Synthesis of (S)-, (R)-, and (rac)-2-amino-3,3-bis(4-fluorophenyl)propanoic acids and an evaluation of the DPP IV inhibitory activity of Denagliptin diastereomers

Article abstract of DOI:10.1016/j.tet.2008.08.097

The non-proteinogenic amino acid (2S)-2-amino-3,3-bis(4-fluorophenyl)propanoic acid [(S)-1] is a key intermediate required for the synthesis of Denagliptin (2a). Denagliptin is a dipeptidyl peptidase IV (DPP IV) inhibitor that is being developed for the treatment of type-2 diabetes mellitus. A diastereoselective, cost-efficient synthetic procedure for (S)-1 was developed by alkylating a Ni(II) glycine equivalent derived from (S)-2-[(N-benzylprolyl) amino] benzophenone [(S)-BPB]. The alkylated product was then decomposed to isolate the target amino acid (S)-1 (ee >99%) and ligand (S)-BPB, which can be reused in subsequent reactions. The enantiomer (R)-1 and racemate (rac)-1 were synthesized from their corresponding Ni(II) glycine equivalents. Denagliptin diastereomers (2), derived from the key intermediates (S)-1, (R)-1, and (rac)-1 were synthesized, and their dipeptidyl peptidase IV inhibitory activities were investigated. These findings are important in the design and synthesis of DPP IV inhibitors.

Full text of DOI:10.1016/j.tet.2008.08.097

Tetrahedron 64 (2008) 10512–10516
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of (S)-, (R)-, and (rac)-2-amino-3,3-bis(4-fluorophenyl)propanoic
acids and an evaluation of the DPP IV inhibitory activity of Denagliptin
diastereomers
,
Guanghui Deng a, Deju Ye a, Yuanyuan Li b, Lingyan He a, Yu Zhou a c, Jiang Wang a, Jia Li b,
,
,
*
Hualiang Jiang a d, Hong Liu a
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Graduate School of the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
b National Center for Drug Screening, Shanghai Institute of Materia Medica, Graduate School of the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China
c School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, China
d School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 May 2008
Received in revised form 28 August 2008
Accepted 29 August 2008
Available online 6 September 2008
The non-proteinogenic amino acid (2S)-2-amino-3,3-bis(4-fluorophenyl)propanoic acid [(S)-1] is a key
intermediate required for the synthesis of Denagliptin (2a). Denagliptin is a dipeptidyl peptidase IV (DPP
IV) inhibitor that is being developed for the treatment of type-2 diabetes mellitus. A diastereoselective,
cost-efficient synthetic procedure for (S)-1 was developed by alkylating a Ni(II) glycine equivalent de-
rived from (S)-2-[(N-benzylprolyl) amino] benzophenone [(S)-BPB]. The alkylated product was then
decomposed to isolate the target amino acid (S)-1 (ee >99%) and ligand (S)-BPB, which can be reused in
subsequent reactions. The enantiomer (R)-1 and racemate (rac)-1 were synthesized from their corre-
sponding Ni(II) glycine equivalents. Denagliptin diastereomers (2), derived from the key intermediates
(S)-1, (R)-1, and (rac)-1 were synthesized, and their dipeptidyl peptidase IV inhibitory activities were
investigated. These findings are important in the design and synthesis of DPP IV inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
S
F
Recently, glucagon-like peptide-1 (GLP-1)1 and dipeptidyl pep-
tidase IV (DPP IV)2 have been intensively researched for use in the
treatment of type-2 diabetes mellitus. GLP-1 is an incretin hormone
secreted by the intestinal L-cells in response to food ingestion. It is
a novel pharmacological target with multiple antihyperglycemic
actions. The glucoregulatory actions of GLP-1 include enhancement
of glucose-dependent insulin secretion, inhibition of glucagon se-
cretion, delayed gastric emptying, and reduction in food intake.
However, GLP-1 is rapidly degraded in circulation at its N-terminus
by DPP IV.3 In order to potentiate the glucose-lowering actions of
GLP-1, it is essential to inhibit DPP IV by inhibitors, which have now
been of great importance in the treatment of type-2 diabetes.
The chiral amino acid (S)-1 ((2S)-2-amino-3,3-bis(4-fluoro-
phenyl)propanoic acid, Figure 1) is a key intermediate in the
synthesis of Denagliptin (2a),4 a well-known DPP IV inhibitor as
oral medication of type-2 diabetes being developed by Glaxo-
SmithKline. The original procedure used for the synthesis of (S)-1
S
F
CN
NH2
NH2
N
HO
HCl
O
O
F
F
F
2a, Denagliptin, (S,S,S)
2b, (S,S, rac)
1
2c, (S,S,R)
O
N
O
O
N
N
O
N
N
Ni
O
O
Ni
Ni
O
N
N
O
N
O
N
(R)-3
Figure 1. Structures of compounds 1, 2 (Denagliptin), and Ni(II) complexes 3 and 4.
(S)-3
4
* Corresponding author. Tel.: þ86 21 5080 7042; fax: þ86 21 5080 7088.
E-mail address: hliu@mail.shcnc.ac.cn (H. Liu).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.097

G. Deng et al. / Tetrahedron 64 (2008) 10512–10516
10513
employed seven steps. In this procedure, asymmetric azidation was
mediated by an oxazolidinone employed as a chiral auxiliary;5 this
procedure was considered unsatisfactory because of its duration as
well as safety issues related to the stability of the azide in-
termediates generated. Patterson et al.6 reported the synthesis of
(S)-1 by the asymmetric phase transfer catalytic (PTC) alkylation of
N-(diphenylmethylene) glycerin tert-butyl ester; this reaction
exhibited low enantioselectivity (ee¼60% before crystallization).
We aimed to carry out a structure–activity relationship (SAR) study
of Denagliptin. For this study, this elucidation required key in-
termediate amino acids such as (S)-1, (R)-1, and (rac)-1. Therefore,
it is necessary to develop strategies that are safe, inexpensive, and
that produce minimal waste for the preparation of (S)-1, (R)-1, and
(rac)-1. The Ni(II) complexes (S)-37 (commercially available),
(R)-3,8 and 49 (Fig. 1) were selected as nucleophilic glycine equiv-
alents for C-alkylation due to their preferred control to induce S- or
R-configuration. In this study, we present an efficient protocol for
the preparation of compound (S)-1 and the synthesis of (R)-1 and
(rac)-1 in two steps. In the first step, the Ni(II) complexes were
alkylated by 4,40-difluorobenzhydryl chloride (5) in the presence of
NaH dissolved in the solvent DMF. In the second step, the alkylated
products were decomposed with HCl to yield the desired amino
acids (ee ꢀ99%; Scheme 1). These synthesized amino acids were
subsequently utilized for the synthesis of Denagliptin diastereomers.
The inhibitory activity of these diastereomers against DPP IV was
then evaluated. Interestingly, the bioassay results revealed that
Denagliptins 2a and 2b (derived from (S)-1 and (rac)-1, respectively)
exhibited high inhibitory activities; however, diastereomer 2c
(derived from (R)-1) exhibited extremely low DPP IV inhibitory
activity.
(S)-3 with 4,40-difluorobenzhydryl chloride 5 was challenging due
to steric hindrance, the bulk of the two individual compounds and
the unexpected influence of the high electronegative F atoms on
the aromatic ring of chloride 5. Table 1 summarizes the interesting
experimental results of the alkylation of (S)-3 with chloride 5 with
different bases in various solvents under a series of conditions.
However, when previously described protocols were used,7–9 7 of
the 12 solvent/base pairs (entries 1–7) yielded disappointing re-
sults since the desired alkylated product 6 was not obtained, even
though strong inorganic bases such as lithium diisopropylamide
(LDA) and KOtBu and organic bases such as 1,8-diazabicyclo[5,4,0]undec-
7-ene (DBU) (entries 3, 6, and 7) were employed. Experiments were
continuously carried out until the discovery of the DMF/NaH pair.
With this pair, a moderate yield (35%) of the alkylated complex 6
was obtained (entry 8). Subsequently, when the reaction was
allowed to run for a longer time period (entry 9) or at a higher
temperature (entry 10), a significant decrease in product yield was
observed. The product dr value was not different from that ob-
served initially. Under these conditions (entries 9 and 10), com-
pound (S,S)-6 was gradually converted to a new product, which was
identified to be derived by ring cleavage 8 by liquid chromatogra-
phy-mass spectrometry (LC-MS) ([MþH]þ¼701) and nuclear
magnetic resonance (NMR) analysis. This byproduct 8 was identi-
fied to be responsible for the decreased yield (entries 9 and 10).
Alkylation generated an excellent yield when the reaction was
performed using 1 equiv of complex (S)-3, 1.3 equiv of chloride 5,
and 3 equiv of NaH at ꢁ20 ꢂC. The reaction proceeded smoothly
until its completion in 2.5 h (98:2 dr); purification using flash
chromatography yielded the product (S,S)-6 in >99:1 dr (86%
combined yield; entry 11). By simulating the conditions under
which optimum results were previously obtained, the experiment
was successfully repeated with (R)-3 as the starting material in-
stead of (S)-3 (entries 12 vs 11).
F
Thereafter, (S,S)-6 and (R,R)-6 were dissolved in methanol so-
lution, followed by decomposition in 6 N HCl under reflux. The
reaction was considered complete when the solution changed color
from red to green. The reaction was completed in 30 min. The
isolation of the desired amino acid 1 from the reaction mixture
comprising water, NiCl2, and ligand 2-[(N-benzylprolyl)amino]-
benzophenone (BPB, 7), is difficult due to the solubility of 1 both in
water and in organic solvents. The classical methods elaborated by
Belokon et al.7,10 involving the use of Dowex resin or ethylene di-
amine tetraacetic acid (EDTA) were employed to isolate 1. Un-
fortunately, these procedures yielded unacceptably low yields of
product 1 (below 20%). Excellent yields of the products (S)-1$HCl
Cl
F
Chloride 5
F
O
N
N
O
N
N
O
DMF, 20 °C, NaH
>86%
O
N
*
Ni
*
Ni
O
N
O
*
F
(S)-3
(R)-3
(S,S)-6
(R,R)-6
23
23
([
a]
þ55.1; lit.6 ee >99%11) and (R)-1$HCl ([
a
]
ꢁ55.2; ee >99%)
Gly, Ni2+
D
D
6 N HCl
were successfully obtained by column chromatography performed
using a reusable C18-reversed phase (230–400 mesh) silica gel
column, a widely used apparatus in the manufacturing industry. In
this method, NiCl2 was completely separated from the mixture
using water as the eluent. Compound (S)-1 was collected by 50%
MeOH in water eluent, and BPB (7) was recycled using the MeOH
eluent. The C18-reversed phase silica gel could be washed with
MeOH
>95%
Recovery >96%
HCl.
O
N
F
NH
*
NH2
*
HO
O
+
NiCl2
O
(S)-7
(R)-7
(S)-1-HCl, ee >99%
(R)-1-HCl, ee >99%
MeOH and reused. Further, the chiral ligand (S)-7 was recovered
23
F
(recovered (S)-7 [
duction costs.
a
]
ꢁ133.2, c 0.5 lit.12); this merits lower pro-
D
Scheme 1. Synthesis of (2S)-2-amino-3,3-bis(4-fluorophenyl)propanoic acid (S)-1$HCl.
Using similar protocols as those used for (S)-1 and (R)-1, a high
yield (95%) of (rac)-1 was easily obtained by using Ni(II) complex 4
and chloride 5 (Scheme 2). Ni(II) complex 4 was prepared using
a one-pot procedure in our published paper.9a The reaction was ter-
minated by the addition of water saturated with NH4Cl to form a red
precipitate that was pure enough to be decomposed by 6 N HCl. By
following the classical method developed by Belokon et al.10 (yield,
71%) or by our established method that employed reusable C18-re-
2. Results and discussion
The successful preparation of (S)-amino acids by the electronic
re-side attack of the Ni (II) complex of (S)-3 enolate double bond
using simple halides was widely published by Belokon et al.;8
however, the preparation of (R)-amino acids through the Ni(II)
complex of (R)-3 has been rarely reported.8b In this study, it was
versed phase silica gel, (rac)-1 and ligand 2-[N-(a-picolyl) amino]-
observed that the alkylation of the a-carbon atom of the complex
benzophenone (PABP, 10) could be easily separated (yield, 98%).

10514
G. Deng et al. / Tetrahedron 64 (2008) 10512–10516
Table 1
Alkylation of Ni(II) complex (S)-3 with chloride 5 under various conditions
F
F
+
Cl
F
O
N
N
O
O
N
N
N
N
O
O
N
N
HO
Ni
N
O
O
5
Ni
Ni
Ni
+
O
N
O
N
O
N
O
F
F
F
(S,R)-6
(S,S)-8
(S)-3
(S,S)-6
Entry
Solvent/base
Temp (ꢂC)
Time (h)
Yielda of (S,S)-6
dr (S,S)-6:(S,R)-6b
1
2
3
4
DCM/NaOH
THF/BuLi
THF/LDA
rt
7
1.5
1.5
3
0.1
24
24
2
5
2
2.5
2.5
0
0
0
0
0
0
0
35%
d
ꢁ78 to rt
d
ꢁ78 to rt
d
MeCN/NaH
DMF/NaOH
DMF/DBU
DMF/KOtBu
DMF/NaH
DMF/NaH
DMF/NaH
DMF/NaH
DMF/NaH
ꢁ10 to rt
d
5
rt
0
d
6c
7c
8c
9c
10c
11d
12f
d
ꢁ10
d
0
97:3
97:3
95:5
98:2
98:2
0
rt
20%
10%
ꢁ20
70% (86%e)
75 (89%e)
ꢁ20
a
Yield of product (S,S)-6 isolated by column chromatography.
Determined by chiral Daicel optical density (OD)-H column analysis of the crude product 6 [80:20 hexane/isopropanol, 11.4 min (S,S), 12.7 min (S,R)] before purification.
b
The configurations were assigned according to optical data and information from literature.7,9b
c
Using a 1:1.3:2 mixture of Ni(II) complex (S)-3, chloride 5, and base.
Using a 1:1.3:3 mixture of Ni(II) complex (S)-3, chloride 5, and base.
d
e
Combined yield.
f
Using (R)-3 as the starting Ni(II) complex to yield (R,R)-6.
By following procedures similar to those described in a pub-
lished patent application,5 we synthesized Denagliptins 2a, 2b, and
2c from (S)-1, (rac)-1, and (R)-1, respectively. These three com-
pounds were assayed in vitro to determine the inhibitory activities
of DPP IV. The results are shown in Table 2. The inhibition rates
were observed to be 97.7%, 95.3%, and 13.1% for 2a, 2b, and 2c,
3. Conclusion
In summary, an efficient protocol for the synthesis of (S)-1 (ee
>99%), (R)-1 (ee >99%), and (rac)-1 in two steps from chloride 5
and the corresponding Ni(II) complexes was established. Using this
optimized protocol, the overall yields of the optically pure amino
acids, namely, (S)-1, (R)-1, and (rac)-1, generated were 82%, 85%,
and 95%, respectively. Further, the chiral auxiliary 7 and the achiral
auxiliary 10 were recoverable and reusable for the further prepa-
ration of Ni (II) complexes. The biological evaluation of the Dena-
gliptin diastereomers 2a–c indicate that the S-configuration of the
amino acid portion of Denagliptin is vital for its DPP IV inhibitory
activity and the R-configuration results in low inhibitory activity.
These findings have useful applications in the design and synthesis
of DPP IV inhibitors.
respectively; further, the median inhibition concentration (IC50
)
value of 2b was 0.33-fold less than that of 2a. These results
revealed that the absolute S-configuration of the amino acid resi-
due is vital for the inhibitory activity of Denagliptin (Table 2). These
findings have useful applications in the design and synthesis of
DPP IV inhibitors.
F
O
N
N
O
N
N
DMF, Chloride 4,
O
O
4. Experimental section
4.1. General methods
Ni
-10 °C, NaH
Ni
O
N
O
N
97%
F
All the reagents were used as obtained, unless otherwise stated.
Solvents were evaporated under reduced pressure and below 50 ꢂC
if no description was noted. Melting points were measured in
9
4
Gly, Ni2+
6 N HCl
MeOH
N
Recovery, 98%
Table 2
98%
DPP IV inhibition study of compounds, 2a, 2b, and 2c
O
NH O
F
HCl.
NH2
Entry
Inhibition rate
IC50 (nM)
HO
(%, 50 mM)
NiCl
2536
377
>48,800
38
+
2
2a
2b
97.7
95.3
13.1
NDb
O
10
( )-1
2c
LAF-237a
F
a
LAF-2374 was used as a control.
Not determined.
b
Scheme 2. Preparation of (rac)-1.

G. Deng et al. / Tetrahedron 64 (2008) 10512–10516
10515
capillary tube without correction. 1H and 13C NMR spectra were
recorded on a 300 MHz instrument. Chemical shifts were reported
in parts per million (ppm) upfield from TMS. Proton coupling pat-
terns were described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). High resolution mass spectra
(HRMS) were obtained on a Fourier Transform Mass Spectrometer.
Optical rotations were measured on PE-341 polarimeter.
6.89 (m, 6H), 7.07–7.11 (m, 3H), 7.34–7.36 (m, 3H), 7.47–52 (m, 3H),
7.63 (d, 3H), 7.85–89 (m, 1H), 7.95–8.01 (m, 1H), 8.77 (d, J¼9.0 Hz,
3H). 13C NMR (CDCl3, 75 MHz):
d
¼56.5, 76.0, 114.9, 115.0, 115.1,
115.2, 121.6, 123.7, 123.9, 126.7, 127.4, 127.5, 127.6, 129.4, 129.5,
129.8, 129.9, 130.2, 132.8, 132.9, 133.6, 133.7, 134.6, 134.7, 134.9,
140.4, 143.5, 146.6, 153.1, 160.4, 161.3, 162.8, 163.7, 169.4, 171.7,
176.4. HRMS (ESI) [MþH]þ found m/z 619.1145, calcd for
[C40H33F2N3NiO3þH]þ: 619.1139.
4.2. General procedure for the preparation of (S,S)-6, (R,R)-6,
and 9 described as those for BPB-Ni(II)-(2S)-2-amino-3,3-
bis(4-fluorophenyl)propanoic acid (S,S)-6
4.3. General procedure for the preparation of (S)-1, (R)-1, and
(rac)-1 described as those for (2S)-2-amino-3,3-bis(4-
fluorophenyl)propanoic acid hydrochloride (S)-1$HCl
To a stirred solution of nickel compound (S)-313 (8.936 g,
17.94 mmol) in DMF (60 ml) was added 4,40-difluorobenzhydryl
chloride 5 (4.708 g, 19.73 mmol) in one portion under an argon
atmosphere. The mixture was cooled to ꢁ20 ꢂC and NaH (2.15 g,
53.82 mmol, 60% suspension in oil) was added without stirring. The
air was evacuated with a vacuum pump and the flask was filled
with argon. Then the stirring was commenced and the reaction
proceeded smoothly to completion in 2.5 h (98:2 dr). Then the
reaction mixture was neutralized by slow addition of acetic acid
(approximately 3.3 ml) under stirring and cooling. Finally, the re-
action mixture was slowly poured into icewater (500 ml). After 12 h
the precipitate was filtered. The crude red product was purified by
silica gel chromatography with eluent (petroleum ether/ethyl
acetate¼1:1) to afford pure red product (S,S)-6 8.81 g (70% yield).
The unreacted raw material (S)-3 (2.2 g) obtained from the chro-
matography was reused repeating the procedure described above
to afford an additional 2.0 g of product (S,S)-6. Totally, 10.81 g (86%
yield) of pure product (S,S)-6 was obtained in >99:1 dr (DAICEL OD-
A one-neck flask was charged with (S,S)-6 (10 g, 14.31 mmol)
followed by MeOH (300 ml). The resulting slurry was reflux and
then 25 ml 6 N HCl was added in one portion. The mixture was
continued refluxing for 30 min while the red color disappeared and
became green. The reaction was cooled to room temperature and
then evaporated to dryness. Water (20 ml) was added to the residue
to form a clear solution then this solution was separated by column
chromatography on C18-reversed phase (230–400 mesh) silica gel.
Pure water as eluent was employed to remove the green NiCl2 and
excess HCl, then MeOH/water (1:1) was used to obtain optically
pure product (S)-1$HCl (4.3 g, 95%). The ligand BPB (7) decomposed
from (S,S)-6 was recovered by MeOH eluent (5.8 g, 96%), and the
column chromatography was washed by 100 ml MeOH for further
use. Rf¼0.36 (DCM/MeOH¼5:1). Mp 162–163 ꢂC (lit.6 mp 149 ꢂC).
23
23
[a
]
þ55.1 (c 2, MeOH, lit.6 [
a
]
þ56.3), ee >99%. 1H NMR (CD3OD,
D
D
300 MHz):
d
¼4.43 (d, J¼10.5 Hz, 1H), 4.80 (d, J¼10.5 Hz, 1H), 7.05 (t,
J¼9.0 and 8.7 Hz, 2H), 7.14 (t, J¼9.0 and 8.7 Hz, 2H), 7.39–7.43 (m,
H HPLC COLUMN). Rf¼0.23 (petroleum ether/ethyl acetate¼1:1).
2H), 7.50–7.54 (m, 2H). 13C NMR (CDCl3, 75 MHz):
d¼53.7, 57.9,
23
Mp 213–215 ꢂC. [
a
]
D
þ1991.9 (c 0.49, CHCl3). 1H NMR (CDCl3,
116.9, 117.1, 117.6, 117.8, 131.7, 131.8, 131.9, 136.0, 136.6, 163.0, 165.4,
171.3. HRMS (ESI) [MþH]þ found m/z 278.0993, calcd for
[C15H13F2NO2þH]þ: 278.0993.
300 MHz):
d
¼1.71–1.75 (m, 2H), 2.01–2.07 (m, 1H), 2.28–2.54 (m,
2H), 2.96–2.99 (m, 1H), 3.34–3.38 (m, 1H), 3.46 (d, J¼12.9 Hz, 1H),
4.28 (d, J¼12.6 Hz, 1H), 4.33 (d, J¼3.0 Hz, 1H), 4.71 (d, J¼3.6 Hz, 1H),
6.66–6.79 (m, 5H), 7.05–7.19 (m, 6H), 7.25–7.39 (m, 5H), 7.42–7.49
(m, 3H), 8.03 (d, J¼7.2 Hz, 2H), 8.28 (d, J¼8.7 Hz, 1H). 13C NMR
4.3.1. (2R)-2-Amino-3,3-bis(4-fluorophenyl)propanoic acid
hydrochloride (R)-1$HCl
Compound (R)-1$HCl was prepared from (R,R)-6 using a pro-
cedure similar to that described for the preparation of (S)-1$HCl as
(CDCl3, 75 MHz):
d
¼23.1, 31.0, 56.8, 57.3, 63.6, 70.6, 74.1,114.7,114.9,
123.1, 126.0, 127.0, 127.8, 128.7, 128.8, 128.9, 129.0, 129.5, 129.7,
129.8,131.5,131.7,131.8,132.6,133.2,133.7,134.0,135.2,136.3,143.0,
160.1, 161.3, 162.6, 163.7, 171.5, 176.9, 180.3. HRMS (ESI) [MþNa]þ
found m/z 722.1702, calcd for [C40H33F2N3NiO3þNa]þ: 722.1741.
23
a white solid. Rf¼0.36 (DCM/MeOH¼5:1). Mp 181–183 ꢂC. [
a]
D
ꢁ55.2 (c 0.52, MeOH), ee >99%. 1H NMR (CD3OD, 300 MHz):
¼4.26
d
(d, J¼9.6 Hz, 1H), 4.47 (d, J¼9.3 Hz, 1H), 6.97–7.11 (m, 4H), 7.33–7.44
(m, 4H). 13C NMR (CDCl3, 75 MHz):
d
¼53.7, 59.2, 116.8, 117.0, 117.4,
4.2.1. (2R)-BPB-Ni(II)-(2S)-2-amino-3,3-bis(4-fluorophenyl)
propanoic acid (R,R)-6
117.7, 131.7, 131.8, 131.9, 132.0, 136.5, 137.2, 162.9, 165.3, 172.3. HRMS
(ESI) [MþH]þ found m/z 278.0993, calcd for [C15H13F2NO2þH]þ:
278.0995.
Compound (R,R)-6 was prepared from (R)-3 using a procedure
similar to that described for the preparation of (S,S)-6 as a red solid.
23
Rf¼0.23 (petroleum ether/ethyl acetate¼1:1). Mp 144–146 ꢂC. [
a]
D
ꢁ
4.3.2. rac-2-Amino-3,3-bis(4-fluorophenyl)propanoic acid
hydrochloride rac-1$HCl
2006 (c 0.57, CHCl3). 1H NMR (CDCl3, 300 MHz):
d
¼1.70–1.74 (m, 1H),
1.98–2.09 (m,1H), 2.23–2.57 (m, 2H), 2.88–3.01 (m,1H), 3.34–3.38 (m,
1H), 3.46 (d, J¼12.6 Hz, 1H), 4.28 (d, J¼12.6 Hz, 1H), 4.33 (d, J¼3.0 Hz,
1H), 4.71 (d, J¼3.3 Hz, 1H), 6.65–6.79 (m, 5H), 7.05–7.19 (m, 6H), 7.24–
7.33(m, 5H),7.40–7.48(m, 3H), 8.03(d,J¼7.2 Hz, 2H), 8.28(d, J¼8.7 Hz,
Compound (rac)-1$HCl was prepared from 9 using a procedure
similar to that described for the preparation of (S)-1$HCl as a white
solid. Rf¼0.36 (DCM/MeOH¼5:1). Mp 186–187 ꢂC. 1H NMR (CD3OD,
300 MHz):
d
¼4.26 (d, J¼9.6 Hz, 1H), 4.47 (d, J¼9.0 Hz, 1H), 7.01 (t,
1H). 13C NMR (CDCl3, 75 MHz):
d
¼23.1, 31.0, 56.8, 57.3, 63.6, 70.6, 74.1,
J¼8.7 and 9.0 Hz, 2H), 7.09 (t, J¼9.0 and 8.7 Hz, 2H), 7.32–7.35 (m,
114.7, 114.9, 123.1, 126.0, 127.0, 127.8, 128.7, 128.8, 129.0, 129.1, 129.5,
129.7, 129.8, 131.5, 131.7, 131.8, 132.6, 133.2, 133.7, 134.0, 135.2, 136.3,
142.9,160.1,161.3,162.6,163.7,171.5,177.0,180.3. HRMS(ESI) [MþNa]þ
found m/z 722.1736, calcd for [C40H33F2N3NiO3þNa]þ: 722.1741.
2H), 7.39–7.44 (m, 2H). 13C NMR (CDCl3, 75 MHz):
d
¼53.7, 60.6,
116.7, 116.8, 117.3, 117.5, 131.7, 131.8, 132.1, 132.2, 137.1, 138.0, 162.7,
165.2. 172.3. HRMS (ESI) [MþH]þ found m/z 278.0993, calcd for
[C15H13F2NO2þH]þ: 278.0993.
4.2.2. PABP-Ni(II)-2-amino-3,3-bis(4-fluorophenyl)
propanoic acid 9
4.3.3. (2S,4S)-1-[(2S)-2-Amino-3,3-bis(4-fluorophenyl)propanoyl]-
4-fluoropyrrolidine-2-carbonitrile hydrochloride (2a)
Compound 9 was prepared from complex 4 using a procedure
similar to that described for the preparation of (S,S)-6 as a red solid.
The reaction temperature was controlled at ꢁ10 ꢂC. Rf¼0.24 (pe-
troleum ether/ethyl acetate¼1:1). Mp >290 ꢂC. 1H NMR (CDCl3,
According to the procedures described in the published patent
application,5 2a was prepared as a white solid. Rf¼0.47 (DCM/
23
MeOH/acetic acid¼10:1:1). Mp 183–184 ꢂC. [
a
]
D
þ119 (c 0.51,
MeOH). 1H NMR (CD3OD, 300 MHz):
d
¼2.23–2.53 (m, 2H), 3.06 (q,
300 MHz):
d
¼4.27 (d, J¼2.1 Hz, 1H), 4.80 (d, J¼2.4 Hz, 1H), 6.80–
1H), 3.75–3.86 (m, 1H), 4.53 (d, J¼11.4 Hz, 1H), 4.86–4.90 (m, 2H),

10516
G. Deng et al. / Tetrahedron 64 (2008) 10512–10516
5.32–35 (m, 1H), 7.01 (t, J¼8.7 Hz, J¼8.7 Hz, 2H), 7.22 (t, J¼8.7 and
8.7 Hz, 2H), 7.34 (q, J¼5.1, 3.6, and 5.4 Hz, 2H), 7.65 (q, J¼5.4, 3.6,
this work, and buffer (100 mmol/l HEPES, pH 7.5, 0.1 mg/ml BSA) in
a total reaction volume of 100 l. Liberation of AMC was monitored
using an excitation wavelength of 360 nm and an emission wave-
length of 460 nm. The enzyme used in these studies was soluble
human protein produced in a baculovirus expression system (Bac-
To-Bac; Life Technologies).
m
and 5.1 Hz, 2H). 13C NMR (CDCl3, 75 MHz):
d
¼37.2, 46.8, 54.8, 54.3,
56.2, 92.7, 94.5, 117.6, 117.8, 118.0, 118.4, 132.0, 132.1, 132.3, 132.4,
134.8, 134.9, 163.2, 163.3, 165.6, 165.7, 169.4. HRMS (ESI) [MþNa]þ
found m/z 396.1285, calcd for [C20H18F3N3OþNa]þ: 396.1300.
4.3.4. (2S,4S)-1-[(ꢃ)-2-Amino-3,3-bis(4-fluorophenyl)propanoyl]-
4-fluoropyrrolidine-2-carbonitrile hydrochloride (2b)
Acknowledgements
Compound 2b was prepared as a white solid using a procedure
We gratefully acknowledge financial support from the State Key
Program of Basic Research of China (Grant 2002CB512802), the
National Natural Science Foundation of China (Grants 20372069,
29725203, and 20472094), and the 863 Hi-Tech Program (Grants
2006AA020402, 2006AA020602).
similar to 2a. Rf¼0.47 and 0.33 (DCM/MeOH/acetic acid¼10:1:1).
23
Mp 174–175 ꢂC. [
a]
–31 (c 0.51, MeOH). 1H NMR (CD3OD,
D
300 MHz):
d
¼1.75–1.95 (m, 1H), 2.2–2.44 (m, 2H), 2.81–3.18 (m,
1H), 3.81–4.20 (m, 1H), 4.52 (t, J¼11.1 and 11.1 Hz, 2H), 4.91–5.05
(m, 1H), 5.13–5.24 (m, 1H), 6.98–7.09 (m, 2H), 7.22 (t, J¼8.7 and
8.4 Hz, 2H), 7.34 (q, J¼4.8, 3.6, and 5.1 Hz, 2H), 7.66 (q, J¼5.1, 3.3,
and 5.1 Hz, 2H). 13C NMR (CDCl3, 75 MHz):
d
¼37.5, 46.9, 54.2, 56.2,
References and notes
92.4, 94.3, 117.1, 117.3, 117.7, 118.5, 132.0, 132.1, 132.2, 132.4, 134.5,
135.7, 163.2, 165.5, 169.3, 175.7. HRMS (ESI) [MþH]þ found m/z
374.1501, calcd for [C20H18F3N3OþH]þ: 374.1480.
1. Scheen, A. J. Rev. Med. Liege 2007, 62, 217–221.
2. For recent papers, see: (a) Hanson, R. L.; Goldberg, S. L.; Brzozowski, D. B.;
Tully, T. P.; Cazzulino, D.; Parker, W. L.; Lyngberg, O. K.; Vu, T. C.; Wong, M. K.;
Patel, R. N. Adv. Synth. Catal. 2007, 349, 1369–1378; (b) Pei, Z.; Li, X.; von-
Geldern, T. W.; Longenecker, K.; Pireh, D.; Stewart, K. D.; Backes, B. J.; Lai, C.;
Lubben, T. H.; Ballaron, S. J.; Beno, D. W. A.; Kempf-Grote, A. J.; Sham, H. L.;
Trevillyan, J. M. J. Med. Chem. 2007, 50, 1983–1987; (c) Feng, J.; Zhang, Z.;
Wallace, M. B.; Stafford, J. A.; Kaldor, S. W.; Kassel, D. B.; Navre, M.; Shi, L.;
Skene, R. J.; Asakawa, T.; Takeuchi, K.; Xu, R.; Webb, D. R.; Gwaltney, S. L.
J. Med. Chem. 2007, 50, 2297–2300.
3. (a) Svensson, A. M.; Ostenson, C. G.; Efendic, S.; Jansson, L. Clin. Sci. 2007, 112,
345–351; (b) Sonne, D. P.; Engstrøm, T.; Treiman, M. Regul. Pept. 2008, 146, 243–
249.
4. Thomas, J. M.; vonGeldern, T. W. Drug Dev. Res. 2006, 67, 627–642.
5. Haffner, C. D.; Durham, N.; Randhawa, A. S.; Reister, S. M.; Lenhard, J. M.
(Glaxosmithklin Company), PCT Int. Appl. WO 2004/0171848 A1, 2004.
6. Patterson, D. E.; Xie, S.; Jones, L. A.; Osterhout, M. H.; Henry, C. G.; Roper, T. D.
Org. Process Res. Dev. 2007, 11, 624–627.
7. (a)Soloshonok, V. A.; Yamada, T.; Ueki, H.; Moore,A. M.; Cook, T. K.; Arbogast, K. L.;
Soloshonok, A. V.; Martina, C. H.; Ohfuneb, Y. Tetrahedron 2006, 62, 6412–6419;
(b) Soloshonok, V. A.; Ueki, H. J. Am. Chem. Soc. 2007, 129, 2426–2427;
(c) Tararov, V. I.; Savel’eva, T. F.; Kuznetsov, N. Y.; Ikonnikov, N. S.; Orlova, S. A.;
Belokon, Y. N.; North, M. Tetrahedron: Asymmetry1997, 8, 79–83; (d) Saghiyan, A. S.;
Dadayan, S. A.; Petrosyan, S. G.; Manasyan, L. L.; Geolchanyan, A. V.; Djamgaryan,
S. M.; Andreasyan, S. A.; Maleev, V. I.; Khrustalev, V. N. Tetrahedron: Asymmetry
2006, 17, 455–467.
8. (a) Sagiyan, A. S.; Oganesyan, A. M.; Ambartsumyan, A. A.; Mangasaryan, K. A.;
Avetisyan, A. A.; Belokon, Y. N. Aiastani kimiakan andes/Khimicheskii zhurnal
Armenii 2000, 53, 27–36; (b) Belokon, Y. N. Pure Appl. Chem. 1992, 64, 1917–
1924.
9. (a) Deng, G.; Wang, J.; Zhou, Y.; Jiang, H.; Liu, H. J. Org. Chem. 2007, 72, 8932–
8934; (b) Soloshonok, V. A.; Cai, C.; Yamada, T.; Ueki, H.; Ohfune, Y.; Hruby, V. J.
J. Am. Chem. Soc. 2005, 127, 15296–15303.
10. Belokon, Y. N.; Kochetkov, K. A.; Churkina, T. D.; Ikonnikov, N. S.; Larionov, O. V.;
Harutyunyan, S. R.; Vyskocil, S.; North, M.; Kagan, H. B. Angew. Chem., Int. Ed.
2001, 40, 1948–1951.
4.3.5. (2S,4S)-1-[(2R)-2-Amino-3,3-bis(4-fluorophenyl)propanoyl]-
4-fluoropyrrolidine-2-carbonitrile hydrochloride (2c)
Compound 2c was prepared using a procedure similar to 2a as
a white solid. Rf¼0.33 (DCM/MeOH/acetic acid¼10:1:1). Mp 175–
23
176 ꢂC. [
a
]
D
ꢁ199 (c 0.51, MeOH). 1H NMR (CD3OD, 300 MHz):
d
¼1.78–1.92 (m, 2H), 2.22–2.44 (m, 1H), 2.62–2.95 (m, 1H), 4.04–
4.11 (m, 1H), 4.32 (d, J¼9.6 Hz, 2H), 4.50 (d, J¼11.4 Hz, 1H), 4.97 (t,
J¼11.4 and 11.7 Hz, 1H), 5.09–5.26 (m, 1H), 7.05 (t, J¼8.4 and 8.4 Hz,
2H), 7.21 (t, J¼8.4 and 9.0 Hz, 2H), 7.33 (m, 2H), 7.65 (q, 2H). 13C
NMR (CDCl3, 75 MHz):
d¼37.5, 47.0, 54.0, 56.4, 60.7, 92.4, 94.3,117.2,
117.4, 117.8, 118.9, 132.2, 132.3, 132.38, 132.41, 132.5, 134.4, 135.6,
163.2, 165.6, 169.3, 175.7. HRMS (ESI) [MþNa]þ found m/z 396.1295,
calcd for [C20H18F3N3OþNa]þ: 396.1300.
4.4. Determination of the optical purity of 1$HCl11
The 1$HCl (0.1 mg) in methanol (20 ml) was added to the solu-
tion of Marfey’s reagent (1 mg, 10 equiv) in acetone (0.1 ml) and
10 ml of Et3N. The reaction mixture was heated for 2 h at 40 ꢂC.
Then the above solution was diluted to 0.8 ml, and 5
lution was injected on the HPLC column (ZORBAX SB-C18,
m) at a flow rate of 1 ml/min, mobile phase 0.5%
ml of this so-
4.6ꢄ150 mm, 5
m
TFA in water; detection, 340 nm, 8 nm bandwidth; temperature
40 ꢂC; retention times, desired diastereomer (S): 4.267 min, un-
desired (R): 6.278 min (the retention times were determined by the
(rac)-1).
11. For details, see: the enantiomeric excess of (S)-1$HCl was determined by de-
rivatization with Marfey’s reagent, followed by HPLC analysis. Sypniewski, M.;
Penke, B.; Simon, L.; Rivier, J. J. Org. Chem. 2000, 65, 6595–6600.
12. Belokon, Y. N.; Tararov, V. I.; Maleev, V. I.; Saveleva, T. F.; Ryzhov, M. G. Tetra-
hedron: Asymmetry 1998, 9, 4249–4252.
13. Compound (S)-3 is commercially available or prepared as: Ueki, H.; Ellis, T. K.;
Martin, C. H.; Boettiger, T. U.; Bolene, S. B.; Solos honok, V. A. J. Org. Chem. 2003,
68, 7104–7107.
14. Lankas, G. R.; Leiting, B.; Roy, R. S.; Eiermann, G. J.; Beconi, M. G.; Biftu, T.; Chan, C.;
Edmondson, S.; Feeney, W. P.; He, H.; Ippolito, D. 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–
2994.
4.5. Assay for DPP IV inhibition14
To measure the activity of DPP IV, a continuous fluorometric
assay was employed using Gly-Pro–AMC, which is cleaved by the
enzyme to release the fluorescent aminomethylcoumarin (AMC). A
typical reaction contained 50 pmol/l enzyme, 50
mmol/l Gly-Pro–
AMC, different concentrations of the compounds synthesized in