Identification of 8-aminoadenosine derivatives as a new class of human concentrative nucleoside transporter 2 inhibitors

Article abstract of DOI:10.1021/ml500343r

Purine-rich foods have long been suspected as a major cause of hyperuricemia. We hypothesized that inhibition of human concentrative nucleoside transporter 2 (hCNT2) would suppress increases in serum urate levels derived from dietary purines. To test this hypothesis, the development of potent hCNT2 inhibitors was required. By modifying adenosine, an hCNT2 substrate, we successfully identified 8-aminoadenosine derivatives as a new class of hCNT2 inhibitors. Compound 12 moderately inhibited hCNT2 (IC₅₀ = 52 ± 3.8 μM), and subsequent structure-activity relationship studies led to the discovery of compound 48 (IC₅₀ = 0.64 ± 0.19 μM). Here we describe significant findings about structural requirements of 8-aminoadenosine derivatives for exhibiting potent hCNT2 inhibitory activity.

Full text of DOI:10.1021/ml500343r

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Letter
Identification of 8-Aminoadenosine Derivatives as a New Class
of Human Concentrative Nucleoside Transporter 2 Inhibitors
Kazuya Tatani, Masahiro Hiratochi, Yoshinori Nonaka, Masayuki Isaji, and Satoshi Shuto
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml500343r • Publication Date (Web): 28 Jan 2015
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Identification of 8-Aminoadenosine Derivatives as a New Class of
Human Concentrative Nucleoside Transporter 2 Inhibitors
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Kazuya Tatani*,†,‡, Masahiro, Hiratochi†, Yoshinori, Nonaka†, Masayuki, Isaji†, Satoshi, Shuto‡,§
†Central Research Laboratories, Kissei Pharmaceutical Co., Ltd., 4365-1, Kashiwabara, Hotaka, Azumino, Nagano
399-8304, Japan
‡Faculty of Pharmaceutical Science, Hokkaido University, Kita-12, Nishi-6, Kita-Ku, Sapporo 060-0812, Japan
§Center for Research and Education on Drug Discovery, Hokkaido University, Kita-12, Nishi-6, Kita-Ku, Sapporo
060-0812, Japan
8-aminoadenosine derivatives, human concentrative nucleoside transporter 2, dietary purines, hyperuricemia, gout
ABSTRACT: Purine-rich foods have long been suspected as a major cause of hyperuricemia. We hypothesized that inhibi-
tion of human concentrative nucleoside transporter 2 (hCNT2) would suppress increases in serum urate levels derived
from dietary purines. To test this hypothesis, the development of potent hCNT2 inhibitors was required. By modifying
adenosine, an hCNT2 substrate, we successfully identified 8-aminoadenosine derivatives as a new class of hCNT2 inhibi-
tors. Compound 12 moderately inhibited hCNT2 (IC50 = 52 ± 3.8 μM), and subsequent structure-activity relationship stud-
ies led to the discovery of compound 48 (IC50 = 0.64 ± 0.19 μM). Here we describe significant findings about structural
requirements of 8-aminoadenosine derivatives for exhibiting potent hCNT2 inhibitory activity.
Hyperuricemia is a condition in which serum urate lev-
els are abnormally elevated. It is widely accepted that
sustained hyperuricemia is the primary risk factor for
urate deposition diseases, such as gouty arthritis, tophi,
urinary stones, and renal damage.1 Prospective cohort
studies conducted in healthy men or patients with asymp-
tomatic hyperuricemia demonstrated that serum urate
levels exceeding 7.0 mg/dL are closely associated with an
increased risk for developing gouty arthritis.2,3 Moreover,
a retrospective study of patients with gout revealed that
the frequency of recurrent gouty arthritis decreases when
serum urate levels are kept low.4 These findings suggest
the importance of proactively reducing serum urate levels
to prevent the onset of gouty attacks.
be a promising approach for treating hyperuricemia.
Maintaining a strict diet with low purine foods over a long
period is difficult, however, because nutrition is impaired
and quality of life is decreased. Therefore, we attempted
to develop a therapeutic drug as an alternative to restrict-
ing the intake of dietary purines.
We focused on the absorption paths of purines in the
gastrointestinal tract. Dietary purines comprise various
forms, such as DNA, RNA, nucleoproteins, oligonucleo-
tides, purine nucleotides, purine nucleosides, and purine
bases. Most of these forms are poorly absorbed directly
via the plasma membrane because of their high molecular
weights and/or highly hydrophilic nature. Thus, carrier-
mediated transport likely plays an important role in the
gastrointestinal absorption of dietary purines. Nucleoside
transporter proteins have been extensively studied as po-
tential carriers.9-13 Currently, they are classified into
roughly two groups in mammals.14,15 One group contains
concentrative nucleoside transporters (CNT1-3), which
mediate unidirectional sodium- and/or proton-dependent
transport.16 The second group contains equilibrative nu-
cleoside transporters (ENT1-4), which mediate bidirec-
tional sodium-independent transport (facilitated diffu-
sion).17 Although distribution of the seven transporters
varies depending on the tissue, in epithelia, CNTs and
Purine-rich foods have long been suspected as a major
cause of hyperuricemia. Recent epidemiologic studies
reported that high levels of meat and seafood consump-
tion are associated with high levels of serum urate and an
increased risk of developing gout.5,6 Another study
demonstrated that ingesting yeast RNA for several days
induces hyperuricemia in healthy subjects.7 Conversely, a
low purine diet effectively lowers serum urate levels in
patients with hyperuricemia.8 Together, these findings
suggest that dietary purines increase serum urate levels
and suppression of the absorption of dietary purines may
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ENTs are mostly localized in the apical and basolateral
dependent inosine uptake in COS-7 cells transiently ex-
membranes, respectively.14,15,18,19 Therefore, CNTs are
thought to be more important carriers for the absorption
of purine nucleosides in the gastrointestinal tract.
pressing hCNT2,25 however, revealed unexpectedly weak
inhibitory effects (IC50 > 1000 μM). Thus, to test our hy-
pothesis, the development of more potent hCNT2 inhibi-
tors was required. Here we describe the discovery of a
new class of potent hCNT2 inhibitors along with signifi-
cant findings about structural requirements for exhibiting
potent hCNT2 inhibitory activity.
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Extensive studies have revealed the inherent substrate
selectivity of CNTs (Table 1).14,15,18 CNT1 recognizes pyrim-
idine nucleosides and adenosine, and adenosine is trans-
ported in a high-affinity and low-capacity manner.16,20,21
Hence, CNT1 is a pyrimidine nucleoside-specific trans-
porter. CNT2 is the preferred purine nucleoside trans-
porter because it transports purines and uridine.16,22,23
CNT3 has much broader substrate selectivity, transport-
ing both purine and pyrimidine nucleosides.16,24 Based on
these findings, CNT2 and/or CNT3 are likely to be the key
transporters for the uptake of purine nucleosides in the
gastrointestinal tract. Therefore, we performed quantita-
tive real-time reverse transcription-polymerase chain re-
action (RT-PCR) to confirm the expression profiles of
human CNT2 (hCNT2) and CNT3 (hCNT3) in the gastro-
intestinal tract.25 In the small intestine, hCNT2 was abun-
dantly expressed, whereas hCNT3 was only weakly ex-
pressed. Furthermore, analysis of the distribution pat-
terns in the gastrointestinal tract revealed that hCNT2
was expressed mostly in the duodenum of the upper small
intestine, followed by the jejunum; and hCNT3 and
hCNT2 expression were comparable in the ileum, though
the expression was weak overall. These findings suggest
that hCNT2 is a central carrier responsible for the absorp-
tion of purine nucleosides in the gastrointestinal tract.
Hence, we selected hCNT2 as our drug target candidate
and hypothesized that its inhibitors would suppress the
increase in serum urate levels derived from dietary pu-
rines.
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Figure 1. Structures of known hCNT2 inhibitors.
We investigated the conversion of adenosine, an hCNT2
substrate, into an inhibitor by its derivatization. In an
initial study, we synthesized a series of adenosine deriva-
tives modified at the 2-, 8-. 5'-, or N6-position (Figure 2)
and evaluated their inhibitory effects on sodium-
dependent inosine uptake in COS-7 cells transiently ex-
pressing hCNT2. Among the base-modified adenosines 1-
12, only 8-(benzylamino)adenosine 12 exhibited an inhibi-
tory effect greater than 50% at 100 μM (IC50 = 52 ± 3.8
μM). On the other hand, the sugar-modified adenosines
13-24 were inactive or almost inactive (IC50 > 100 μM),
consistent with a previous report demonstrating that the
5'-hydroxy group in the ribofuranosyl moiety is essential
for recognition by hCNT2.29 Thus, we performed struc-
ture-activity relationship (SAR) studies of the 8-modified
adenosines.
Table 1. Substrate selectivities of hCNTs.14,15,18
nucleoside transporters
substrates
CNT1
CNT2
CNT3
adenosine
guanosine
inosine
T
T
T
T
purine
nucleosides
NT
NT
T
T
T
T
cytidine
NT
NT
T
T
pyrimidine
nucleosides
thymidine
uridine
T
T
T
T
nucleobases
NT
NT
NT
T: transported, NT: not transported
Unlike human ENT inhibitors, there were few reports
about CNT inhibitors at the beginning of our research.
Among them, 2'-deoxy-5-fluorouridine,26 phlorizin,27,28
and 7,8,3'-trihydroxyflavone28 are reported to be strong or
moderate hCNT2 inhibitors (Figure 1). Evaluation of the
inhibitory effects of these compounds on sodium-
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ACS Medicinal Chemistry Letters
Scheme 1. Synthesis of 8-substituted adenosinesa
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Figure 2. Structures of modified adenosines evaluated in an
initial study.
The 8-substituted adenosines evaluated in this study
were synthesized from commercially available 8-
bromoadenosine 25 as a common starting material
(Scheme 1). Aromatic nucleophilic substitution reactions
of compound 25 at the 8-position with appropriate
amines and BnSH gave 8-aminoadenosine derivatives 9-12,
27, 28, 32-48 and 8-(benzylsulfanyl)adenosine 31, respec-
tively.30 O-Silylation of compound 25 and subsequent Pd-
catalyzed coupling reaction with PhNH2 followed by
treatment with NH4F gave 26. O-Acetylation of com-
pound 25 and subsequent Sonogashira coupling with
aReagents and conditions: (a) aqueous R3R5NH, EtOH, 100
°C, in a sealed tube; (b) R3R5NH, i-Pr2NEt, EtOH or PrOH,
120 °C, in a sealed tube; (c) R3R5NH, i-Pr2NEt, EtOH, 150 °C,
ethynylbenzene
gave
2',3',5'-tri-O-acetyl-8-
(phenylethynyl)adenosine, which was then reduced under
catalytic hydrogenation condition before treatment with
NaOMe to afford 29. O-Silylation of compound 25 and
subsequent aromatic nucleophilic substitution reaction at
8-position with BnOH followed by treatment with NH4F
gave 30.
in
a
sealed tube, microwave irradiation; (d) tert-
butyldimethylsilyl chloride, imidazole, DMF, ambient temp.;
(e) PhNH2, tris(dibenzylideneacetone)dipalladium(0), tert-
BuONa, (±)-BINAP, toluene, 80 °C; (f) NH4F, MeOH, reflux;
(g) Ac2O, Et3N, 4-dimethylaminopyridine, MeCN, ambient
temp.; (h) ethynylbenzene, (Ph3P)4Pd, CuI, Et3N, DMF, 80 °C;
(i) H2, 10% Pd-C, AcOEt, ambient temp.; (j) NaOMe, MeOH,
ambient temp.; (k) BnOH, tert-BuOK, 40 °C then (l) NH4F,
MeOH, reflux; (m) BnSH, i-Pr2NEt, EtOH, 120 °C, in a sealed
tube.
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The inhibitory effects of analogs of 12 were investigated In conclusion, we identified a new class of potent hCNT2
1
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5
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8
(Table 2). Both deletion and elongation of the methylene
linker between the nitrogen and benzene ring decreased
the inhibitory activity (compound 12 vs 26-28). Replace-
ment of the nitrogen with carbon, oxygen, or sulfur was
also disadvantageous (compound 12 vs 29-31). Methyla-
tion of the nitrogen or at the benzylic position led to a
complete loss of activity (compound 12 vs 32-34). These
findings indicate that, in the 8-substituent of compound
12, the methyleneamino (-CH2NH-) structure was essen-
tial for the inhibitory effect, and therefore we performed
SAR studies on derivatives of compound 12 by modifying
the terminal phenyl moiety (Table 3). Introduction of a
substituent at the 3- or 4-position on the benzene ring
improved the inhibitory activity regardless of the elec-
tronic nature (compound 12 vs 36, 37, 39, 40, 42, and 43).
In contrast to the 2-chloro derivative 38, both the 2-
methyl derivative 35 and 2-methoxy derivative 41 were
less active than compound 12, suggesting a spatial limita-
tion around the position in the binding mode. An elec-
tron-withdrawing group on the benzene ring might be
preferred because all chloro derivatives exhibited more
potent activity than the corresponding methyl or methoxy
derivatives. On the other hand, replacement of the phenyl
group with a 1- or 2-naphthyl group also enhanced the
inhibitory activity (compound 44 and 45), regardless of
the substituted position. Based on these results, a space
around the phenyl moiety that accommodates rather
large substituents might be present in the binging mode.
Therefore, we designed biphenyl derivatives 46-48. The
biphenyl-2-yl derivative 46 was inactive, as expected. The
biphenyl-3-yl derivative 47 exhibited 3-fold more potent
inhibitory activity than compound 12, but the activity did
not surpass naphthyl derivatives 44 and 45. The introduc-
tion of a biphenyl-4-yl group, however, was highly effec-
tive, and the biphenyl-4-yl derivative 48 exhibited 81-fold
more potent inhibitory activity than the parent com-
pound 12. In addition, compound 48 exhibited inhibitory
activity 1500-fold more potent than that of 2'-deoxy-5-
fluorouridine, phlorizin, and 7,8,3'-trihydroxyflavone,
which are well-known hCNT2 inhibitors.
inhibitors. Starting with a natural substrate, adenosine,
we successfully identified 8-aminoadenosine derivative 12
and subsequent SAR studies led to the discovery of com-
pound 48 (IC50 = 0.64 ± 0.19 μM). Conformational analy-
sis data of compound 48 suggested the preference for anti
conformers, and therefore valuable information for specu-
lating the bioactive conformation was provided. The in-
hibitor 48 can be a prototype compound for the develop-
ment of conceptually new drugs for the treatment of
hyperuricemia and gout.
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Table 2. Inhibitory Effects on Sodium-dependent In-
osine Uptake in COS-7 Cells Transiently Expressing
hCNT2.
compd
12
R6
Inh.%a
58
IC50 (µM)b
PhCH2NH-
52 ± 3.8
26
PhNH-
36
289 ± 87
27
Ph(CH2)2NH-
Ph(CH2)3NH-
Ph(CH2)2-
39
233 ± 19
28
26
536 ± 89
29
<5
-
-
-
-
-
-
30
PhCH2O-
<5
31
PhCH2S-
<5
32
PhCH2N(Me)-
(R)-PhCH(Me)NH-
(S)-PhCH(Me)NH-
<5
33
<5
34
<5
Finally, we performed a conformational analysis of the
most potent compound 48 by the density functional theo-
ry (DFT) B3LYP/6-31g* method in Spartan'10 to get insight
into the bioactive conformation. The calculations demon-
strated that compound 48 is likely to take predominantly
the anti conformation rather than the syn conformation,
of which energy difference was 3.27 kcal/mol.31 The pref-
erence for the anti conformers to the syn conformers
would be due to the intramolecular hydrogen bonding
formed by the N-H of the 8-substituent with the 5’-
hydroxy oxygen and the ribose ring oxygen. These con-
formational analysis results are in accord with structure-
activity relationship results that the N-H moiety in the 8-
substituents is essential for exhibiting the inhibitory ac-
tivity against hCNT2.
2'-deoxy-5- fluorouridine
phlorizin
17
14
-
-
-
7,8,3'-trihydroxyflavone
<5
aInhibition% at 100 µM. If more than 20% inhibition was
observed, the IC50 value was calculated. bConcentration of
each compound required to inhibit inosine uptake by 50%.
Data represent mean ± standard error of the mean of at least
three independent experiments unless otherwise stated.
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ACS Medicinal Chemistry Letters
Corresponding Author
Table 3. Inhibitory Effects on Sodium-dependent In-
1
2
3
4
osine Uptake in COS-7 Cells Transiently Expressing
hCNT2.
*Phone: +81-263-82-8820. Fax: +81-263-82-8827. E-mail:
kazuya_tatani@pharm.kissei.co.jp
Author Contributions
5
6
The manuscript was written through contributions of all
authors.
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8
ACKNOWLEDGMENT
9
We thank Mr. Shinjiro Watanabe for providing biological
activity data. We thank Dr. Tomonaga Ozawa for performing
conformational analysis.
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compd
12
R7
IC50 (µM)a
52 ± 3.8
65 ± 5.9
19 ± 2.3
11 ± 4.2
5.7 ± 1.2
5.4 ± 1.3
11 ± 2.8
>100
ABBREVIATIONS
phenyl
RNA, ribonucleic acid; DNA, deoxyribonucleic acid; CNT,
concentrative nucleoside transporter; ENT, equilibrative
nucleoside transporter; RT-PCR, reverse transcription-
polymerase chain reaction
35
2-methylphenyl
3-methylphenyl
4-methylphenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
1-naphthyl
36
37
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41
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13 ± 5.7
36 ± 13
43
44
45
5.7 ± 1.1
5.3 ± 1.7
>100
2-naphthyl
46
47
biphenyl-2-yl
biphenyl-3-yl
biphenyl-4-yl
21 ± 9.8
0.64 ± 0.19
48
2'-deoxy-5- fluorouridine
phlorizin
>1000b
>1000b
>1000b
7,8,3'-trihydroxyflavone
aConcentration of each compound required to inhibit inosine
uptake by 50%. Data represent mean ± standard error of the
mean of at least three independent experiments unless oth-
erwise stated. bInhibition was less than 50% at 1000 μM.
ASSOCIATED CONTENT
Supporting Information. Synthetic procedures and charac-
terization data of all synthesized compounds, assay method,
and conformational analysis data of compound 48. This ma-
terial is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
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ACS Medicinal Chemistry Letters
Identification of 8-Aminoadenosine Derivatives as a New Class of Human Concentrative Nucleoside Transporter 2
Inhibitors
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