Synthesis of 3,3-dimethylglutamic acid derivatives as DPP-IV inhibitors and evaluation of their chemical stability

Article abstract of DOI:10.1002/jccs.201190049

A novel five-step synthesis of Boc-3,3-dimethylglutamic acid α-ethyl ester 11 is reported. All the steps are high yielding and simple to carry out. By use of the 3,3-dimethylglutamic acid building block, we successfully discovered a novel class of DPP-IV inhibitors, Glu-Pro-Nitrile dipeptide mimics 2, with high potency (IC₅₀ < 40 nM). The consequence of 3,3-dimethyl substituent on the rate of intramolecular cyclization between N-terminal amine and 5-position amide bond in different buffer solutions was also evaluated.

Full text of DOI:10.1002/jccs.201190049

108
Journal of the Chinese Chemical Society, 2011, 58, 108-117
Synthesis of 3,3-Dimethylglutamic Acid Derivatives as DPP-IV Inhibitors
and Evaluation of Their Chemical Stability
Tsu Hsu, Ting-Yueh Tsai, Ya-Ju Tseng, Mei-Chun Chiou, Cheng-Tai Lu,
Yu-Sheng Chao and Weir-Torn Jiaang*
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
No. 35, Keyan Rd., Zhunan Town, Miaoli Country 350, Taiwan, R.O.C.
Received August 25, 2010; Accepted November 29, 2010; Published Online December 20, 2010
A novel five-step synthesis of Boc-3,3-dimethylglutamic acid a-ethyl ester 11 is reported. All the
steps are high yielding and simple to carry out. By use of the 3,3-dimethylglutamic acid building block, we
successfully discovered a novel class of DPP-IV inhibitors, Glu-Pro-Nitrile dipeptide mimics 2, with high
potency (IC50 < 40 nM). The consequence of 3,3-dimethyl substituent on the rate of intramolecular
cyclization between N-terminal amine and 5-position amide bond in different buffer solutions was also
evaluated.
Keywords: Glutamic acid; Dipeptidyl peptidase IV; Inhibitor; Chemical stability.
INTRODUCTION
Dipeptidyl peptidase IV (DPP-IV, also known as
intramolecular cyclization pathway, further development
of a series of compounds with dimethyl substituents at the
b-position of the P2 site glutamic acid was performed (2,
Table 1). These derivatives showed potent DPP-IV inhibi-
tion with IC50 values of < 40 nM and excellent selectivity
over the closely related enzymes, DPP8, DPP-II and FAP.4
The potency and selectivity of compound 1 and several rep-
resentative compounds 2 are shown in Table 1. In this arti-
cle, we describe our first investigations on the synthesis of
3,3-dimethylglutamic acid and the discovery of 3,3-di-
methylglutamic acid dipeptide mimics as potent DPP-IV
inhibitors with increased chemical stability. Additionally, a
CD26) (EC 3.4.14.5) is a prolyl dipeptidase involved in the
in vivo degradation of two insulin-sensing hormones,
glucagon-like peptide 1 (GLP-1) and glucose-dependent
insulinotropic polypeptide (GIP), by cleaving at the pep-
tide bond of the penultimate position.1 Thus inhibition of
DPP-IV extends the half-life of endogenously secreted
GLP-1, which in turn enhances insulin secretion and im-
proves the glucose tolerance.2 Therefore, DPP-IV has be-
come a validated target for the treatment of type II diabetes,
and several inhibitors of DPP-IV have been developed as
oral antidiabetic agents.3
Our program has produced compound 1 (Fig. 1) that
features a L-glutamic acid at the P2 site and imparts the in-
hibitor with a superior DPP-IV inhibition (IC50 = 2 nM).4
Often this nitrile group is able to bind covalently with
serine 630 forming a high-affinity imidate complex.5 How-
ever, the electrophilic nitrile group also makes this com-
pound unstable due to reaction with the primary amine of
the P2 amino acid forming inactive cyclic imidate (X =
NH) and/or its diketopiperazine hydrolysis product (X = O,
Fig. 1).6,7 Magnin et al. reported that increasing the degree
of branching at the b-position of the alkylglycine substi-
tuent increases solution stability.7 To circumvent the known
O
N
N
H2N
CN
O
1
R
O
R
N
O
N
Cyclization
N
CN
NH2
H2N
R
N
H
X
CN
O
X= NH, O
Fig. 1. Glutamic acid derivatives as DPP-IV inhibitors
and their cyclization products.
* Corresponding author. E-mail: wtjiaang@nhri.org.tw

Synthesis of Boc-3,3-Dimethylglutamic Acid
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 109
of the imine of a-amino ester to bulky Michael acceptor
mesityl oxide, but the corresponding ester 4 cannot be syn-
thesized using the methyl 3,3-dimethylacrylate as Michael
acceptor. We speculated that this phenomenon was proba-
bly due to the electronic effect on the Michael acceptor.
Therefore, we chose highly reactive diethyl isopropyli-
denemalonate 8 as Michael acceptor and this approach suc-
cessfully established racemic and optically pure 3,3-di-
methylglutamic acid a-ethyl ester 11b in five and six steps,
respectively, with high yields.
Table 1. Potency and selectivity of compounds 1 and 2
W = 1o or 2o amines
O
W
N
H2N
CN
O
2
IC50 (mM)
Compd
W
DPP-IV DPP8 DPP-II
FAP
1.2
1
0.002
1.2
8.4
F3C
N
N
2a
0.012
10
>20
>20
>20
N
RESULTS AND DISCUSSION
N
2b
2c
0.012
0.014
1.9
15
>20
>20
N
Preparation of racemic Boc-3,3-dimethylglutamic acid
As shown in Scheme I, the Schiff’s base 7a, after
lithiation with lithium bromide (LiBr) and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU), did react with diethyl iso-
propylidenemalonate 8 to give triester 9a.8a This com-
pound was too labile to be purified by column chromatog-
raphy. After aqueous ammonium chloride work-up, com-
pound 9a was used for the next reaction.8 We found that
triacid compound generated from triester 9a was reluctant
to be decarboxylated. We tried a few decarboxylation con-
ditions, however these conditions gave no or poor product
yields of 10 after esterification and cyclization reactions
(Scheme I). Fortunately, this yield can be improved by use
O
>20
N
comparative study to investigate aqueous solution stability
of these analogues is also presented.
Several reports described the Michael addition of the
imines of a-amino ester with a,b-unsaturated esters in the
presence of DBU or other nonionic strong bases.8 This pro-
cess offered a useful method for establishing 3-methyl glu-
tamate 3 (Fig. 2) and its derivatives.8a But this approach did
not work for the synthesis of 3,3-dimethyl glutamate 4 as
no product was observed (LC-Mass detected). However,
the synthetic approach successfully prepared the ketone
analogue 5.8b Krebs et al. developed a variation of the ester
enolate Claisen rearrangement for the synthesis of g,d-un-
saturated amino acid 6.9 Using the amino acid 6, we can
synthesize the desired racemic Boc-3,3-dimethylglutamic
acid a-t-butyl ester 11a (an analogue of 11b shown in
Scheme I) in four steps from 6, but the total yield was poor
(10%). As ketone 5 can be synthesized by the 1,4 addition
Scheme I
CO2Et
O
O
CO2Et
O
a
O
EtO
OEt
N
+
N
O
O
7a
8
9a
b,c
O
O
HO
R'
d,e
O
O
R
O
O
N
Boc N
H
H
O
Boc
OH
OMe
N
H
N
10
11b
(Racemate)
O
O
3
4
5
R = H, R'= OMe
6
R = CH3, R' = OMe
R = CH3, R' = CH3
Synthesis of racemic 3,3-dimethyl glutamic acid derivative 11b:
(a) DBU, LiBr, THF; (b) 48% HBr, reflux; (c) H2SO4 (cat.),
EtOH, reflux, then Na2CO3, 50% (3 steps); (d) (Boc)2O, 4-
DMAP; (e) NaOH(aq), THF, 81% (2 steps).
Fig. 2. Synthesis of highly substituted glycine re-
ported by literatures.

110 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Hsu et al.
of 48% HBr under reflux for 48 h and concentrated in
vacuo to give amino diacid salt. The crude diacid product,
without isolation, was treated with H2SO4 (cat.) in EtOH to
afford amino diester salt, and then subsequent cyclization
after work-up by neutralization provided racemic cyclic
3,3-dimethylpyroglutamate 10 in 50% yield for three steps.
The treatment of 10 with (Boc)2O in the presence of 4-di-
methylaminopyridine (4-DMAP) in dichloromethane fol-
lowed by selectively hydrolyzing the N-Boc protected am-
ide bond using NaOH in THF/H2O afforded racemic Boc-
3,3-dimethylglutamic acid a-ethyl ester 11b in 81% yield
for two steps.
ger base lithium diisopropylamide (LDA) was used instead
of DBU.11 The lithiated 7b generated by LDA in THF at
–78 oC then reacted with diester 8 and warmed up to room
temperature to yield two diastereomers, more polar 9b frac-
tion and less polar 9c fraction, in 70% yield, with a dia-
stereomeric ratio 86:14 as determined by chromatography
isolation. In addition, when (1R,4R)-camphor11 instead of
(1R,2R,5R)-2-hydroxypinan-3-one was used as a chiral
auxiliary, the Michael addition of camphor imine with 8 un-
der identical conditions gave only 10% of the Michael
adduct (Scheme not shown).
The imine and triester of the major isomer 9b were
hydrolyzed using 48% HBr followed by decarboxylation in
a one-pot manner to give optically active 10 in a poor yield
(5%). The yield can be improved by stepwise approach
(Scheme II). Cleavage of the chiral auxiliary using 15% cit-
ric acid at RT10 or NH2OH/AcOH in ethanol reflux11c gave
12 in a poor yield. The more promising hydrolysis condi-
tion of the chiral imine 9b was treated with 2N HCl in
Preparation of optically active Boc-3,3-dimethylglut-
amic acid
Once we successfully synthesized racemic 11b by
Michael addition approach. We tried to synthesize enantio-
merically pure 3,3-dimethylglutamic acid 11b by asym-
metric Michael addition (Scheme II). Firstly, commercial
available (1R,2R,5R)-2-hydroxypinan-3-one was used as a
chiral auxiliary.10 If DBU was used as the base, the Michael
addition of lithiated chiral imine 7b with diethyl isopropyl-
idenemalonate 8 gave no reaction at room temperature.
This indicates that DBU is not a strong enough base to
lithiate imine 7b in the presence of LiBr. Therefore, stron-
o
EtOH/1,4-dioxane at 60 C for 18 h to give diester 12 in
90% yield. The diester 12 could be converted to cyclic
3,3-dimethylpyroglutamate enantiomer 10 by in situ hy-
drolysis, decarboxylation, esterification and concomitant
cyclization in 45% yield. Optically active 10 was trans-
formed to optically active Boc-3,3-dimethylglutamic acid
a-ethyl ester 11b by two-step reaction as racemic 10
(Scheme I) was treated.
Scheme II
The closely related analogues of racemic 10 were also
discovered by Yamazaki et al. in 1976 and Mackman et al.
in 1997.12 Compared to their synthetic approach, our first
approach (Scheme I) affords higher total yield and is sim-
ple to carry out,13 and the secondary approach (Scheme II)
produces 10 with modest enantiomeric excess (9b:9c =
86:14, 72% ee).
CO2Et
O
O
CO2Et
O
a
2
EtO
OEt
O
+
N
N
HO
O
O
HO
8
7b
polar 9b, less polar 9c
(9b:9c=86:14)
b
Preparation of 3,3-dimethylglutamic acid-based DPP-
IV inhibitors
O
O
O
HO
Next, a series of 3,3-dimethylglutamic acid-based
DPP-IV inhibitors with (2S)-cyanopyrrolidines at the P1
site can be prepared from the building block 11b. The rep-
resentative compound 2a was synthesized as shown in
Scheme III. DCC coupling of racemate 11b or optically ac-
tive 11b with heterobicyclic amine 13 was followed by
saponification to give acid 14 in 80% yield (2 steps). For
racemic 14, acid 14 was then coupled with L-prolinamide
followed by dehydration of the amides to provide two
e
c, d
O
O
O
O
O
N
H
O
Boc N
H
N
H
O
O
12
Chiral 10
Chiral 11b
Synthesis of optically active 3,3-dimethyl pyroglutamate 11b: (a)
o
LDA, THF, -78 C to rt, 70%; (b) 9b, 2 N HCl in EtOH/1,4-
dioxane, then Na2CO3, 90%; (c) 48% HBr, reflux, 48 h; (d) H2SO4
(cat.), EtOH, reflux, then Na2CO3, 45% (2 steps); (e) Scheme I,
steps d and e.

Synthesis of Boc-3,3-Dimethylglutamic Acid
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 111
Scheme III
O
O
F3C
HO
N
N
N
a,b
NH
F3C
N
N
N
+
O
OH
N
Boc N
H
Boc N
H
O
O
13
11b
14
O
c,d
F3C
N
N
N
N
N
H3N
CN
TFA
O
16
+
O
O
O
F3C
F3C
F3C
O
N
N
N
N
N
N
N
N
F3C
e
f
N
N
N
+
N
N
N
N
N
N
N
N
N
H3N
CN
Boc N
H
CN
Boc N
H
CN
O
TFA
O
O
H3N
NH
TFA
O
O
2a
15b
more polar fraction
15a
17
less polar fraction
Trace amount
+
41%
82%
44%
0%
from 11b
from chiral 11b
O
H3C
N
H
18
o
Synthetic strategy of compounds 2a and 16: (a) DCC, HOBt, 1,4-dioxane/CH2Cl2; (b) NaOH, MeOH, 0 C to rt, 80% (2 steps); (c)
L-Prolinamide, EDC, HOBt, 1,4-dioxane/CH2Cl2; (d) Imidazole, POCl3, pyridine, isolated yield (2 steps); (e) CF3COOH; (f)
CF3COOH, CH3CN.
diastereomers, less polar glutamic acid derivative 15a and
more polar glutamic acid derivative 15b in 41% and 44%
isolated yields, respectively. For optically active 14, chro-
matography gave only less polar fraction glutamic acid de-
rivative 15a in 82% yield. Removal of the Boc protecting
group of 15a with neat trifluoroacetic acid (TFA) yielded
the desired 3,3-dimethylglutamic acid derivative 2a (TFA
salt) without further purification. However, more polar
glutamic acid derivative 15b was treated with TFA under
identical condition to give 3,3-dimethylglutamic acid de-
rivative 16 and by-product 17, with a ratio of 3:1 as deter-
mined by LC-MS (compound 17, Mw: 501). The proposed
mechanism for the formation of 17 is that the nitrile group
of 15b trapped the tert-butyl carbocation followed by hy-
drolysis to produce amide compound 17. Therefore, the
search for scavengers of alkylating agents is required.
When the thioanisole was used as a scavenger for the inter-
mediate carbocation, its ability to suppress the formation of
17 is not effective. The more promising result was ob-
served when acetonitrile (CH3CN) was applied as co-sol-
vent with TFA, and thus, nitrile group of CH3CN partici-
pated in the reaction by acting as acceptor of the leaving
carbocation group. As expected, this approach slashed the
yield of 17 and produced another by-product 18 (Mw: 115,
detected by LC-MS).
Since Zhao et al. reported that in the case of proline
substrates (Pro in P1-position), DPP-IV has an absolute re-
quirement for the S-configuration at both the penultimate
and N-terminal amino acid residues.14 A comparison of the
inhibitory activity of compounds 2a and 16 against DPP-
IV, compound 2a (IC50 = 12 nM) showed at least 100-fold
more potent than 16 (IC50 = 1702 nM) as DPP-IV inhibi-
tor.4 It is speculated that the assignment of (2S) configura-
tion of 3,3-dimethylglutamic acid is the more active com-
pound 2a and (2R) configuration of 3,3-dimethylglutamic
acid is the less active compound 16.

112 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Hsu et al.
The chemical stability of 3,3-dimethylglutamic acid-
based DPP-IV inhibitors
product; its molecular weight is 409.2. Based on this mo-
lecular weight, we proposed intermediate imine 21 as the
chemical structure of this new product. The ratio of 2a/21
is decreased gradually as time goes by. When the 40 mM
Tris buffer was diluted to 5 mM, the cyclized compound 19
generated quickly (Scheme IV). It means that the interme-
diate 21 can be stablized in highly concentrated Tris-HCl
buffer solution. The half-life of 2a in 5 mM Tris buffer (t1/2
= 48 h) is longer than that of 2a in 40 mM Tris–HCl buffer
(t1/2 = 15 h).
Among these analogues of 2a,4 compound 2b with
2,3-dihydro-1H-isoindole at 5-position of 3,3-dimethyl-
glutamic acid increased a slight stability (t1/2 = 6 h) com-
pared to 2a (t1/2 = 3 h) in PBS buffer. We speculated that
electronic effect made the difference in stability between
compounds 2a and 2b. The polar triazole group in 2a in-
duced the secondary amine of piperazine less nucleophilic,
therefore compound 2a is less stable than 2b. The most sta-
ble compound that we have developed in this class is inhib-
itor 2c with methoxymethyl group at the 2-position of pyr-
rolidine. Because of the steric hindrance imposed by the
methoxymethyl group and the intrinsic nucleophilicity of
pyrrolidine, the ability for 2c to cyclize to compound 19
was significantly reduced (Scheme IV), no cyclized prod-
ucts was found after 24 h in 40 mM PBS buffer (LC-MS de-
tected). Importantly, compound 2c (IC50 = 14 nM) is
equipotent with heterobicyclic 2a (IC50 = 12 nM) and 2,3-
dihydro-1H-isoindole 2b (IC50 = 12 nM) as DPP-IV inhibi-
tor (Table 1).4 Because of the steric size imposed by the
fully branched carbon at the b-position of the glutamic
acid, the amino group of this class of inhibitors 2 did not
intramolecularly cyclize with the nitrile to form a cyclic
amidine or diketopiperazine during the period of investiga-
tion (Fig. 1).
After the discovery that 2a was a potent inhibitor of
DPP-IV in vitro, the aqueous stability of this compound
need to be investigated before testing its in vivo efficacy.
The aqueous solution stability of 2a was determined by
monitoring the disappearance of 2a on reverse-phase LC-
MS. Firstly, we investigated the chemical stability of 2a in
40 mM phosphate buffer saline (PBS) buffer (pH 7.4) at
room temperature. HPLC mass spectral analysis revealed
that the amino group of 2a quickly proceeded to undergo
intramolecular cyclization with 5-position carbonyl group
to produce cyclized compound 19 and the leaving second-
ary amine 20. This cyclization reaction was complete after
7 h (Scheme IV). In addition, the cyclization rate depends
on the salt concentration of PBS buffer: the half-life of 2a
in 5 mM PBS (t1/2 = 16 h) is longer than that of 2a in 40 mM
PBS (t1/2 = 3 h). We postulated that the accelerated rate of
cyclization results from a gem-dialkyl moiety located on
b-position of glutamic acid.15
Scheme IV
F3C
O
N
N
NH
F3C
a
N
+
N
N
N
O
N
H
N
CN
N
O
N
H2N
CN
O
20
Mw: 192.14
19
Mw: 235.28
2a
Mw: 427.42
c
b
N
N
N
N
CN
F3C
O
N
N
21
Mw: 409.41
CONCLUSIONS
The aqueous stability of 2a in PBS and Tris-HCl buffer: (a) 40
mM PBS, pH = 7.4; (b) 40 mM Tris buffer, pH = 8.3; (c) Diluted
the 40 mM Tris buffer to 5 mM.
We demonstrated a facile method for the synthesis of
3,3-dimethylglutamic acid 11b. The decarboxylation of the
Michael adduct 9 was the key step in the preparation of 11b
and this compound was employed as a building block for
the synthesis of a series of 3,3-dimethylglutamic acid-
based DPP-IV inhibitors with high potency against DPP-
IV. The aqueous stability of these DPP-IV inhibitors in dif-
ferent buffer solutions was also evaluated. To date, inhibi-
tor 2c is the most stable compound in this class. The high
stability of 2c is most likely due to steric hindrance im-
Since compound 2a is not stable in PBS buffer, the
cyclization rate of 2a in the DPP-IV assay condition (Ex-
perimental Section), Tris–HCl buffer solution (pH 8.3),
should be evaluated.16 Compound 2a is more stable in 40
mM Tris–HCl buffer (t1/2 = 15 h) than in 40 mM PBS buffer
(t1/2 = 3 h), and no cyclized product 19 was found (Scheme
IV). However, HPLC mass spectral analysis revealed a new

Synthesis of Boc-3,3-Dimethylglutamic Acid
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 113
posed by the methoxymethyl group at the 2-position of
pyrrolidine and the inherent nucleophilicity of pyrrolidine.
over MgSO4 and concentrated. The residue was purified by
chromatography on silica gel (40% to 60% EtOAc/hexanes
gradient) to give 10 (5.71 g, 50% in three steps) as a color-
less oil. 1H NMR (300 MHz, CDCl3): d 6.95-6.67 (m, 1H),
4.28-4.20 (m, 2H), 3.93 (s, 1H), 2.23 (s, 2H), 1.34 (s, 3H),
1.31 (t, J = 7.2 Hz, 3H), 1.06 (s, 3H); 13C NMR (100 MHz,
CDCl3): d 14.49, 23.79, 28.05, 40.03, 45.69, 61.60, 65.86,
170.78, 176.98; MS (ESI) m/z: 186 [M+H]+, 208 [M+Na]+.
Racemic 2-tert-butoxycarbonylamino-3,3-dimethyl-
pentanedioic acid 1-ethyl ester (11b)
EXPERIMENTAL SECTION
All commercial chemicals and solvents are reagent
grade and were used without further treatment unless oth-
erwise noted. 1H NMR spectra were obtained with a Varian
Mercury-300 or a Varian Mercury-400 spectrometer. Chem-
ical shifts were recorded in parts per million (ppm, d) and
were reported relative to the solvent peak or TMS. LC/MS
data were measured on an Agilent MSD-1100 ESI-MS/MS
System. High resolution mass spectra (EI) were measured
on an MAT-95XL HRMS. Microanalyses were carried out
on a Heraeus VarioIII-NCH analyzer. The rotation values
of optically active compounds were measured using a P-
1020 Automatic Polarimeter. Flash column chromatogra-
phy was done using silica gel (Merck Kieselgel 60, No.
9385, 230-400 mesh ASTM). Reactions were monitored by
TLC using Merck 60 F254 silica gel glass backed plates (5 ×
10 cm); zones were detected visually under ultraviolet irra-
diation (254 nm) or by spraying with phosphomolybdic
acid reagent (Aldrich) followed by heating at 80 °C. All
starting materials and amines were commercially available
unless otherwise indicated.
To a stirred solution of 10 (5.71 g, 20.0 mmol) in
CH2Cl2 (40 mL) was added di-tert-butyl dicarbonate (4.58
g, 21.0 mmol) followed by 4-dimethylaminopyridine (0.18
g, 1.50 mmol), and the reaction mixture was stirred at room
temperature overnight. The mixture was diluted with
CH2Cl2 (200 mL), washed sequentially with saturated so-
dium bicarbonate, aqueous citric acid and brine, and then
dried over MgSO4. The organic layer was concentrated and
purified by chromatography on silica gel (10% to 40%
EtOAc/hexanes gradient) to give the N-Boc-protected
lactam as a colorless oil.
To a stirred solution of the N-Boc-protected lactam in
THF (40 mL) was added 1N NaOH (40 mL) at 15 oC, and
the reaction mixture was allowed to warm to room tempera-
ture. After being stirred at room temperature for 2 h, the
mixture was acidified to pH = 2 with aqueous citric acid.
The volatiles were removed under reduced pressure and the
residue was partitioned between water and CH2Cl2 (300
mL). The organic layer was washed with brine, dried over
MgSO4 and concentrated. The residue was purified by
chromatography on silica gel (20% to 60% EtOAc/CH2Cl2
gradient) to give racemic 11b (4.91 g, 81% in two steps) as
a colorless oil. 1H NMR (400 MHz, CDCl3): d 5.40 (d, J =
7.6 Hz, 1H), 4.35 (d, J = 7.6 Hz, 1H), 4.21 (q, J = 7.2 Hz,
2H), 2.43-2.21 (m, 2H), 1.44 (s, 9H), 1.29 (t, J = 7.2 Hz,
3H), 1.10 (s, 3H), 1.07 (s, 3H); 13C NMR (100 MHz,
CDCl3): d 14.37, 24.03, 24.27, 28.48, 37.25, 43.83, 60.33,
61.63, 80.76, 156.25, 171.56, 176.11; MS (ESI) m/z: 308
[M+Na-H2O]+, 326 [M+Na]+.
3,3-Dimethyl-5-oxo-pyrrolidine-2-carboxylic acid ethyl
ester (10)
To a mixture of lithium bromide (4.86 g, 56.0 mmol),
imine 7a (6.85 g, 40.0 mmol) and diethyl isopropylidene-
malonate 8 (8.01 g, 40.0 mmol) in dry THF (50 mL) was
added DBU (7.31 g, 48.0 mmol) dropwisely at room tem-
perature under nitrogen. After being stirred at room tem-
perature for 1 h, the mixture was quenched with saturated
aqueous ammonium chloride and extracted with CH2Cl2
(250 mL). The combined extracts were washed with brine,
dried over MgSO4 and concentrated to give racemic 9a as a
colorless oil. This intermediate was used for the next reac-
tion without further purification.
To the above 9a was added hydrobromic acid (100
mL, 48 wt. % in water) and the reaction mixture was heated
to 160 oC for 48 h. After cooling to room temperature, the
volatiles were removed in vacuo to afford brown oil, which
was dissolved in EtOH (150 mL) and treated with sulfuric
acid (5 mL). The mixture was heated to reflux overnight.
After cooling to room temperature, the mixture was parti-
tioned between aqueous sodium bicarbonate and CH2Cl2
(300 mL). The organic phase was washed with brine, dried
More polar 2-ethoxycarbonyl-4-{(1R,2R,5R)-2-hydroxy-
2,6,6-trimethyl-bicyclo[3.1.1]hept-3-ylideneamino}-3,3-
dimethyl-pentanedioic acid diethyl ester (9b) and less
polar 2-ethoxycarbonyl-4-{(1R,2R,5R)-2-hydroxy-2,6,6-
trimethyl-bicyclo[3.1.1]hept-3-ylideneamino}-3,3-di-
methyl-pentanedioic acid diethyl ester (9c)
LDA (14.0 mmol) was prepared at 0 oC by addition of

114 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Hsu et al.
n-BuLi (8.75 mL, 1.6 M in hexanes) to a solution of diiso-
propylamine (2.10 mL, 15.0 mmol) in THF (20 mL) under
nitrogen. The solution was cooled to -78 oC and imine 7b
(1.77 g, 7.0 mmol) in THF (10 mL) was added. The mixture
was stirred for 10 min and then diethylisopropylidene-
malonate (1.38 mL, 7.0 mmol) was added. The reaction
mixture was allowed to warm to room temperature over-
night. The reaction was quenched with water (50 mL) and
extracted with CH2Cl2 (80 mL). The organic phase was
washed with brine, dried over MgSO4 and concentrated.
The residue was purified by chromatography on silica gel
(20% to 40% EtOAc/hexane gradient) to give the more po-
lar product 9b (1.90 g, 60%) as a colorless oil and the less
mic acid (20 mL, 48 wt. % in water) was heated to 160 oC
for 48 h. After cooling to room temperature, the volatiles
were removed in vacuo to afford a brown oil, which was
dissolved in EtOH (30 mL) and treated with sulfuric acid (1
mL). The mixture was heated to reflux overnight. After
cooling to room temperature, the mixture was partitioned
between aqueous sodium bicarbonate and CH2Cl2 (150
mL). The organic phase was washed with brine, dried over
MgSO4 and concentrated. The residue was purified by
chromatography on silica gel (40% to 60% EtOAc/hexane
gradient) to give optically active 10 (0.22 g, 45%) as a col-
orless oil. 1H NMR spectrum and mass data of optically ac-
tive 10 is identical to that of racemic 10. The specific rota-
tion of optically active 10 is [a] 2D4 = +22 (c 0.6, CH2Cl2).
Optically active 2-tert-butoxycarbonylamino-3,3-di-
methyl-pentanedioic acid 1-ethyl ester (Chiral 11)
Optical active 10 was transformed to optically active
Boc-(2S)-3,3-dimethylglutamic acid a-ethyl ester 11b by
two-step reaction as described in the preparation of ra-
cemic 11b. 1H NMR spectrum and mass data of optically
active 11b are identical to that of racemic 11b.
1
polar product 9c (0.32 g, 10%) as a white solid. 9b: H
NMR (400 MHz, CDCl3): d 4.54 (s, 1H), 4.22-3.89 (m,
6H), 3.74 (s, 1H), 2.80-2.51 (m, 2H), 2.34-1.88 (m, 5H),
1.56-1.44 (m, 4H), 1.38-1.14 (m, 18H), 0.79 (s, 3H); 13C
NMR (100 MHz, CDCl3): d 14.17, 14.26, 14.36, 21.40,
21.84, 22.94, 27.48, 28.25, 28.35, 33.78, 38.39, 38.44,
40.19, 50.06, 57.01, 60.86, 61.08, 61.18, 67.24, 76.80,
168.46, 168.78, 170.51, 179.97; MS (ESI) m/z: 454
[M+H]+, 476 [M+Na]+. 9c: Mp = 57-59 oC; 1H NMR (400
MHz, CDCl3): d 4.34 (s, 1H), 4.20-3.89 (m, 6H), 3.74 (s,
1H), 2.59-2.44 (m, 2H), 2.51-1.90 (m, 5H), 1.50-1.44 (m,
4H), 1.36-1.18 (m, 18H), 0.76 (s, 3H); 13C NMR (100
MHz, CDCl3): d 14.18, 14.34, 14.39, 21.94, 22.95, 22.90,
27.53, 28.31, 28.56, 34.12, 38.56, 38.90, 40.39, 50.28,
57.69, 60.91,61.36, 61.41, 68.21, 77.08, 168.85, 169.00,
170.56, 179.72; MS (ESI) m/z 454 [M+H]+, 476 [M+Na]+.
Mixture of cis,trans-3,3-dimethyl-5-oxo-pyrrolidine-
2,4-dicarboxylic acid diethyl ester (12)
2-tert-Butoxycarbonylamino-3,3-dimethyl-5-oxo-5-(3-
trifluoromethyl-5,6-dihydro-8H-[1,2,4]triazolo[4,3-a]-
pyrazin-7-yl)-pentanoic acid (14)
To a mixture of 11b (0.61 g, 2.0 mmol), 13 (0.38 g,
2.0 mmol) and 1-hydroxybenzotriazole hydrate (HOBt hy-
drate, 0.27 g, 2 mmol) in CH2Cl2 (10 mL) was added N,N¢-
dicyclohexylcarbodiimide (DCC, 0.52 g, 2.5 mmol). The
reaction mixture was stirred at ambient temperature for 9
days and most of DCU was removed by filtration. The fil-
trate was diluted with CH2Cl2 (40 mL) and washed sequen-
tially with saturated aqueous sodium bicarbonate (20 mL),
0.5 N aqueous citric acid (20 mL) and brine (20 mL). The
organic phase was dried over magnesium sulfate, filtered,
and concentrated under reduced pressure to yield the crude
material as a viscous oil. The crude material was used for
the next reaction without further purification.
To a stirred solution of polar 9b (1.35 g, 2.98 mmol)
in EtOH (10 mL) was added hydrogen chloride solution (5
mL, 4.0 M in 1,4-dioxane) and the reaction mixture was
heated to 80 oC overnight. After cooling to room tempera-
ture, the mixture was partitioned between aqueous sodium
bicarbonate and CH2Cl2 (150 mL). The organic phase was
washed with brine, dried over MgSO4 and concentrated.
The residue was purified by chromatography on silica gel
(20% EtOAc/hexanes to 50% EtOAc/CH2Cl2) to give cis/
trans isomers of 12 (0.69 g, 90%) as a colorless oil. MS
(ESI) m/z: 258 [M+H]+, 280 [M+Na]+.
To a stirred solution of the above coupling product in
THF (10 mL) was added 1N NaOH (4.0 mL) at 15 oC, and
the reaction mixture was allowed to warm to room tempera-
ture. After being stirred at room temperature for 2 h, the
mixture was acidified to pH = 2 with aqueous citric acid.
The volatiles were removed under reduced pressure and the
residue was partitioned between water and CH2Cl2 (100
mL). The organic layer was washed with brine, dried over
Optically active 3,3-dimethyl-5-oxo-pyrrolidine-2-car-
boxylic acid ethyl ester (Chiral 10)
To a solution of 12 (0.69 g, 2.68 mmol) in hydrobro-

Synthesis of Boc-3,3-Dimethylglutamic Acid
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 115
MgSO4 and concentrated to afford 14 (0.72 g, 80% in two
steps) as a white foamy solid. Compound 14 exists as a
mixture of rotamers. 1H NMR (400 MHz, CDCl3): d 5.75-
5.56 (m, 1H), 5.11 (d, J = 16.4, 1H), 5.01 (d, J = 16.4, 1H),
4.43 (t, J = 8.8, 1H), 4.31-3.92 (m, 4H), 2.70 (d, J = 14.8,
1H), 2.52 (d, J = 14.8, 1H), 1.39 (s, 9H), 1.08 (s, 3H), 1.05
(s, 3H); MS (ESI) m/z: 350 [M-Boc+H]+, 450 [M+H]+, 472
[M+Na]+.
2.68-2.50 (m, 2H, overlapped with DMSO), 2.28-1.93 (m,
4H), 1.42-1.20 (m, 9H), 1.18-1.01 (m, 6H). MS (ESI) m/z:
472 [M-Boc+H]+, 528 [M+H]+, 550 [M+Na]+; Anal. Calcd
for (C23H32F3N7O4×0.33H2O): C, 51.73; H, 6.17; N, 18.38.
1
Found: C, 51.72; H, 6.29; N, 18.14. 15b: H NMR (300
MHz, DMSO-d6): d 7.23-7.16 (m, 1H), 5.08-4.92 (m, 1H),
4.92-4.78 (m, 1H), 4.78-4.58 (m, 1H), 4.57-4.36 (m, 1H),
4.28-3.82 (m, 4H), 3.81-3.50 (m, 2H), 2.78-2.50 (m, 2H,
overlapped with DMSO), 2.20-1.82 (m, 4H), 1.34 (s, 9H),
1.18-0.97 (m, 6H). MS (ESI) m/z: 472 [M-Boc+H]+, 528
[M+H]+, 550 [M+Na]+; Anal. Calcd for (C23H32F3N7O4×
0.5H2O): C, 51.49; H, 6.20; N, 18.27. Found: C, 51.62; H,
6.32; N, 17.74.
[1-((2S)-2-Cyanopyrrolidine-1-carbonyl)-2,2-dimethyl-
4-oxo-4-(3-trifluoromethyl-5,6-dihydro-8H-[1,2,4]tri-
azolo[4,3-a]pyrazin-7-yl)-butyl]-carbamic acid tert-
butyl ester (15a) and [1-((2S)-2-cyanopyrrolidine-1-
carbonyl)-2,2-dimethyl-4-oxo-4-(3-trifluoromethyl-5,6-
dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl)-butyl]-
carbamic acid tert-butyl ester (15b)
Optically active (2S)-1-[2-amino-3,3-dimethyl-5-oxo-5-
(3-trifluoromethyl-5,6-dihydro-8H-[1,2,4]triazolo[4,3-
a]pyrazin-7-yl)-pentanoyl]-pyrrolidine-2-carbonitrile
trifluoroacetate (2a)
To a mixture of 14 (0.72 g, 1.6 mmol), L-prolinamide
(0.18 g, 1.6 mmol) and 1-hydroxybenzotriazole hydrate
(HOBt hydrate, 0.22 g, 1.6 mmol) in CH2Cl2 (5 mL) was
added N-ethyl-N¢-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC, 0.31 g, 1.6 mmol). The reaction mix-
ture was stirred at ambient temperature overnight and was
diluted with CH2Cl2 (40 mL). The solution was washed se-
quentially with saturated aqueous sodium bicarbonate (20
mL), 0.5 N aqueous citric acid (20 mL) and brine (20 mL),
dried over magnesium sulfate, filtered, and concentrated
under reduced pressure to yield the crude amide product as
a white foamy solid, which was used for the next reaction
without further purification.
A solution of 15a (0.35 g, 0.66 mmol) in trifluoacetic
acid (2 mL) was stirred at room temperature for 10 min and
concentrated in vacuo to afford compound 2a (0.36 g,
100% overall yield) as a colorless oil. Compound 2a exists
as a mixture of rotamers. 1H NMR (300 MHz, CDCl3): d
5.21-4.76 (m, 3H), 4.50 (d, J = 13.2 Hz, 1H), 4.36-4.20 (m,
2H), 4.17-4.02 (m, 2H), 3.89-3.80 (m, 1H), 3.77-3.69 (m,
1H), 2.84-2.72 (m, 2H), 2.46-2.02 (m,4H), 1.33 (s, 3H),
1.27 (s, 3H); MS (ESI) m/z: 428 [M+H]+, 450 [M+Na]+.
HRMS (FAB) m/z calcd for C18H25F3N7O2 428.2016, found
428.2021. Compound 2a HCl salt: [a] 2D4 = -44 (c 0.1,
MeOH).
To a mixture of the above crude product and imida-
zole (0.12 g, 1.8 mmol) in pyridine (5 mL) cooled to -35 oC
was added phosphoryl chloride (0.64 g, 4.2 mmol) under
nitrogen. After being stirred for 30 min at -35 oC, the mix-
ture was evaporated to dryness in vacuo. The resulting
browm solid was dissolved in CH2Cl2 (40 mL) and washed
with 0.5 N aqueous citric acid (20 mL). The organic phase
was dried over magnesium sulfate, filtered, and concen-
trated under reduced pressure to yield the crude material as
a viscous oil. The crude material was purified by chroma-
tography by using a Biotage system (FLASH12i, silica gel,
eluting with 60% EtOAc/hexane) to give the less polar
product 15a (0.35 g, 41%) and the more polar product 15b
(0.37 g, 44%). Compounds 15a and 15b exist as a mixture
of rotamers. 15a: 1H NMR (300 MHz, DMSO-d6): d 7.19-
6.95 (m, 1H), 5.10-4.92 (m, 1H), 4.92-4.80 (m, 1H), 4.78-
4.70 (m, 1H), 4.56 (d, J = 8.7 Hz, 1H), 4.29-3.52 (m, 6H),
Optically active (2S)-1-[2-amino-3,3-dimethyl-5-oxo-5-
(3-trifluoromethyl-5,6-dihydro-8H-[1,2,4]triazolo[4,3-
a]pyrazin-7-yl)-pentanoyl]-pyrrolidine-2-carbonitrile
hydrochloride (16)
To a solution of 15b (0.37 g, 0.70 mmol) in aceto-
nitrile (2 mL) was added trifluoacetic acid (2 mL) and the
mixture was stirred at room temperature for 10 min. The re-
action mixture was concentrated in vacuo and the residue
was redissolved in EtOAc (5 mL). To this solution was
treated with hydrogen chloride solution (5 mL, 1.0 M in di-
ethyl ether) with stirring. The precipitate was collected,
washed with EtOAc, and vacuum-dried to give the com-
pound 16 (0.29 g, 90% overall yield) as a white solid, mp:
175-177 oC. Compound 16 exists as a mixture of rotamers.
1H NMR (400 MHz, D2O): d 5.19-4.81 (m, 3H, overlapped
with HOD), 4.52-4.44 (m, 1H), 4.42-4.26 (m, 2H), 4.25-

116 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011
Hsu et al.
4.08 (m, 1H), 4.07-3.06 (m, 1H), 3.94-3.84 (m, 1H), 3.74-
3.62 (m, 1H), 2.81 (s, 1H), 2.77 (s, 1H), 2.41-2.40 (m, 2H),
2.23-2.12 (m, 2H), 1.34-1.10 (m, 6H); MS (ESI) m/z: 428
[M+H]+, 450 [M+Na]+. HRMS (FAB) m/z calcd for
C18H25F3N7O2 428.2016, found 428.2022. [a] 2D4 = -75 (c
0.1, MeOH).
with 40 mM Tris buffer, pH 8.3.
ACKNOWLEDGEMENTS
We would like to thank Dr. Xin Chen for the purifica-
tion of DPP-IV used in this study. National Health Re-
search Institutes, Taiwan, financially supported the study.
Optically active (2S)-1-[2-amino-5-(1,3-dihydro-isoin-
dol-2-yl)-3,3-dimethyl-5-oxo-pentanoyl]-pyrrolidine-2-
carbonitrile trifluoroacetate (2b)
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Synthesis of Boc-3,3-Dimethylglutamic Acid
J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 117
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