Synthesis, biological evaluation and structural determination of β-aminoacyl-containing cyclic hydrazine derivatives as dipeptidyl peptidase IV (DPP-IV) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.01.111

Inhibitors of dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes. A series of β-aminoacyl-containing cyclic hydrazine derivatives were synthesized and evaluated as DPP-IV inhibitors. One member of this series, (R)-3-amino-1-(2-benzoyl-1,2-diazepan-1-yl)-4-(2,4,5-trifluorophenyl)but an-1-one (10f), showed potent in vitro activity, good selectivity and in vivo efficacy in mouse models. Also, the binding mode of compound 10f was determined by X-ray crystallography.

Full text of DOI:10.1016/j.bmcl.2007.01.111

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2622–2628
Synthesis, biological evaluation and structural determination
of b-aminoacyl-containing cyclic hydrazine derivatives as
dipeptidyl peptidase IV (DPP-IV) inhibitors
Jin Hee Ahn,a,* Mi Sik Shin,a Mi Ae Jun,a Sun Ho Jung,a Seung Kyu Kang,a
Kwang Rok Kim,a Sang Dal Rhee,a Nam Sook Kang,a Sun Young Kim,b
Sang-Kwon Sohn,b Sung Gyu Kim,b Mi Sun Jin,c Jie Oh Lee,c
Hyae Gyeong Cheona and Sung Soo Kima,*
aBioorganic Science Division, Korea Research Institute of Chemical Technology, Yuseong-Gu, Daejeon 305-600, Republic of Korea
bNamyang Research Center, Yungjin Pharm. Co., Ltd, Musong-dong, Kyungki-do, Republic of Korea
cKorea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon 305-701, Republic of Korea
Received 12 November 2006; revised 21 January 2007; accepted 31 January 2007
Available online 8 February 2007
Abstract—Inhibitors of dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes. A series of
b-aminoacyl-containing cyclic hydrazine derivatives were synthesized and evaluated as DPP-IV inhibitors. One member of this ser-
ies, (R)-3-amino-1-(2-benzoyl-1,2-diazepan-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (10f), showed potent in vitro activity, good
selectivity and in vivo efficacy in mouse models. Also, the binding mode of compound 10f was determined by X-ray crystallography.
Ó 2007 Elsevier Ltd. All rights reserved.
Glucagon-like peptide-1 (GLP-1)1 is an incretin hor-
mone secreted from the L cells of the small intestine in
response to food intake. This hormone plays several bio-
logical roles including the stimulation of insulin secre-
tion, inhibition of glucagon secretion, retardation of
gastric emptying, induction of satiety and stimulating
the regeneration and differentiation of islet b-cells.2
However, GLP-1 (GLP-1[7-36]amide) is rapidly degrad-
ed in vivo (lifetime: about 1 min) through the action of
dipeptidyl peptidase IV (DPP-IV), which cleaves a
dipeptide from the N-terminus to give the inactive
GLP[9-36]amide.3
thus increases insulin secretion,5 which can ameliorate
hyperglycaemia in type 2 diabetes. A number of small
molecule inhibitors of DPP-IV have been described6
and several of these, including Vildagliptin (LAF237),7a
Saxagliptin (BMS477118)7b and Sitagliptin (MK-0431),8
are in late-stage of clinical development or approved by
the U.S. Food and Drug Administration (Fig. 1).
Since DPP-IV is a dipeptidase that selectively binds
substrates with proline at the P1 position, many of its
inhibitors investigated to date possess 5-membered het-
erocyclic rings (e.g., pyrrolidine, thiazolidine, cyano-
pyrrolidine and cyano-thiazolidine) that serve as proline
mimics. A number of DPP-IV inhibitors contain cyano-
pyrrolidine ring.6 We have recently described several
novel 5-membered proline surrogates that are based on
pyrazolidine9,10 and pyrazoline.11 Our compounds con-
taining the pyrazolidine template showed moderate in
vivo efficacy as well as in vitro activity against DPP-
IV.9a,10 A group at Merck recently described a series
of structurally novel b-amino amide derivatives that
have good in vitro potency and in vivo efficacy.8 Among
these, MK-0431, which is approved by FDA, shows
excellent selectivity and in vivo efficacy. This
DPP-IV is a serine protease cleaving the N-terminal
dipeptide with a preference for L-proline or L-alanine
at the penultimate position.4 This protease is expressed
in many tissues and body fluids, and exists as either a
membrane-bound or a soluble enzyme. Inhibition of
DPP-IV increases the level of circulating GLP-1 and
Keywords: DPP-IV inhibitor; Diabetes; Cyclic hydrazine.
*
Corresponding authors. Tel.: +82 42 860 7076; fax +82 42 860
7160(S.S.K.); e-mail addresses: jhahn@krict.re.kr; sungsuk@krict.
re.kr
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.111

J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2622–2628
2623
OH
F
H3PO4
NH2
F
O
HO
H
N
O
CN
CN
O
N
N
N
N
N
NH2
F
N
CF3
Saxagliptin (BMS477118)
Vildagliptin (LAF-237)
Sitagliptin (MK-0431)
Figure 1. Molecular structures of representative DPP-IV inhibitors.
F
F
F
F
O
HCl
NH2
F
F
F
a,b
O
O
O
O
R
N
N
OH
OMe
NH2
O
N
N
F
F
n
HN
4
5
R
n = 1 -3
c,d
Figure 2. The design of new DPP-IV inhibitor, b-aminoacyl-contain-
ing cyclic hydrazine derivatives.
F
F
F
F
Boc
F
e,f
NH
O
NH2 O
observation prompted us to explore the properties of
new DPP-IV inhibitors that possess our basic pyrazoli-
dine structure and contain appended b-amino acid
moiety (Fig. 2, n = 1). In this effort, we also investigated
related compounds that have 6- and 7-membered cyclic
hydrazine core structures (Fig. 2, n = 2 or 3). We now
wish to report the synthesis of b-aminoacyl-containing
cyclic hydrazine derivatives and their biological evalua-
tion as DPP-IV inhibitors (Fig. 2).
OH
OMe
F
6
7
Scheme 2. Reagents and conditions: (a) 2,2-dimethyl-1,3-dioxane-4,6-
dione, DMAP, N,N-diisopropylethylamine, trimethylacetylchloride,
acetonitrile, rt, 95%; (b) MeOH, toluene, reflux, 89%; (c) MeOH,
ammonium acetate, reflux, 92%; (d) chloro(1,5-cyclooctadiene)rhodi-
um (I) dimer, (R)-(À)-1-[(S)-2-(bis(4-trifluoromethylphenyl)phosphino)
ferrocenyl ethyl-di-tert-butylphosphine, H2 (100 psi), trifluoroethanol,
68%; (e) Boc2O, CH2Cl2, rt, 93%; (f) LiOH, THF, MeOH, H2O, rt,
99%.
A series of b-aminoacyl-containing cyclic hydrazine
derivatives were synthesized by using the route shown
in Schemes 1–3. Cyclic hydrazines (3, n = 1–3), including
pyrazolidine 2HCl (n = 1), were produced by employing
a procedure we developed earlier (Scheme 1).12 Di-tert-
butyldihydrazodiformate (1) was treated with dib-
romoalkanes in the presence of Et4NBr in toluene under
reflux, followed by deprotection of Boc using 4 M HCl
in dioxane to produce the cyclic hydrazine as their bis-
HCl salts (3).
F
n
F
Boc
NH
O
a
HN NH
2HCl
N
HN
F
n
3
8
n = 1 -3
c
(R)-3-BocNH-4-(2,4,5-trifluorophenyl)butanoic acid (7)8
was synthesized according to a modification of one
described by the Merck group13 (Scheme 2). 2-(2,4,5-
Trifluorophenyl)acetic acid (1) was coupled with 2,2-di-
methyl-1,3-dioxane-4,6-dione (Meldrum’s acid) using
pivaloyl chloride as an activating reagent to produce
the coupling product, followed by methanolysis to yield
the b-keto ester (5). Reaction of 5 with ammonium
acetates produced the corresponding b-enamino-ester,
which was converted to the chiral b-(R)-amino ester
(6) by asymmetric catalytic hydrogenation with the
b
F
F
F
HCl
NH2
F
Boc
F
c
NH
O
O
R
N
N
N
N
F
n
n
R
10
n = 1 -3
9
n = 1 -3
Scheme 3. Reagents and conditions: (a) compound 7, triethylamine,
EDCI, CH2Cl2, rt, 47–96%; (b) electrophiles phenylisocyanate,
p-toluenesulfonyl chloride, and benzoyl chloride, CH2Cl2, triethyl-
amine, rt; (c) HCl, dioxane, rt, 75–90%.
n
n
a
b
HN NH
Boc Boc
chloro(1,5-cyclooctadiene)rhodium (I) dimer and (R)-
(À)-1-[(S)-2-(bis(4-trifluoromethylphenyl)phosphino)
ferrocenyl ethyl-di-tert-butylphosphine under H2
(100 psi) in trifluoroethanol. Protection of the chiral
b-(R)-amino ester (6) with Boc2O, and hydrolysis with
LiOH produced the desired (R)-3-BocNH-4-(2,4,5-tri-
fluorophenyl)butanoic acid (7).
HN NH
2HCl
N
N
Boc
Boc
1
2
3
Scheme 1. Reagents and conditions: (a) 50% NaOH, dibromopropane
(n = 1), dibromobutane (n = 2), dibromopentane (n = 3), Et4NBr,
toluene, reflux, 6 h, 78–95%; (b) 4M HCl, dioxane, rt, 12 h, 89–98%.

2624
J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2622–2628
Table 1. Inhibitory activity of cyclichydrazine derivatives with
b-aminoacyl group against DPP-IV
shown in Scheme 3. The cyclic hydrazines (3, n = 1–3)
were coupled with b-amino acid (7) in the presence of
EDCI to produce the coupling product (8) which upon
reaction with electrophiles yielded the corresponding
N-substituted derivatives (9). Boc-deprotection with
HCl transformed these substances into the desired
b-aminoacyl-containing cyclic hydrazine derivatives
(10).14
a
IC50 (nM)
Compound
Structure
F
HCl
NH2
F
O
10a
3850
N
F
F
HN
HCl
NH2
F
The b-aminoacyl-containing cyclic hydrazine derivatives
(10a–10f) were evaluated in vitro for their inhibition of
DPP-IV. MK-0431 was used as a reference compound.
Unsubstituted pyrazolidine (R = H, 10a) showed weak
inhibitory activity (IC50 = 3.85 lM). However, N-substi-
tuted pyrazolidines (10b–10d) exhibited improved in
vitro activities, among those pyrazolidines, benzoyl-
substituted pyrazolidine (10d, IC50 = 350 nM) showed
O
N
N
10b
990
F
O
HN
F
HCl
NH2
F
O
N
N
10c
500
O
F
S
I= 12.5 nM
I= 25 nM
O
I= 50 nM
F
I= 100 nM
I= 200 nM
I= 400 nM
HCl
F
NH2
O
N
N
10d
350
F
O
0.8
0.6
0.4
0.2
0
F
HCl
F
NH2
O
O
N
N
10e
50
F
O
F
HCl
NH2
F
N
N
10f
70
50
0
0.05
0.1
1 / S
0.15
0.2
F
O
Figure 3. Inhibition kinetics of DPP-IV by 10f. Different concentra-
tions of 10f were incubated in the presence of various concentrations of
Ala-Pro-AFC. The initial rates of the reaction were measured, and the
results were expressed as a Lineweaver–Burk plot. The data are
represented as means of three separated experiments.
MK-0431
a IC50 values were determined from direct regression curve analysis.
a nearly 10-fold more potency than that of the parent
pyrazolidine (R = H, 10a). Changing the pyrazolidine
ring to the larger 6- and 7-membered cyclic hydrazines
The synthesis of the b-aminoacyl cyclic hydrazine deriv-
atives (10) was achieved by using the 3-step procedure
Table 2. Selectivity of Compounds 10e and 10f towards DPP-IV related enzymes
a
DPP-IV IC50 (nM)
Compound
DPP-2 (nM)
DPP-8 (nM)
DPP-9 (nM)
10e
10f
50
70
72,000
18,190
>100,000
89,120
>100,000
48,660
APN (nM)
>100,000
>100,000
POP (nM)
>100,000
>1,000,000
Trypsin (nM)
>100,000
>1,000,000
Elastase (nM)
>100,000
>100,000
10e
10f
aIC50 values were determined from direct regression curve analysis.

J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2622–2628
2625
(10e and 10f), found in 10e and 10f, resulted in improved
100
75
50
25
0
***
***
inhibition. Compounds 10e and 10f were the most po-
tent DPP-IV inhibitors (IC50 = 50 and 70 nM, respec-
tively) in this series, showing 5–7 times greater activity
than the 5-membered pyrazolidine (10d). Thus, we used
those compounds (10e and 10f) as representative com-
pounds for further development (Table 1).
***
Compounds 10e and 10f were investigated for their
selectivity towards a variety of DPP-IV related pepti-
dases, including DPP-2, DPP-8, DPP-9, APN, POP,
trypsin and elastase. Selectivity over DPP-8 and DPP-
9 was considered particularly important because inhibi-
tion of these enzymes has been ascribed to toxicity in
preclinical species.15 The results of this effort (Table 2)
showed that 10e displayed a greater than 1400-fold
selectivity over all the enzymes tested including DPP-8
and DPP-9. Also, compound 10f showed good to excel-
lent selectivity against most of the isozymes tested.
control MK0431
10e
10f
1 hr
100
75
50
25
0
***
**
**
To identify the enzyme-inhibition kinetic patterns of
compound 10f, we carried out inhibition kinetic analyses
with porcine kidney DPP-IV. As shown in Figure 3,
compound 10f showed a competitive inhibition pattern
well fitted to a Lineweaver–Burk plot and had a Ki value
of 56.2 nM. Similar experiments showed that 10e is also
a competitive inhibitor (Ki = 44 nM, data not shown).
control MK0431
10e
10f
6 hr
Figure 5. In vivo inhibitory effect of compounds 10e and 10f in
C57BL/6J mice. Each compound was orally administered in a single
dose of 50 mg/kg. The data are represented as means SEM (n = 7).
**P < 0.01 versus control; ***P < 0.005 versus control.
a
b
100
100
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
1999.0
2399.4
2799.8
3200.2
3600.6
4001.0
1999.0
2399.4
2799.8
3200.2
3600.6
4001.0
Mass (m/z)
Mass (m/z)
GLP-1 only
GLP-1 + DPP-IV
c
d
100
100
90
80
70
60
50
40
30
20
10
90
80
70
60
50
40
30
20
10
0
0
1999.0
2399.4
2799.8
3200.2
3600.6
4001.0
1999.0
2399.4
2799.8
3200.2
3600.6
4001.0
Mass (m/z)
Mass (m/z)
GLP-1+DPP-IV +10 M10f
GLP-1+DPP-IV+ 1 M10f
Figure 4. Effect of compound 10f on GLP-1 degradation by DPP-IV in vitro. GLP-1 (15 lM) was incubated with porcine kidney DPP-IV (1 lU) in
the presence of 10 or 1 lM 10f.

2626
J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2622–2628
The effect of compound 10f on DPP-IV-mediated
degradation of GLP-1 was probed in vitro by using
GLP-1 peptide (NH2-7-36) and DPP-IV. The extent
of degradation of GLP-1 was analyzed by employing
MALDI-TOF mass spectrometry. The results
(Fig. 4) showed that compound 10f blocks 85% and
46% DPP-IV-mediated degradation of GLP-1 over a
24 h period when used in concentrations of 10 and
1 lM, respectively.
better and longer inhibition of DPP-IV activity than
MK-0431 or 10e (ca. 65% DPP-IV inhibition).
On the basis of its in vivo DPP-IV inhibition in normal
mice, compound 10f was subjected to in vivo evaluation
by assessing its ability to improve glucose intolerance in
TallyHo mice.16 The results (Fig. 6) showed that glucose
levels were significantly reduced in a dose-dependent
manner from 10 mg/kg (52% reduction) to 50 mg/kg
(77% reduction) in treated animals when 10f was admin-
istered 30 min before an oral glucose challenge (2 g/kg).
Compounds 10e and 10f were evaluated in vivo for their
ability to reduce DPP-IV activity in normal C57BL/6J
mice (Fig. 5). Oral administration of compounds 10e
and 10f, at high dose (50 mg/kg), resulted in ca 75%
and 90% inhibition of plasma DPP-IV activity after
1 h, respectively. After 6 h, compound 10f exhibited
The positive results arising from studies with 10f stimu-
lated an investigation aimed at determining the crystal
structure of recombinant human DPP-IV, complexed
with this inhibitor.
Lean
a
b
750
Vehicle
700
10f(10mg/kg)
650
600
550
500
450
400
350
300
250
200
150
100
50
70000
60000
*
50000
*
40000
30000
20000
10000
0
B6
vehicle
Treatment
10f
0
20
40
60
80
100
120
Time (min)
Lean
600
550
500
450
400
350
300
250
200
150
100
50
Vehicle
10f(50mg/kg)
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
*
*
*
*
0
B6
vehicle
10f
Treatment
0
20
40
60
80
100
120
Time (min)
Figure 6. (a) Effects of compound 10f on plasma glucose levels in oral glucose tolerance test in TallyHo mice. (b) Glucose AUC was determined from
0 to 120 min. The percent-inhibition values for each treatment were generated from the AUC data normalized to the 0.5% CMC (carboxyl methyl
cellulose) challenged controls. The data are represented as mean SEM (n = 7). *P < 0.05 versus vehicle in Student’s t test.

J. H. Ahn et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2622–2628
2627
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.01.111.
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Figure 7. Compound 10f bound to DPP-IV. The overlay of 10f (green)
and MK-0431 (purple) shows the different binding mode. Coordinate
for the structure of DPP-IV in complex with 10f has been deposited
with the RCSB protein databank, PDB ID 2OLE.
As can be seen by viewing the binding site region in the
complex (pictured in Fig. 7), the b-aminoacyl group has
a similar conformation as that of MK-0431.8 However,
the cyclic hydrazine moiety in 10f has different binding
modes with the carbonyl oxygen of its benzoyl moiety
forming water-bridged hydrogen bonding interactions
with the side chains of His 126 and Ser 209 and the car-
bonyl group of Glu205.
In conclusion, this investigation has led to the design,
synthesis and biological evaluation of a series of
b-aminoacyl-containing cyclic hydrazine derivatives.
In this series, 10e and 10f were found to be compet-
itive inhibitors of DPP-IV and to display excellent
selectivity over related isozymes. The in vivo efficacy
of 10f was demonstrated by its inhibition of plasma
DPP-IV activity and its suppression of blood glucose
elevation. Also, the binding mode of 10f in the bind-
ing site of DPP-IV was determined by using X-ray
crystallography. The cyclic hydrazine derivatives eval-
uated in this effort are members of an interesting
chemical class that possess two nitrogens suitable
for diverse derivatization. Further studies are under-
way to optimize this compound class for the treat-
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Acknowledgments
This research was supported by the Center for Biologi-
cal Modulators of the 21st Century Frontier R&D
Program, the Ministry of Science and Technology,
Korea. We thank the staff of the 4A and 6B beam lines
of Pohang Accelerator Laboratory.

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Simmons, B. L.; Williams, M.; Feng, X.; Yang, C. PCT
Int. WO2004/083212 A1, 2004 (Merck & Co., Inc.).
[1,2]diazepanÆ2HCl (3.36 g, 18 mmol), EDCI (3.45 g,
18 mmol) and triethylamine (8.36 mL, 60 mmol) in
CH2Cl2 (50 mL) was stirred for 12 h at room temperature.
The reaction mixture was diluted with brine and CH2Cl2.
The organic layer was dried and evaporated. The residue
was purified by silica gel column chromatography to give
[3-[1,2]diazepan-1-yl-3-oxo-1-(2,4,5-trifluorobenzyl)pro-
14. Compound 10e; A mixture of 3-(R)-tert-butoxycarbonyla-
mino-4-(2,4,5-trifluorophenyl)butyric acid (2 g, 6 mmol),
hexahydro pyridazineÆ2HCl (2.86 g, 18 mmol), EDCI
(3.45 g, 18 mmol) and triethylamine (8.36 mL, 60 mmol)
in CH2Cl2 (50 mL) was stirred for 12 h at room temper-
ature. The reaction mixture was diluted with brine and
CH2Cl2. The organic layer was dried and evaporated. The
residue was purified by silica gel column chromatography
to give [3-oxo-3-(tetrahydropyridazin-1-yl)-1-(2,4,5-triflu-
orobenzyl)propyl]carbamic acid tert-butyl ester (1.6 g,
1
pyl]carbamic acid tert-butyl ester (2.2 g, 90%). H NMR
(CDCl3, 200 MHz) d 7.07–7.03 (m, 1H), 6.71–6.87 (m,
1H), 5.63 (d, J = 8.4 Hz, 1H), 4.15–4.13 (m, 1H), 3.64 (t,
J = 5.0 Hz, 1H), 3.46 (t, J = 5.0 Hz, 1H), 3.93–3.89 (m,
4H), 2.74 (dd, J = 13.1, 5.4 Hz, 1H), 2.58 (dd, J = 19.8,
4.6 Hz, 1H), 1.71–1.56 (m, 6H), 1.36 (s, 9H). To a mixture
of (R)-[3-[1,2]diazepan-1-yl-3-oxo-1-(2,4,5-trifluoroben-
zyl)propyl]carbamic acid tert-butyl ester (200 mg,
0.482 mmol) and Et3N (97 mg, 0.962 mmol)in CH2Cl2
(10 mL) was added benzoyl chloride (101 mg, 0.722 mmol)
and the mixture was stirred for 3 h at room temperature.
The solvents were evaporated, and the residue was purified
by silica gel column chromatography to give (R)-tert-butyl
4-(2-benzoyl-1,2-diazepan-1-yl)-4-oxo-1-(2,4,5-trifluor-
ophenyl)butan-2- ylcarbamate (210 mg, 84%). 1H NMR
(CDCl3, 200 MHz) d 7.61–7.37 (m, 5H), 7.20–7.02 (m,
1H), 7.01–6.83 (m, 1H), 5.50-5.20 (m, 1H), 4.45–3.99 (m,
3H), 3.98–2.40 (m, 5H), 1.90–1.50 (m, 6H), 1.37 (s, 9H).
To a solution of (R)-tert-butyl 4-(2-benzoyl-1,2-diazepan-
1-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-ylcarbamate
(220 mg, 0.423 mmol) in EtOAc (2 mL) was added 4M-
HCl/1,4-dioxane (2 mL) and the mixture was stirred for
16 h at room temperature. The solvents were evaporated,
and the residue was crystallized with ether to give (R)-3-
amino-1-(2-benzoyl-1,2-diazepan-1-yl)-4-(2,4,5-trifluor-
ophenyl)butan-1-one hydrochloride (143 mg, 75%). 1H
NMR (DMSO-d6, 500 MHz) d 8.41–8.20 (br s, 2H), 7.70–
7.22 (m, 7H), 4.25–3.89 (m, 1H), 3.83–3.68 (m, 2H), 3.55–
3.30 (m, 2H), 3.18–2.67 (m, 4H), 1.82–1.46 (m, 6H);
HRMS (free base, C22H24F3N3O2): calcd, 419.1821 found,
419.1837. Analytical HPLC method A: tR = 3.17 min
(99.9% purity); method B: tR = 6.31 min (98.4% purity).
15. Lankas, G. R.; Leiting, B.; Sinha Roy, R.; Eiermann, G.
J.; Biftu, T.; Chan, C.-C.; Edmondson, S. D.; Feeney, W.
P.; He, H.; Ippolito, E. E.; Kim, D.; Lyons, K. A.; Ok, H.
O.; Patel, R. A.; Petrov, A. N.; Pryor, K. A.; Qian, X.;
Reigle, L.; Woods, A.; Wu, J. K.; Zaller, D.; Zhang, X.;
Zhu, L.; Weber, A. E.; Thornberry, N. A. Diabetes 2005,
54, 2988.
1
66%). H NMR (CDCl3, 300 MHz) d 7.11–7.02 (m, 1H),
6.92–6.83 (m, 1H), 5.53 (d, J = 8.5 Hz, 1H), 4.18–4.11 (m,
1H), 3.68–3.12 (m, 3H), 2.88–2.67 (m, 6H), 1.72–1.58 (m,
4H), 1.37 (s, 9H). To a mixture of (R)-[3-oxo-3-(tetrahyd-
ropyridazin-1-yl)-1-(2,4,5-trifluorobenzyl) propyl]carbam-
ic acid tert-butyl ester (300 mg, 0.747 mmol) and Et3N
(208 lL, 1.495 mmol) in CH2Cl2 (10 mL) was added
benzoyl chloride (158 mg, 1.121 mmol) and the mixture
was stirred for 2 h at room temperature. The solvents were
evaporated, and the residue was purified by silica gel
column chromatography to give (R)-[3-(2-benzoyltetra-
hydropyridazin-1-yl)-3-oxo-1-(2,4,5-trifluorobenzyl)pro-
pyl] carbamic acid tert-butyl ester (375 mg, 99%). 1H
NMR (CDCl3, 300 MHz) d 7.61–7.40 (m, 5H), 7.11–7.02
(m, 1H), 6.92–6.83 (m, 1H), 5.30–5.50 (m, 1H), 4.70–4.50
(m, 1H), 4.13–3.95 (m, 2H), 3.10–2.40 (m, 5H), 1.90–1.54
(m, 4H), 1.37 (s, 9H); LC-MS m/e (relative intensity) 506
(M+H)+. To a solution of (R)-[3-(2-benzoyltetrahydropy-
ridazin-1-yl)-3-oxo-1-(2,4,5-trifluorobenzyl)propyl]carbam-
ic acid tert-butyl ester (360 mg, 0.712 mmol) in EtOAc
(2 mL) was added 4M-HCl/1,4-dioxane (2 mL) and the
mixture was stirred for 12 h at room temperature. The
solvents were evaporated, and the residue was crystallized
with ether to give (R)-3-amino-1-(2-benzoyltetrahydropy-
ridazin-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one
HCl
(290 mg, 92%). 1H NMR (DMSO-d3, 300 MHz) d 8.16
(br s, 3H), 7.60–7.21 (m, 7H), 4.40–4.20 (m, 1H), 3.80–3.60
(m, 2H), 2.96–2.86 (m, 6H), 2.09–1.68 (m, 4H); HRMS
(free base, C21H22F3N3O2): calcd, 405.1664 found,
405.1638. Analytical HPLC method A: tR = 3.46 min
(99.1% purity); method B: tR = 6.06 min (95.8% purity).
Compound 10f; A mixture of 3-(R)-tert-butoxycarbonyla-
mino-4-(2,4,5-trifluorophenyl)butyric acid (2 g, 6 mmol),
16. Sung, Y. Y.; Lee, Y. S.; Jung, W. H.; Kim, H. Y.; Cheon,
H. G.; Yang, S. D.; Rhee, S. D. Biochem. Biophys. Res.
Commun. 2005, 338, 1779.