Novel hypoglycemic dihydropyridones serendipitously discovered from O- versus C-alkylation in the synthesis of VMAT2 antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.07.129

Vesicular monoamine transporter type 2 (VMAT2) is a newly emerging target for both diagnostic and therapeutic applications in diabetes mellitus. In pursuit of novel VMAT2 antagonists, we identified a potent hypoglycemic agent with a novel dihydropyridone

Full text of DOI:10.1016/j.bmcl.2008.07.129

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5111–5114
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel hypoglycemic dihydropyridones serendipitously discovered
from O- versus C-alkylation in the synthesis of VMAT2 antagonists
Yuli Xie a, Anthony Raffo a, Masanori Ichise b, Shixian Deng a, Paul E. Harris a, Donald W. Landry a,
*
a Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA
b Department of Radiology, Columbia University Medical Center, New York, NY 10032, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Vesicular monoamine transporter type 2 (VMAT2) is a newly emerging target for both diagnostic and
therapeutic applications in diabetes mellitus. In pursuit of novel VMAT2 antagonists, we identified a
potent hypoglycemic agent with a novel dihydropyridone scaffold. Several analogs were designed and
synthesized. A preliminary structure activity relationship (SAR) showed that the dihydropyridone scaf-
fold is required for the activity.
Received 2 July 2008
Revised 24 July 2008
Accepted 28 July 2008
Available online 7 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
VMAT2
Hypoglycemic
SARs
Dihydropyridone
Diabetes mellitus is a growing epidemic affecting hundreds of
millions of people worldwide.1 Despite a recent explosion of new
classes of hypoglycemic agents, the medical need remains largely
unmet and innovative diagnostics and therapeutics are still ur-
gently needed. We have been particularly interested in the vesicu-
In an effort to generate novel VMAT2 antagonists, we attempted
to synthesize compound 1 (Fig. 1), a simplified analog of DTBZ. As
shown in Scheme 1, veratraldehyde 2 was treated with ammonium
acetate and converted into b-amino acid 3 by condensation with
malonic acid. Protection with Boc anhydride and subsequent con-
densation with potassium malonate methyl ester led to b-keto
ester 4. Alkylation with isobutyl bromide in the presence of potas-
sium carbonate afforded a mixture of 5 and 6 in moderate yield.
After removal of the Boc group, the mixture was treated with
sodium bicarbonate in methanol to yield the cyclized products 7
and 8 quantitatively. Compound 9 was obtained from reduction
of 7 with sodium borohydride and then further converted to 1
and its diastereoisomers with lithium aluminum hydride.
Racemic compound 1 and its diastereoisomers were evaluated
for their ability to improve glucose tolerance by intraperitoneal
glucose tolerance tests (IPGTTs)7 in rats. The new analogs were less
potent than TBZ possibly due to diminished affinity for VMAT2
(Fig. 2). This poor result halted our further study of compound 1.
However, during random screens of intermediates generated in
the course of the synthesis of 1, we found that compound 8, a novel
dihydropyridone resulted from the competing O- versus C-alkyl-
ation of enolic b-keto ester 4 followed by cyclization, showed
potent hypoglycemic effect. As illustrated in Figure 2, compound
8 decreased the AUC IPGTT (the area under the blood glucose con-
centration–time curve) by 45% at the dose of 2 mg/kg compared to
26% for TBZ.
lar monoamine transporter type
2 (VMAT2) as a potential
diagnostic and therapeutic target for diabetes.
VMAT is a member of the vesicular transporter family responsi-
ble for the uptake and secretion of monoamine neurotransmitters
in neurons and endocrine cells.2 Two isoforms of VMAT (types
1 and 2) have been cloned, and interestingly, the insulin-producing
beta cells in the pancreas only express the VMAT2 isoform.3 We
have recently demonstrated the feasibility of noninvasive mea-
surement of beta cell mass both in humans and in rodents by pos-
itron emission tomography (PET) using VMAT2 as the biomarker
and its specific antagonist dihydrotetrabenazine (DTBZ) as the tra-
cer.4,5 More strikingly, our studies have further shown that VMAT2
plays an important functional role in the regulation of insulin
secretion in beta cells.6 VMAT2 antagonist tetrabenazine (TBZ)
and its active metabolite DTBZ (Fig. 1) are potent hypoglycemic
agents that stimulate insulin secretion in vitro and improve glu-
cose tolerance in normal and diabetic rats.6 VMAT2 antagonists,
therefore, have both diagnostic and therapeutic potential in the
management of diabetes mellitus.
Prompted by this surprising result, we designed and
* Corresponding author. Tel.: +1 212 3055839; fax: +1 212 3053475.
E-mail address: dwl1@columbia.edu (D.W. Landry).
synthesized several analogs of 8.8 As outlined in Scheme 2,
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.129

5112
Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5111–5114
OH
OH
O
N
H
H
O
O
H
O
O
N
H
N
O
O
1
TBZ
DTBZ
Figure 1. The structure of TBZ, DTBZ and compound 1.
O
1. NH4Ac,EtOH/H2O
50˚C,3h
O
OH
O
O
1. Boc2O/NaOH,rt,24h
O
H
O
OCH3
O
CO2CH3
O
O
O
2. CDI,THF
2. Melonic acid
80˚C,24h
NH2
3 65%
,rt, 2d
CO2K
NHBoc
4 57%
2
O
O
O
O
OCH3
O
O
O
N
H
O
NHBoc
1.TFA,CH2Cl2
rt, 0.5h
Br
5
34%
7
O
2. NaHCO3,MeOH
rt, 24h
O
K2CO3/TBAI
acetone,reflux,3h
O
O
OCH3
O
O
N
H
O
NHBoc
O
6
44%
8
OH
OH
NaBH4
MeOH,rt,1h
LiAlH4
THF,reflux,4h
O
O
N
H
O
N
H
O
O
9
91%
1
74%
Scheme 1. The synthesis of compound 1.
veratraldehyde 2 was first condensed with ethyl acetoacetate,
and spontaneous cyclization yielded lactone 10. Using potas-
sium carbonate as the base, O-alkylation of 10 with methyl
bromide or isobutyl bromide provided 11 and 12. Similarly,
15 and 16 were prepared from dihydroisoquinoline 13 via con-
densation with dimethyl 1,3-acetonedicarboxylate followed by
cyclization and alkylation. DDQ-induced aromatization and
acidic hydrolysis of 8 afforded 17 and 18, respectively. Analogs
prepared as above were tested for their hypoglycemic activities
in rats using the IPGTT protocol. Interestingly, the hypoglyce-
mic effects of these compounds were only seen following glu-
cose stimulation. Results in Figure 2 demonstrated that the
dihydropyridone scaffold in 8 is essential for the hypoglycemic
activity. Replacement with dihydropyrone (11, 12), oxidation, or
hydrolysis of 8 (17, 18) resulted in the total loss of activities.
Interestingly, the rigid derivatives 15 and 16 were active but
less potent in opposition to the SAR trend seen in compound
1 and DTBZ.
and the uptake of [11C]DTBZ in pancreas was monitored by PET
scan in the presence of excess of 8. Contrary to our expectation,
8 did not significantly displace [11C]DTBZ in the endocrine pan-
creas (Fig. 3), suggesting a weak binding of 8 to VMAT2 relative
to DTBZ. VMAT2 is known to possess multiple binding sites.10
Our results do not rule out the possibility that 8 binds to VMAT2
at different binding sites. We have previously shown that TBZ and
DTBZ act directly on beta cells of the endocrine pancreas to in-
crease insulin secretion following glucose challenge,6 and give
rise to a hypoglycemic activity, but due to abundant expression
of VMAT2 in brain, these compounds also cause central nervous
system side effects. Interestingly, we observed that the treatment
with 8 (at 2 mg/kg) was devoid of tonic seizure commonly asso-
ciated with equal doses of TBZ or DTBZ, supporting that com-
pound
8 works through a different mechanism. Another
possibility consistent with our observations is that 8 may act as
a competitive inhibitor of dopamine at VMAT2 on beta cell vesi-
cles. DTBZ and its analogs are structurally similar to a class of
quinolizine alkaloids previously shown to inhibit dipeptidyl pep-
tidase IV (DPP-IV),11 but 8 tested negative against DPP-IV in vitro
To test whether the strong hypoglycemic effect of 8 is con-
ferred by binding to VMAT2 in beta cells, we performed a PET
study in rats.9 The animals were treated with radiolabeled DTBZ,
at concentrations up to 10 l
M (data not shown).12 Therefore, the

Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5111–5114
5113
Figure 3. Effect of post-injection of compound 8 on biodistribution of [11C]DTBZ in
pancreas. PET scans were performed on anesthetized 12–14-week-old Lewis male
rats injected with radioligand [ lCi/gm body weight,
11C]DTBZ (0 min, 0.5–1.0
specific activity >2000 mCi/mol) and cold compound 8 (30 min, 2 mg/kg). The
fraction of radioligand in pancreas relative to the total amount of injected was
calculated and plotted versus time after injection. Error bars indicate SEM (n = 3).
The area under the curve (AUC) from 30 min to 90 min was calculated by the
trapezoidal rule for each experiment. The statistical significance of difference
between the average AUC baseline and AUC compound 3 was p > 0.05 as calculated
by the method of Student.
mechanism underlying the anti-hyperglycemic effect of 8 remains
to be determined.
Figure 2. Glucose tolerance tests of novel hypoglycemic compounds. Six hours
fasted Lewis rats were administered the drugs intravenously (À30 min, 2 mg/kg)
followed by intraperitoneal glucose injection (0 min, 2 g/kg), and blood glucose
levels were monitored for 120 min. Top panel: the blood glucose concentration
versus time curves are presented for three repeat experiments with vehicle, TBZ,
and compound 8 performed in a single rodent. Bottom panel: results are presented
as area under the curve. The area under the curve (AUC IPGTT) was calculated by
the trapezoidal rule. Error bars indicate SEM (n = 5). The statistical significance of
the difference between drug and vehicle was calculated by the method of Student. A
p value of less than 0.005 is shown by an asterisk. The p value of the difference in
between the average AUC IPGTT of TBZ and compound 8 was 0.030.
In summary, we have completed the synthesis of a novel analog
of VMAT2 antagonist DTBZ (compound 1). Despite the disappoint-
ing results of 1, a highly potent hypoglycemic agent (compound 8)
was serendipitously discovered. Subsequent modification revealed
the requirement of a novel dihydropyridone core structure for the
activity. Mechanism studies show that compound 8 may not work
through VMAT2 and DPP-IV. More detailed studies of 8 are cur-
rently being pursued.
R
O
O
O
O
O
O
CHO
RBr
O
O
O
O
O
O
O
OEt
K2CO3/TBAI
NaH,THF,
-5˚C, 1.5h
O
acetone,reflux,3h
11
, R=CH3 87%
83%
10
2
12, R=CH2CH(CH3)2
61%
O
R
O
O
H3CO2C
CO2CH3
1.
N
O
O
RBr
K2CO3/TBAI
O
O
NaHCO3,H2O,10˚C
N
O
O
O
N
O
2. NaOH(50%),rt,24h
acetone,reflux,3h
13
14
56%
15
, R=CH3 78%
16,R=CH2CH(CH )
3 2 64%
O
O
O
DDQ
HCl
O
O
O
O
O
O
O
N
H
O
N
H
N
H
O
CH2Cl2,rt,24h
MeOH,rt,24h
17
18 95%
76%
8
Scheme 2. The synthesis of analogs of compound 8.

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Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5111–5114
3.86 (s, 3H), 3.64–3.60 (m, 2H), 2.71–2.47 (m, 2H), 2.02–1.96 (m, 1H), 0.96–0.93
Acknowledgments
(dd, 6H); 13C NMR (75 MHz, CD3OD) d 170.0, 168.9, 149.4, 149.1, 133.5, 119.0,
111.4, 109.3, 93.8, 75.1, 56.3, 56.2, 55.0, 37.3, 28.1, 19.5, 19.4; ESI-MS (M++H):
306.3.15: 1H NMR (300 MHz, CDCl3) d 6.58 (s, 1H), 6.54 (s, 1H), 5.15 (s, 1H),
4.71–4.65 (m, 2H), 3.80 (s, 3H), 3.79 (s, 3H), 3.67 (s, 3H), 2.81–2.60 (m, 4H),
2.48–2.37 (m, 1H); 13C NMR (75 MHz, CD3OD) d 168.3, 167.4, 148.0(2C), 127.4,
127.3, 111.3, 108.6, 94.6, 56.3, 56,2, 56.0, 54.1, 38.4, 37.2, 29.4; ESI-MS (M++H):
290.1.
This work was supported by grants from the PHS, NIH, NIDDK 2
RO1 DK63567-05 (P.E.H.), and the Columbia University DERC (5
P30 DK063608-05).
9. PET study protocol: PET scans were performed on 12–14-week-old Lewis
male rats. Prior to imaging, the animals were anesthetized by isoflurane
inhalation. After a transmission scan of the area of interest had been acquired
(used to perform attenuation correction of the emission data), the radioligand
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(m, 2H), 6.84 (s, 1H), 5.45 (br, 1H), 5.07(s, 1H), 4.69–4.63 (dd, 1H), 3.87 (s, 3H),
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