Discovery of N-(2,3,5-triazoyl)mycophenolic amide and mycophenolic epoxyketone as novel inhibitors of human IMPDH

Article abstract of DOI:10.1016/j.bmcl.2013.07.016

Syntheses of ten derivatives of mycophenolic acid (MPA) at C-6′ position, and structure-activity relationship study among these derivatives, MPA and mycophenolic hydroxamic acid (MPHA) led to discovery of N-(2,3,5-triazolyl)mycophenolic amide 4, (7′S) mycophenolic epoxyketone 9 and (7′R) mycophenolic epoxyketone 10 having potent inhibitory activity against human inosine-5′-monophosphate dehydrogenase (IMPDH) type I and II as well as antiproliferative activity on human leukemia K562 cells. Compounds 4, 9, and 10 showed induction activity of erythroid differentiation in K562 cells. Inhibitory effects of 4 and 10 against IMPDH were attenuated by supplemental guanosine in K562 cells. In contrast, attenuation effect by supplemental guanosine was not significant in the case of 9. Compound 9 weakly inhibited the enzyme activity of HDAC in the nuclear lysate of K562 cells at 10 μM. These observations suggest that the primary target of 4, 9, and 10 is IMPDH, whereas compound 9 partially inhibits a certain type of HDAC.

Full text of DOI:10.1016/j.bmcl.2013.07.016

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5140–5144
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-(2,3,5-triazoyl)mycophenolic amide and
mycophenolic epoxyketone as novel inhibitors of human IMPDH
⇑
Kazuhiro Sunohara, Shinya Mitsuhashi, Kengo Shigetomi, Makoto Ubukata
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
a r t i c l e i n f o
a b s t r a c t
Syntheses of ten derivatives of mycophenolic acid (MPA) at C-60 position, and structure–activity relation-
ship study among these derivatives, MPA and mycophenolic hydroxamic acid (MPHA) led to discovery of
N-(2,3,5-triazolyl)mycophenolic amide 4, (70S) mycophenolic epoxyketone 9 and (70R) mycophenolic
epoxyketone 10 having potent inhibitory activity against human inosine-50-monophosphate dehydroge-
nase (IMPDH) type I and II as well as antiproliferative activity on human leukemia K562 cells. Compounds
4, 9, and 10 showed induction activity of erythroid differentiation in K562 cells. Inhibitory effects of 4 and
10 against IMPDH were attenuated by supplemental guanosine in K562 cells. In contrast, attenuation
effect by supplemental guanosine was not significant in the case of 9. Compound 9 weakly inhibited
Article history:
Received 13 May 2013
Revised 5 July 2013
Accepted 12 July 2013
Available online 19 July 2013
Keywords:
Mycophenolic acid
Erythroid differentiation
Mycophenolic hydroxamic acid
Inosine-50-monophosphate dehydrogenase
(IMPDH)
the enzyme activity of HDAC in the nuclear lysate of K562 cells at 10 lM. These observations suggest that
the primary target of 4, 9, and 10 is IMPDH, whereas compound 9 partially inhibits a certain type of
HDAC.
Histone deacetylase (HDAC)
Ó 2013 Elsevier Ltd. All rights reserved.
Inosine-50-monophosphate dehydrogenase (IMPDH) is an en-
zyme that catalyzes the NAD-dependent oxidation of inosine
monophosphate (IMP) to xanthosine monophosphate (XMP), the
first committed and rate-limiting step in guanine nucleotide bio-
synthesis.1 We found mycophenolic acid (1), known as an inhibitor
of IMPDH, to be a latent agonist of peroxisome proliferator acti-
K562 cell proliferation, inductive activity on erythroid differentia-
tion in K562 cells, and inhibitory activity against human IMPDH
in vitro.
Since one of the molecular targets for cancer therapy is zinc
dependent HDAC16 and Zn2+ is classified as borderline acid in
HASB rule,7 substitution of hydroxyl group as hard base of
hydroxamic acid in 2 with heterocyclic group might afford excel-
lent dual inhibitor for HDAC1 and IMPDH II. Six MPA derivatives
3–8, each having heterocyclic group were designed for dual inhib-
itor having the zinc binding moiety. Other MPA derivatives de-
signed here were compounds 9 and 10 which have terminus
epoxyketone known as zinc binding group in trapoxin,8 and com-
pound 12 having thiol group recognized as zinc binding group in
FK228.9 Thioacetate 11 was also selected as a candidate of a
masked dual-inhibitor for HDAC and IMPDH (Fig. 1).
7-O-TBS-mycophenolic acid (13) was obtained by treating 1
with 3 equiv of TBSOTf in the presence of Et3N in CH2Cl2, followed
by selective hydrolysis using AcOH/THF/H2O (1:9:1) in 95% yield.
The condensation reaction of 13 with each heterocyclic amine fol-
lowed by deprotection using TBAF afforded desired products 3–8
as shown in Table 1. The reaction of 13 with 2-aminopyridine gave
corresponding amide in 39.6% yield, when 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride (EDC) was used as a
condensation reagent, and deprotection of TBS group afforded 3
as a final product in 78.6%. The result suggests that more effective
activation of carboxylic acid is needed due to poor nucleophilicity
of heterocyclic amine. We therefore used ethyl chlorocarbonate
(ClCOOEt) for syntheses of 4, 6, and 7, oxalyl chloride [(COCl)2]
vated receptor
c (PPARc
).2 This finding led us to perform a chal-
lenge for conversion of molecular target of 1 by derivatization.
To develop dual inhibitors for human IMPDH and histone deacetyl-
ase (HDAC), we synthesized hydroxamic acid derivatives of 1,
mycophenolic hydroxamic acid (MPHA, 2), 7-O-acetyl MPHA, and
7-O-lauroyl MPHA which were found to be new tubulin-specific
HDAC inhibitors in culture cells (Fig. 1).3 The inhibitory effect of
2 on IMPDH was however 1-order of magnitude less than that of
1, and the selectivity of 1 toward human IMPDH type II was lost
after such derivatization (selectivity index of 1: 1.6, 2: 0.83, IMPDH
I/IMPDH II IC50 ratio).4 Transplantation therapy and chemotherapy
could be improved with IMPDH II specific inhibitors.5 Therefore,
discovery of new type of dual inhibitor for HDAC and IMPDH II
would provide important information for drug discovery for trans-
plantation therapy and chemotherapy. Herein, we describe design,
syntheses of new MPA heterocyclic amide derivatives 3, 4, 5, 6, 7, 8,
terminus epoxide derivatives 9, 10, thiol derivatives 11, 12, and
their structure–activity relationships for inhibitory effect on
⇑
Corresponding author. Tel./fax: +81 11 706 3638.
E-mail addresses: m-ub@for.agr.hokudai.ac.jp, sol999hokudai@yahoo.com
(M. Ubukata).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.016

K. Sunohara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5140–5144
5141
Figure 1. The structures of MPA (1), MPHA (2), and new derivatives 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.
Table 1
Synthesis of heterocyclic amide derivatives
OTBS
O
Me
OH
Me
Me
O
R1
OH
1) conditions
2) TBAF, THF
O
O
O
O
OMe
OMe
Me
7-O-TBS MPA (13)
MPA heterocyclic amide derivatives
Condition
EDC, HOBt, 2-aminopyridine, CH2Cl2
ClCOOEt, Et3N, CH2Cl2, 0 °C, then 3-amino-1,2,4-triazole in DMSO, rt
(COCl)2, cat. DMF, CH2Cl2, 0 °C to rt then 4-amino-1,2,4-triazole in DMF, 0 °C
ClCOOEt, Et3N, CH2Cl2, 0 °C, then 2-aminothiazole in DMSO, rt
ClCOOEt, Et3N, CH2Cl2, 0 °C, then 2-amino-1,3,4-thiadiazole in DMSO, rt
CDI, DMF, rt, then 5-aminotetrazole in DMSO, 40 °C
Final product
Total yield (%)
3
4
5
6
7
8
31
93
92
85
88
87
for 5, and 1,10-carbodiimidazole (CDI) for 8 as optimized reagents,
and satisfactory results were obtained as shown in Table 1.
Figure 2 shows synthesis of (70S) mycophenolic epoxyketone 9
and (70R) mycophenolic epoxyketone 10 via kinetic resolution by
Sharpless asymmetric epoxidation.10 Esterification of 13 and sub-
sequent DIBAL reduction afforded 14, which was converted to
racemic alcohol 15. Kinetic resolution of (60R,70S) epoxy alcohol
lowed by deprotection using TBAF gave desired (70S) epoxyketone
9, and a pure sample was subjected to biological evaluation after
recrystallization from EtOAc/hexane. Kinetic resolution of 15 with
D
-(+)-DIPT afforded (60S,70R) epoxy alcohol, which was subjected to
Dess–Martin oxidation and deprotection of the silyl group to give
(70R) epoxyketone 10 as a pure compound after recrystallization.
Aldehyde 14 was converted to mycophenolic thioacetate 11 in 3
steps and mycophenolic thiol 12 in 4 steps as shown in Figure 3.
Sodium borohydride reduction of aldehyde 14 gave alcohol 16,
was successfully achieved by treating 15 with
D
-(ꢀ)-DIPT, Ti(OiPr)4,
t
and BuOOH. Dess–Martin oxidation of (60R,70S) epoxy alcohol fol-

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K. Sunohara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5140–5144
Figure 2. Syntheses of (70S) mycophenolic epoxyketone (9) and (70R) mycophenolic epoxyketone (10).
which was converted to 17 in 81% yield by Mitsunobu reaction
using thioacetate.11 Deprotection of silyl ether 17 with TBAF affor-
ded compound 11, which was converted to thiol 12 by
methanolysis.
Compound 1 induces differentiation or apoptosis depending on
the cell type. When K562 cells were cultured in the presence of
IMPDH inhibitors, the cells differentiate into late erythroblast
and acquire the capability to synthesize hemoglobin.4 As shown
in Table 2, 3-amino-1,2,4-triazole derivative 4 exhibited most po-
tent antiproliferative activity (IC50 = 0.48
10 showed comparable antiproliferative activity (IC50 = 2.9
for 9 and 2.3 M for 10) against K562 cells. Similar tendency was
lM). Compounds 9 and
l
M
l
observed on differentiation-inducing activity of these compounds
in K562 cells.
Figure 3. Syntheses of mycophenolic thioacetate (11) and mycophenolic thiol (12).

K. Sunohara et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5140–5144
5143
With these three effective MPA analogs in hand, we first deter-
mined comparable effective dose of 1, 2, 4, 9, and 10 on antiprolif-
erative activity against K562 cells. Antiproliferative activities of 2
10
9
8
7
6
5
4
3
2
1
0
at 2.5
to that of 1 at 1
novo GMP biosynthesis pathway, hence guanosine recovers
depletion of GMP pool via salvage pathway.4 Addition of 50
lM, 4 at 1
lM, 9 at 5
lM, and 10 at 5 lM were similar level
lM as shown in Figure 4. IMPDH inhibitor blocks de
lM
of guanosine to each sample attenuated antiproliferative activities
of 1, 2, 4, and 10. In contrast, attenuation effect by supplemental
guanosine was not significant in the case of 9. Furthermore,
sodium butyrate, an inhibitor of HDAC12 did not show the attenu-
ation in antiproliferative activity by supplemental guanosine under
our assay conditions. These results suggested that compounds 2, 4,
and 10 are effective inhibitors of IMPDH as well as 1 in cultured
cells. Table 3 shows inhibitory activities of 1, 2, 4, 9, and 10 against
human IMPDH I and II in vitro. The inhibitory activity of compound
4 was comparable to 1, however, selectivity for IMPDH II was lost
(selectivity index of 4: 1.1, 1: 1.6, IMPDH I/IMPDH II IC50 ratio).
(70S) Epoxyketone 9 and (70R) epoxyketone 10 showed comparable
activities to 2, whereas selectivity for IMPDH II slightly increased
(selectivity index of 9: 1.7, 10: 1.8, 2: 0.8, IMPDH I/IMPDH II IC50
ratio).
SB
1
2
4
9
10
[1 µM] [5 µM] [5 µM]
[1 µM] [2.5 µM]
[1 mM]
Figure 4. Effects of guanosine on proliferation of K562 cells. K562 cells were
treated by the indicated-compounds with 50 lM guanosine (closed bar) or without
guanosine (open bar). Assays were performed as described previously.4 SB: sodium
butyrate, –: dimethyl sulfoxide (vehicle).
Table 3
IMPDH inhibitory activity of MPA derivatives
In conclusion, we succeeded in converting 1 into 10 new MPA
derivatives and found three distinctive antiproliferative agents 4,
9, and 10 against K562 cells. K562 cell proliferation inhibited by
4 or 10 was recovered by supplemental guanosine similar to the
case of IMPDH inhibitors 1 and 2. Compounds 4 and 10 showed a
similar tendency in attenuation of K562 cell differentiation-induc-
ing activity by guanosine as did 1 and 2 (data not shown). These
observations indicate that IMPDH is the primary target of 4 and
10 at the cellular level. Compounds 4 and 10 showed potent inhib-
itory activities against IMPDH in vitro as expected. Compound 9
showed similar inhibitory activity against IMPDH as did 10,
whereas K562 cell differentiation-inducing activity of 9 was weak-
er than that of 10. Moreover, attenuation by guanosine was very
weak on antiproliferation activity of 9. These results indicate that
compound 9 has the other molecular target as well as IMPDH. It
is noteworthy that compound 9 has the same S configuration at
the terminus epoxide as did trapoxin, an irreversible inhibitor of
HDAC. We, therefore, hypothesized that compound 9 was a dual
inhibitor against HDAC and IMPDH II. Compound 9 weakly inhib-
ited the enzyme activity of HDAC in the nuclear lysate3 of K562
Compound
IC50
(l
M)a
Selectivity indexc
IMPDH I
IMPDH II
1
2
4
9
0.019 0.001b
0.352 0.018b
0.105 0.008
0.558 0.061
0.680 0.047
0.012 0.001b
0.424 0.085b
0.098 0.005
0.330 0.045
0.387 0.025
1.6
0.83
1.1
1.7
1.8
10
a
b
c
Errors are expressed as SE (initial cell number: 0.5 ꢁ 105 cells/ml).
Data from Ref. 4.
IC50 of human IMPDH I/IC50 of hIMPDH II.
activities against IMPDH, whereas 9 might partially inhibit a cer-
tain type of HDAC. 7-O-Lauroyl derivative of 2 inhibited HDAC at
the cellular level as described in the previous paper.3 Evaluations
of these inhibitors and their 7-O-acyl derivatives against various
type of HDACs and other molecular targets are our next challenge.
The present work may provide an important information regarding
development of a new type of drug lead for cancer and organ
transplantation.
cells at 10 lM, and the relative inhibitory activities of 1, 2, 9, and
10 were 3%, 76%, 19%, and 15% respectively as shown in Supple-
mentary Figure. Antiproliferative effects of compounds 4, 9, and
10 in K562 cells could eventually be explained by the inhibitory
Acknowledgment
We are grateful to L. Hedstrom (Brandeis University, USA) for
providing plasmids for expression of human IMPDH type I and II.
Table 2
Antiproliferative effect of MPA derivatives in K562 cells
Supplementary data
a
Compound
IC50
(
lM)
Differentiationc at 30
lM
0.19 0.01b
2.1 0.2b
26 1.7
0.48 0.08
31 2.1
+++ at 1.0
+++ at 2.5
+
+++ at 1.0
+
l
l
Md
Md
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.07.
016.
1
2
3
4
5
6
l
Me
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22 1.9
7
8
9
10
11
12
27 1.7
34 1.9
2.9 0.47
2.3 0.32
26 1.7
+
+
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