Synthesis, biological assay in vitro and molecular docking studies of new imidazopyrazinone derivatives as potential dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.08.002

A series of novel imidazopyrazinone derivatives were synthesized and evaluated with regard to their ability to inhibit dipeptidyl peptidase IV (DPP-IV) in vitro. Of these compounds (2R)-4-oxo-4-[2-(3-carbamoylbenzyl)- hexahydro-3-oxoimidazo [1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan- 2-amine fumaric acid (17h, IC₅₀ = 78 nM) was shown to effectively inhibit the activity of the dipeptidyl peptidase IV enzyme. Molecular docking studies were also performed to illustrate the binding mode of compounds 15c and 17h. Favorable interactions were identified from the binding of inhibitor 15c with DPP-IV. By analogy to the binding mode of compound 15c, it seems that the introduction of a substituted benzyl moiety onto the imidazopyrazinone could remarkably improve the inhibitory activity of compound 17h.

Full text of DOI:10.1016/j.ejmech.2010.08.002

European Journal of Medicinal Chemistry 45 (2010) 4953e4962
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological assay in vitro and molecular docking studies of new
imidazopyrazinone derivatives as potential dipeptidyl peptidase IV inhibitors
,
a
Yanyun Zhu a, Shuang Xia a, Mingjie Zhu b, Weiyin Yi b, Jiagao Cheng a, Gonghua Song a , Zhong Li ,
*
,
Peng Lu b
*
a Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, 130 meilong Road, Shanghai 200237, China
b Shanghai Sun-Sail Pharmaceutical Science and Technology Co., Shanghai, 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel imidazopyrazinone derivatives were synthesized and evaluated with regard to their ability
to inhibit dipeptidyl peptidase IV (DPP-IV) in vitro. Of these compounds (2R)-4-oxo-4-[2-(3-carba-
moylbenzyl)-hexahydro-3-oxoimidazo [1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine
fumaric acid (17h, IC50 ¼ 78 nM) was shown to effectively inhibit the activity of the dipeptidyl peptidase IV
enzyme. Molecular docking studies were also performed to illustrate the binding mode of compounds 15c
and 17h. Favorable interactions were identified from the binding of inhibitor 15c with DPP-IV. Byanalogy to
the binding mode of compound 15c, it seems that the introduction of a substituted benzyl moiety onto the
imidazopyrazinone could remarkably improve the inhibitory activity of compound 17h.
Received 13 March 2010
Received in revised form
18 June 2010
Accepted 1 August 2010
Available online 12 August 2010
Keywords:
Dipeptidyl peptidase IV inhibitor
Imidazopyrazinone
Anti-diabetic agent
Molecular docking
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
In the past few years, we have endeavored to synthesize novel
dipeptide surrogates containing a piperazine mimic. These dipep-
Type 2 diabetes mellitus (T2DM) is a severe and increasingly
prevalent disease. According to the IDF Diabetes Atlas, the esti-
mated number for 2010 is about 285 million [1]. T2DM is the most
prevalent abnormality related to glucose homeostasis. Individuals
with T2DM have a relative insulin deficiency due to insulin resis-
tance or abnormalities. Glucagon-like peptide-1 (GLP-1) and
glucose-dependent insulinotropic peptide (GIP) are two important
incretin hormones released from the intestine in response to
nutrient ingestion. Both peptides stimulate insulin secretion in
a strictly glucose-dependent manner [2]. In addition, GLP-1 also
suppresses the secretion of glucagon, slows gastric emptying,
reduces food intake, and stimulates the regeneration and differ-
tide analogues have exhibited strong inhibition of DPP-IV. These
observations prompted us to explore the new DPP-IV inhibitors
that contain our basic piperazine structure. Herein, we describe the
synthesis, in vitro and molecular docking simulations of several
novel imidazopyrazinone compounds for used as potential DPP-IV
inhibitors.
2. Synthesis
The imidazopyrazinone analogues described in this report as
DPP-IV inhibitors were synthesized using standard peptide
couplings of
The Merck researchers provided two excellent routes to
synthesis -amino acid 7 [7,8], in this report, another flexible route
b-amino acids with fused heterocycles [6].
entiation of islet b-cells [2,3]. Unfortunately, GLP-1 can be deacti-
vated rapidly (<2 min) by plasma DPP-IV via the cleavage of two
amino acids from its N-terminus [4]. The inhibition of DPP-IV leads
to increased levels of endogenous GLP-1 and GIP [2]. Therefore,
DPP-IV has become a validated target for the treatment of type 2
diabetes, and several inhibitors of DPP-IV are currently launched in
certain countries or undergoing late-stage clinical trials [5].
b
without crystallization was described, which could conveniently
apply to lab investigation. Our initial synthetic efforts were focused
on producing the b-amino acids (Scheme 1). 2,4,5-Trifluorobenzoic
acid was converted to acetyl chloride, then the mono-anion of bis
[trimethylsilyl] malonate was synthesized from bis[trimethylsilyl]
malonate and n-BuLi, and added to acetyl chloride to produce
keto acid 1 according to a known method [9]. The enantioselective
reduction of 1 to
-hydroxy acid 2 was carried out using (þ)-dii-
sopinocampheylchloroborane ((þ)-DIP-Cl) in 64% yield and 92% ee.
b-
b
* Corresponding author. Tel.: þ86 21 64253140; fax: þ86 21 64252603.
E-mail address: ghsong@ecust.edu.cn (G. Song).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.002

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Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
Scheme 1. Synthesis of
b
-amino acid 7. (a) ClCOCOCl, DMF, CH2Cl2, rt; (b) n-BuLi, THF, ꢀ60 ꢁC, 40%; (c) (þ)-DIP-Cl, TEA, CH2Cl2, 0 ꢁC, 64%; (d) BnONH2, EDC, HOBT, THF, rt, 60%; (e)
PPh3, DIAD, THF, rt, 72%; (f) LiOH, H2O, THF, MeOH, 0 ꢁC, 93%; (g) 10% Pd/C, H2, MeOH, rt, 92%; (h) Boc2O, TEA, H2O, THF, rt, 91%.
Compound 3 was then prepared from
b
-hydroxy acid 2 and O-
ofNaHinTHF. Finally, after theremovalofBoc-protection groupof 12
in HCl/Et2O solution obtained target free amines 13.
benzyl hydroxylamine using N-(3-dimethylaminopropyl)-N0-eth-
ylcarbodiimidehydrochloride (EDC) and N-hydroxybenzotrizole
Synthetic efforts then focused on coupling b-amino acids 5 and
(HOBT) as coupling reagents. Ring closure of 3 to
achieved using a Mitsunobu reaction [7]. Subsequently, ring
opening of -lactam 4 to key intermediate 5 was carried out with
LiOH in a mixed solvent system of THF/H2O [10]. The desired
amino acid 7 was obtained by catalytic hydrogenation (Pd/C) and
reaction with Boc2O.
Various heterocycles 13 were synthesized via the routes depicted
in Scheme 2. Compound 8 was easily accessible in two steps starting
from piperazine-2-carboxylic acid through known procedure. [11]
Subsequently, the conversion of 8 to primary amine 9 was
completed underastandard Mitsunobureaction condition, followed
by ammonolysis in refluxing methylamine ethanol solution. The key
intermediate 11 was successfully obtained in excellent yield by
refluxing 10 in THF with NaH. The BuchwaldeHartwig reactions of
11 with aryl bromides proceeded in the conditions of [1,10-bis
(diphenylphosphino)ferrocene] dichloropalladium [PdCl2(dppf)]/
NaO-t-Bu/toluene. The intermolecular amidation of alkyl or benzyl
halides with lactams 11 werecarried out successfully in the presence
b
-lactam 4 was
7 with heterocycles of type 13 to provide compounds 14 and 16
(Scheme 3). The desired compounds 15aee and 17aeo (Table 1)
were obtained after deprotection of the amine. Analogue 17h was
synthesized from compound 16h by oxidation with DMSO and
H2O2 and then deprotection of the amine (Scheme 4). For in vitro
evaluation, the HCl salts of compounds 15 and 17 were converted to
their fumaric acid salts [6].
b
b
-
3. Results and discussion
3.1. Inhibitory activity against DPP-IV in vitro
The derivatives 15aee and 17aeo were evaluated for their
inhibition of DPP-IV in vitro [12]. (Table 1) Soluble DPP-IV was
purified from human seminal fluid as described in reference [13,
14], and sitagliptin was used as a reference compound.
As a first-in-class drug, sitagliptin (IC50 ¼ 18 nM) shows excel-
lent selectivity and oral bioavailability. To study the bioisosteres of
sitagliptin, the triazolopiperazine subunit of sitagliptin was
replaced by an oxazolopyrazinone or imidazopyrazinone group.
Table 1 lists data for the initial series of DPP-IV inhibitors con-
taining oxazolopyrazinone, imidazopyrazinone, as well as 2-alkyl
or 2-benzyl substituted imidazopyrazinone moieties. The unsub-
stituted imidazopyrazinone analogue 15b (IC50 ¼ 1060 nM) showed
micromolar activity and was 2-fold more active than the oxazolo-
pyrazinone derivative 15a. Introducing a methyl group at the 2
position of the imidazopyrazinone (15c) led to about a 12-fold
decrease in potency. However, the 2-ethyl-imidazopyrazinone
derivative 15d (IC50 ¼ 520 nM) exhibited about a 2-fold increase in
potency over 15b, while the 2-benzyl analogue 15e (IC50 ¼ 300 nM)
showed significant improvement in activity (about 4-fold). Further
investigations involved introducing various substitutions onto the
benzyl group. Unfortunately, the 4-fluorobenzyl analogue 17a
(IC50 ¼ 1236 nM) was less active than 10e (4-fold), and the 4-
methylbenzyl and 4-chlorobenzyl derivatives (17bec) lost their
potency completely. Interestingly, the 4-methoxylbenzyl derivative
Boc
N
O
O
Boc
Ref. 5
a
N
OH
Boc
HN
OH
N
N
N
Boc
NH
O
9
8
c
f
b
Boc
HN
Boc
Boc
N
N
N
NH2
Boc
NH
N
N
O
10
11
d or e
N
R2
N
N
O
R2
N
O
12
13
Scheme 2. Synthesis of heterocycles 13. (a) PPh3, DIAD, THF, 93%; (b) MeNH2, EtOH,
77%; (c) NaH, THF, reflux, 90%; (d) aryl bromides, PdCl2(dppf), NaO-t-Bu, tol-
uene,15e98%; (e) benzylhalides, NaH, THF, reflux, 48e90%; (d) HCl/Et2O, DCM, 71e99%.
17d (IC50 ¼ 360 nM) exhibited
a potency similar to that of
compound 15e. To investigate the effect that the position and

Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
4955
F
F
F
F
R
HN 1O
a
F
R1
HN
NH
O
X R2
N
+
OH
N
X R2
O
F
N
5 R1=OBn
7 R1=Boc
14 R1=OBn
16 R1=Boc
O
13
F
F
NH2
O
c
b
N
X R2
F
N
.
trans-C4H4O4
O
15 and 17
Scheme 3. Synthesis of 15 and 17. (a) EDC, HOBT, CH2Cl2, rt, 30e98%; (b) 10% Pd/C, H2, MeOH, rt; or TFA, CH2Cl2, rt; (c) EtOH, Et2O, fumaric acid, rt, 45e98%.
quantity of methoxy groups had on the potency of inhibition,
compounds 17eeg were prepared. The 2-methoxybenzyl, 3-
methoxybenzyl and 3,5-dimethoxybenzyl analogues displayed
about a 3-fold increase in vitro activity compared with compound
15e. It is interesting that the 3-CONH2- analogue 17h (IC50 ¼ 78 nM)
showed the most potent activity and is comparable to the approved
drug Sitagliptin (IC50 ¼ 18 nM).
3.2. Molecular docking studies
With In vitro activity against DPP-IV enzyme in hand, it is
thought worth-while to do molecular docking studies to support
the potency of inhibition. Docking studies were performed to
illustrate the binding mode of these compounds. The X-ray struc-
ture of DPP-IV was downloaded from the Protein Data Bank (PDB
code: 1N1M) [6, 14] and was modified by adding hydrogen atoms
and removing water and cocrystallized inhibitors using sybyl 7.0
[15]. Compounds with weak activity (15c) and potent activity (17h)
were built and minimized within sibyl 7.0. Then, all of the inhibi-
tors’ stereo isomers were docked into the active site pocket of DPP-
IV by using GOLD version 3.2 with the ChemScore function used to
evaluate the docking results [16]. All atoms within 10 Å of the
ligand in the 1N1M structure were considered in the docking study.
The docking results are depicted in Fig. 1.
We then turned our attention to introducing various 2-aryl
functional groups onto the imidazopyrazinone. Table 1 shows the
enzyme assay data for derivatives 17ieo, but unfortunately these
analogs lost in vitro activity.
Table 1
In vitro activity against DPP-IV.a
F
F
It can be seen from Fig. 1 that all of the isomeric structures can
interact with DPP-IV’s binding pocket. Many interactions are
evident in the binding of 15c with DPP-IV. The 2,4,5-tri-
fluorophenyl moiety fully occupies the S1 hydrophobic pocket and
NH2
O
N
X R2
F
N
forms a pep interaction with the side chain of Tyr662. The (3R)-b-
.
trans-C4H4O4
amino group forms an N-H$$$O hydrogen bond with the side
chain of Glu205. The carbonyl group interacts with the side chain
of Tyr547 via an OeH/O hydrogen bond. The imidazopyrazinone
portion of the molecule could be experiencing a hydrophobic
interaction with the phenyl group of Phe357, but no clear inter-
action could be identified in this portion of the inhibitor, so it may
serve as an area of the structure that can be further modified for
increased activity.
In comparison to compound 15c, the introduction of
a substituted benzyl moiety to the fused piperazine should
remarkably improve the inhibitory activity, as demonstrated by
compound 17h. It is clear from the docking results that both
isomeric structures of 17h have favorable interactions with the
active site of DPP-IV. The 2,4,5-trifluorophenyl moiety fully
occupies the S1 hydrophobic pocket and interacts with the side
O
Entry
X
R2
Percent inhibition (at 10
m
M)
IC50 (nM)
15a
15b
15c
15d
15e
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
17k
17l
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
e
93.5
97.1
87.0
95.4
96.5
95.2
ꢀ0.8
0.6
98.7
99.6
98.9
98.9
98.9
1.5
2300
1060
1240
460
300
1236
NTb
NT
360
101
124
114
78
NT
NT
NT
NT
NT
NT
NT
11
H
Me
Et
Bn
4-FPhCH2
4-MePhCH2
4-ClPhCH2
4-MeOPhCH2
2- MeOPhCH2
3- MeOPhCH2
3,5-Di-MeOPhCH2
3-CONH2PhCH2
Pyridin-2-yl
4-CF3Ph
2,4,5-Tri-FPh
4-FPh
4-ClPh
chain of Tyr662 via a pep interaction. The (3R)-b-amino group
2.5
1.3
1.6
can form two NeH/O hydrogen bonds with the side chains of
Glu205 and Tyr662. The carbonyl group of the imidazopyrazinone
moiety can interact with the side chain of Ser209 via an OeH/O
hydrogen bond. A weak CeH/O hydrogen bonding interaction
could also be found for both isomeric structures between the
phenyl ring and the phenol group of Tyr585. In addition to the
above interactions shared by both isomers, the 8aR isomer enjoys
another OeH/O hydrogen bonding interaction to the phenol of
Tyr547.
17m
17n
17o
ꢀ0.6
ꢀ0.8
2.2
4-CNPh
4-MeSO2Ph
Sitagliptin
a
The percent inhibition and IC50 results are an average of three independent
titrations, unless otherwise noted, having calculated standard errors below 15%.
b
NT ¼ not tested.

4956
Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
F
F
Boc
F
F
HN
O
HN
a
N
F
Boc
N
NH
O
N
+
N
O
F
N
OH
O
NC
7
16h
NC
F
F
NH2
O
d
c
b
N
N
O
F
N
.
trans-C4H4O4
17h
H2NOC
Scheme 4. Synthesis of 17h. (a) EDC, HOBT, CH2Cl2, rt, 95%; (b) DMSO, 30%H2O2, CH2Cl2, 0 ꢁC; (c) TFA, CH2Cl2, rt; (d) EtOH, Et2O, fumaric acid, rt, 67%.
Fig. 1. Docking results obtained by combining compounds 15c and 17h with DPP-IV enzyme. (A) The interactions of compound 15c (8aR) with the key residues of DPP-IV; (B) the
interactions of compound 15c (8aS) with the key residues of DPP-IV; (C) the interactions of compound 17h (8aR) with the key residues of DPP-IV; (D) the interactions of compound
17h (8aS) with the key residues of DPP-IV. The figures were prepared using PyMol (www.pymol.org).
4. Conclusion
5. Experimental
In summary, we have synthesized a series of novel 2-alkyl, 2-
benzyl, and substituted 2-alkyl imidazopyrazinone derivatives. Some
of the target compounds, such as compounds 15e (IC50 ¼ 300 nM)
and 17h (IC50 ¼ 78 nM), showed potent in vitro activity against DPP-
IV. Molecular docking studies showed good binding of these
compounds to the active site of DPP-IV. Further investigations on the
optimizationofthese twoleadingcompoundsandindividualepimers
of compound 17h, are being carried out in our laboratory.
5.1. General
Tetrahydrofuran was dried over sodium. DMF was dried over
calcium hydride. Other chemicals and solvents were purchased
from commercial suppliers and used without further purification.
Melting points were recorded on a B-540 melting point apparatus
and are uncorrected. 1H NMR and 13C NMR spectra were recorded
on a Varian Mercury-400 MHz spectrometer. Chemical shifts are

Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
4957
given in ppm relative to tetramethylsilane (TMS) as an internal
standard (0 ppm). Mass spectra were (LRMS and HRMS) recorded
on a Finnigan MAT 95 mass spectrometer.
2.86 (m, 1H), 3.50e3.62 (m, 1H), 4.92 (m, 2H), 6.87e6.93 (m, 2H),
7.33e7.40 (m, 5H).
5.6. N-benzyloxy-4-(2,4,5-trifluorophenyl)-3(R)-aminobutanoic
acid 5
5.2. 4-(2,4,5-Trifluorophenyl)-3-oxobutanoic acid 1
LiOH$H2O (0.5 g, 12 mmol) was added to a solution of 4 (3.2 g,
10 mmol) in THF/H2O (1:1, 20 mL) at 0 ꢁC. After the reaction was
complete, the pH was adjusted to 10 with 2 N NaOH and the
aqueous phase was extracted with methyl-tert-butyl ether. The
aqueous phase was then acidified to pH ¼ 5 with 2 N HCl and
extracted with EtOAc. The combined organic layers were washed
with brine, dried, filtered, and concentrated to give the title
compound (3.1 g, 93%) as a white solid: mp 109e111 ꢁC; 1H NMR
To a solution of 2-(2,4,5-trifluorophenyl)acetic acid (10.0 g,
53.0 mmol) in CH2Cl2 (200 mL) was added DMF (1.0 mL) and
(COCl)2 (8.4 mL, 106.0 mmol). After stirring for 2 h, the solution was
concentrated in vacuo. Separately, a solution of n-BuLi (40.0 mL,
100 mmol) was slowly added at ꢀ60 ꢁC under an N2 atmosphere to
a solution of bis[trimethylsilyl] malonate (25.0 g, 100 mmol) in THF
(50 mL). After being stirred for 10 min, a solution of acetyl chloride
in THF (30 mL) was poured into the mixture at 0 ꢁC. The reaction
mixture was quenched with 5% aqueous NaHCO3, acidified to
pH ¼ 5 with 2 N HCl, and then extracted with EtOAc. The combined
organic layers were washed with brine, dried, filtered, and
concentrated to give the title compound (4.9 g, 40%) as a yellow
(CDCl3, 300 MHz)
d 2.39e2.87 (m, 4H), 4.48 (m, 1H), 4.68 (m, 2H),
4.90e4.98 (m, 1H), 6.82e7.07 (m, 2H), 7.26e7.35 (m, 5H).
5.7. 4-(2,4,5-Trifluorophenyl)-3(R)-aminobutanoic acid 6
solid: mp 65e67 ꢁC. 1H NMR (CDCl3, 400 MHz)
6.91e6.98 (m, 1H), 7.18e7.26 (m, 1H).
d 4.43 (s, 4H),
10% Pd/C (1.6 g) was added to a solution of 5 (2.6 g, 7.7 mmol) in
MeOH (50 mL), and then the mixture was submitted to an atmo-
sphere of hydrogen gas (40 psi H2) for 12 h. The catalyst was filtered
off and the solvent was evaporated. The crude material was
recrystallized from CH2Cl2 and hexane to yield compound 6 (2.1 g,
92%) as a white solid: mp 169e178 ꢁC; 1H NMR (CF3COOD,
5.3. 4-(2,4,5-Trifluorophenyl)-3(S)-hydroxybutanoic acid 2
Compound 1 (2.0 g, 8.6 mmol) and Et3N (870 mg, 8.6 mmol)
were dissolved in CH2Cl2 (20 mL) at ꢀ60 ꢁC under an N2 atmo-
sphere. After being stirred for 5 min, (þ)-DIP-Cl (3.3 g, 10.0 mmol)
was added. The mixture was stirred at 0 ꢁC for 2 h under an N2
atmosphere and quenched with H2O. The pH of the reaction
mixture was adjusted to 10 with 2 N NaOH, and the aqueous phase
was extracted with methyl-tert-butyl ether. The aqueous phase was
then acidified to pH ¼ 5 with 2 N HCl and extracted with EtOAc. The
combined organic layers were washed with brine, dried, filtered,
and concentrated to give the title compound (1.3 g, 64%) in 92% ee
(Chiracel AD-H, 95% hexanes, 5% IPA, 1% TFA, 1 mL/min, 35 ꢁC,
tr ¼ 20.5 min (S-2), 22.8 (R-2)). 1H NMR (CDCl3, 400 MHz)
400 MHz)
d
1.23 (dd, J1 ¼12.3 Hz, J2 ¼1.0 Hz, 2H), 2.88e2.95 (m,
2H), 3.04e3.18 (m, 2H), 4.02e4.17 (m, 1H), 6.96 (m, 1H), 7.03 (m,
1H); MS (ESI) m/z: 234 [M þ 1].
5.8. N-Boc-4-(2,4,5-trifluorophenyl)-3(R)-aminobutanoic acid 7
To a solution of 6 (2.1 g, 7.1 mmol) in 1,4-dioxane/H2O (1:1,
40 mL) was added Et3N (5 mL) and Boc2O (2.0 g, 9.2 mmol). After
the reaction was complete, the reaction mixture was acidified to
pH ¼ 5 with 2 N HCl, and extracted with EtOAc. The combined
organic layers were washed with brine, dried, filtered, and
concentrated to give the title compound (2.4 g, 91%) as a white
d
2.48e2.62 (m, 2H), 2.79 (d, J ¼ 6.0 Hz, 2H), 4.24e4.29 (m, 1H), 6.91
solid: mp 114e115 ꢁC; 1H NMR (CDCl3, 300 MHz)
d 1.34e1.42 (m,
(s, 1H), 7.18e7.30 (m, 1H).
9H), 2.60 (m, 2H), 2.88 (d, J ¼ 13.6 Hz, 2H), 4.10e4.18 (m, 1H),
6.86e6.94 (m, 1H), 7.01e7.10 (m, 1H).
5.4. N-benzyloxy-4-(2,4,5-trifluorophenyl)-3(S)-
hydroxybutanamide 3
5.9. Di-tert-butyl 2-((1,3-dioxoisoindolin-2-yl)methyl)-piperazine-
1,4-dicarboxylate 9
To a solution of 2 (0.8 g, 3.6 mmol) in THF (20 mL) was added O-
benzylhydroxyamine hydrochloride (1.4 g, 11.5 mmol), K2CO3
(2.1 g, 15.3 mmol) and HOBT (10 mg, 0.1 mmol), followed by EDC
(0.8 g, 4.0 mmol) added slowly over 15 min at 20e30 ꢁC. The
resulting mixture was stirred for 3 h at 20e22 ꢁC, quenched with
H2O, diluted with CH2Cl2, washed with 2 N HCl and brine, dried,
filtered, concentrated and recrystallized from CH2Cl2 and hexane to
yield the title compound (0.7 g, 60%) as a white solid: mp
Compound 8 was prepared following the literature procedure.
[13] To the solution of 8 (15.0 g, 47.5 mmol) in anhydrous THF
(250 mL) at 10 ꢁC was added PPh3 (16.2 g, 61.8 mmol) and phtha-
limide (9.1 g, 61.8 mmol). DIAD (12.0 mL, 61.8 mmol) was then
added slowly to the reaction mixture at 10 ꢁC. The reaction was
stirred at 10 ꢁC for additional 2 h. After the reaction was completed,
the reaction mixture was treated with 1 N HCl (100 mL) and then
extracted with ethyl acetate. The extract was washed with 5%
aqueous NaHCO3 (100 mL), dried and concentrated in vacuo. The
residue was purified by silica gel chromatography to give the title
compound (19.9 g, 93%) as a white solid. mp 95e96 ꢁC; 1H NMR
99e105 ꢁC; 1H NMR (CDCl3, 300 MHz)
d 2.18e2.76 (m, 5H),
4.18e4.20 (m, 1H), 4.78e4.91 (m, 2H), 6.87e6.93 (m, 1H), 7.06e7.12
(m, 1H), 7.37e7.44 (m, 5H).
5.5. N-benzyloxy-4(R)-[1-methyl-(2,4,5-trifluorobenzyl)]-2-
oxoazetidine 4
(CDCl3, 400 MHz)
d 1.02 (m, 9H), 1.44 (m, 9H), 2.73e2.84 (m, 1H),
2.93e3.01 (m, 1H), 3.28e3.40 (m, 1H), 3.55 (m, 1H), 3.65e4.15 (m,
4H), 4.48e4.66 (m, 1H), 7.67e7.84 (m, 4H).
Triphenylphosphine (530 mg, 2.0 mmol) was added to a solu-
tion of compound 3 (530 mg, 1.6 mmol) in THF (30 mL) at 10 ꢁC.
DIAD (404 mg, 2.0 mmol) was then added slowly to the reaction
mixture at a rate that maintained the internal reaction temperature
at <10 ꢁC. The reaction was stirred at 10 ꢁC for additional 2 h. After
the reaction was complete, the reaction mixture was concentrated
in vacuo. The crude product was purified by silica gel chromatog-
raphy to give the title compound (400 mg, 72%) as a white solid: mp
5.10. Di-tert-butyl 2-(aminomethyl)piperazine-1,4-dicarboxylate
(10)
The solution of 9 (14.1 g, 31.7 mmol) in EtOH (100 mL) was
treated with methylamine alcohol solution (32%, 100 mL) and
heated at reflux for 4 h, upon cooling to room temperature and
concentrated. The resulting residue was dissolved in H2O (50 mL),
89e91 ꢁC, 1H NMR (CDCl3, 300 MHz)
d 2.33 (m, 1H), 2.67 (m, 2H),

4958
Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
acidified to pH ¼ 3 with 2 N HCl and extracted with methyl-tert-
butyl ether. The pH was adjusted to 10 with 2N NaOH, and the
mixture was extracted with ethyl acetate. The extract was dried and
filtered, the solvent was removed under reduced pressure togive the
title compound (7.7 g, 77%) as a white solid. Mp 92e93 ꢁC; 1H NMR
5.14. General acylation procedure for compounds 14aee and
16aeo
To a solution of b-amino acid 5 or 7 (1.0 mmol) in CH2Cl2 (5 mL),
fused heterocycles 13 (1.2 mmol), EDC (1.2 mmol), HOBT
(0.1 mmol) and Et3N (3.0 mmol) were added. The reaction mixture
was stirred for 12 h at room temperature, quenched with H2O,
diluted with CH2Cl2, washed with 2N HCl, 2N NaOH and brine, and
dried over with anhydrous MgSO4. The solvent was removed in
vacuo and the residue was purified by silica gel chromatography to
give the desire compound.
(CDCl3, 400 MHz)
d 1.45 (s, 18H), 2.73e2.82 (m, 5H), 2.89e2.95 (m,
4H), 3.82e4.16 (m, 3H); MS (ESI) m/z: 316.2 (M þ H)þ.
5.11. tert-Butyl-hexahydro-3-oxoimidazo[1,5-a]pyrazine-7(1H)-
carboxylate (11)
The solution of 10 (7.7 g, 24.2 mmol) in anhydrous THF (50 mL)
was added dropwise to a suspension of NaH (60% dispersion in
mineral oil, 0.7 g, 48.4 mmol) in THF (50 mL) at 0 ꢁC. The reaction
mixture was heated at reflux for 3 h. Upon cooling to room
temperature, the mixture was poured into ice H2O (50 mL), and
extracted with ethyl acetate. The extracts were washed with H2O
and brine, dried, filtered, and concentrated to give the desired
product (5.3 g, 90%). mp 212e213 ꢁC; 1H NMR (CDCl3, 400 MHz)
5.14.1. (3R)-3-(Benzyloxyamino)-1-[hexahydro-3-oxo-oxazolo[1,5-
a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one 14a
Yield: 56%. 1H NMR (CDCl3, 300 MHz)
d 2.26e3.31 (m, 1H), 2.41
(m, 1H), 2.59e2.66 (m, 2H), 2.79e3.05 (m, 4H), 3.52e3.72 (m, 2H),
3.77e3.97 (m, 2H), 4.40 (m, 1H), 4.76e5.30 (m, 3H), 6.85e6.93 (m,
1H), 6.99e7.08 (m, 1H), 7.22e7.36 (m, 5H).
5.14.2. (3R)-3-(Benzyloxyamino)-1-[hexahydro-3-oxoimidazo[1,5-
d
1.44 (s, 9H), 2.68e2.87 (m, 3H), 3.03 (m, 1H), 3.53 (t, J ¼ 12.0 Hz,
a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one 14b
1H), 3.62e3.71 (m, 1H), 3.76 (d, J ¼ 12.8 Hz, 1H), 3.98 (m, 2H),
Yield: 100%. 1H NMR (CDCl3, 300 MHz)
d 2.17e2.31 (m, 1H),
5.18e5.36 (m, 1H); MS (ESI) m/z: 264.2 (M þ Na)þ.
2.48e2.65 (m, 2H), 2.76e3.08 (m, 5H), 3.51e3.85 (m, 5H), 4.60e4.71
(m, 4H), 6.85e6.93 (m, 1H), 7.05e7.26 (m, 1H), 7.30e7.36 (m, 5H).
5.12. General procedure for the synthesis of compound 12
Method A: the compound 11 (480 mg, 2.0 mmol) was dissolved
in toluene (20 mL) under nitrogen. Then aryl bromides (4.0 mmol),
NaO-t-Bu (384 mg, 4.0 mmol) and PdCl2(dppf) (10 mg, 0.01 mmol)
were added to the mixture and refluxing for 12e48 h. Upon cooling,
the mixture was concentrated and the residue was dissolved in H2O
(30 mL), extracted with ethyl acetate. The extract was washed with
brine, dried, filtered, and concentrated. The residue was purified by
column chromatography to give the compounds 12.
5.14.3. (3R)-3-(Benzyloxyamino)-1-[2-methyl-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)
butan-1-one 14c
Yield: 30%. 1H NMR (CDCl3, 400 MHz)
d 2.52e2.69 (m, 3H),
2.73e2.84 (m, 3H), 2.83e2.88 (m, 3H), 2.96 (m, 2H), 3.47e3.55 (m,
4H), 3.92 (m, 2H), 4.65e4.76 (m, 2H), 6.88 (m,1H), 7.26 (m,1H), 7.34
(m, 5H); MS (ESI) m/z 476 [M þ 1].
Method B: benzylhalide (2.0 mmol) was added to the solution of
11 (241 mg, 1.0 mmol) in anhydrous THF (10 mL). Then the mixed
solution was added dropwise to a suspension of NaH (60% disper-
sion in mineral oil, 4.0 mmol) in THF (20 mL) at 0 ꢁC. The mixture
was refluxing for 3e8 h. Upon cooling, the mixture was poured into
ice H2O (50 mL), extracted with ethyl acetate. The combined
extracts were washed with H2O and brine, dried, filtered, and
concentrated to give the desired products 12 as yellow solids.
5.14.4. (3R)-3-(Benzyloxyamino)-1-[2-ethyl-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)
butan-1-one 14d
Yield: 100%. 1H NMR (CDCl3, 400 MHz)
d 1.09e1.13 (m, 3H),
2.13e2.31 (m, 2H), 2.58e2.64 (m, 2H), 2.80e3.01 (m, 5H), 3.24e3.30
(m, 2H), 3.42e3.63 (m, 4H), 3.84 (m, 1H), 4.64 (m, 3H), 6.89 (m, 1H),
7.07 (m, 1H), 7.28e7.34 (m, 5H).
5.14.5. (3R)-3-(Benzyloxyamino)-1-[2-benzyl-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)
butan-1-one 14e
5.13. General procedure for the synthesis of compound 13
Yield: 97%. 1H NMR (CDCl3, 400 MHz)
d 2.04e2.14 (m, 2H),
To a solution of 12 (1.0 mmol) in CH2Cl2 (15 mL) was added HCl/
Et2O solution (1.7 mL, 4.0 N, 6.8 mmol). After being stirred over-
night, the precipitated solids were filtered and washed with Et2O to
give the desired compounds.
2.52e2.78 (m, 2H), 2.78e2.87 (m, 4H), 2.99e3.32 (m, 1H),
3.47e3.52 (m, 1H), 3.53e3.65 (m, 1H), 3.73 (m, 1H), 3.89 (m, 1H),
4.37 (m, 2H), 4.38e4.54 (m, 1H), 4.79 (m, 2H), 6.89 (m, 1H), 7.08 (m,
1H), 7.19e7.37 (m, 10H).
5.13.1. Hexahydro-2-(4-trifluoromethylphenyl)-imidazo[1,5-a]
5.14.6. tert-Butyl[(1R)-3-[2-(4-fluorobenzyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-trifluorobenzyl)
propyl]carbamate 16a
pyrazin-3-one
Mp 233e235 ꢁC; 1H NMR (CF3COOD, 400 MHz)
d 3.29e3.36 (m,
2H), 3.54e3.72 (m, 2H), 3.76 (m, 1H), 3.86 (m, 1H), 4.11e4.29 (m,
Yield: 83%. 1H NMR (CDCl3, 300 MHz)
d 1.36 (m, 9H), 2.52e2.60
2H), 4.48 (m, 1H), 7.48 (d, J ¼ 8.6 Hz, 2H), 7.60 (d, J ¼ 8.5 Hz, 2H); 13C
(m, 3H), 2.82e3.12 (m, 5H), 3.47e3.57 (m, 2H), 3.63e3.70 (m, 1H),
3.93 (m, 1H), 4.02e4.11 (m, 1H), 4.29e4.42 (m, 2H), 5.59 (m, 1H),
6.82e6.89 (m, 1H), 7.02 (m, 3H), 7.19e7.24 (m, 2H).
NMR (DMSO-d6, 100 MHz)
d 37.3, 42.3, 45.7, 47.1, 50.3, 117.2, 126.3,
126.4, 143.9, 155.3, 160.3; HRMS (ESI): C13H14N3OF3Na, calc.
308.0987; found 308.0992.
5.14.7. tert-Butyl[(1R)-3-[2-(4-methylbenzyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16b
5.13.2. Hexahydro-2-benzyl-imidazo[1,5-a]pyrazin-3-one
Mp 214e215 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d 2.74 (t,
J ¼ 8.0 Hz, 1H), 2.82 (dt, J2 ¼ 9.6 Hz, J2 ¼ 4.0 Hz, 1H), 2.91 (dd,
J2 ¼ 12.4 Hz, J2 ¼ 4.8 Hz, 1H), 3.16e3.30 (m, 4H), 3.78 (dd,
J1 ¼14.8 Hz, J2 ¼ 3.8 Hz, 1H), 3.92e3.97 (m, 1H), 4.30 (t, J ¼ 17.2 Hz,
2H), 7.22e7.27 (m, 3H), 7.33e7.37 (m, 2H); 13C NMR (DMSO-d6,
Yield: 68%. 1H NMR (CDCl3, 400 MHz)
d 1.37 (s, 9H), 2.34 (s, 3H),
2.53e2.60 (m, 3H), 2.80e2.96 (m, 5H), 3.08 (m, 1H), 3.27e3.32 (m,
1H), 3.52 (m, 1H), 3.68 (m, 1H), 3.91e3.97 (m, 1H), 4.03e4.10 (m,
1H), 4.35 (m, 2H), 4.58 (m, 1H), 6.87e6.92 (m, 1H), 6.99e7.07 (m,
1H), 7.14 (s, 4H).
100 MHz)
d 37.4, 42.2, 44.9, 45.4, 47.3, 47.9, 127.7, 128.1, 129.0, 137.6,
158.8; HRMS (ESI): C13H17N3ONa, calc. 254.1269; found 254.1277.

Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
4959
5.14.8. tert-Butyl[(1R)-3-[2-(4-chlorobenzyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16c
(m, 3H), 4.64e4.85 (m, 1H), 6.90 (m, 1H), 7.05e7.10 (m, 1H), 7.59 (d,
J ¼ 8.8 Hz, 2H), 7.67 (d, J ¼ 8.8 Hz, 2H).
Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d
1.35 (s, 9H), 2.38e2.67
5.14.16. tert-Butyl[(1R)-3-[2-(2,4,5-trifluorophenyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16k
(m, 3H), 2.78e3.12 (m, 5H), 3.28e3.34 (m, 1H), 3.50e3.58 (m, 1H),
3.64e3.70 (m, 1H), 3.91e3.97 (m, 1H), 4.03e4.10 (m, 1H), 4.30e4.37
(m, 2H), 4.58e4.62 (m, 1H), 6.89 (m, 1H), 7.05 (m, J ¼ 8.4 Hz, 1H),
7.18 (d, J ¼ 8.4 Hz, 2H).
Yield: 55%. 1H NMR (CDCl3, 400 MHz)
d 1.42e1.49 (m, 9H),
2.55e2.65 (m, 3H), 2.90e3.15 (m, 4H), 3.48e3.59 (m, 1H),
3.72e3.78 (m, 2H), 3.94e4.00 (m, 2H), 3.09e4.10 (m, 1H),
4.62e4.83 (m, 1H), 6.90 (m, 1H), 7.05e7.10 (m, 1H), 7.32 (m, 1H),
7.52 (m, 1H).
5.14.9. tert-Butyl[(1R)-3-[2-(4-methoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16d
Yield: 75%. 1H NMR (CDCl3, 400 MHz)
d
1.36 (m, 9H), 2.44e2.64
5.14.17. tert-Butyl[(1R)-3-[2-(4-fluorophenyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16l
(m, 3H), 2.77e3.12 (m, 5H), 3.27e3.32 (m, 1H), 3.49e3.54 (m, 1H),
3.65e3.70 (m, 1H), 3.80 (s, 3H), 3.92e3.96 (m, 1H), 4.06e4.08 (m,
1H), 4.29e4.37 (m, 2H), 5.56e5.60 (m, 1H), 6.89 (m, 1H), 7.05 (m,
1H), 7.18 (d, J ¼ 8.4 Hz, 2H), 7.31 (d, J ¼ 8.4 Hz, 2H).
Yield: 34%. 1H NMR (CDCl3, 400 MHz)
d 1.42e1.49 (m, 9H),
2.53e2.65 (m, 3H), 2.92e3.20 (m, 4H), 3.40e3.48 (m, 2H),
3.72e3.78 (m, 2H), 3.90e4.02 (m, 3H), 4.62e4.83 (m, 1H), 6.91 (m,
1H), 7.01e7.08 (m, 3H), 7.46e7.52 (m, 2H).
5.14.10. tert-Butyl[(1R)-3-[2-(2-methoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16e
5.14.18. tert-Butyl[(1R)-3-[2-(4-chlorophenyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16m
Yield: 94%.1H NMR (CDCl3, 300 MHz)
d 1.36 (s, 9H), 2.44e2.65 (m,
3H), 2.83e3.08 (m, 5H), 3.33e3.40 (m, 1H), 3.48e3.56 (m, 1H), 3.66
(m, 1H), 3.83 (s, 3H), 3.90e3.95 (m, 1H), 4.09 (m, 1H), 4.44 (s, 2H),
4.55e4.60(m,1H), 6.86e6.95(m, 3H), 7.06 (m,1H), 7.22e7.29(m, 2H).
Yield: 75%, mp 207e208 ꢁC. 1H NMR (CDCl3, 400 MHz)
d 1.38 (m,
9H), 2.22e2.70 (m, 3H), 2.88e3.14 (m, 4H), 3.43e3.49 (m, 1H),
3.70e3.78 (m, 2H), 3.90e4.13 (m, 3H), 4.65e4.84 (m, 1H), 5.40 (m,
1H), 6.90 (m, 1H), 7.05e7.10 (m, 1H), 7.28 (d, J ¼ 8.8 Hz, 2H), 7.49 (d,
J ¼ 8.8 Hz, 2H).
5.14.11. tert-Butyl[(1R)-3-[2-(3-methoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-trifluorobenzyl)
propyl]carbamate 16f
Yield: 83%. 1H NMR (CDCl3, 300 MHz)
d
1.36 (s, 9H), 2.40e2.68
5.14.19. tert-Butyl[(1R)-3-[2-(4-cyanophenyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16n
(m, 3H), 2.82e3.14 (m, 5H), 3.29e3.35 (m, 1H), 3.51e3.57 (m, 1H),
3.60e3.65 (m,1H), 3.69 (m,1H), 3.80 (s, 3H), 3.94 (m,1H), 4.05e4.11
(m, 1H), 4.36 (s, 2H), 4.59 (m, 1H), 6.78e6.84 (m, 3H), 6.88 (m, 1H),
7.06 (m, 1H), 7.22e7.27 (m, 1H).
Yield: 97%, mp 99e100 ꢁC. 1H NMR (CDCl3, 300 MHz)
d 1.38 (m,
9H), 2.57e2.67 (m, 3H), 2.81e3.05 (m, 4H), 3.70e3.84 (m, 1H),
3.95e4.17 (m, 4H), 4.73 (m, 1H), 4.89e4.94 (m, 1H), 5.30e5.38 (m,
1H), 6.88e6.96 (m, 1H), 7.07e7.25 (m, 2H), 7.60e8.00 (m, 1H),
8.00e8.67 (m, 1H).
5.14.12. tert-Butyl[(1R)-3-[2-(3,5-dimethoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16g
Yield: 50%. 1H NMR (CDCl3, 300 MHz)
d
1.36 (s, 9H), 2.39e2.66
5.14.20. tert-Butyl[(1R)-3-[2-(4-(methylsulfonyl)phenyl)-
hexahydro-3-oxoimidazo [1,5-a]pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16o
(m, 3H), 2.80e3.12 (m, 5H), 3.30e3.37 (m, 1H), 3.49e3.59 (m, 1H),
3.60e3.65 (m, 1H), 3.62e3.75 (m, 1H), 3.77 (s, 6H), 3.80e3.96 (m,
1H), 4.00e4.11 (m,1H), 4.27e4.37 (m, 2H), 4.60 (m,1H), 6.38 (s, 3H),
6.84e6.93 (m, 1H), 7.01e7.08 (m, 1H).
Yield: 49%, mp 227e230 ꢁC. 1H NMR (CDCl3, 400 MHz)
d 1.35 (m,
9H), 2.52e2.68 (m, 3H), 2.92 (m, 2H), 2.97e3.16 (m, 5H), 3.48e3.55
(m, 1H), 3.76e3.82 (m, 2H), 3.98e4.11 (m, 3H), 4.68e4.87 (m, 1H),
5.38 (m, 1H), 6.90 (m, 1H), 7.05e7.10 (m, 1H), 7.75 (d, J ¼ 8.6 Hz, 2H),
7.90 (d, J ¼ 8.7 Hz, 1H).
5.14.13. tert-Butyl[(1R)-3-[2-(3-cyanobenzyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16h
Yield: 95%. 1H NMR (CDCl3, 400 MHz)
d
1.36 (m, 9H), 2.52e2.71
5.15. General acylation procedure for compounds 15aee
(m, 3H), 2.81e3.27 (m, 5H), 3.39 (m, 1H), 3.65e3.79 (m, 1H),
3.89e3.98 (m, 1H), 4.02 (m, 1H), 4.06e4.17 (m, 1H), 4.37e4.50 (m,
2H), 4.54 (m, 1H), 6.89 (m, 1H), 7.05 (m, 1H), 7.45e7.62 (m, 4H).
To a solution of 15aee (0.5 mmol) in MeOH was added 10% Pd/C
(500 mg). The mixture was submitted to an atmosphere of
hydrogen (40 psi H2) for 12 h. The catalyst was filtered off, and the
filtrate evaporated to give a crude material that was recrystallized
from CH2Cl2 and hexane to yield compounds 15aee. To a solution of
fumaric acid (0.2 N) in ethanol, a solution of the above-prepared
compounds (0.2 N) in ethanol was added via a pipet. Anhydrous
Et2O was added in a dropwise manner to the reaction until it
became cloudy. The cloudy white heterogeneous mixture was
stirred vigorously at room temperature for 20 min, filtered, and
washed with Et2O to give the desired compounds as white solids.
5.14.14. tert-Butyl[(1R)-3-[2-(pyridin-2-yl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-trifluorobenzyl)
propyl]carbamate 16i
Yield: 96%. 1H NMR (CDCl3, 300 MHz)
d 1.42e1.49 (m, 9H),
2.47e2.71 (m, 3H), 2.88e3.15 (m, 4H), 3.48e3.53 (m, 1H),
3.70e3.82 (m, 2H), 3.94e4.13 (m, 3H), 4.64e4.87 (m, 1H),
6.86e6.94 (m, 1H), 7.02e7.10 (m, 1H), 7.60e7.69 (m, 4H).
5.14.15. tert-Butyl[(1R)-3-[2-(4-(trifluoromethyl)phenyl)-
hexahydro-3-oxoimidazo [1,5-a]pyrazin-7(8H)-yl]-3-oxo-1-(2,4,5-
trifluorobenzyl)propyl]carbamate 16j
5.15.1. (2R)-4-Oxo-4-[hexahydro-3-oxo-oxazolo[1,5-a]pyrazin-7
(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine fumaric acid 15a
Yield: 55%, mp 74e75 ꢁC. 1H NMR (CF3COOD, 300 MHz)
Yield: 51%. 1H NMR (CDCl3, 400 MHz)
d
1.48 (m, 9H), 2.53e2.67
(m, 3H), 2.92e3.15 (m, 4H), 3.52 (m, 1H), 3.76 (m, 2H), 3.96e4.11
d
3.01e3.23 (m, 6H), 3.89e4.19 (m, 5H), 4.45e4.80 (m, 3H),

4960
Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
6.93e7.01 (m, 4H); 13C NMR (DMSO-d6, 100 MHz)
52.94, 56.48, 66.62, 106.28, 120.13, 121.84, 135.56, 147.49, 156.64,
169.22; HRMS (ESI) calcd for C16H18F3N3O3 (357.1300 g/mol), found
(MHþ) 358.1386.
d
32.78, 48.51,
44.84, 45.56, 46.64, 48.45, 50.60, 105.83, 114.19, 119.06, 123.46,
130.52, 134.02, 139.32, 160.34, 161.48, 163.91, 168.62, 160.48; HRMS
(ESI) calcd for
465.1920.
C
23H24F4N4O2 (464.1835 g/mol), found (MHþ)
5.15.2. (2R)-4-Oxo-4-[hexahydro-3-oxoimidazo[1,5-a]pyrazin-7
(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine fumaric acid 15b
Yield: 50%, mp 236e238 ꢁC. 1H NMR (CF3COOD, 400 MHz)
5.16.2. (2R)-4-Oxo-4-[2-(4-methylbenzyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17b
d
3.04e3.40 (m, 9H), 3.78e4.13 (m, 5H), 6.98e7.13 (m, 4H); 13C NMR
Yield: 53%, mp 118e119 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
d 2.31
(DMSO-d6, 100 MHz)
d
32.48, 36.97, 41.41, 44.98, 48.46, 52.88,
(s, 2H), 2.33e2.72 (m, 2H), 2.78e3.10 (m, 5H), 3.24e3.28 (m, 2H),
3.47e3.75 (m, 3H), 3.84e3.86 (m, 2H), 4.19e4.38 (m, 2H),
4.45e4.48 (m, 1H), 6.85e6.89 (m, 1H), 7.07e7.12 (m, 4H), 7.23e7.28
53.32, 106.47, 111.76, 119.99, 121.29, 136.11, 147.96, 161.16, 169.04;
HRMS (ESI) calcd for C16H19F3N4O2 (356.1460 g/mol), found (MHþ)
357.1543.
(m, 1H); 13C NMR (DMSO-d6, 100 MHz)
d 31.81, 35.58, 41.34, 44.97,
45.49, 47.04, 48.27, 50.84, 56.47, 106.30, 120.25, 121.27, 128.18,
129.57, 134.66, 136.83, 146.08, 147.38, 149.97, 159.08, 168.39; HRMS
(ESI) calcd for C24H27F3N4O2 (460.2086 g/mol), found (MHþ)
461.2173.
5.15.3. (2R)-4-Oxo-4-[2-methyl-hexahydro-3-oxoimidazo[1,5-a]
pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine fumaric
acid 15c
Yield: 71%, mp 91e92 ꢁC. 1H NMR (CF3COOD, 400 MHz)
d
3.16e3.46 (m, 10H), 3.40e3.46 (m, 1H), 3.55 (m, 2H), 3.71 (m, 1H),
4.28 (m, 3H), 7.21e7.30 (m, 4H); 13C NMR (DMSO-d6, 100 MHz)
24.13, 33.41, 36.27, 37.61, 43.43, 49.76, 54.14, 66.37, 106.27, 120.14,
5.16.3. (2R)-4-Oxo-4-[2-(4-chlorobenzyl)-hexahydro-3-oxoimidazo
[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine
fumaric acid 17c
d
122.41, 136.34, 150.30, 155.23, 163.57, 168.72; HRMS (ESI) calcd for
C17H21F3N4O2 (370.1617 g/mol), found (MHþ) 371.20.
Yield: 60%, mp 137e138 ꢁC.1H NMR (CDCl3, 400 MHz)
d
2.38e2.74 (m, 3H), 2.76e2.88 (m, 3H), 2.91e3.12 (m, 2H),
3.29e3.35 (m, 1H), 3.49e3.58 (m, 2H), 3.76e3.92 (m, 2H),
4.27e4.38 (m, 2H), 4.44e4.51 (m, 1H), 6.91 (m, 1H), 7.09 (m, 1H),
5.15.4. (2R)-4-Oxo-4-[2-ethyl-hexahydro-3-oxoimidazo[1,5-a]
pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine fumaric
acid 15d
7.16 (m, 2H), 7.28 (m, 2H); 13C NMR (DMSO-d6, 100 MHz)
d 31.27,
34.54, 41.34, 44.99, 46.41, 46.63, 48.29, 50.85, 106.48, 116.11, 120.18,
128.97, 130.03, 132.31, 136.91, 146.25, 147.53, 150.14, 159.04, 168.39;
HRMS (ESI) calcd for C23H24ClF3N4O2 (480.1540 g/mol), found
(MHþ) 481.1606.
Yield: 45%, mp 99e100 ꢁC. 1H NMR (CF3COOD, 400 MHz)
d 1.16
(t, J ¼ 3.3 Hz, 3H), 2.95e3.33 (m, 10H), 3.40e4.20 (m, 6H),
4.20e4.40 (m, 1H), 6.89e6.99 (m, 1H), 7.03 (m, 4H); 13C NMR
(DMSO-d6,100 MHz) d 12.81, 32.06, 36.87, 38.18, 41.35, 43.48, 46.09,
47.26, 48.37, 50.57, 106.23, 120.05, 135.72, 147.46, 149.91, 158.91,
168.76; HRMS (ESI) calcd for C18H23F3N4O2 (384.1773 g/mol), found
(MHþ) 385.1841.
5.16.4. (2R)-4-Oxo-4-[2-(4-methoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17d
Yield: 55%, mp 123e124 ꢁC. 1H NMR (CDCl3, 300 MHz)
5.15.5. (2R)-4-Oxo-4-[2-benzyl-hexahydro-3-oxoimidazo[1,5-a]
pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine fumaric
acid 15e
d 2.30e2.46 (m, 3H), 2.59e2.67 (m, 2H), 2.59e2.91 (m, 3H),
3.21e3.32 (m, 1H), 3.47e3.55 (m, 2H), 4.64e3.71 (m, 1H), 3.79 (s,
3H), 3.93 (m, 1H), 4.28e4.37 (m, 2H), 4.58 (m, 1H), 6.84e6.92 (m,
3H), 7.06 (m, 1H), 7.16 (m, 2H); 13C NMR (DMSO-d6, 100 MHz)
Yield: 50%, mp 94e95 ꢁC. 1H NMR (CF3COOD, 400 MHz)
d
2.71e3.27 (m, 8H), 3.49e3.54 (m, 1H), 3.76e4.06 (m, 4H),
4.33e4.45 (m, 3H), 6.85e6.91 (m, 1H), 6.97e7.25 (m, 6H); 13C NMR
(DMSO-d6, 100 MHz) 31.40, 34.88, 41.36, 44.99, 46.63, 47.31, 48.18,
d 25.92, 32.93, 36.30, 41.26, 44.93, 46.72, 48.57, 50.59, 55.49, 62.48,
106.43, 114.39, 120.13, 129.55, 135.73, 147.30, 158.96, 169.10; HRMS
(ESI) calcd for C24H27F3N4O3 (476.2035 g/mol), found (MHþ)
477.2111.
d
60.56, 106.12, 120.29, 120.89, 127.69, 128.12, 129.02, 136.30, 137.78,
147.59, 159.11, 169.91; HRMS (ESI) calcd for C23H25F3N4O2
(446.1930 g/mol), found (MHþ) 447.2015.
5.16.5. (2R)-4-Oxo-4-[2-(2-methoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17e
5.16. General acylation procedure for compounds 17aeg and 17ieo
Yield: 83%, mp 133e134 ꢁC. 1H NMR (D2O, 400 MHz)
To a solution of 16aeg and 16ieo (0.3 N) in CH2Cl2 was added
TFA (10 equiv). The mixture was stirred for 12 h at room tempera-
ture, and evaporated to dryness. The resulting residue was dis-
solved in 2 N NaOH (10 mL) and extracted with CH2Cl2 (3 ꢂ 20 mL).
The combined organic layers were washed with brine, dried
(Na2SO4), filtered, concentrated and recrystallized from CH2Cl2 and
hexane to yield desire compounds. The HCl salts of these
compounds were converted to their fumaric acid salts using the
procedure described in Section 5.15.
d 2.40e2.70 (m, 2H), 2.78e2.90 (m, 5H), 3.27 (m, 1H), 3.47 (m, 2H),
3.60e3.68 (m, 5H), 3.76 (m, 1H), 4.16e4.26 (m, 3H), 6.48 (s, 1H),
6.85 (m, 1H), 6.92 (m, 1H), 6.97 (m, 1H), 7.01e7.14 (m, 2H), 7.23 (m,
1H); 13C NMR (DMSO-d6, 100 MHz)
d 32.72, 36.28, 41.32, 42.22,
45.03, 46.14, 48.42, 50.94, 55.82, 56.48, 106.23, 111.26, 120.85,
125.37, 128.82, 135.55, 157.49, 159.19, 168.17; HRMS (ESI) calcd for
C24H27F3N4O3 (476.2035 g/mol), found (MHþ) 477.2104.
5.16.6. (2R)-4-Oxo-4-[2-(3-methoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin -7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17f
5.16.1. (2R)-4-Oxo-4-[2-(4-fluorobenzyl)-hexahydro-3-oxoimidazo
[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine
fumaric acid 17a
Yield: 69%, mp 131e133 ꢁC. 1H NMR (D2O, 400 MHz)
d 2.40e2.90
(m, 8H), 3.30 (m, 1H), 3.46e3.52 (m, 1H), 3.52e3.66 (m, 5H), 3.79
(m, 1H), 4.13e4.25 (m, 3H), 6.42 (m, 1H), 6.71e6.81 (m, 3H), 7.00
(m, 1H), 7.10 (m, 1H), 7.20 (m, 1H); 13C NMR (DMSO-d6, 100 MHz)
Yield: 45%, mp 123e125 ꢁC. 1H NMR (CF3COOD, 300 MHz)
d
2.83e2.86 (m, 1H), 3.00e3.42 (m, 9H), 3.60e3.65 (m, 1H),
3.80e4.17 (m, 3H), 3.50e3.58 (m, 1H), 4.46e4.62 (m, 2H), 7.02e7.40
(m, 6H); 13C NMR (D2O, 100 MHz)
30.82, 33.50, 40.00, 41.45,
d
32.71, 36.66, 41.28, 44.97, 45.68, 47.28, 48.51, 50.59, 55.41, 56.48,
d
106.23, 113.00, 113.71, 120.19, 130.12, 135.77, 139.41, 159.11, 159.90,

Y. Zhu et al. / European Journal of Medicinal Chemistry 45 (2010) 4953e4962
4961
168.76, 169.04; HRMS (ESI) calcd for C24H27F3N4O3 (476.2035 g/
mol), found (MHþ) 477.2118.
(m, 4H), 7.48e7.62 (m, 4H); 13C NMR (DMSO-d6, 100 MHz)
34.43, 41.22, 44.75, 45.43, 46.02, 48.08, 49.53,106.39,119.06,120.34,
126.35, 128.95, 135.35, 139.47, 147.97, 155.96, 168.59; HRMS (ESI)
calcd for C22H22ClF3N4O2 (466.1383 g/mol), found (MHþ) 467.1443.
d
31.27,
5.16.7. (2R)-4-Oxo-4-[2-(3,5-dimethoxybenzyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17g
5.16.13. (2R)-4-Oxo-4-[2-(4-cyanophenyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17n
Yield: 60%, mp 136e137 ꢁC. 1H NMR (D2O, 400 MHz)
d 2.40e3.13
(m, 8H), 3.51e3.73 (m, 3H), 3.79e3.87 (m, 7H), 4.00 (m, 1H),
4.30e4.45 (m, 3H), 6.58 (m, 2H), 6.60 (m, 1H), 6.63 (m, 1H), 7.23 (m,
Yield: 98%, mp 147e148 ꢁC. 1H NMR (CF3COOD, 400 MHz)
1H), 7.31 (m, 1H); 13C NMR (DMSO-d6, 100 MHz)
d
32.97, 36.81,
d
2.93e3.25 (m, 8H), 3.77e3.83 (m, 1H), 4.01e4.22 (m, 4H), 7.02 (m,
1H), 7.11e7.17 (m, 2H), 7.70 (m, 1H), 7.78 (m, 2H); 13C NMR (DMSO-
d6, 100 MHz) 31.16, 34.29, 40.14, 41.18, 45.85, 48.23, 48.91, 65.52,
41.30, 44.99, 45.73, 47.42, 48.58, 50.6, 51.08, 55.56, 99.27, 105.98,
120.14, 121.82, 135.76, 140.23, 145.17, 147.46, 155.41, 159.12, 161.60,
168.74; HRMS (ESI) calcd for C25H29F3N4O4 (506.2141 g/mol), found
(MHþ) 507.2209.
d
103.68, 106.26, 117.31, 119.77, 133.39, 135.68, 144.28, 155.62, 168.86,
170.39; HRMS (ESI) calcd. for C23H22F3N5O2 (457.1726 g/mol), found
(MHþ) 458.1801.
5.16.8. (2R)-4-Oxo-4-[2-(pyridin-2-yl)-hexahydro-3-oxoimidazo
[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine
fumaric acid 17i
5.16.14. (2R)-4-Oxo-4-[2-(4-(methylsulfonyl)phenyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17o
Yield: 95%, mp 115e116 ꢁC. 1H NMR (CF3COOD, 400 MHz)
d
2.94e3.40 (m, 8H), 3.90e4.02 (m, 3H), 4.17 (m, 3H), 4.29 (m, 1H),
Yield: 94%, mp 171e173 ꢁC. 1H NMR (CF3COOD, 400 MHz)
4.41e4.46 (m, 1H), 6.97e7.04 (m, 1H), 7.09e7.18 (m, 2H), 7.32 (d,
J ¼ 8.8 Hz, 1H), 7.56 (t, J ¼ 6.8 Hz, 1H), 8.42e8.49 (m, 2H); 13C NMR
d
2.93e3.46 (m, 10H), 3.76e3.81 (m, 1H), 4.02e4.28 (m, 5H),
4.58e4.82 (m, 1H), 6.99 (m, 1H), 7.10 (m, 2H), 7.15 (m, 1H), 7.80 (m,
2H), 8.02 (m, 2H); 13C NMR (DMSO-d6, 100 MHz)
29.68, 38.86,
(DMSO-d6, 100 MHz)
d
32.99, 36.84, 41.13, 44.98, 45.64, 48.47,
d
49.03, 49.55, 50.00, 106.20, 112.23, 118.00, 119.99, 121.86, 135.90,
137.81, 147.93, 152.64, 155.34, 169.13; HRMS (ESI) calcd for
C21H22F3N5O2 (433.1726 g/mol), found (MHþ) 434.1890.
44.82, 46.03, 48.31, 50.14, 60.58, 106.47, 117.83, 120.61, 129.41,
133.38, 134.86, 130.13, 147.65, 155.88, 167.16, 168.48; HRMS (ESI)
calcd for C23H25F3N4O4S (510.1549 g/mol), found (MHþ) 511.1624.
5.16.9. (2R)-4-Oxo-4-[2-(4-(trifluoromethyl)-phenyl)-hexahydro-
3-oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17j
5.17. (2R)-4-oxo-4-[2-(3-carbamoylbenzyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17h
Yield: 87%, mp 183e185 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
d
2.53e3.05 (m, 9H), 3.57e3.79 (m, 4H), 3.94e4.00 (m, 1H),
7.51e7.57 (m, 2H), 7.67 (m, 2H), 7.75 (m, 2H), 7.89e7.94 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) 31.39, 34.62, 41.14, 44.82, 46.71, 48.23,
To a solution of 16h (280 mg, 0.5 mmol) in CH2Cl2 (20 mL),
DMSO (1 mL) and H2O2 (30%, 5 mL) were added at 0 ꢁC. After 1 h,
the mixture was poured into H2O (30 mL) and extracted with
CH2Cl2 (3 ꢂ 50 mL). The combined organic layers were washed with
brine, dried (Na2SO4), filtered, and concentrated. The residue was
then submitted to amine deprotection conditions and converted to
its fumaric acid salt using the procedure in Section 5.9, which gave
compound 17h (202 mg, 67%), mp 221e223 ꢁC. 1H NMR (D2O,
d
49.72, 55.31, 106.20, 116.99, 120.19, 122.03, 122.35, 123.64, 126.34,
144.18, 145.30, 147.70, 150.29, 155.55, 168.47; HRMS (ESI) calcd for
C23H22F6N4O2 (500.1647 g/mol), found (MHþ) 501.1706.
5.16.10. (2R)-4-Oxo-4-[2-(2,4,5-trifluorophenyl)-hexahydro-3-
oxoimidazo[1,5-a] pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17k
400 MHz)
d 2.60e2.89 (m, 9H), 3.23 (m, 1H), 3.55e3.63 (m, 2H),
3.72e3.80 (m, 1H), 4.14e4.20 (m, 3H), 6.46 (s, 2H), 6.95 (m, 1H),
Yield: 73%, mp 120e121 ꢁC. 1H NMR (DMSO-d6, 400 MHz)
7.06 (m, 1H), 7.25e7.31 (m, 2H), 7.45 (s, 1H), 7.52 (m, 1H); 13C NMR
d
2.50e2.69 (m, 3H), 2.76e3.05 (m, 5H), 3.56e3.78 (m, 4H), 3.92 (m,
1H), 4.39e4.58 (m, 1H), 7.48e7.70 (m, 2H), 7.76e7.97 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) 31.39, 34.58, 41.18, 45.21, 48.22, 49.14,
(DMSO-d6, 100 MHz)
d 30.84, 33.72, 40.30, 41.43, 44.91, 45.55,
46.84, 48.42, 50.90, 64.20, 105.79, 119.05, 126.64, 129.20, 131.69,
134.93, 137.22, 160.56, 169.64, 172.67; HRMS (ESI) calcd for
C24H26F3N5O3 (489.1988 g/mol), found (MHþ) 490.2053.
d
50.67, 106.71, 113.43, 120.19, 120.24, 128.63, 145.27, 147.77, 151.02,
153.76, 155.96, 158.96, 168.54; HRMS (ESI) calcd for C22H20F6N4O2
(486.1490 g/mol), found (MHþ) 487.1556.
References
5.16.11. (2R)-4-Oxo-4-[2-(4-fluorophenyl)-hexahydro-3-
oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17l
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d
2.58e2.79 (m, 3H), 3.01e3.21 (m, 5H), 3.43e3.45 (m, 1H),
3.72e4.02 (m, 4H), 4.77e4.86 (m, 1H), 6.95 (m, 1H), 7.01e7.06 (m,
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33.46, 39.91, 41.36, 44.86, 47.05, 48.45, 50.01, 106.89, 115.78, 119.10,
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oxoimidazo[1,5-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)
butan-2-amine fumaric acid 17m
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Yield: 98%, mp 163e165 ꢁC. 1H NMR (CF3COOD, 400 MHz)
d
3.16e3.41 (m, 8H), 3.50e3.64 (m,1H), 3.94e4.40 (m, 5H), 7.21e7.38

4962
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