Synthesis and biological evaluation of immunosuppressive agent DZ2002 and its stereoisomers

Article abstract of DOI:10.1016/j.bmc.2008.09.017

DZ2002 and its related stereoisomers were efficiently synthesized. The optical data of (R)- and (S)-DZ2002 were disclosed here for the first time. Their inhibitory potency was evaluated on SAHase and MLR assay in the mean time. In accordance with respecti

Full text of DOI:10.1016/j.bmc.2008.09.017

Bioorganic & Medicinal Chemistry 16 (2008) 9212–9216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of immunosuppressive agent DZ2002
and its stereoisomers
a
c,
*
Yang-Ming Zhang a,b, , Yu Ding c, , Wei Tang c, Wei Luo b, Min Gu b, Wei Lu a, , Jie Tang , Jian-Ping Zuo
,
*
Fa-Jun Nan b,
*
a Department of Chemistry and Institute of Medicinal Chemistry, East China Normal University, 3663 North Zhong Shan Road, Shanghai 200062, China
b The National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road,
Shanghai 201203, China
c Department of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2008
Revised 1 September 2008
Accepted 5 September 2008
Available online 9 September 2008
DZ2002 and its related stereoisomers were efficiently synthesized. The optical data of (R)- and (S)-
DZ2002 were disclosed here for the first time. Their inhibitory potency was evaluated on SAHase and
MLR assay in the mean time. In accordance with respective inhibitory potency of SAHase, the immuno-
suppressive potency order was demonstrated as (S)-DZ2002 > (Rac)-DZ2002 > (R)-DZ2002 > (Keto)-
DZ2002. These results indicate (S)-configuration of 2-chiral center in DZ2002 is important for binding
with SAHase.
Keywords:
DZ2002
Ó 2008 Elsevier Ltd. All rights reserved.
Stereoisomers
Synthesis
Immunosuppressive potency
S-Adenosyl-L-homocysteine hydrolase
1. Introduction
mycin, neplanocin A, MDL28,842, C3-Ado, etc., are irreversibly
trapped in the active site of SAHase utilizing its 30-oxidative activ-
S-Adenosylmethionine (AdoMet)-dependent biological trans-
methylations are involved in a variety of important physiologic
processes, including most methylations of proteins,1 lipids,2 nu-
cleic acids,3 and small molecules.4 These reactions are primarily
catalyzed by AdoMet-dependent methyltransferases, which con-
ity. Type II inhibitors, such as 60-bromo-50,60-didehydro-60-deoxy-
60-fluorohomoadenosine, act by permanently altering the active
site of the enzyme using its 50-hydrolytic activity. Among three
types of inhibitors, type 1 inhibitors have been explored most
extensively. However, irreversible inhibitors manifest severe toxic-
ity and side effects due to the nature of irreversible binding with
the enzyme, which circumscribe their development to clinically
useful drugs.4 In yet another aspect, type III inhibitors can revers-
ibly bind to the open form of the enzyme, maintaining a similar po-
tency with much reduced toxicity.
vert AdoMet to S-adenosyl-
then hydrolyzed by S-adenosyl-
L
-homocysteine (AdoHcy). AdoHcy is
-homocysteine hydrolase (SAH-
L
ase) to prevent feedback inhibition of transmethylations.4–9 Inhib-
iting SAHase leads to accumulation of intracellular AdoHcy and
most intracellular transmethylations cease or halt. Since lympho-
cytes seem more dependent on transmethylation reactions than
most other cell types for their functions,10 SAHase is regarded as
a suitable target for the design of immunosuppressive, anti-inflam-
matory, and antiviral agents.4
Within recent years, 4-(6-aminopurine-9-yl)-2-hydroxybutyric
acid methyl ester (DZ2002), has been recognized as a potent
reversible inhibitor of SAHase, whose reversibility was proved by
the rapid restoration of SAHase activity after treatment discontin-
uation.5–9 Biological studies show that DZ2002 reduces DNFB-in-
duced delayed-type hypersensitivity responses,6 suppresses
OVA-induced lymphocyte proliferation and Th1-type cytokine pro-
duction,8 ameliorates MOG35-55-induced experimental autoim-
Generally, three types of SAHase inhibitors have been catego-
rized based on binding mechanism: the irreversible type I, type
II, and reversible type III inhibitors.4 Type I inhibitors, like aristero-
mune encephalomyelitis (EAE).9 DZ2002 is regarded as
a
* Corresponding authors. Tel.: +86 21 50806600x2507 (J.-P. Zuo); tel./fax: +86 21
50800954 (F.-J. Nan).
E-mail addresses: jpzuo@mail.shcnc.ac.cn (J.-P. Zuo), fjnan@mail.shcnc.ac.cn
(F.-J. Nan).
promising therapeutic agent for immune-related diseases. The
presence of 2-chiral center allows DZ2002 to exist in two optical
forms, both of which have the same physical and chemical
properties in an achiral environment, but probably have different
These authors contribute equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.017

Y.-M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 9212–9216
9213
biological properties. However, the lack of disclosed optical infor-
mation on DZ2002 from reported data makes it difficult to develop
a practical manufacturing process.11 All of mentioned above intri-
gued us to synthesize all its related stereoisomers with an aim to
investigate the necessity of chiral center in DZ2002. Here, we de-
scribed efficient approaches to prepare DZ2002 and its related ster-
eoisomers. Their inhibitory activity against SAHase and
immunosuppressive potency, were examined in the mean time.
The ketone form of DZ2002, a possible existing form in vivo, was
also prepared and examined (Fig. 1).
OH
O
O
OH
O
a
c
b
HO
O
O
OH
O
OH
O
O
O
2
1
(S)-Malic acid
NH2
NH2
NH2
N
N
N
N
N
N
N
e
O
O
N
H
N
d
N
N
N
O
OTs
O
2. Chemistry
HO
3
O
O
Different from patented synthesis of DZ2002, which either
commenced with the reaction of adenine and 3-hydroxybutyrolac-
tone,11a or utilized 9-(3,4-O-isopropylidene-3,4-dihydroxybu-
tyl)adenine as starting material,11b,11c our synthesis started with
cheap and commercially available optical malic acid (Scheme 1).
In brief, (S)-malic acid was first transformed to acetonide 1. Aceto-
nide 1 was reduced to alcohol 2,12 which was immediately con-
verted to tosylate 3. Coupling of tosylate 3 with adenine gave
compound 4 in a yield of 32% after silica-gel chromatograph. Com-
pound 4 was readily transformed to (S)-DZ2002 upon spontaneous
deprotection and esterification in a methanol solution of HCl. Be-
cause of the rigid conformation of acetonide during reaction se-
quences, 2-chiral center was highly reserved and potential
racemization was avoided. Optical test results of newly prepared
MeO
O
4
(S)-DZ2002
Scheme 1. Reagents and conditions: (a) 2,2-dimethoxypropane, cat. p-TSA, 6 h,
61%; (b) (CH3)2SꢁBH3, THF, 0 °C–room temperature, 12 h, quantitative yield; (c) TsCl,
pyridine, 0 °C–room temperature, 5 h, 67%; (d) K2CO3, adenine, 18-crown-6, DMF,
50 °C, 12 h, 32%; (e) AcCl, MeOH, 0 °C–room temperature, 5 h, 50%.
NH2
NH2
N
N
NH2
N
N
N
N
N
c
N
N
CO2Me
a
b
N
O
N
O
PPh3
N
H
a]
25 +19.2 (c 0.3, MeOH/H2O = 1:1, v/v), ee 92%. Fol-
D
CN
sample were, [
lowing the same process, (R)-DZ2002 was synthesized smoothly
OH
OMe
25
from (R)-malic acid, [
94%.
a]
ꢀ17.9 (c 0.3, MeOH/H2O = 1:1, v/v), ee
D
6
5
Attempts to synthesize (Keto)-DZ2002 by direct oxidation of
(S)- or (R)-DZ2002 with Jones agent, PCC or IBX, failed and an alter-
native approach was employed (Scheme 2). Ester 5, prepared via a
Michael-type addition of adenine to ethyl acrylate, was hydrolyzed
in refluxing 3 N HCl solution.13 The obtained acid 6 was then cou-
pled with easily accessible (triphenylphosphoranylidene)acetoni-
trile in the presence of EDCI to afford compound 7.14 After
ozonolylsis of compound 7 in methanol, the desired (Keto)-
DZ2002 was obtained cleanly. Upon treatment with NaBH4 in
methanol, (Rac)-DZ2002 was obtained in a yield of 60% after sil-
ica-gel chromatograph.
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
e
d
N
N
O
N
O
HO
O
O
PPh3
MeO
MeO
NC
(Rac)-DZ2002
(Keto)-DZ2002
7
Scheme 2. Reagents and conditions: (a) EtONa, benzene, reflux, 12 h, 83%; (b) 3 N
HCl, reflux, 3 h, 95%; (c) EDCI, DMAP, DMF, room temperature, 12 h, 71%; (d) O3,
MeOH, ꢀ78 °C, 20 min, 88%; (e) NaBH4, MeOH, room temperature, 30 min, 60%.
3. Biological assay
The inhibitory potency against SAHase was measured by DTNB-
coupled assay of SAHase hydrolytic activity, a mature and reliable
method developed by Lozada-Ramirez,15 later further proved by
Wu et al16. Test results (Fig. 2) showed that (S)-DZ2002 was the
strongest inhibitor among four compounds (IC50 = 21.2 lM, Table
1). The order of enzyme inhibition potency was (S)-
.016
(Keto)-DZ2002
.014
(R)-DZ2002
(Rac)-DZ2002
(S)-DZ2002
Control
.012
.010
.008
.006
.004
.002
0.000
Z2002 > (Rac)-DZ2002 > (R)-DZ2002 > (Keto)-Z2002.
NH2
N
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
N
N
N
(S)
(R)
HO
HO
HO
MeO
O
O
O
O
O
0
100
200
300
400
500
600
MeO
MeO
MeO
Time (Sec)
(S)-DZ2002
(R)-DZ2002
(Rac)-DZ2002 (Keto)-DZ2002
Figure 2. Inhibition of SAHase by (S)-, (R)-, (Rac)-, and (Keto)-DZ2002. Data are
Figure 1. DZ2002 and related stereoisomers.
representative of three independent experiments.

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Y.-M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 9212–9216
Table 1
Inhibitory value on SAHase of each compound
Compound
IC50
(l
M)
CC50 (lM)
(S)-DZ2002
(Rac)-DZ2002
(R)-DZ2002
21.2
24.4
61.3
>1000
>1000
>1000
>1000
(Keto)-DZ2002
193.9
IC50, concentration, at which the lymphocyte proliferation was inhibited by 50%;
CC50, concentration, at which cell viability was reduced by 50%.
The immunosuppressive potency of each compound was exam-
ined by mixed lymphocyte reaction (MLR) (Fig. 3).6 Respective sup-
pressive potency appeared to match the inhibitory potency of the
SAHase. The order of immunosuppressive activity in vitro was in
accordance with that of inhibitory potency of enzyme. Of the four
compounds, (S)-DZ2002 displayed the most potent immunosup-
pressive activity in vitro.
4. Discussion
Chirality is an important issue for current pharmaceuticals
because the body always recognizes chirality. In most times,
both chiral forms of a drug can be taken safely without serious
side effects. In some cases, one chiral form is active and the
other is not. In our DZ2002 case, (S)-, (R)-, and (Rac)-DZ2002
were evaluated for their biological efficacy. The results indicated
that maintenance of 2-chiral center in the molecule as (S)-con-
figuration is important for its binding affinity. Further evidence
in support of this hypothesis comes from simplified docking re-
sults of (S)-, (R)-, and (Keto)-DZ2002 into the active site of SAH-
ase (Fig. 4). The crystal structure of SAHase complexed with
neplanocin (PDB code: 1Ii4)16 was chosen to represent the en-
zyme structure and the computation was performed on Discov-
ery StudioTM. When the conformation of adenine rings were
fixed for a simplicity reason, their aliphatic side chains orien-
tated differently, resulting in different numbers of hydrogen
bonds interacted with SAHase, a critical factor determining affin-
ity with the enzyme, particularly for reversible inhibitors. The
side chain of (S)-DZ2002 interacted with Asp55, Asp131, and
Asp301 via four hydrogen bonds. The side chain of (R)-DZ2002
interacted via three hydrogen bonds with Asp131, Asp186, and
Asp190 and the side chain of (Keto)-DZ2002 interacted via only
two hydrogen bonds with Asp157 and Asp346. Combined these
Figure 4. (S)-, (R)-, and (Keto)-DZ2002 docked into the active site of SAHase. For
clarity, only the residues providing the main hydrogen bond interactions are shown.
Hydrogen bonds are represented as dotted lines.
100
(Keto)-DZ2002
(R)-DZ2002
results together, we determined the critical role of (S)-configura-
tion of 2-hydroxy group in DZ2002 for binding with SAHase.
In summary, we described a practical approach to prepare
optical DZ2002 and related isomers. The optical data of (R)-
and (S)-DZ2002 were disclosed here for the first time. (Keto)-
DZ2002 and (Rac)-DZ2002 were also synthesized conveniently.
Biological studies demonstrated that their immunosuppressive
potency was in accordance with respective inhibitory potency
of SAHase, following the order of (S)-DZ2002 > (Rac)-
DZ2002 > (R)-DZ2002 > (Keto)-DZ2002. (S)-DZ2002 displayed the
strongest inhibitory potency against SAHase and immunosup-
pressive potency in vitro. These experimental results indicate
that 2-chiral center in the molecule of DZ2002 is necessary
and important for its activity, possibly by affecting the formation
of hydrogen bonds with the enzyme. Therefore, our results pro-
vide useful information for quality control in development of
practical manufacturing process.
(Rac)-DZ2002
80
(S)-DZ2002
60
40
20
0
25
50
100
Concentration (μM)
Figure 3. (S)-, (R)-, (Rac)-, and (Keto)-DZ2002 (three concentrations: 25, 50, and
100 M) suppressed cell proliferation in mixed lymphocyte reaction. Data are
representative of three independent experiments.
l

Y.-M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 9212–9216
9215
5.1.4. (2R)-4-(6-Aminopurine-9-yl)-2-hydroxybutyric acid
methyl ester [(R)-DZ2002]
5. Experimental
In the same manner, (R)-DZ2002 was obtained as a white solid:
1H NMR (DMSO-d6): d 8.14 (1H, s), 8.07 (1H, s), 7.18 (2H, br s), 5.71
(1H, br d, J = 5.7 Hz), 4.24 (2H, t, J = 6.9 Hz), 4.01 (1H, m), 3.56 (3H,
5.1. General procedure
1H (300 MHz) and 13C (75 MHz) NMR spectra were performed
on a Varian Mercury-VX300 spectrometer, using residue CHCl3,
DMSO or H2O as internal standard. HRMS (ESI) were recorded on
a waters-micromass Q-TOF Ultima Global electrospray mass spec-
trometer. Melting point was measured on a ShengGuang WRR
Melting Points apparatus and was not corrected. Optical rotation
data was recorded on a Perkin-Elmer Model 341 Polarimeter. ee
values were determined by HPLC: Chiralcel OJ-H column; n-hex-
ane/2-propanol (90:10); 1.0 mL/min; 263 nm; tR (R) 69.2 min; tR
(S) 98.1 min.
s), 2.21 (1H, m), 2.06 (1H, m); HR ESIMS m/z 252.1100 [M+H]+
25
(calcd. for C10H14N5O3 252.1097); [
= 1:1, v/v); ee 94%.
a
]
D
ꢀ17.9 (c 0.3, MeOH/H2O
5.1.5. 3-(6-Aminopurine-9-yl)-propionic acid methyl ester (5)
To a mixture of adenine (5.0 g, 37.0 mmol) in absolute ethanol
(130 mL) and dry benzene (16 mL) was added sodium metal
(60 mg, 1.6 mmol) carefully at room temperature. Upon the disap-
pearance of sodium, ethyl acrylate was added dropwise. The result-
ing mixture was heated to reflux for 12 h, and then cooled to room
temperature. The solvent was removed under reduced pressure.
The obtained residue was titrated with cold ethanol. The precipi-
tate was collected by filtration, washed with cold ethanol and dried
in vacuum. Compound 5 was obtained (7.2 g, 83%) as an off-white
solid: 1H NMR (CDCl3) d 8.36 (1H, s), 7.90 (1H, s), 5.71 (2H, br s),
4.50 (2H, t, J = 6.3 Hz), 4.13 (2H, q, J = 7.2 Hz), 2.92 (2H, t,
J = 6.3 Hz), 1.22 (2H, t, J = 7.2 Hz).
5.1.1. (4S)-Toluene-4-sulfonic acid 2-(2,2-dimethyl-5-oxo-
[1,3]dioxolan-4-yl)-ethyl ester (3)
To a solution of compound 2 (60.0 g, 375 mmol), prepared from
(S)-malic acid,12 in pyridine (300 mL), was added p-toluenesulfonyl
chloride (71.5 g, 375 mmol) at 0 °C. The mixture was stirred at that
temperature for 1 h and then at room temperature for 4 h. The sol-
vent was removed under reduced pressure and the residue was
dissolved in ethyl acetate (600 mL). The organic solution was
washed with HCl solution (5%), satd NaHCO3 solution, and brine,
dried on MgSO4 and concentrated. Crude product was purified by
silica-gel chromatograph eluting with petroleum ether/ethyl ace-
tate (5:1). Compound 3 was obtained (67%) as a colorless oil: 1H
NMR (CDCl3) d 7.80 (2H, d, J = 8.1 Hz), 7.35 (2H, d, J = 8.4 Hz),
4.43 (1H, dd, J = 4.5 and 7.8 Hz), 4.23 (2H, m), 2.45 (3H, s), 2.22
(1H, m), 2.02 (1H, m), 1.57 (3H, s), 1.51 (3H, s).
5.1.6. 3-(6-Aminopurine-9-yl)-propionic acid (6)
A mixture of compound 5 (4 g, 17 mmol) and aqueous 3 N HCl
solution was heated to reflux for 3 h and was cooled to room tem-
perature. The mixture pH was adjusted to 3 by careful addition of
solid NaOH. The precipitate was collected by filtration, washed
with cold water and dried in vacuum. Compound 6 was obtained
(3.35 g, 95%) as a white solid: 1H NMR (D2O) d 8.42 (1H, s), 8.37
(1H, s), 4.58 (2H, t, J = 6.3 Hz), 3.02 (2H, t, J = 6.3 Hz).
5.1.2. (5S)-5-[2-(6-Aminopurine-9-yl)-ethyl]-2,2-dimethyl-
[1,3]dioxolan-4-one (4)
5.1.7. 5-(6-Aminopurine-9-yl)-3-oxo-2-(triphenyl-k5-
A mixture of compound 3 (72.0 g, 229 mmol), adenine (62.0 g,
459 mmol), K2CO3 (92.0 g, 665 mmol) and 18-crown-6 (2.0 g,
7.6 mmol) in dry DMF (360 mL) was heated to 50 °C for 12 h. The
mixture was cooled to room temperature and filtered. The solid
was washed with ethyl acetate. The combined organic solution
was concentrated to remove most of DMF to afford a brown oil.
The crude product was purified by silica-gel chromatograph elut-
ing with chloroform/methanol (20:1, then 10:1). Compound 4
was obtained (20.0 g, 32%) as a pale yellow solid: 1H NMR
(DMSO-d6) d 8.14 (2H, s), 7.19 (2H, br s), 4.67 (1H, dd, J = 4.2 and
7.8 Hz), 4.27 (2H, t, J = 7.2 Hz), 2.40 (1H, m), 2.23 (1H, m), 1.56
(3H, s), 1.52 (3H, s).
phosphanylidene)-pentanenitrile (7)
A mixture of compound 6 (1.3 g, 6 mmol), (triphenylphosphor-
anylidene)acetonitrile (3.8 g, 13 mmol),14 EDCI (1.3 g, 7 mmol) and
DMAP (77 mg, 0.6 mmol) in dry DMF (35 mL) was allowed to stir at
room temperature for 12 h. The mixture was diluted with water
(35 mL) and extracted with ethyl acetate (3ꢂ 40 mL). The com-
bined organic phase was washed with water and brine, dried on
MgSO4, and concentrated to afford the title product. Compound 7
was obtained (2.2 g, 71%) as a pale yellow powder: 1H NMR (CDCl3)
d 8.38 (1H, s), 7.75 (1H, s), 7.65 (4H, m), 7.49 (11H, m), 5.54 (2H, br
s), 4.53 (2H, t, J = 6.0 Hz), 3.32 (2H, t, J = 6.0 Hz).
5.1.8. 3-(6-Aminopurine-9-yl)-2-oxo-propionic acid methyl
ester [(Keto)-DZ2002]
5.1.3. (2S)-4-(6-Aminopurine-9-yl)-2-hydroxybutyric acid
methyl ester [(S)-DZ2002]
Ozone was bubbled into a mixture of compound 7 (300 mg,
0.6 mmol) in dichloromethane (20 mL) and methanol (20 mL) at
ꢀ78 °C until the color turned pale blue and then for additional
10 min. N2 was then bubbled through to remove excess ozone. Sil-
ica-gel (1.2 g) was added to the mixture and was then concentrated
until a free-running solid was obtained. The solid was transferred
to a silica-gel column and eluted with chloroform/methanol
(15:1). After removal of solvents, (Keto)-DZ2002 was obtained
(134 mg, 88%) as a white solid: 1H NMR (CDCl3): d 8.34 (1H, s),
7.90 (1H, s), 5.58 (2H, br s), 4.54 (2H, t, J = 6.0 Hz), 3.86 (3H, s),
3.53 (2H, t, J = 6.0 Hz); 13C NMR (DMSO-d6): d 190.90, 160.07,
155.88, 152.24, 149.43, 140.95, 118.70, 52.54, 38.27, 37.64; HR
ESIMS m/z 272.0758 [M+Na]+ (calcd. for C10H11N5NaO3 272.0760).
Acetyl chloride (7.0 mL, 98 mmol) was added dropwise to a
solution of compound 4 (18.0 g, 65 mmol) in methanol (540 mL)
at 0 °C within 30 min. The mixture was then allowed to stir at
room temperature for 3 h. NaHCO3 (14 g, 167 mmol) was added
carefully to neutralize the generated acid. The mixture was filtered.
To the filtrate was added silica-gel (60 g) and concentrated under
reduced pressure until a freely running solid was obtained. This so-
lid was transferred to a silica-gel column and eluted with chloro-
form/methanol (15:1, then 10:1). The obtained crude product
was further purified by recrystallization in methanol. (S)-DZ2002
was obtained (8.1 g, 50%) as white crystalline powders: 1H NMR
(DMSO-d6): d 8.13 (1H, s), 8.07 (1H, s), 7.18 (2H, br s), 5.69 (1H,
br d, J = 5.7 Hz), 4.24 (2H, t, J = 6.9 Hz), 4.01 (1H, m), 3.56 (3H, s),
2.23 (1H, m), 2.06 (1H, m); 13C NMR (DMSO-d6): d 174.53,
156.63, 153.06, 150.18, 141.67, 119.49, 67.97, 52.22, 40.34,
5.1.9. (2Rac)-4-(6-Aminopurine-9-yl)-2-hydroxybutyric acid
methyl ester [(Rac)-DZ2002]
A mixture of (Keto)-DZ2002 (38 mg, 0.15 mmol) in methanol
(20 mL) was added NaBH4 (7 mg, 0.18 mmol). The mixture was
stirred for 30 min at room temperature. Silica-gel (120 mg) was
34.21; HR ESIMS m/z 274.0921 [M+Na]+ (calcd. for C10H13N5NaO3
25
274.0916); mp 162–164 °C; [
a
]
D
+19.2 (c 0.3, MeOH/H2O = 1:1);
ee 92%.

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Y.-M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 9212–9216
added to the mixture and concentrated until a free-running solid
was obtained. The solid was transferred to a silica-gel column
and eluted with chloroform/methanol (20:1). Removal of solvents
afforded (Rac)-DZ2002 (23 mg, 60%) as a white solid: 1H NMR
(DMSO-d6): d 8.13 (1H, s), 8.07 (1H, s), 7.19 (2H, br s), 5.72 (1H,
br d, J = 5.7 Hz), 4.24 (2H, t, J = 6.6 Hz), 4.01 (1H, m), 3.56 (3H, s),
2.23 (1H, m), 2.05 (1H, m); HR ESIMS m/z 274.0936 [M+Na]+ (calcd.
for C10H13N5NaO3 274.0916).
glass fiber filters and incorporated radioactivity was counted using
a Beta Scintillation Counter (MicroBeta Trilux).
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (Grants 30725049 to F.N.), the Shanghai Commission
of Science and Technology (Grant 064319014 to J.Z.), National Nat-
ural Science Foundation of China (No. 30572195 to J.Z.), and Pro-
gram for New Century Excellent Talents in University (NCET to
W.L.). We thank for Dr. Zhan-Li Wang and Ms. Cui-Fang Lin of Neo-
trident Inc. for their kindly help on docking computation.
5.2. SAHase and MLR assay
5.2.1. Reagents and animals
S-Adenosyl-
erythrocytes, S-adenosyl-
L
-homocysteine hydrolase (SAHase) from rabbit
-homocysteine (SAH), and 5,5-dithi-
L
References and notes
obis-2-nitrobenzoic acid (DTNB) were obtained from Sigma. RPMI
1640 medium was purchased from Gibco BRL/Life Technologies
Inc. (Gaithersburg, MD), fetal calf serum (FCS) was obtained from
Hyclone Laboratories (Logan, UT), [3H]thymidine was provided by
GE Healthcare.
BALB/c and C57BL/6 mice were purchased from the Shanghai
Experimental Animal Center of the Chinese Academy of Sciences.
The animals were housed in specific pathogen-free conditions.
All experiments were carried out according to the National Insti-
tutes of Health Guide for Care and Use of Laboratory Animals and
were approved by the Bioethics Committee of the Shanghai Insti-
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(200
and 10
l
L) containing 50
lM SAH, 100 lM DTNB, 0.066 U SAHase,
lM compound was measured using a spectraMAX-190
spectrophotometer, and O.D. at 412 nm (maximum absorbance of
the product HCY-TNB) was continuously read every minute from
0 to 8 min. All operational works proceeded at 37 °C.
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5.2.3. Mixed lymphocyte reaction (MLR)
BALB/c splenic lymphocytes (1 ꢂ 107cells/mL) were
c-irradi-
ated with a 60Co source for 7 min, and then cultured with fresh
C57BL/6 splenic lymphocytes and compound in a 96-well plate.
After a 72 h incubation at 37 °C in a 5% CO2 water jacketed CO2
incubator, cells were pulsed with 1
l
Ci/well of [3H]thymidine
and incubated for another 24 h. Then cells were harvested onto