Structure-Activity Relationships of Neplanocin A Analogues as S -Adenosylhomocysteine Hydrolase Inhibitors and Their Antiviral and Antitumor Activities

Article abstract of DOI:10.1021/acs.jmedchem.5b00553

On the basis of the potent inhibitory activity of neplanocin A (1) against S-adenosylhomocysteine (AdoHcy) hydrolase, we analyzed the comprehensive structure-activity relationships by modifying the adenine and carbasugar moiety of 1 to find the pharmacoph

Full text of DOI:10.1021/acs.jmedchem.5b00553

Article
pubs.acs.org/jmc
Structure−Activity Relationships of Neplanocin A Analogues as
S‑Adenosylhomocysteine Hydrolase Inhibitors and Their Antiviral
and Antitumor Activities
Girish Chandra,†,‡,∥ Yang Won Moon,§,∥ Yoonji Lee,§,∥ Ji Yong Jang,§ Jayoung Song,† Akshata Nayak,†
Kawon Oh,†,§ Varughese A. Mulamoottil,§ Pramod K. Sahu,† Gyudong Kim,† Tong-Shin Chang,§
Minsoo Noh,† Sang Kook Lee,† Sun Choi,*,§ and Lak Shin Jeong*,†
†Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
‡Department of Chemistry, School of Chemical and Physical Sciences, Central University of Bihar, Gaya, Bihar, 823001, India
§College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Korea
S
* Supporting Information
ABSTRACT: On the basis of the potent inhibitory activity of neplanocin
A (1) against S-adenosylhomocysteine (AdoHcy) hydrolase, we analyzed
the comprehensive structure−activity relationships by modifying the
adenine and carbasugar moiety of 1 to find the pharmacophore in the
active site of the enzyme. The introduction of 7-deazaadenine instead of
adenine eliminated the inhibitory activity against the AdoHcy hydrolase,
while 3-deazaadenine maintained the inhibitory activity of the enzyme,
indicating that N-7 is essential for its role as a hydrogen bonding acceptor.
The substitution of hydrogen at the 6′-position with fluorine increased the inhibitory activity of the enzyme. The one-carbon
homologation at the 5′-position generally decreased the inhibitory activity of the enzyme, indicating that steric repulsion exists. A
molecular docking study also supported these experimental data. In this study, 6′-fluoroneplanocin A (2) was the most potent
inhibitor of AdoHcy hydrolase (IC50 = 0.24 μM). It showed a potent anti-VSV activity (EC50 = 0.43 μM) and potent anticancer
activity in all the human tumor cell lines tested.
Recently, 7-deazaneplanocin A (3) was reported to show
potent antiviral activity against the hepatitis C virus (HCV) and
cowpox along with enzymatic stability, indicating that the 7-
deazaadenine moiety may serve as a good template for the
design of antiviral agents.9,10 Additionally, 3-deazaneplanocin A
(4) is a powerful inhibitor of AdoHcy hydrolase with potent
and selective in vitro and in vivo antiviral activities.11 The one-
carbon homologated analogues of 1 also exhibited a potent
inhibitory activity against AdoHcy hydrolase with potent
antiviral activities.12
Thus, it is of great interest to determine the systematic
structure−activity relationships of AdoHcy hydrolase inhibitors
by modifying the 5′ or 6′ position of the carbasugar moiety
and/or the N6, 3, or 7 position of the purine moiety of the
carbocyclic nucleosides, 1−4. Herein, we report the stereo-
selective synthesis of the neplanocin A analogue 5, using
stereoselective epoxidation, the regio- and stereoselective
fluorination, and their AdoHcy hydrolase inhibitory activity
along with their antiviral and anticancer activities.13
INTRODUCTION
■
S-Adenosyl-L-homocysteine (AdoHcy) hydrolase catalyzes the
interconversion of AdoHcy to adenosine and L-homocysteine
(Hcy).1,2 The inhibition of AdoHcy hydrolase accumulates
AdoHcy in the cell, which triggers the negative feedback
inhibition to suppress the S-adenosyl-L-methionine (SAM)
dependent transmethylation.1,2 Because SAM-dependent trans-
methylation plays an essential role in forming the capped
methylated structure at the 5′-terminus of viral mRNA,
AdoHcy hydrolase has been an attractive target for the
development of a broad-spectrum of antiviral agents.3−5
Neplanocin A (1)6 is a representative carbocyclic nucleoside
and is known to be one of the most potent inhibitors against
AdoHcy hydrolase, and it exhibits potent and wide-ranging
antiviral activities (Figure 1).7
However, despite the potent antiviral activities against several
RNA and DNA viruses, its high cytotoxicity has hindered it
from being further developed as a clinical agent.5,7 To search
for new carbocyclic nucleosides with a therapeutic index better
than 1, many modifications have been made on the carbasugar
ring and on the purine base. Among these, fluoroneplanocin A
(2) was discovered as a highly potent inhibitor of AdoHcy
hydrolase, exhibiting both the type I mechanism based
reversible cofactor (NAD) depletion and the type II mechanism
based irreversible inhibition.8
Received: April 8, 2015
© XXXX American Chemical Society
A
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Figure 1. Rationale for the design of the target nucleoside 5.
a
Scheme 1
a
Reagent and conditions: (a) NaBH4, CeCl3·7H2O, 0 °C to rt, 1 h; (b) adenine (for 1) or 7-deaza-6-chloropurine (for 3), Ph3P, DIAD, THF, 0 °C
to rt, 15 h; (c) NH3, MeOH, 100 °C, 24 h for 1; NH3, MeOH, 120 °C, 40 h for 3; (d) 2 N HCl, THF, MeOH, 55 °C, 15 h followed by NaHCO3
solution; (e) MeNH2 (40% in H2O), MeOH, 80 °C, 15 h; (f) 1,3-dioxane, 1 N HCl, 100 °C, 50 h.
Scheme 2
B
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After the synthesis of the epoxides, 9a and 9b, we attempted
the nucleophilic opening of the epoxide with fluoride by
employing the same conditions used in the synthesis of 2.13
However, unlike the successful openings of the one-carbon
homologated α- and β-epoxides with fluoride (n-Bu4NH2F3,
KHF2), the same fluorination reactions of 9a and 9b did not
give the desired fluorinated compounds, 10a and 10b,
respectively. The bulky trityl protecting group may have
prevented the approach of the fluoride anion to the epoxide.
To circumvent this problem, we increased the nucleophilicity
of the fluoride by changing the polarity of the solvent. To
increase the polarity of the solvent, we used a room
temperature ionic liquid (RTIL)17 as the polar solvent in the
fluorination reaction. The treatment of the α-epoxide 9a with n-
Bu4NH2F3 and KHF2 in the presence of 1-butyl-3-methyl-
imidazolium tetrafluoroborate ([bmim][BF4]) at 140 °C in a
glass seal tube produced the desired fluorinated derivative 10a
but in only a 19% yield. The low yield was attributed to the
decomposition of the ionic liquid at high temperature; thus, we
changed the ionic liquid [bmim][BF4] to 1-butyl-3-methyl-
imidazolium hexafluorophosphate ([bmim][PF6]), which pro-
duced the desired fluoro derivative 10a in a 70% yield.
However, the β-epoxide 10b was totally inert under various
reaction conditions, such as the use of different ionic liquids,
the increase of the reaction temperature, and microwave
irradiation. The lack of reactivity may be attributed to the steric
hindrance caused by the bulky trityl and the isopropylidene
groups that make it difficult for the incoming fluoride
nucleophile to approach from the concave side.
RESULTS AND DISCUSSION
■
Chemistry. First, we synthesized the 7-deazaneplanocin A
analogues, 5a and 5b, and the known analogues, 1 and 3,
starting from the key intermediate 6, which was efficiently
synthesized from D-ribose according to our previously
published procedure (Scheme 1).14
The key intermediate 6 was reduced with NaBH4 in the
presence of CeCl3·7H2O to give the glycosyl donor 7 as a single
diastereomer. The condensation of 7 with adenine under the
standard Mitsunobu conditions was followed by the removal of
the protecting groups under acidic conditions to yield 1.6 The
glycosyl donor 7 was also condensed with 3-deaza-6-
chloropurine to produce the protected nucleoside 8. The
treatment of 8 with methanolic ammonia was followed by
acidic hydrolysis to yield 3.10 Compound 8 was then converted
to the 7-deaza-N6-methylneplanocin A (5a) by treating it with a
40% methylamine solution that was followed by acidic
hydrolysis. Compound 8 was also converted to the 7-
deazahypoxanthine derivative 5b via heating with 1 N HCl in
1,4-dioxane.
Second, we synthesized compound 2 and 6′-fluoro-7-
deazaneplanocin A derivatives 5c−e by employing stereo-
selective epoxidation followed by regio- and stereoselective
nucleophilic fluorination as the key steps, as shown in Scheme
2.
For the regio- and stereoselective nucleophilic fluorination,
α-epoxide 9a was favored over β-epoxide 9b because of the
steric factor. The epoxidation of the α-allylic alcohol 7 was
expected to produce α-epoxide 9a as a major product, based on
the directing effect15 of the α-hydroxyl group; however, the
epoxidation with m-CPBA at room temperature yielded the
desired α-epoxide 9a with a modest degree of selectivity (9a/9b
= 3/1, 96% total yield was based on the recovered starting
material). This result indicated that the steric factor created by
the bulky trityl group competed with the electronic factor of the
α-hydroxyl group. There have been many attempts to improve
the selectivity of the epoxidation. Because of the acid-labile
trityl protecting group, the epoxidation with m-CPBA was
conducted in the presence of NaHCO3 to neutralize the m-
chlorobenzoic acid, which is a byproduct of the reaction. This
condition resulted in an improved total yield (80%) but a poor
selectivity (9a/9b = 1.67/1). The configurations of two
diastereomeric epoxides 9a and 9b were confirmed via the
careful analysis of the 1H NMR data and 1D NOE experiments.
Two isomers showed the typical diagnostic coupling constants
of the boat conformation of the bicyclo[3.1.0]hexane system,
which has previously been elucidated using X-ray crystallog-
The fluorodiol 10a was converted to another glycosyl donor
13, as illustrated in Scheme 3. The oxidation of 10a with
a
Scheme 3
16
1
raphy and H NMR experiments. The JH1,H6 value in the α-
epoxide 9a should be greater than zero because of the cis
relationship, while the corresponding coupling constant in the
β-epoxide 9b must be zero because of the H1−C−C−H6
dihedral angle that has a trans configuration close to 90°.
Thus, the H-6 in 9a should split into a doublet, whereas the H-
6 in 9b should appear as a singlet. Indeed, the 1H NMR analysis
of 9a and 9b indicated that the H-6 of 9a was split into a
doublet at δ 3.43 and the H-6 of 9b was a singlet at δ 3.64. In
the 1D NOE experiments, irradiation of the H-6 in 9a
enhanced the H-1 proton peak by 4.14%, while the same
irradiation in 9b produced only a 2.84% enhancement. This
experiment confirmed that the H-1 and H-6 of 9a are in a cis
configuration, while those of 9b are in a trans configuration.
a
Reagents and conditions: (a) DMSO, EDCI, TFAA, pyridine,
benzene, 0 °C to rt, 15 h; (b) MsCl, Et3N, MC, 0 °C, 2 min; (c)
NaBH4, CeCl3·7H2O, MeOH, 0 °C to rt, 1 h.
DMSO in the presence of EDCI produced β-hydroxyketone 11
as the major product (74%) and α,β-unsaturated ketone 12 as
the minor product (7%). Compound 11 was smoothly
converted to 12 by treating it with MsCl and triethylamine.13
The Luche18 reduction of 12 yielded the glycosyl donor 13,
which can readily condense with the purine bases.
For the synthesis of compound 2, the glycosyl donor 13 was
first condensed with 6-chloropurine or adenine using the
Mitsunobu conditions; however, this failed to produce the
desired condensed product in a satisfactory yield (Scheme 4).
C
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a
Scheme 4
a
Reagent and conditions: (a) MsCl, Et3N, MC, 0 °C, 40 min; (b) adenine, Cs2CO3, DMSO, 80 °C, 15 h; (c) 2 N HCl, THF, MeOH, 55 °C, 15 h;
(d) 7-deaza-6-chloropurine, Ph3P, DIAD, THF, 0 °C to rt, 15 h; (e) NH3, MeOH, 120 °C, 40 h for 5c and MeNH2 (40% in H2O), MeOH, 80 °C,
15 h for 5d; (f) HSCH2CH2OH, NaOMe, MeOH, 80 °C, 15 h.
Thus, the direct SN2 condensation using the mesylate 14 was
employed. The condensation of 14 with the adenine anion,
prepared by treating adenine with Cs2CO3 in DMSO yielded
the desired nucleoside 15 in good yield. The removal of the
protecting groups of 15 was achieved with 2 N HCl in
methanol and THF at 55 °C to give the final compound 2.8
Then, 7-deaza-6-chloropurine was successfully condensed with
the glycosyl donor 13 under the standard Mitsunobu
conditions to yield the condensed nucleoside 16 in an excellent
yield. The treatment of 16 with methanolic ammonia or 40%
aqueous methylamine in MeOH, followed by the acidic
hydrolysis yielded the 7-deazafluoroneplanocin A (5c) or the
7-deaza-N6-methyfluoroneplanocin A (5d), respectively. Com-
pound 16 was converted to the desired 7-deazahypoxanthine
derivative 5e, in addition to the formation of the undesired
oxathiolanyl derivative 17, by treating with 2-mercaptoethanol
and sodium methoxide at 80 °C, followed by the removal of the
protecting groups under acidic conditions. The structure of 17
reported procedure.13 The condensation of 1813 with 6-
chloropurine or 7-deaza-6-chloropurine using the Mitsunobu
conditions yielded the condensed derivative, 20 or 21,
respectively. These compounds were then treated with
trifluoroacetic acid (TFA), and the resulting 6-chloro
derivatives 23 and 24 with ammonia in t-BuOH to yield
homoneplanocin A (5f)13 and 7-deazahomoneplanocin A (5g),
respectively. The glycosyl donor 18 was also condensed with
N,N-(BOC)2-3-deazaadenine to produce the protected nucleo-
side 22, which was converted to the final 3-deazahomonepla-
nocin A (5h), using a similar procedure as in the preparation of
5f and 5g. The condensation of 19 with 6-chloropurine
produced the 6-chloro derivative 25, whose protecting groups
were removed to give 26. Finally, the treatment of 26 with
ammonia in t-BuOH yielded fluorohomoneplanocin A (5i).
However, the desired 3-deazafluorohomoneplanocin A was not
obtained because the condensation of 19 with 3-deaza-6-
chloropurine,11 6-azido-3-deazapurine,19 or N,N-(BOC)2-3-
deazaadenine19 resulted in almost exclusive formation of the
N7-derivatives. The N7-regioisomers were confirmed by
comparing their UV or NOE data with those of the reported
N7-isomers.11,19
1
was confirmed using H NMR, which exhibited four extra
protons at δ 3.51 (t, J = 6.4 Hz, 2 H) and δ 3.85 (t, J = 6.4 Hz,
2 H).
The homoneplanocin A analogues, 5f−i were synthesized
from D-ribose as shown in Scheme 5. D-Ribose was converted to
the glycosyl donors, 18 and 19, according to our previously
AdoHcy Hydrolase Inhibitory Activity. All the final
compounds 1−5 were assayed for inhibitory activity against
D
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a
Scheme 5
a
Reagent and conditions: (a) 6-chloropurine or 7-deaza-6-chloropurine or 3-deaza-N-BOC-adenine, Ph3P, DIAD, THF, 0 °C to rt, 15 h; (b) TFA,
THF, rt, 18 h; (c) NH3, t-BuOH, 120 °C, 24 h; (d) 6-chloropurine, Ph3P, DIAD, THF, 0 °C to rt, 15 h; (e) 2 N HCl, THF, MeOH, 55 °C, 15 h; (f)
NaOMe, MeOH, 45 °C, 4 h.
Table 1. AdoHcy Hydrolase Inhibitory Activity of the Neplanocin A Analogues 1−5
compd
1 (neplanocin A)
X, Y, Z, R1, R2
IC50 (μM)
X = H, Y = N, Z = N, R1 = NH2, R2 = OH
X = F, Y = N, Z = N, R1 = NH2, R2 = OH
X = H, Y = CH, Z = N, R1 = NH2, R2 = OH
X = H, Y = N, Z = CH, R1 = NH2, R2 = OH
X = H, Y = CH, Z = N, R1 = NHMe, R2 = OH
X = H, Y = CH, Z = N, R1 = OH, R2 = OH
X = F, Y = CH, Z = N, R1 = NH2, R2 = OH
X = F, Y = CH, Z = N, R1 = NHMe, R2 = OH
X = F, Y = CH, Z = N, R1 = OH, R2 = OH
X = H, Y = N, Z = N, R1 = NH2, R2 = CH2OH
X = H, Y = CH, Z = N, R1 = NH2, R2 = CH2OH
X = H, Y = N, Z = CH, R1 = NH2, R2 = CH2OH
X = F, Y = N, Z = N, R1 = NH2, R2 = CH2OH
0.47
2 (fluoroneplanocin A)
3 (7-deazaneplanocin A)
4 (3-deazaneplanocin A)
5a (7-deaza series)
0.24
>100
0.44
>100
>100
>100
>100
>100
0.48
5b (7-deaza series)
5c (7-deaza series)
5d (7-deaza series)
5e (7-deaza series)
5f (homoneplanocin A)
5g (7-deazahomoneplanocin A)
5h (3-deazahomoneplanocin A)
5i (fluorohomoneplanocin A)
>100
3.76
0.91
AdoHcy hydrolase using the recombinant AdoHcy hydrolase
protein, which was obtained from E. coli BL21 that harbored
the plasmid pET14b containing the DNA-encoding human
placental AdoHcy hydrolase (Table 1).8 All the final
compounds were preincubated with AdoHcy hydrolase at
various concentrations for 10 min at 37 °C, and the hydrolytic
activity of the enzyme was determined using a 5,5′-dithiobis-2-
nitrobenzoate (DTNB) coupled assay as described by Lozada-
Ramirez et al.20
The effect of the purine base modification (Y, Z) was first
analyzed to determine its inhibitory activity against AdoHcy
hydrolase. The 7-deazaadenine derivatives did not exhibit
inhibitory activity, whereas the 3-deazaadenine and adenine
derivatives exhibited potent inhibitory activity. In the 7-
deazaadenine series, substitution of the N6-amino group (R1)
with the N6-methylamino or the 6-hydroxyl group did not
improve the inhibitory activity. Additionally, the effect of the
fluorine atom (X) on the sugar moiety was examined. The
introduction of fluorine (X = F) at the 6′-position generally
E
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Table 2. Anticancer Activity of the Neplanocin A Analogues 1−5
IC50 (μM)
a
b
c
d
e
f
compd
A549
HCT116
MDA-MB-231
PC3
SK-hep-1
SNU-638
1 (X = H, Y = N, Z = N, R1 = NH2, R2 = OH)
2 (X = F, Y = N, Z = N, R1 = NH2, R2 = OH)
3 (X = H, Y = CH, Z = N, R1 = NH2, R2 = OH)
4 (X = H, Y = N, Z = CH, R1 = NH2, R2 = OH)
5a (X = H, Y = CH, Z = N, R1 = NHMe, R2 = OH)
5b (X = H, Y = CH, Z = N, R1 = OH, R2 = OH)
5c (X = F, Y = CH,Z = N, R1 = NH2, R2 = OH)
5d (X = F, Y = CH, Z = N, R1 = NHMe, R2 = OH)
5e (X = F, Y = CH, Z = N, R1 = OH, R2 = OH)
5f (X = H, Y = N, Z = N, R1 = NH2, R2 = CH2OH)
5g (X = H, Y = CH, Z = N, R1 = NH2, R2 = CH2OH)
5h (X = H, Y = N, Z = CH, R1 = NH2, R2 = CH2OH)
5i (X = F, Y = N, Z = N, R1 = NH2, R2 = CH2OH)
Ara-C
1.22
0.9
0.4
3.7
4.5
0.7
1.20
2.8
1.4
1.4
0.8
2.2
1.84
10.44
0.26
>100
>100
0.94
>100
>100
1.72
32.03
17.94
2.90
2.42
0.79
0.30
49.54
>100
0.94
>100
>100
2.64
26.26
8.01
6.95
1.37
7.7
1.62
9.39
1.17
0.72
99.18
>100
1.39
>100
>100
6.12
42.09
28.89
8.07
2.12
1.2
1.40
0.91
>100
>100
0.96
>100
>100
2.31
30.19
28.16
3.73
3.98
0.3
>100
>100
16.84
>100
>100
8.95
>100
>100
1.3
>100
>100
>100
>100
>100
>100
50.51
34.01
76.48
11.70
1.20
g
etoposide
0.4
1.9
ND
2.8
a
b
c
d
A549: human lung cancer cell line. HCT-116: human colon cancer cell line. MDA-MB-231: human breast cancer cell line. PC3: human prostate
e
f
g
cancer cell line. SK-hep-1: human liver cancer cell line. SNU-638: human stomach cancer cell line. ND: not determined.
Figure 2. Structure of the AdoHcy hydrolase complexed with fluoroneplanocin A (2). (A) Key pharmacophores at the active site (red, hydrogen
bond acceptor; blue, hydrogen bond donor; yellow, hydrophobe; gray sphere, exclusion volume). The cocrystallized ligand is displayed using sticks
with the carbon color of gray, and its N-3 and N-7 positions are marked with red circles. The His353 and Asp190 residues are represented using
sticks with their carbons in white. (B) Monomer structure of the enzyme. The secondary structures of the catalytic, cofactor binding, and C-terminal
domains are colored in light blue, sky blue, and orange, respectively, while the hinge region is in pink. The locations of the His353 and Asp190
residues in the hinge region are depicted as the spheres. The bound ligand and cofactor are displayed as green balls-and-sticks and green blue sticks,
respectively.
increased the inhibitory activity against the AdoHcy hydrolase.
For example, the 6′-fluoro derivatives 2 and 5i were more
potent than the corresponding 6′-H analogues, 1 and 5f,
respectively. Finally, the effect of the homologation (R2) was
studied. Compound 2 (IC50 = 0.36 μM) exhibited a more
potent inhibitory activity than the corresponding 5′-homo
derivative 5i (IC50 = 0.91 μM), indicating that homologation
slightly decreased the inhibitory activity. Among the com-
pounds tested, compound 2 exhibited the most potent
inhibitory activity against the AdoHcy hydrolase.
(HIV, MT-4 cells) 1, picornavirus (PC, CB-1 cell), and
vesicular stomatitis virus (VSV, HeLa cells).21 Half of the
synthesized compounds exhibited toxicity-dependent antiviral
activities. Among the compounds tested, 5c showed potent
anti-PC activity (EC50 = 2.34 μM) without cytotoxicity up to
100 μM, although it was inactive against AdoHcy hydrolase.
However, compound 2 showed the most potent inhibitory
activity against the AdoHcy hydrolase and exhibited potent
anti-VSV activity (EC50 = 0.43 μM) without cytotoxicity up to
40 μM. Because most of the synthesized compounds exhibited
high cytotoxicity in various cell lines, they were tested for
anticancer activity in various cancer cell lines.
Antiviral Activity. All of the synthesized compounds 1−5
were tested for antiviral activity against herpes simplex virus
(HSV, Vero cell) 1 and 2, human immunodeficiency virus
F
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Figure 3. Binding mode comparison of the 3- or 7-deaza and the homologated neplanocin A analogues. The docked poses of compounds 1A, 4B,
4C, 5f, and 3D are represented as balls-and-sticks, and their carbon atoms are colored in purple, sky blue, yellow, and gray, respectively. The
secondary structure of the AdoHcy hydrolase is colored light blue, and the interacting residues are depicted as sticks. For a comparison of the
carbocyclic ring conformation, the binding modes of (E) 1 and 5f, (F) 2 and 5i, and (G) 4 and 5h are aligned. The colors of the carbon atoms of 2,
5i, and 5h are in pink, orange, and slate, respectively. The H-bonds are displayed as black dashed lines, and the nonpolar hydrogens are not displayed
for clarity.
Anticancer Activity. All of the synthesized neplanocin A
analogues 1−5 were also assayed for anticancer activity against
several human cell lines, such as human lung cancer cell line
(A549), human colon cancer cell line (HCT-116), human
breast cancer cell line (MDA-MB-231), human prostate cancer
cell line (PC3), human liver cancer cell line (SK-hep-1), and
human stomach cancer cell line (SNU-638).22
analogues, we utilized the X-ray crystal structure of AdoHcy
hydrolase complexed with the most potent compound 2 (PDB
code 3NJ4).8c The pharmacophore analysis of the complex
structure clearly supports the activity differences between the 3-
deaza and the 7-deaza compounds. As shown in Figure 2, the
N-7 position of the ligand functions as a hydrogen bond
acceptor, whereas the N-3 position does not play a role in the
inhibitory activity.
As shown in Table 2, all of the adenine, 3-deaza- and 7-
deazaadenine derivatives except for the N6-methyladenine
derivatives, 5a and 5d, and hypoxanthine derivatives, 5b and
5e, exhibited potent anticancer activity in all of the human
cancer cell lines tested. In the base-modified series (Y, Z), the
anticancer activity is in the following order: 4 (Y = N, Z = CH)
> 1 (Y = N, Z = N) > 3 (Y = CH, Z = N). Introduction of the
fluorine (X = F) at the sugar moiety generally exhibited an
improved anticancer activity over the corresponding hydrogen
derivatives (X = H). For example, the 6′-F derivatives 2, 5c, and
5i exhibited an improved anticancer activity over the
corresponding 6′-H derivatives 1, 3, and 5f, respectively. The
one-carbon homologation at the 5′-position generally decreased
the anticancer activity substantially. For example, the 5′-
CH2OH analogues 5f, 5g, 5h, and 5i were less potent than the
corresponding 5′-OH analogues 1, 3, 4, and 2, respectively.
Among the tested compounds, compound 4 exhibited the most
potent anticancer activity against the human colon cancer
(HCT116), breast cancer (MDA-MB-231), and liver cancer
(SK-Hep-1) cell lines; however, compound 2 exhibited the
most potent anticancer activity against the human lung cancer
(A549) and prostate cancer (PC3) cell lines. Compound 1 was
the most potent in the stomach cancer cell (SNU-638) line.
Molecular Modeling Study. To investigate the inter-
actions between the AdoHcy hydrolase and the synthesized
For a more detailed comparison of the binding modes,
flexible docking studies were performed using Glide (Schro-
̈
dinger, LLC, NY, USA).
Compound 1 and its 3-deaza derivative 4, which both
showed inhibitory activities with the IC50 values of 0.47 μM and
0.44 μM, respectively, both bound well at the active site of the
AdoHcy hydrolase with their adenine or 3-deaza adenine parts
while maintaining the H-bonding interactions with Thr57,
Glu59, and His353 (Figure 3A and Figure 3B). However, the 7-
deaza derivative 3, whose inhibitory activity was drastically
diminished, could not maintain the H-bonding with His353
(Figure 3D). All of the 7-deaza compounds lost the H-bonding
with His353 (Figure S1) and exhibited much lower docking
scores compared with the neplanocin A and 3-deaza neplanocin
A analogues (Figure S2). The interaction energies between the
ligands and His353, especially the H-bonding and Coulombic
scores, are important for their binding. Therefore, the N-7 of
the adenine ring, which H-bonds with His353, functions as a
critical pharmacophore for the enzyme inhibition.
For the homologated compounds, the structure−activity
relationships are more complicated. The 5′-homo derivative 5f,
which has a similar inhibitory activity as compared with 1, binds
easily at the active site, retaining the H-bonding network of the
adenine portion (Figure 3C). Additionally, its carbocyclic ring
G
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Journal of Medicinal Chemistry
Article
spectra (CDCl3, CD3OD) were recorded on Varian Unity Inova 376
MHz instrument. The chemical shifts were reported as parts per
million (δ) relative to the solvent peak. Optical rotations were
determined on Jasco III in appropriate solvent. UV spectra were
recorded on a U-3000 instrument made by Hitachi in methanol or
water. Infrared spectra were recorded on FT-IR (FTS-135) instrument
made by Bio-Rad. Melting points were measured on a B-540 made by
Buchi. Elemental analyses (C, H, and N) were used to determine
purity of all synthesized compounds, and the results were within
0.4% of the calculated values, confirming ≥95% purity. Reactions
were checked with TLC (Merck precoated 60F254 plates). Spots were
detected by viewing under a UV light, colorizing with charring after
dipping in anisaldehyde solution with acetic acid, sulfuric acid, and
methanol. Column chromatography was performed on silica gel 60
(230−400 mesh, Merck). Reagents were purchased from Aldrich
Chemical Co. Solvents were obtained from local suppliers. All the
anhydrous solvents were distilled over CaH2, P2O5, or sodium/
benzophenone prior to the reaction.
(1S,4R,5S)-4,5-(O-Isopropylidenedioxy)-3-(trityloxymethyl)-
cyclopent-2-ene-1-ol (7). To a stirred solution of 6 (4.0 g, 9.38
mmol) in MeOH (80 mL, HPLC grade) were added CeCl3·7H2O
(3.49 g, 9.38 mmol) and NaBH4 (0.36 g, 9.38 mmol) at 0 °C, and the
mixture was stirred at rt for 1 h, quenched with water (50 mL), and
evaporated to a half volume. The mixture was extracted with EtOAC
(50 mL × 3), and the combined organic layers were washed with brine
(50 mL), dried over anhydrous MgSO4, and evaporated. The crude
residue was purified by flash column chromatography (hexanes/ethyl
acetate = 4:1) to give 7 (3.20 g, 80%) as a white solid, whose
spectroscopic data were identical with those of authentic sample.14
(1S,4R,5S)-1-(6-Chloro-7-deaza-purine-9-yl)-4,5-(O-isopropy-
lidenedioxy)-3-(trityloxymethyl)-2-cyclopentene (8). To a
stirred solution of PPh3 (1.60 g, 6.13 mmol) in anhydrous THF (25
mL) was added DIAD (1.2 mL, 6.13 mmol) dropwise at 0 °C, and the
mixture was stirred at the same temperature for 10 min. To this
solution was added 7 (1.05 g, 2.45 mmol) in anhydrous THF (30 mL)
at 0 °C, and the resulting solution was stirred at rt for 30 min. To this
solution was added 6-chloro-7-deazapurine (0.38 g, 2.45 mmol), and
the mixture was further stirred at rt for 15 h. The solvent was
evaporated and the residue was purified by flash silica gel column
chromatography (hexanes/ethyl acetate = 4:1) to afford 8 (0.88 g, with
DIAD byproduct) as white foam: 1H NMR (400 MHz, CDCl3) δ 1.31
(s, 3H), 1.45 (s, 3H), 3.83−3.87 (m, 1H), 4.01−4.05 (m, 1H), 4.61 (d,
J = 5.6 Hz, 1H), 5.24 (d, J = 5.6 Hz, 1H), 5.89 (s, 1H), 6.02 (br s, 1H),
6.61 (d, J = 3.2 Hz, 1H), 7.08 (d, J = 3.6 Hz, 1H), 7.23−7.32 (m,
10H), 7.45−7.47 (m, 5H), 8.69 (s, 1H). HRMS (ESI) calculated for
C34H31ClN3O3: 564.1981. Found (M + H)+: 564.2054. Anal. Calcd for
C34H31ClN3O3: C, 72.40; H, 5.36; N, 7.45. Found: C, 72.41; H, 5.43;
N, 7.05.
showed very similar conformation as 1 (Figure 3E). The region
where the 5′-CH2OH group binds is tolerable for the one-
carbon homologation. However, the homologation of the
fluorinated or 3-deaza analogues decreased the activities lightly.
Interestingly, the docking results showed that these com-
pounds, 5i and 5h, could not maintain the carbocyclic ring
conformation, resulting in the loss of H-bonding with the
Asp190 (Figure 3F, Figure 3G).
Thus, we confirmed that the His353 and Asp190 residues,
which are located at the hinge region (Figure 2B), play critical
roles in the inhibitory activities: (i) N-7 of the adenine ring H-
bonds with the His353, and this interaction is essential for the
activity; (ii) the one-carbon homologation at the 5′-position is
tolerable at the binding site, but it reduces the activities for the
fluorinated or 3-deaza analogues. This is because the H-
bonding with Asp190 is critical for the inhibitory activity, and it
requires a suitable carbocyclic ring conformation. These results
agree with our previous work that identified these hinge region
residues as hot spots for the open-to-closed domain motion,23
which suggests their importance for enzymatic function.
CONCLUSIONS
■
The neplanocin A analogues 1−5 were synthesized from D-
ribose, tested for their inhibitory activity against AdoHcy
hydrolase, and tested for antiviral and anticancer activities. The
introduction of fluorine at the 6′-position during the synthesis
of the key fluorocarbasugars 13 and 19 was achieved by
stereoselective epoxidation, stereo- and regioselective nucleo-
philic fluorination, and the simultaneous oxidation−elimination
reaction. All of the glycosyl donors 7, 13, 18, and 19 were
condensed with 6-chloropurine or adenine using the
Mitsunobu conditions or via the direct SN2 reaction to give
the neplanocin A analogues 1−5. All of the carbocyclic
nucleosides exhibited very potent inhibitory activity against
AdoHcy hydrolase except for the 7-deazaneplanocin A
analogues. Among these compounds, compound 2 exhibited
the most inhibitory activity (IC50 = 0.24 μM) against the
AdoHcy hydrolase. The pharmacophore analysis of the
complex that was docked into the structure of the AdoHcy
hydrolase clearly showed that the N-7 position of the ligand
functions as a key hydrogen bond acceptor, whereas the N-3
position does not play a role in the inhibitory activity. Most of
the compounds exhibited toxicity-dependent antiviral activity
(1S,2R,5S)-5-(6-Aminopurine-9-yl)-3-hydroxymethyl-
cyclopent-3(4)-ene-1,2-diol (1). Compound 7 (1.00 g, 2.45 mmol)
was condensed with adenine (0.798 g, 2.45 mmol), using the same
procedure used in the preparation of 8 to give the trityl derivative
(0.88 g, with DIAD byproduct) as white foam. To a solution of the
trityl derivative (0.88 g) in MeOH and THF (10 mL, 1:1 v/v) was
added 2 N HCl (0.2 mL) at 0 °C, and the mixture was heated at 55 °C
for 15 h. After cooling to rt, solvents were evaporated and the residue
was neutralized with saturated NaHCO3 solution (5 mL). The mixture
was evaporated and coevaporated with toluene and the crude residue
was purified by flash silica gel column chromatography (CH2Cl2/
MeOH = 4:1) to give 1 (1.0 g, 64%) as a white solid, whose
spectroscopic data were identical to those of authentic sample.6
(1 S , 2R , 5S ) - 5 - ( 7 - D e a z a - 6 - a m i n op u r i n e - 9 - y l ) - 3 -
hydroxymethylcyclopent-3(4)-ene-1,2-diol (3). A solution of 8
(0.30 g, 0.98 mmol) in methanolic ammonia (10 mL) was heated at
120 °C for 40 h in a steel bomb. After cooling to rt, the mixture was
evaporated, and the residue was purified by flash silica gel column
chromatography (CH2Cl2/MeOH = 9:1) to give the 6-amino
derivative (0.17 g) as white foam. To a solution of the 6-amino
derivative (0.17 g) in MeOH and THF solution (10 mL, 1:1 v/v) was
added 2 N HCl (0.2 mL) at 0 °C, and the mixture was heated at 55 °C
except for 2 (EC50 = 0.43 μM against VSV) and 5c (EC50
=
2.34 μM against PC). All of the N6-adenine derivatives
exhibited potent anticancer activity in all of the human cancer
cell lines tested, among which the 6′-F-adenine derivative 2 (X
= F, Y = N, Z = N) and the 6′-H-3-deazaadenine derivative 4
(X = H, Y = N, Z = CH) exhibited the most potent anticancer
activity. This comprehensive structure−activity relationship
analysis study and the identification of the pharmacophore at
the active site of the AdoHcy hydrolase will assist in the
therapeutic development of carbocyclic nucleosides as antiviral
and anticancer compounds.
EXPERIMENTAL SECTION
■
General Methods. 1H NMR spectra (CDCl3, CD3OD, or DMSO-
d6) were recorded on Varian Unity Invoa 400 MHz instrument. The
1H NMR data are reported as peak multiplicities: s for singlet, d for
doublet, dd for doublet of doublets, t for triplet, q for quartet, br s for
broad singlet, and m for multiplet. Coupling constants are reported in
hertz. 13C NMR spectra (CDCl3, CD3OD, or DMSO-d6) were
recorded on Varian Unity Inova 100 MHz instrument. 19F NMR
H
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Article
for 15 h. After cooling to rt, solvents were evaporated and the residue
was neutralized with saturated NaHCO3 solution (5 mL). The mixture
was evaporated and coevaporated with toluene and the crude residue
was purified by flash silica gel column chromatography (CH2Cl2/
MeOH = 4:1) to give 3 (0.17 g, 65%) as a white solid, whose
spectroscopic data were identical to those of authentic sample.10
(1S,2R,5S)-5-(6-N-Methylamine-7-deazapurine-9-yl)-3-
hydroxymethylcyclopent-3(4)-ene-1,2-diol (5a). To a solution of
8 (0.28 g, 0.92 mmol) in MeOH (20 mL) was added aqueous MeNH2
(20 mL, 40% solution in H2O), and the mixture was heated at 80 °C
for 15 h in a steel bomb. Removal of the protecting groups using the
same procedure used in the preparation of 3 and purification by silica
gel column chromatography (CH2Cl2/MeOH = 4:1) afforded 5a (0.1
for C28H28O5Na: 467.193. Found 467.1823 (M + Na)+. Anal. Calcd
for C28H28O5: C, 75.65; H, 6.35. Found: C, 75.89; H, 6.67.
(1S,2S,3R,4R,5S)-2-Fluoro-4,5-(O-isopropylidenedioxy)-1-
(trityloxymethyl)cyclopent-1,3-diol (10). To a solution of 9a (1.4
g, 3.15 mmol) in dry DMF (2 mL) were added 1-butyl-3-
methylimidazolium hexafluorophosphate (2 mL), KHF2 (0.62 g, 7.88
mmol), and tetrabutylammonium dihydrogen trifluoride (2.38 g, 7.88
mmol), and the mixture was heated in a glass sealed tube at 140 °C for
72 h. After cooling to rt, the reaction mixture was diluted with water
(50 mL) and extracted with EtOAc (50 mL × 3). The combined
organic layers were washed with brine (25 mL), dried over anhydrous
MgSO4, and evaporated. The crude residue was purified by flash silica
gel column chromatography (hexanes/ethyl acetate = 2:1) to give 10
25
25
1
g, 72%) as a white solid: mp 168−169 °C; [α]D −90.8° (c 0.12,
(1.13 g, 70%) as a white foam: [α]D −9.5° (c 0.70, CH2Cl2); H
NMR (400 MHz, CDCl3) δ 1.35 (s, 3H), 1.52 (s, 3H), 2.59 (d, J = 9.6
Hz, 1H), 2.94 (s, 1H), 3.24 (dd, J = 2.8, 9.6 Hz, 1H), 3.40 (dd, J = 0.8,
9.6 Hz, 1H), 4.22 (dd, J = 2.8, 6.0 Hz, 1H), 4.32−4.41 (m, 1H), 4.53
(dt, J = 2.0, 6.0 Hz, 1H), 4.70 (dd, J = 7.2, 7.6 Hz, 1H), 7.23−7.33 (m,
9 H), 7.40−7.43 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 24.6, 26.2,
63.9 (d, JC−F = 6.0 Hz), 71.8 (d, JC−F = 22.2 Hz), 76.0, 76.2, 79.6 (d,
JC−F = 18.4 Hz), 88.0, 103.8 (d, JC−F = 193.1 Hz), 112.3, 127.5, 128.8,
128.9, 143.5; 19F NMR (376 MHz) δ −219. HRMS (ESI) calculated
for C28H29FO5: 487.2003. Found (M + Na)+: 487.1895. Anal. Calcd
for C28H29FO5: C, 72.40; H, 6.29. Found: C, 72.76; H, 6.01.
CH3OH); UV (MeOH) λmax 275.5 nm; 1H NMR (400 MHz, CDCl3)
δ 3.06 (s, 3H), 4.19 (pseudo t, J = 5.6 Hz, 1H), 4.29−4.30 (m, 2H),
4.59 (d, J = 6.8 Hz, 1H), 5.61 (m, 1H), 5.82−5.83 (m, 1H), 6.56 (d, J
= 3.6 Hz, 1H), 7.03 (d, J = 3.6 Hz, 1H), 8.15 (s, 1H); 13C NMR (100
MHz, CD3OD) δ 28.9, 61.1, 66.6, 75.1, 80.2, 100.9, 105.9, 123.4,
127.6, 150.8, 151.3, 153.0, 159.4. HRMS (ESI) calculated for
C13H16N4O3: 276.1225. Found (2M + Na)+: 575.2340. Anal. Calcd
for C13H16N4O3: C, 56.51; H, 5.84; N, 20.28. Found: C, 56.87; H,
5.54; N, 20.01.
(1S,2R,5S)-5-(7-Deazahypoxanthine-9-yl)-3-hydroxymethyl-
cyclopent-3(4)-ene-1,2-diol (5b). To a solution of 8 (0.30 g, 0.98
mmol) in 1,4-dioxane (15 mL) was added 1 N HCl (10 mL), and the
mixture was heated at 100 °C for 50 h. After evaporating solvents, the
residue was neutralized with saturated NaHCO3 solution (20 mL) and
evaporated. The residue was purified by flash silica gel column
chromatography (CH2Cl2/MeOH = 6:1) to give 5b (0.4 g, 29%) as a
(2R,3S,4S,5R)-2-Fluoro-3-hydroxy-4,5-(O-isopropylidene-
dioxy) -3-(trityloxymethyl)cyclopentanone (11) and (4R,5R)-2-
Fluoro-4,5-(O-isopropylidenedioxy)-3-(trityloxymethyl)-
cyclopent-2-ene-1-one (12). To a stirred solution of 10 (0.56 g,
1.21 mmol) in dry DMSO (10 mL) and benzene (12 mL) were added
EDCI (0.70g, 3.63 mmol), pyridine (0.15 mL, 1.81 mmol), and TFAA
(0.097 mL, 1.81 mmol), and the reaction mixture was stirred at rt for
15 h, quenched with water (15 mL), and extracted with EtOAc (30
mL). The organic layer was washed with brine (10 mL), dried over
anhydrous MgSO4, and evaporated. The crude residue was purified by
flash silica gel column chromatography (hexanes/ethyl acetate = 5:1 to
3:1) to give 11 (0.411 g, 74%) and 12 (0.04 g, 7%) as colorless liquids.
25
white solid: mp 242−243 °C; [α]D −34.0° (c 0.10, CH3OH);
UV(MeOH) λmax 261.0 nm; 1H NMR (400 MHz, CD3OD) δ 4.21 (t,
J = 5.7 Hz, 1H), 4.31−4.32 (m, 2H), 4.60−4.61 (m, 1H), 5.67 (br s,
1H), 5.83 (d, J = 1.5 Hz, 1H), 6.70 (d, J = 3.5 Hz, 1H), 7.08 (d, J = 3.6
Hz, 1H), 7.96 (s, 1H); 13C NMR (100 MHz, CD3OD + D2O) δ 60.9,
66.7, 74.9, 80.2, 104.4, 110.2, 123.9, 127.7, 144.7, 150.1, 150.9, 162.4.
HRMS (ESI) calculated for C12H13N3O4: 263.0906. Found (2M +
Na)+: 549.1703. Anal. Calcd for C12H13N3O4: C, 54.75; H, 4.98; N,
15.96. Found: C, 54.56; H, 5.04; N, 15.58.
25
1
Compound 11. [α]D −66.8° (c 0.25, CH2Cl2); H NMR (400
MHz, CDCl3) δ 1.27 (s, 3H), 1.48 (s, 3H), 2.79 (s, 1H), 2.99 (dd, J =
2.0, 8.8 Hz, 1H), 3.77 (d, J = 8.8 Hz, 1H), 4.03 (dd, J = 4.4, 6.4 Hz,
1H), 4.38−4.40 (m, 1H), 5.32 (dd, J = 0.8, 49.6 Hz, 1 H), 7.22−7.26
(m, 4H), 7.29−7.36 (m, 11H); 13C NMR (100 MHz, CDCl3) δ 24.0
(d, JC−F = 3 Hz), 26.2 (d, JC−F = 2.9), 62.4 (d, JC−F = 6.6), 63.1(d, JC−F
= 4.4), 78.8−78.9 (m), 89.4, 94.6 (dd, JC−F = 20.6, 26.4 Hz), 113.5 (d,
JC−F = 25.6), 127.3, 127.4, 128.0, 128.1, 128.6, 142.3, 146.9, 200.2 (d,
JC−F = 13.1); 19F NMR (376 MHz) δ −223.66 . HRMS (ESI)
calculated for C28H27FO5Na: 485.1841. Found (M + Na)+: 485.1734.
Anal. Calcd for C28H27FO5: C, 72.71; H, 5.88. Found: C, 72.78; H,
5.90.
(1R,2S,3R,4R,5R)-4,5-(O-Isopropylidenedioxy)-2(3)oxirane-3-
(trityloxymethyl)cyclopent-1-ol (9a) and (1R,2R,3R,4R,5S)-4,5-
(O-Isopropylidenedioxy)-2(3)oxirane-3-(trityloxymethyl)-cy-
clopent-1-ol (9b). To a suspension of 7 (4.03 g, 9.40 mmol) and
NaHCO3 (1.60 g) in anhydrous CH2Cl2 (60 mL) was added m-CPBA
(6.45 g, 37.4 mmol) in anhydrous CH2Cl2 (60 mL) at 0 °C, and the
reaction mixture was stirred at rt for 72 h and quenched with saturated
NaHCO3 solution (100 mL). The aqueous layer was extracted with
methylene chloride, and the organic layer was washed with brine (50
mL), dried over anhydrous MgSO4, and evaporated. The residue was
purified by flash silica gel column chromatography (hexanes/ethyl
acetate = 3:1) to give the α-epoxy derivative 9a (2.2 g, 50%) as a white
foam and the β-epoxy derivative 9b (0.82 g, 17%) as a white foam with
recovered starting material 7 (1.0 g).
25
Compound 12. [α]D +6.6° (c 0.30, CH2Cl2); 1H NMR (400
MHz, CDCl3) δ 1.38 (s, 3H), 1.41 (s, 3H), 4.13 (dd, J = 16.0, 31.6 Hz,
1H), 4.23 (dd, J = 2.0, 15.2 Hz, 1H), 4.41 (J = 2.4, 6.0 Hz, 1H) 5.21
(pseudo t, J = 5.6 Hz, 1H), 7.23−7.33 (m, 10 H), 7.47−7.50 (m, 5H);
13C NMR (100 MHz, CDCl3) δ 26.4, 27.9, 58.3 (d, JC−F = 1.5 Hz),
74.0 (d, JC−F = 6.6), 74.8 (d, JC−F = 6.6), 88.0, 115.6, 127.6, 128.1,
25
1
Compound 9a. [α]D + 30.7° (c 8.25, CH2Cl2); H NMR (400
MHz, CDCl3) δ 1.32 (s, 3H), 1.58 (s, 3H), 2.77 (d, J = 11.2 Hz, AB
pattern, 1H), 3.33 (d, J = 11.2 Hz, AB pattern, 1H), 3.43−3.44 (m,
1H), 3.52 (d, part of AB pattern, J = 11.2 Hz, 1H), 4.09−4.16 (m,
1H), 4.52 (dt, J1 = 6.8, J2 = 0.8 Hz, 1H), 4.72 (d, J = 6.8 Hz, 1H),
7.22−7.32 (m, 10H), 7.41−7.44 (m, 5H); 13C NMR (100 MHz,
CDCl3) δ 26.2, 26.6, 61.4, 66.1, 66.6, 67.6, 70.0, 79.1, 87.3, 114.7,
127.4, 128.1, 128.8, 143.9. HRMS (ESI) calculated for C28H28NaO5:
467.1829. Found (M + Na)+: 467.1826. Anal. Calcd for C28H28O5: C,
75.65; H, 6.35. Found: C, 75.54; H, 6.45.
128.3, 128.8, 143.5, 146.4, 153.0 (d, JC−F = 289.1 Hz), 192.4 (d, JC−F
=
16.9); 19F NMR (376 MHz) δ −136.76. HRMS (ESI) calculated for
C28H25FO4Na: 467.1734. Found (M + Na)+: 467.1626. Anal. Calcd
for C28H25FO4: C, 75.66; H, 5.67. Found: C, 75.98; H, 5.32.
(4R,5R)-2-Fluoro-4,5-(O-isopropylidenedioxy)-3-
(trityloxymethyl)cyclopent-2-ene-1-one (12). To a stirred
solution of 11 (0.4 g, 0.866 mmol) in methylene chloride (20 mL)
was added Et3N (0.49 mL, 3.463 mmol), and the mixture was stirred at
rt for 10 min. To this solution was added a solution of MsCl (0.15 mL,
0.952 mmol) in anhydrous methylene chloride (10 mL) at −5 °C, and
the mixture was stirred at the same temperature for 5 min, quenched
with water (10 mL), and extracted with methylene chloride (25 mL).
The organic layer was washed with brine (15 mL), dried over
anhydrous MgSO4, and evaporated. The crude residue was purified by
flash silica gel column chromatography (hexanes/ethyl acetate = 3:1)
25
Compound 9b. [α]D +8.0° (c 9.35, CH2Cl2); 1H NMR (400
MHz, CDCl3) δ 1.38 (s, 3H), 1.42 (s, 3H), 2.81 (d, J = 5.2 Hz, 1H),
3.20 (d, J = 10.8 Hz, 1H), 3.64 (s, 1H), 3.73 (d, J = 10.1 Hz, 1H), 4.10
(t, J = 5.6 Hz, 1H), 4.54 (t, J = 5.6 Hz, 1H), 4.82 (dd, J1 = 5.6 Hz, J2 =
0.8 Hz, 1H), 7.21−7.31 (m, 10H), 7.45−7.47 (m, 5H); 13C NMR
(100 MHz, CDCl3) δ 24.9, 26.4, 60.6, 64.1, 68.1, 68.6, 79.8, 80.3,
113.4, 127.2, 128.1, 128.3, 128.7, 129.0, 143.9. HRMS (ESI) calculated
I
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Journal of Medicinal Chemistry
Article
to give 12 (0.38 g, 81%) as a colorless liquid, whose spectroscopic data
were identical with those of authentic sample.8
(m, 10H), 7.48−7.51 (m, 5H), 8.67 (s, 1H); 19F NMR (376 MHz) δ
−126.53. HRMS (ESI) calculated for C34H30ClFN3O3: 582.1883.
Found (M + H)+: 582.1956. Anal. Calcd for C34H29ClFN3O3: C,
70.16; H, 5.02; N, 7.22. Found: C, 70.56; H, 5.42; N, 7.02.
(1R,4R,5S)-2-Fluoro-4,5-(O-isopropylidenedioxy)-3-
(trityloxymethyl)cyclopent-2-ene-1-ol (13). To a solution of 12
(1.23 g, 2.76 mmol) in MeOH (25 mL) were added CeCl3·7H2O
(1.02 g, 274 mmol) and NaBH4 (0.1 g, 263 mmol) at 0 °C, and the
mixture was stirred at rt for 1 h. The reaction mixture was quenched
with water (30 mL) and evaporated to half volume. The residue was
diluted with water (20 mL) and extracted with ethyl acetate (50 mL ×
2). The combined organic layers were washed with brine (30 mL),
dried over anhydrous MgSO4, and evaporated. The crude residue was
purified by flash silica gel column chromatography (hexanes/ethyl
(1S,2R,5S)-5-(7-Deaza-6-aminopurine-9-yl)-4-fluoro-3-
hydroxymethylcyclopent-3(4)-ene-1,2-diol (5c). Compound 16
(0.28 g, 0.8 mmol) was converted to 5c (0.1 g, 49% for three steps) as
a white solid, according to the same procedure used in the preparation
25
of 3: mp 215−216 °C; [α]D −213.3° (c 0.13, CH3OH); UV
1
(MeOH) λmax 271 nm; H NMR (400 MHz, CD3OD) δ 4.14−4.18
(m, 1H), 4.36 (dt, J = 1,6, 5.6 Hz, 1H), 4.40 (d, J = 12.4 Hz, 1H),
4.75−4.80 (m, 1H), 5.68 (m, 1H), 6.66 (d, J = 3.6 Hz, 1H), 7.13 (d, J
= 3.6 1H), 8.08 (s, 1H); 13C NMR (100 MHz, CD3OD) 54.2, 62.6 (d,
JC−F = 17.8 Hz), 70.9 (d, JC−F = 34 Hz), 75.9 (d, JC−F = 5.5 Hz), 101.2,
104.6, 121.2, 124.0, 150.9, 152.1, 155.9 (d, J = 285.6 Hz), 158.7; 19F
NMR (376 MHz) −130.59. HRMS(ESI) calculated for C12H14FN4O3:
281.0972. Found (M + H)+: 281.1045. Anal. Calcd for C12H14FN4O3:
C, 51.43; H, 4.68; N, 19.99. Found: C, 51.03; H, 4.98; N, 19.90.
(1S,2R,5S)-5-(7-Deaza-6-N-methylaminopurine-9-yl)-4-fluo-
ro-3-hydroxymethylcyclopent-3(4)-ene-1,2-diol (5d). Com-
pound 16 (0.30 g, 0.52 mmol) was converted to 5d (0.106 g, 40%
for 3 steps) as a white solid, according to the same procedure used in
25
acetate = 3:1) to give 13 (1.195 g, 97%) as a colorless thick oil: [α]D
+19.4° (c 5.00, CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 1.41 (s, 3H),
1.42 (s, 3H), 2.78 (d, J = 9.6 Hz, 1H), 3.74−3.78 (m, 1H), 3.91 (d, J =
12.0 Hz, 1H), 4.38−4.42 (m, 1H), 4.68 (superimposed ddd, J = 3.6,
9.6 Hz, 1H), 5.12 (pseudo t, J = 6.0 HZ, 1H), 7.21−7.31 (m, 9H),
7.45−7.48 (m, 6H); 13C (100 MHz, CDCl3) δ 26.8, 27.9, 56.4, 69.3
(d, JC−F = 20.9 Hz), 74.0 (d, JC−F = 7.7 Hz), 78.9 (d, JC−F = 9.3 Hz),
87.3, 112.5, 115.9 (d, JC−F = 4.6 Hz), 127.3, 128.0, 128.8, 143.9, 157.9
(d, JC−F = 287.9 Hz); 19F NMR (376 MHz) δ −128.34. HRMS (ESI)
calculated for C28H27FO4Na: 469.1890. Found (M + Na)+: 469.1783.
Anal. Calcd for C28H27FO4: C, 75.32; H, 6.09. Found: C, 75.87; H,
6.49.
25
the preparation of 5a: mp 114−116 °C; [α]D −159.8° (c 0.10,
CH3OH); UV (MeOH) λmax 274 nm; 1H NMR (400 MHz, CD3OD)
δ 3.06 (s, 3H), 4.16 (dt, J = 2.4, 12.8 Hz, 1H), 4.34−4.37 (m, 1H),
4.41 (d, J = 12.8 Hz, 1H), 4.76 (pseudo t, J = 6.0 Hz, 1H), 5.66−5.68
(m, 1H), 6.61−6.62 (d, J = 3.6 Hz, 1H), 7.09 (d, J = 4.0 Hz, 1H), 8.14
(1S,4R,5S)-1-(6-Aminopurine-9-yl)-2-fluoro-4,5-(O-isopropy-
lidenedioxy)-3-(trityloxymethyl)-2-cyclopentene (15). To a
stirred solution of 13 (0.1 g, 0.22 mmol) in anhydrous methylene
chloride (10 mL) were added Et3N (0.09 mL, 0.67 mmol) and MsCl
(0.09 mL, 0.34 mmol) at 0 °C, and the reaction mixture was stirred at
rt for 40 min, quenched with water (10 mL), and extracted with
methlyene chloride (20 mL). The organic layer was washed with
saturated NaHCO3 solution (10 mL), dried over anhydrous MgSO4,
and evaporated. The crude residue was purified by flash silica gel
column chromatography (hexanes/ethyl acetate = 4:1) to give 14
(0.11 g, 94%) as colorless oil, which was used for the next step
immediately.
A suspension of adenine (0.06 g, 0.45 mmol) and cesium carbonate
(0.22 g, 0.67 mmol) in anhydrous DMSO (3 mL) was heated at 80 °C
for 30 min under N2 atmosphere. To this solution was added a
solution of 14 (0.11 g, 0.21 mmol) in DMSO (3 mL) dropwise at the
same temperature, and the mixture was heated at 80 °C for 15 h. The
reaction mixture was cooled to room temperature, quenched with
water (5 mL), and extracted with methylene chloride (15 mL). The
organic layer was washed with brine (20 mL), dried over anhydrous
MgSO4, and evaporated. The crude residue was purified by flash silica
gel column chromatography (hexanes/ethyl acetate = 1:1) to give 15
(80 mg, 67%) as a white foam: 1H NMR (400 MHz, CDCl3) δ 1.38 (s,
3H), 1.48 (s, 3H), 3.86 (d, J = 12.6 Hz, 1H), 4.02 (d, J = 12.6, 1H),
4.73 (t, J = 10.4, 1H), 5.52 (s, 2H), 7.24−7.50 (m, 9H), 7.45−7.50 (m,
6H), 7.82 (s, 1H), 8.32 (s, 1H). Anal. Calcd for C33H30FN5O3: C,
70.32; H, 5.36; F, 3.37; N, 12.43. Found: C, 69.99; H, 5.76; N, 12.13.
(1S,2R,5S)-5-(6-Aminopurin-9-yl)-4-fluro-3-hydroxymethyl-
cyclopent-3-ene-1,2-diol (2). To a stirred solution of 15 (0.08 g,
0.14 mmol) in MeOH and THF (10 mL, 1:1 v/v) was added 2 N HCl
(0.1 mL in 0.5 mL H2O), and the mixture was heated at 55 °C for 15
h. The mixture was evaporated and neutralized with methanolic
ammonia. The mixture was evaporated and the crude residue was
purified by flash silica gelcolumn chromatography (CH2Cl2/MeOH =
5.5:1) to give 2 (0.04 g, 88%) as a white solid, whose spectroscopic
data were identical with those of authentic sample.8
(s, 1H); 13C NMR (100 MHz, CD3OD) δ 28.9, 55.1, 63.6 (d, JC−F
=
18 Hz), 71.7 (d, JC−F = 8.7 Hz), 77.0 (d, JC−F = 5.6 Hz), 101.5, 105.9,
122.7 (d, JC−F = 2.8 Hz), 123.6, 151.3, 153.3, 156.8 (d, JC−F = 285.6
Hz), 159.4; 19F NMR (376 MHz) δ −132.0. HRMS (ESI) calculated
for C13H16FN4O3: 295.1133. Found (M + H)+: 295.1206. Anal. Calcd
for C13H16FN4O3: C, 53.06; H, 5.14; N, 19.04. Found: C, 53.46; H,
4.84; N, 19.34.
(1S,2R,5S)-5-(7-Deazahypoxanthine-9-yl)-4-fluoro-3-
hydroxymethylcyclopent-3(4)-ene-1,2-diol (5e) and (1S,2R,5S)-
4-Fluoro-3-(hydroxylmethyl)-5-(spiro[[1,3]oxathiolane-2,4′-
pyrrolo[2,3-d]pyrimidine]-7′(3′H)-yl)cyclopent-3-ene-1,2-diol
(17). To a stirred solution of 16 (0.30 g, 0.52 mmol) in MeOH (10
mL) were added 2-mercaptoethanol (0.14 mL, 2.06 mmol) and
sodium methoxide (0.11g, 2.06 mmol), and the reaction mixture was
heated at 80 °C for 15 h. After cooling to rt, the mixture was
neutralized with acetic acid and evaporated. The residue was purified
by flash silica gel column chromatography (hexanes/ethyl acetate =
6:1) to give the trityl derivative as a white foam, whose protecting
groups were removed using the same procedure used in the
preparation of 3 to give 5e (0.03 g, 9%) as a white solid and 17
(0.9 g, 58%) as a white foam.
Compound 5e. Mp 210−212 °C; [α]D25 −123.0° (c 0.1, CH2Cl2);
UV (MeOH) λmax 259.5 nm; 1H NMR (400 MHz, CD3OD) δ 4.14−
4.18 (m, 1H), 4.35 (pseudo t, J = 5.1 Hz, 1H), 4.39 (d, J = 13.0 Hz,
1H), 4.76 (pseudo t, J = 5.4 Hz, 1H), 5.74 (bs, 1H), 6.71 (d, J = 3.5
Hz, 1H), 7.11 (d, J = 3.5 Hz, 1H), 7.90 (s, 1H); 13C NMR (100 MHz,
CD3OD) δ 55.1, 63.9 (d, JC−F = 18 Hz), 71.8 (d, JC−F = 8.9 Hz), 77.2
(d, JC−F = 5.3 Hz), 104.8, 110.5, 123.0 (d, JC−F = 2.4 Hz), 123.6, 114.9,
151.0, 156.4 (d, JC−F = 285.2 Hz), 162.2; 19F NMR (376 MHz) δ
−132.39. HRMS (ESI) calculated for C12H13FN3O4: 282.0814. Found
(M + H)+: 282.0887. Anal. Calcd for C12H13FN3O4: C, 51.25; H, 4.30;
N, 14.94. Found: C, 5.65; H, 4.70; N, 14.65.
Compound 17. 1H NMR (400 MHz, CDCl3) δ 3.51 (t, J = 6.4 Hz,
2H), 3.85 (t, J = 6.4 Hz, 2H), 4.18 (td, J1 = 12.8 Hz, J2 = 2.4 Hz, 1H),
4.4−4.41 (m. 1H), 4.42−4.43 (m, 1H), 4.79 (pseudo t, J = 6.0 Hz,
1H), 5.8 (bs, 1H), 6.62 (d, J = 3.6 Hz, 1H), 7.38 (d, J = 3.2 Hz, 1H),
8.56 (s, 1H); 19F NMR (376 MHz) −132.34.
Synthesis of 5′-Homoneplanocin A Analogues. 1-
[(1R,2S,3R)-2,3-(O-Isopropylidenedioxy)-4-[2-(tert-
butyldiphenylsilyloxy)ethyl]cyclopent-4(5)en]-(6-chloropur-
ine-9-yl) (20) and Its N7-Isomer.13 To the suspension of 18 (697
mg, 1.59 mmol), PPh3 (1.04 mg, 3.97 mmol), and 6-chloropurine
(0.54 mg, 3.49 mmol) in anhydrous THF (75 mL) was added a
solution of DIAD (0.78 mL, 3.977 mmol) in dry THF (30 mL)
(1S,4R,5S)-1-(6-Chloro-7-deazapurine-9-yl)-2-fluoro-4,5-(O-
isopropylidenedioxy)-3-(trityloxymethyl)-2-cyclopentene (16).
Compound 13 (0.32 g, 0.72 mmol) was condensed with 6-chloro-7-
deazapurine (0.275 g, 1.79 mmol) according to the same procedure
used in the preparation of 8 and purified by flash silica gel column
chromatography (hexanes/ethyl acetate = 4:1) to give 16 (0.28 g, with
1
DIAD byproduct) as a white foam: H NMR (400 MHz, CDCl3) δ
1.37 (s, 3H), 1.50 (s, 3H), 3.82−3.85 (m, 1H), 4.02 (d, J = 12.2 Hz,
1H), 4.67 (t, J = 5.0 Hz, 1H), 5.55 (pseudo t, J = 4.9 Hz, 1H), 5.75
(bs, 1H), 6.66 (d, J = 3.6 Hz, 1H), 7.12 (d, J = 3.6 Hz, 1H), 7.22−7.32
J
DOI: 10.1021/acs.jmedchem.5b00553
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dropwise at 0 °C, and the reaction mixture was stirred at the same
temperature for 15 min and then at room temperature for 12 h. The
solvent was evaporated, and the residue was purified by silica gel
column chromatography (hexanes/ethyl acetate = 1:1) to give 20 (1.0
g, with DIAD byproduct) as a white foam and its N7-isomer (0.08 g,
9%) as a thick liquid.
C13H17N4O3: 277.1301. Found (M + H)+: 277.1320. Anal. Calcd for
C13H16N4O3: C, 56.51; H, 5.84; N, 20.28. Found: C, 56.55; H, 5.98;
N, 20.48.
2,2-Dimethylpropionic Acid 2-[(2R,3S,4S)-4-(6-Chloropur-
ine-9-yl)-5-fluoro-2,3-dihydroxycyclopent-1(5)-en]ethyl Ester
(26).13 To a suspension of 19 (230 mg, 0.76 mmol), PPh3 (0.5 g,
1.90 mmol), and 6-chloropurine (294 mg, 1.90 mmol) in anhydrous
THF (30 mL) was added a solution of DIAD (0.4 mL, 1.90 mmol) in
dry THF (10 mL) at 0 °C, and the reaction mixture was stirred at the
same temperature for 15 min and then at room temperature for 20 h.
The solvent was evaporated, and the residue was purified by silica gel
column chromatography (hexane/ethyl acetate = 2:1) to give 25 (0.3 g
Compound 20. 1H NMR (400 MHz, CDCl3, with DIAD byproduct
impurity) δ 1.03 (s, 3H), 1.08 (s, 3H), 1.27 (s, 9H), 2.54−2.64 (m,
2H), 3.92−3.97 (m, 2H), 4.64 (d, J = 5.2 Hz, 1H), 5.30 (d, J = 6.0 Hz,
1H), 5.59 (d, J = 8.8 Hz, 1H), 7.35−7.45 (m, 6H), 7.64−7.70 (m,
4H), 7.97 (s, 1H), 8.75 (s, 1H).
N7-Isomer. [α]25D 36.25° (c 4.33, CH2Cl2); UV (MeOH) λmax 265.0
1
1
mixed with DIAD byproduct) as a white foam: H NMR (400 MHz,
nm; H NMR (400 MHz, CDCl3) δ 1.07 (s, 9H), 1.33 (s, 3H), 1.43
CDCl3 mixed with DIAD byproduct) δ 1.14 (s, 9H), 1.37 (s, 3H), 1.51
(s, 3H), 2.55−2.73 (m, 2H), 4.23−4.29 (m, 1H), 4.35−4.41 (m, 1H),
4.80 (pseudo t, J = 5.6 Hz, 1h), 5.45 (dt, J = 2.0, 6.4 Hz, 1H), 5.56 (s,
1H), 8.09 (s, 1H), 8.75 (s, 1H); 19F NMR (376 MHz, CDCl3) δ
−135.18.
(s, 3H), 2.51−2.61 (m, 2H), 3.92−4.03 (s, 2H), 4.56 (d, J = 5.2 Hz,
1H), 5.14 (d, J = 5.2 Hz, 1H), 5.71 (s, 1H), 5.99 (s, 1H), 7.37−7.46
(m, 6H), 7.66−7.69 (m, 4H), 8.13 (s, 1H), 8.90 (s, 1H); 13C NMR
(100 MHz, CDCl3) δ 19.40, 26.30, 27.11, 27.62, 29.91, 31.97, 61.65,
67.22, 76.67, 84.68, 85.19, 112.90, 121.67, 128.05, 130.12, 133.55,
135.75, 143.85, 146.60, 152.62, 152.91. HRMS (ESI) calculated for
C31H35ClN4O3Na: 597.2167. Found (M + Na)+: 597.2045.
To a solution of 25 (0.3 g mixed with DIAD byproduct) in
anhydrous THF (10 mL) was added 2 N HCl (5 mL) at 0 °C, and the
reaction mixture was stirred at 45 °C for 18 h. After completion of
reaction (by TLC), the mixture was neutralized with 1 N NaOH
solution and extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous MgSO4, filtered, and
evaporated. The crude residue was purified by silica gel column
chromatography (CH2Cl2/MeOH = 20:1) to give 26 (150 mg, 50%
from 19) as a colorless solid: mp 153−154 °C; [α]D20 −72.28° (c 5.7,
CH2Cl2); UV (MeOH) λmax 261.0 nm; 1H NMR (400 MHz, MeOH-
d4) δ 1.16 (s, 9H), 2.53−2.60 (m, 1H), 2.63−2.71 (m, 1H), 4.25−4.35
(m, 2H), 4.64−4.70 (m, 1H), 4.80 (pseudo t, J = 4.4 Hz, 1H), 5.66−
5.69 (m, 1H), 8.59 (s, 1H), 8.71 (s, 1H); 13C (100 MHz, CDCl3) δ
(1S,2R,5S) 5-(6-Chloropurine-9-yl)-3-hydroxyethylcyclopent-
3-en-1,2-diol (23).13 Compound 20 (1.0 g with DIAD byproduct)
was dissolved in trifluoroacetic acid (5 mL) and water (5 mL) at 0 °C,
and the mixture was stirred at room temperature for 4 h. The mixture
was evaporated and coevaporated with MeOH. The crude residue was
purified by silica gel column chromatography (CH2Cl2/MeOH =
10:1) to give 23 (0.31 g, 66% from 18) as a white solid: mp 152−153
25
1
°C; [α]D −0.34° (c 5.8, MeOH); UV (MeOH) λmax 269.0 nm; H
NMR (400 MHz, CD3OD) δ 2.52−2.55 (m, 2H), 3.80−3.84 (m, 2H),
4.39 (superimposed dd, J = 5.2 Hz, 1H), 4.62 (d, J = 5.6 Hz, 1H),
5.61−5.63 (m, 1H), 5.81−5.83 (m, 1H), 8.56 (s, 1H), 8.72 (s, 1H);
13C NMR (100 MHz, CD3OD) δ 33.61, 60.81, 67.43, 76.32, 78.46,
24.91, 27.53, 39.78, 62.79, 64.53 (d, JC−F = 19.1 Hz), 73.0 (d, JC−F
=
9.2 Hz), 74.82, 126.76 (d, JC−F = 122.38 Hz), 147.58, 151.66, 153.15,
155.84, 160.2, 180.10; 19F NMR (376 MHz, CDCl3) δ −314.85.
HRMS (ESI) calculated for C17H21ClFN4O4: 399.116. Found (M +
H)+: 399.1232.
(1S,2R,5S)-5-(6-Aminopurine-9-yl)-4-fluoro-3-hydroxyethyl-
cyclopent-3(4)-en-1,2-diol (5i).13 A solution of 26 (150 mg, 0.37
mmol) in saturated ammonia in t-BuOH (10 mL) was stirred in a steel
bomb at 120 °C for 1 d. The volatiles were evaporated, and the residue
was purified by silica gel (neutralized with Et3N) column
chromatography (CH2Cl2/MeOH = 10:1) to give adenine derivative
(135 mg, 95%) as a white solid. HRMS (ESI) calculated for
C17H23FN5O4: 380.1657. Found (M + H)+: 380.1730.
126.36, 132.77, 147.04, 149.88, 151.29, 152.90, 153.45. HRMS (ESI)
calculated for C12H13ClN4O3Na: 319.0673. Found (M + Na)+:
319.0562.
(1S,2R,5S)-5-(6-Aminopurine-9-yl)-3-hydroxyethyl-
cyclopent-3-en-1,2-diol (5f).13 A solution of 23 (0.309 g, 0.538
mmol) in saturated ammonia in t-BuOH (10 mL) was stirred in a steel
bomb at 120 °C for 24 h. The volatiles were evaporated, and the
residue was purified by silica gel (neutralized with Et3N) column
chromatography (CH2Cl2/MeOH = 20:1) to give 5f (0.1 g, 67%) as a
white solid: mp 182−183 °C (lit.12 181−182 °C,12 202−203 °C 11);
[α]25 −9.692° (c 5.8, MeOH); UV (MeOH) λmax 267.0 nm; 1H
D
NMR (400 MHz, CD3OD) δ 2.51−2.54 (m, 2H), 3.82 (dt, J1 = 6.4, J2
= 1.6 Hz, 2H), 4.32 (dd, J1 = 5.6, J2 = 4.8 Hz, 1H), 4.60 (d, J = 5.6 Hz,
1H), 5.51−5.52 (m, 1H), 5.77 (d, J = 1.6 Hz, 1H), 8.21 (s, 1H), 8.26
(s, 1H). HRMS (ESI) calculated for C12H15N5O3: 277.1171. Found
(M)+: 277.1131.
To a solution of the adenine derivative (135 mg) in anhydrous
MeOH (3 mL) was added NaOMe (150 mg), and the reaction
mixture was heated at 45 °C for 3 h. After completion of reaction (by
TLC), the mixture was neutralized with glacial acetic acid and
evaporated. The crude residue was purified by silica gel column
chromatography (CH2Cl2/MeOH = 4:1) to give 5i (60 mg, 55%) as a
colorless solid: mp 186−187 °C; [α]D25 −124.9° (c 5.5, MeOH); UV
7-Deazahomoneplanocin A (5g) and 3-deazahomoneplanocin A
(5h) were prepared according to the similar procedure used in the
synthesis of homoneplanocin A (5f).
1
(MeOH) λmax 258.0 nm; H NMR (400 MHz, MeOH-d4) δ 2.48−
(1S,2R,5S)-5-(7-Deaza-6-aminopurine-9-yl)-3-hydroxyethyl-
25
2.54 (m, 2H), 3.76−3.81 (m, 2H), 4.51 (td, J = 1.6, 5.6 Hz, 1H), 4.69
(pseudo t, J = 4.4 Hz, 1H), 5.53−5.56 (m, 1H), 8.17 (s, 1H), 8.18 (s,
1H); 13C (100 MHz, MeOH-d4) δ 28.66, 60.39, 63.6 (d, J = 18.3 Hz),
72.5 (d, J = 9.6 Hz), 75.59 (d, J = 18.3 Hz), 120.58, 121.24 (d, J = 3.6
Hz), 141.78, 150.92, 153.58, 153.92, 157.0 (d, J = 108.4 Hz); 19F
NMR (376 MHz, MeOH-d4) δ −135.0. HRMS (ESI) calculated for
C12H14FNO3: 295.1071. Found (M + H)+: 296.1151. Anal. Calcd for
C12H13FNO3: C, 48.81; H, 4.78; N, 23.72. Found: C, 48.79; H, 4.80;
N, 23.67.
cyclopent-3-en-1,2-diol (5g). Yield: 60%; mp 114−116 °C; [α]D
1
+71.5° (c 0.2, MeOH); UV (MeOH) λmax 272 nm; H NMR (400
MHz, CD3OD) δ 2.49−2.52 (m, 2H), 3.77−3.83 (m, 2H), 4.15 (t, J =
5.2 Hz, 1H) 4.54 (d, J = 5.5 Hz, 1H), 5.59−5.60 (m, 1H), 5.69 (m,
1H), 6.58 (d, J = 3.6 Hz, 1H), 7.07 (s, 1H), 8.07 (s, 1H); 13C NMR
(100 MHz, CD3OD) δ 34.4, 61.7, 67.0, 77.3, 79.8, 101.1, 105.5, 124.1,
128.9, 149.2, 151.6, 152.6, 160.0. HRMS (ESI) calculated for
C13H17N4O3: 277.1301. Found (M + H)+: 277.1311. Anal. Calcd for
C13H16N4O3: C, 56.51; H, 5.84; N, 20.28. Found: C, 56.76; H, 5.44;
N, 19.99.
AdoHcy Hydrolase Assay.20 The reaction mixture (250 μL)
containing 50 mM sodium phosphate (pH 8.0) and AdoHcy hydrolase
(2 μM as monomer; 0.5 μM as tetramer) was preincubated with
various concentrations of compounds for 10 min at 37 °C. The
reaction was initiated by adding 100 μM AdoHcy and was allowed to
run for 20 min. The reaction mixture was further incubated with 200
μM DTNB, and the maximum absorbance of the product 5-thio-2-
nitrobenzoic acid (TNB) was measured at 412 nm using a
spectrophotometer. The molar extinction coefficient for TNB (ε412
(1S,2R,5S)-5-(3-Deaza-6-aminopurine-9-yl)-3-hydroxyethyl-
25
cyclopent-3-en-1,2-diol (5h). Yield: 50%; mp 200 °C (dec); [α]D
1
+22.9° (c 0.2, MeOH); UV (MeOH) λmax 268 nm; H NMR (400
MHz, CD3OD) δ 2.55 (t, J = 6.3 Hz, 1H), 3.81−3.84 (m, 2H), 4.19 (t,
J = 5.5 Hz, 1H), 4.28 (s, 1H), 5.37−5.38 (m, 1H), 5.84−5.85 (m, 1H),
6.97 (d, J = 6.0 Hz, 1H), 7.07 (d, J = 6.0 Hz, 1H), 8.12 (s, 1H); 13C
NMR (100 MHz, CD3OD) δ 34.5, 61.5, 69.0, 76.7, 79.5, 100.3, 127.5,
129.1, 140.8, 141.1, 143.1, 150.3, 154.0. HRMS (ESI) calculated for
K
DOI: 10.1021/acs.jmedchem.5b00553
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
= 13 700 M−1 cm−1) was used to measure TNB formation in all
quantifications.
ASSOCIATED CONTENT
* Supporting Information
Additional figures of molecular modeling, showing binding
modes and docking scores, and an xlsx file containing molecular
formula strings and IC50 values. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jmedchem.5b00553.
■
S
Antiviral Assay. Antiviral assay was briefly described based on
previously published protocols.21 First, 100 CCID50 (50% cell culture
inhibitory dose) per well of 96-well plates was prepared by infecting
Vero cells with 100 μL of the virus. Each well was then diluted with
DME/2% FBS. After the adsorption at 37 °C for 1 h, each well was
concentrated in vacuo and treated with the test compound diluted
with 100 μL of DME/2% FBS in duplicate for each concentration and
further incubated for 3 d. Antiviral activity was measured by MTT
assay, and the concentration of compound responsible for 50%
reduction of viral replication was calculated and expressed as the EC50.
Cytotoxic assay was performed as a control experiment, using mock
instead of virus for infection. Cell viability was measured by MTT
assay, and CC50 (50% cytotoxic concentration) was calculated as the
concentration of compound responsible for 50% reduction of cell
viability.
AUTHOR INFORMATION
Corresponding Authors
*S.C.: phone, 82-2-3277-4503; fax, 82-2-3277-2851; e-mail,
sunchoi@ewha.ac.kr.
*L.S.J.: phone, 82-2-880-7850; fax, 82-2-888-9122; e-mail,
lakjeong@snu.ac.kr.
■
Author Contributions
∥G.C., Y.W.M., and Y.L. contributed equally.
Sulforhodamine B (SRB) Assay.22 Cells (5 × 104 cells/mL) were
treated with various concentrations of compounds in 96-well culture
plates for 72 h, incubated, fixed with 10% trichloroacetic acid, dried,
and stained with 0.4% SRB in 1% acetic acid. After washing the
unbound dye out, the stained cells were dried and resuspended in 10
mM Tris (pH 10.0). The absorbance at 515 nm was measured, and
cell proliferation was calculated using the following equation: cell
proliferation (%) = [(average absorbancecompound − average absorban-
ceday zero)/(average absorbancecontrol − average absorbanceday zero)] ×
100. IC50 values were calculated by nonlinear regression analysis using
TableCurve 2D, versopm 5.01 (Systat Sofrware Inc., Richmond, CA,
USA).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the grants from Midcareer
Research Program (Grant 370C-20130120) (L.S.J.) and
National Leading Research Lab (NLRL) program (Grant
2011-0028885) (S.C.) through the Ministry of Science, ICT &
Future Planning (MSIP) and National Research Foundation
(NRF) of Korea.
Molecular Modeling. Protein Preparation and Active Site
Analysis. The X-ray crystal structure of AdoHcy hydrolase (PDB
code 3NJ4)8c was prepared using the Protein Preparation Wizard in
ABBREVIATIONS USED
■
AdoHcy, S-adenosyl-L-homocysteine; SAM, S-adenosyl-L-me-
thionine; HCV, hepatitis C virus; NOE, nuclear Overhauser
effect; RTIL, room temperature ionic liquid; [bmim][BF4], 1-
butyl-3-methylimidazolium tetrafluoroborate; [bmim][PF6], 1-
butyl-3-methylimidazolium hexafluorophosphate; DTNB, 5,5′-
dithiobis-2-nitrobenzoate; HSV, herpes simplex virus; HIV,
human immunodeficiency virus; PC, picornavirus; VSV,
vesicular stomatitis virus; DIAD, diisopropyl azodicarboxylate;
EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; TFAA,
trifluoroacetic anhydride; SRB, sulforhodamine B; TNB, 5-thio-
2-nitrobenzoic acid; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; FBS, fetal bovine serum; OPLS,
optimized potential for liquid simulation
Maestro, version 9.2 (Schrodinger, LLC, NY, USA). During the
̈
preparation process, bond orders were assigned, hydrogen atoms were
added, and protonation states of the residues at pH 7.4 were generated
by Epik, version 2.6. According to Yamada et al.,24 the His54 residue in
rat appears to be protonated in its NE2 position and the protonation
state of this residue is less influenced by the pH of solution. Therefore,
the His55 residue was prepared to be protonated for the docking
studies. All the hydrogen atoms were energy minimized with the
optimized potential for liquid simulation (OPLS) 2005 force field until
the average root-mean-square deviation for hydrogen atoms reached
0.30 Å. To evaluate the active site features, structure-based
pharmacophore analysis was performed using LigandScout, version
3.1 (Inte:Ligand GmbH, Vienna, Austria).
Ligand Preparation. Three-dimensional structures of the ligand
molecules were prepared by LigPrep, version 2.5, and their
protonation states and tautomeric structures in aqueous solution at
pH 7.4 were generated by Epik, version 2.6, in Maestro. The resulting
structures were energy minimized in implicit solvent with OPLS2005
force field.
Flexible Docking. The ligands were docked to AdoHcy hydrolase
using Glide, version 6.1, in Maestro with the following steps: (i) The
grid for the active site was generated using the centroid of the
cocrystallized ligand, fluoroneplanocin A (2). Since the structure of the
cocrystallized ligand was very similar to that of tested compounds, we
used the grid box size automatically calculated based on the
cocrystallized ligand. (ii) For the initial docking stage, Glide SP
(standard precision) docking was performed retaining a maximum
number of 30 poses per ligand. (iii) Top 5 ranked poses per ligand in
the previous step were selected and re-docked using Glide XP (extra
precision).
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