7.31 (m, 1H), 7.33ꢀ
13
7.43 (m, 2H), 7.47ꢀ7.62 (m, 3H), 7.90 (s, 1H), 9.46 (s, 1H). C NMR (DMSOꢀd6) δ 45.0, 61.8,
71.1, 85.8, 86.6, 114.7, 117.1, 118.2, 126.2, 126.4, 127.9, 129.0, 130.1, 138.6, 140.5, 148.5,
149.8, 151.5, 152.5, 154.2. HRMS calcd for C23H25N6O5 (M+H)+ 465.1881, found 465.1873.
8ꢀ(2ꢀMethoxybiphenylꢀ4ꢀylmethylamino)adenosine (6). The title compound was synthesized
from 33 (370 mg, 1.73 mmol) and 8ꢀbromoadenosine (200 mg, 0.578 mmol) according to the
procedure described for 2. The crude product was purified by flash column chromatography on
NH silica gel (gradient; 8ꢀ15% MeOH in DCM) followed by flash column chromatography on
silica gel (gradient; 10ꢀ17% MeOH in DCM) to afford 6 (257 mg, 93%) as a yellow solid. mp
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137ꢀ139 °C. H NMR (DMSOꢀd6) δ 3.57ꢀ3.72 (m, 2H), 3.75 (s, 3H), 3.97ꢀ4.05 (m, 1H), 4.08ꢀ
5
6
4.21 (m, 1H), 4.51ꢀ4.82 (m, 3H), 5.17 (d, J = 4.0 Hz, 1H), 5.30 (d, J = 6.8 Hz, 1H), 5.87ꢀ6.03 (m,
7
8
9
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2H), 6.53 (br s, 2H), 6.98ꢀ7.08 (m, 1H), 7.13ꢀ7.51 (m, 7H), 7.52ꢀ7.64 (m, 1H), 7.90 (s, 1H). C
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NMR (DMSOꢀd6) δ 45.3, 55.5, 61.8, 71.0, 71.1, 85.9, 86.5, 110.7, 117.1, 119.5, 126.7, 128.0,
128.2, 129.2, 130.2, 138.1, 141.0, 148.6, 149.9, 151.3, 152.5, 156.0. HRMS calcd for
C24H27N6O5 (M+H)+ 479.2037, found 479.2036.
8ꢀ(2ꢀEthoxybiphenylꢀ4ꢀylmethylamino)adenosine (7). To a solution of 39 (350 mg, 0.419
mmol) in MeOH (8.38 mL) was added NH4F (931 mg, 25.1 mmol), and the resulting mixture
was heated under reflux with stirring for 24 h. The reaction mixture was allowed to cool to room
temperature, diluted with DCM (34.0 mL), stirred for 1 h, and then filtered through a pad of
Hyflo SuperꢀCel. The filtrate was concentrated under reduced pressure and the residue was
purified by flash column chromatography on silica gel (gradient; 10ꢀ17% MeOH in DCM) to
give 7 (146 mg, 71%) as a pale yellow solid. mp 128ꢀ130 °C. 1H NMR (DMSOꢀd6) δ 1.24 (t, J =
6.9 Hz, 3H), 3.56ꢀ3.73 (m, 2H), 3.94ꢀ4.21 (m, 4H), 4.61 (d, J = 6.0 Hz, 2H), 4.70ꢀ4.81 (m, 1H),
5.18 (d, J = 4.0 Hz, 1H), 5.30 (d, J = 6.8 Hz, 1H), 5.90ꢀ6.04 (m, 2H), 6.54 (br s, 2H), 6.98ꢀ7.06
(m, 1H), 7.11ꢀ7.55 (m, 7H), 7.58 (t, J = 6.0 Hz, 1H), 7.90 (s, 1H). 13C NMR (DMSOꢀd6) δ 14.6,
45.2, 61.8, 63.5, 71.0, 71.2, 85.9, 86.5, 111.8, 117.1, 119.5, 126.7, 128.0, 128.3, 129.2, 130.3,
138.2, 140.9, 148.6, 149.9, 151.3, 152.5, 155.3. HRMS calcd for C25H29N6O5 (M+H)+ 493.2194,
found 493.2199.
8ꢀ(2ꢀPropoxybiphenylꢀ4ꢀylmethylamino)adenosine (8). The title compound was prepared
from 40 (414 mg, 0.487 mmol) following the procedure described for 7. The crude product was
purified by flash column chromatography on silica gel (gradient; 10ꢀ17% MeOH in DCM) to
afford 8 (236 mg, 96%) as a pale yellow foam. 1H NMR (DMSOꢀd6) δ 0.89 (t, J = 7.4 Hz, 3H),
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1.56ꢀ1.71 (m, 2H), 3.57ꢀ3.72 (m, 2H), 3.92 (t, J = 6.4 Hz, 2H), 3.97ꢀ4.05 (m, 1H), 4.10ꢀ4.18 (m,
1H), 4.61 (d, J = 6.0 Hz, 2H), 4.70ꢀ4.81 (m, 1H), 5.19 (d, J = 4.0 Hz, 1H), 5.30 (d, J = 6.8 Hz,
1H), 5.89ꢀ6.01 (m, 2H), 6.54 (br s, 2H), 6.98ꢀ7.07 (m, 1H), 7.11ꢀ7.18 (m, 1H), 7.23 (d, J = 7.8
Hz, 1H), 7.25ꢀ7.33 (m, 1H), 7.33ꢀ7.64 (m, 5H), 7.90 (s, 1H). 13C NMR (DMSOꢀd6) δ 10.6, 22.1,
45.2, 61.8, 69.3, 71.0, 71.1, 85.9, 86.5, 111.6, 117.1, 119.4, 126.7, 127.9, 128.3, 129.2, 130.2,
138.2, 140.9, 148.6, 149.9, 151.3, 152.5, 155.4. HRMS calcd for C26H31N6O5 (M+H)+ 507.2350,
found 507.2350.
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8ꢀ(2ꢀIsopropoxybiphenylꢀ4ꢀylmethylamino)adenosine (9). The title compound was prepared
from 41 (310 mg, 0.365 mmol) following the procedure described for 7. The crude product was
purified by flash column chromatography on silica gel (gradient; 10ꢀ17% MeOH in DCM) to
afford 9 (180 mg, 97%) as an ivoryꢀcolored foam. 1H NMR (DMSOꢀd6) δ 1.11ꢀ1.24 (m, 6H),
3.55ꢀ3.74 (m, 2H), 3.95ꢀ4.05 (m, 1H), 4.09ꢀ4.18 (m, 1H), 4.48ꢀ4.81 (m, 4H), 5.18 (d, J = 4.0 Hz,
1H), 5.29 (d, J = 6.8 Hz, 1H), 5.88ꢀ6.03 (m, 2H), 6.53 (br s, 2H), 6.96ꢀ7.05 (m, 1H), 7.10ꢀ7.18
13
(m, 1H), 7.19ꢀ7.52 (m, 6H), 7.59 (t, J = 6.0 Hz, 1H), 7.90 (s, 1H). C NMR (DMSOꢀd6) δ 21.8
(2C), 45.2, 61.8, 69.8, 71.0, 71.1, 85.9, 86.5, 113.5, 117.1, 119.6, 126.6, 127.9, 129.1, 129.2,
130.5, 138.4, 140.8, 148.6, 149.9, 151.3, 152.5, 154.1. HRMS calcd for C26H31N6O5 (M+H)+
507.2350, found 507.2345.
8ꢀ(3ꢀHydroxybiphenylꢀ4ꢀylmethylamino)adenosine (10). To a solution of 56 (517 mg, 0.640
mmol) in MeOH (12.8 mL) was added NH4F (1.42 g, 38.4 mmol), and the resulting mixture was
heated under reflux with stirring for 24 h. The reaction mixture was allowed to cool to room
temperature, diluted with DCM (40.0 mL), stirred for 1 h, and then filtered through a pad of
Hyflo SuperꢀCel. The filtrate was concentrated under reduced pressure and the residue was
purified by flash column chromatography on silica gel (gradient; 10ꢀ20% MeOH in DCM) to
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give 10 (291 mg, 98%) as a yellow solid. mp 161ꢀ163 °C. H NMR (DMSOꢀd6) δ 3.53ꢀ3.74 (m,
5
6
7
8
2H), 3.96ꢀ4.05 (m, 1H), 4.09ꢀ4.19 (m, 1H), 4.46ꢀ4.64 (m, 2H), 4.68ꢀ4.84 (m, 1H), 5.18 (d, J =
4.0 Hz, 1H), 5.34 (d, J = 6.8 Hz, 1H), 5.86ꢀ6.04 (m, 2H), 6.55 (br s, 2H), 7.01ꢀ7.13 (m, 2H),
9
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7.22ꢀ7.72 (m, 7H), 7.92 (s, 1H), 10.19 (s, 1H). C NMR (DMSOꢀd6) δ 41.0, 61.8, 70.9, 71.2,
85.9, 86.6, 113.7, 116.6, 117.5, 125.3, 126.5, 127.3, 128.9 (2C), 140.2, 140.3, 148.8, 149.9,
151.7, 152.4, 155.4. HRMS calcd for C23H25N6O5 (M+H)+ 465.1881, found 465.1884.
8ꢀ(3ꢀMethoxybiphenylꢀ4ꢀylmethylamino)adenosine (11). To a solution of 57 (384 mg, 0.468
mmol) in MeOH (9.35 mL) was added NH4F (1.04 g, 28.1 mmol), and the resulting mixture was
heated under reflux with stirring for 26 h. The reaction mixture was allowed to cool to room
temperature, diluted with H2O (40.0 mL), and then stirred for 1 h. The precipitate was collected
by filtration, washed with H2O, and airꢀdried, and then recrystallized from EtOH to give 11 (167
1
mg, 75%) as a pale yellow solid. mp 236ꢀ238 °C (dec). H NMR (DMSOꢀd6) δ 3.57ꢀ3.72 (m,
2H), 3.93 (s, 3H), 3.98ꢀ4.05 (m, 1H), 4.10ꢀ4.19 (m, 1H), 4.51ꢀ4.68 (m, 2H), 4.73ꢀ4.83 (m, 1H),
5.17 (d, J = 4.0 Hz, 1H), 5.33 (d, J = 6.8 Hz, 1H), 5.86 (dd, J = 4.0, 6.0 Hz, 1H), 5.99 (d, J = 7.3
Hz, 1H), 6.51 (br s, 2H), 7.14ꢀ7.26 (m, 2H), 7.28ꢀ7.52 (m, 5H), 7.63ꢀ7.73 (m, 2H), 7.90 (s, 1H).
13C NMR (DMSOꢀd6) δ 40.6, 55.4, 61.8, 70.9, 71.2, 85.9, 86.5, 108.8, 117.1, 118.5, 126.6,
126.8, 127.3, 127.4, 128.9, 140.1, 140.4, 148.6, 149.9, 151.4, 152.5, 157.0. HRMS calcd for
C24H27N6O5 (M+H)+ 479.2037, found 479.2046.
8ꢀ(3ꢀEthoxybiphenylꢀ4ꢀylmethylamino)adenosine (12). To a solution of 58 (322 mg, 0.386
mmol) in MeOH (7.71 mL) was added NH4F (857 mg, 23.1 mmol), and the resulting mixture
was heated under reflux with stirring for 24 h. The reaction mixture was allowed to cool to room
temperature, diluted with DCM (24.0 mL), stirred for 1 h, and then filtered through a pad of
Hyflo SuperꢀCel. The filtrate was concentrated under reduced pressure and the residue was
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triturated in DCM. The precipitate was collected by filtration, washed with DCM, airꢀdried, and
then recrystallized from EtOH to give 12 (120 mg, 63%) as a pale yellow solid. mp 219ꢀ221 °C.
1H NMR (DMSOꢀd6) δ 1.40 (t, J = 6.9 Hz, 3H), 3.56ꢀ3.71 (m, 2H), 3.97ꢀ4.06 (m, 1H), 4.10ꢀ4.27
(m, 3H), 4.52ꢀ4.69 (m, 2H), 4.73ꢀ4.84 (m, 1H), 5.17 (d, J = 4.0 Hz, 1H), 5.34 (d, J = 6.8 Hz,
1H), 5.85 (dd, J = 4.0, 6.0 Hz, 1H), 6.00 (d, J = 7.5 Hz, 1H), 6.50 (br s, 2H), 7.12ꢀ7.52 (m, 7H),
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7.61ꢀ7.74 (m, 2H), 7.90 (s, 1H). C NMR (DMSOꢀd6) δ 14.8, 40.6, 61.8, 63.3, 70.9, 71.2, 85.9,
86.5, 109.5, 117.1, 118.4, 126.7, 126.8, 127.2, 127.3, 128.9, 140.0, 140.4, 148.6, 149.9, 151.5,
152.5, 156.3. HRMS calcd for C25H29N6O5 (M+H)+ 493.2194, found 493.2191.
8ꢀ(3ꢀPropoxybiphenylꢀ4ꢀylmethylamino)adenosine (13). To a solution of 59 (378 mg, 0.445
mmol) in MeOH (8.90 mL) was added NH4F (989 mg, 26.7 mmol), and the resulting mixture
was heated under reflux with stirring for 24 h. The reaction mixture was allowed to cool to room
temperature, diluted with H2O (17.8 mL), and then stirred for 1.5 h. The precipitate was collected
by filtration, washed with MeOH/H2O (1/2), airꢀdried, and then recrystallized from EtOH to give
13 (121 mg, 54%) as a pale yellow solid. mp 211ꢀ213 °C. 1H NMR (DMSOꢀd6) δ 1.05 (t, J = 7.4
Hz, 3H), 1.71ꢀ1.90 (m, 2H), 3.55ꢀ3.72 (m, 2H), 3.97ꢀ4.21 (m, 4H), 4.53ꢀ4.69 (m, 2H), 4.74ꢀ4.83
(m, 1H), 5.19 (d, J = 4.0 Hz, 1H), 5.35 (d, J = 6.8 Hz, 1H), 5.87 (dd, J = 4.1, 5.9 Hz, 1H), 6.00
(d, J = 7.5 Hz, 1H), 6.51 (br s, 2H), 7.11ꢀ7.23 (m, 2H), 7.28 (d, J = 7.8 Hz, 1H), 7.31ꢀ7.50 (m,
13
4H), 7.61ꢀ7.72 (m, 2H), 7.90 (s, 1H). C NMR (DMSOꢀd6) δ 10.6, 22.2, 40.6, 61.8, 69.0, 71.0,
71.2, 85.9, 86.5, 109.5, 117.1, 118.4, 126.8 (2C), 127.0, 127.3, 128.9, 140.0, 140.4, 148.6, 149.9,
151.5, 152.5, 156.4. HRMS calcd for C26H31N6O5 (M+H)+ 507.2350, found 507.2344.
8ꢀ(3ꢀIsopropoxybiphenylꢀ4ꢀylmethylamino)adenosine (14). The title compound was
prepared from 60 (283 mg, 0.333 mmol) following the procedure described for 10. The crude
product was purified by flash column chromatography on silica gel (gradient; 10ꢀ17% MeOH in
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