Design and optimization of purine derivatives as in vivo active PDE10A inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.04.019

Phosphodiesterases are important enzymes regulating signal transduction mediated by second messenger molecules cAMP or cGMP. PDE10A is a unique member in the PDE family because of its selective expression in medium spiny neurons. It is recognized as anti-psychotic drug target. Based on the structural similarity between our previous chemistry work on 8-aminoimidazo[1,2-a]pyrazines and the PDE10A inhibitors reported by Bartolome-Nebreda et al., we initialized a project for developing PDE10A inhibitors. After several rounds of optimization, we were able to obtain a few compounds with good PDE10A enzymatic activity. And after further PDE enzymatic selectivity study, metabolic stability assay and in vivo pharmacological tests we identified two inhibitors as interesting lead compounds with the potential for further PDE10A lead optimizatioin.

Full text of DOI:10.1016/j.bmc.2017.04.019

Accepted Manuscript
Design and Optimization of Purine Derivatives as in-vivo Active PDE10A In-
hibitors
Liu Chen, Danqi Chen, Le Tang, Jing Ren, Jiaojiao Chen, Xuechu Zhen, Yu-
Chih Liu, Chenhua Zhang, Haibin Luo, Jingkang Shen, Bing Xiong
PII:
S0968-0896(16)31503-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.019
BMC 13689
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 December 2016
11 April 2017
12 April 2017
Please cite this article as: Chen, L., Chen, D., Tang, L., Ren, J., Chen, J., Zhen, X., Liu, Y-C., Zhang, C., Luo, H.,
Shen, J., Xiong, B., Design and Optimization of Purine Derivatives as in-vivo Active PDE10A Inhibitors, Bioorganic
&
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.019
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Design and Optimization of Purine Derivatives as in-vivo Active
PDE10A Inhibitors
1
,2,#
2,#
1,2
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3
Liu Chen,
Danqi Chen, Le Tang, Jing Ren, Jiaojiao Chen, Xuechu Zhen,
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2,*
2,*
Yu-Chih Liu, Chenhua Zhang, Haibin Luo, Jingkang Shen, Bing Xiong
1
School of PharmaceuticalSciences, Nanchang University, Nanchang 330006, China
2
Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China
3
Department of Pharmacology, Soochow university College of Pharmaceutical Sciences, Suzhou,
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1
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1
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1
China
4
Shanghai ChemPartner Co., LTD. Building 5, 998 Halei Road, Zhangjiang Hi-Tech Park,
Pudong New Area, Shanghai, 201203, China
5
School of pharmaceutical Science, Sun Yat-Sen University, Guangzhou, 510006, China
#
these authors contributed equally to this work
*
Corresponding authors. (B. X.) Tel: +86 21 50806600 ext. 5412 fax: +86 21 50807088. Email:
bxiong@simm.ac.cn; (J. S.) Tel: +86 21 50806600 ext. 5410. Email: jkshen@simm.ac.cn
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Abstract
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Phosphodiesterases are important enzymes regulating signal transduction mediated by
second messenger molecules cAMP or cGMP. PDE10A is a unique member in the
PDE family because of its selective expression in medium spiny neurons. It is
recognized as anti-psychotic drug target. Based on the structural similarity between

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7
our previous chemistry work on 8-aminoimidazo[1,2-a]pyrazines and the PDE10A
inhibitors reported by Bartolome-Nebreda et al., we initialized a project for
developing PDE10A inhibitors. After several rounds of optimization, we were able to
obtain a few compounds with good PDE10A enzymatic activity. And after further
PDE enzymatic selectivity study, metabolic stability assay and in vivo
pharmacological tests we identified two inhibitors as interesting lead compounds with
the potential for further PDE10A lead optimizatioin.
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Keywords: Phosphodiesterase; PDE10A; purine; inhibitor; anti-psychotic

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Introduction
Through hydrolysis of second messengers cAMP and cGMP, the cyclic nucleotide
phosphodiesterases (PDE) regulate the localization, duration, and amplitude of cyclic
1
-2
nucleotide signaling. Given their important roles in mediating signal transduction,
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-5
PDEs catch considerable attention in drug development. PDE10A is a unique
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-8
member in the PDEs family. Unlike the broad distribution in tissues of other
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-12
members, the expression of PDE10A is limited to brain and testes.
The selective
expression in the medium spiny neurons of the striatum was thought to play critical
roles in regulating the response to external stimuli. Many studies and disclosed
clinical data have shown that inhibiting PDE10A is a potential anti-psychotic
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1
3
approach. A number of PDE10A inhibitors have been developed in the past few
1
4-18
years
and several of them are now in clinical trials against schizophrenia,
including TAK-063 (Takeda, Phase II), OMS-824 (Omeros, Phase II, structure
unknown), PF-2545920 (Pfizer, Phase II) as shown in Figure 1 (Data from Thomson
Reuters Cortellis).
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1
6
7
Figure 1. Representative PDE10A inhibitors
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1
8
9
PDE10A contains two GAF domains and a catalytic domain (The GAF domain is

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named after some of the proteins where it is found, including cGMP-specific
phosphodiesterases, adenylyl cyclases and FhlA.). Although the GAF domains of
PDE10A can bind cAMP, they merely act as regulating domains and do not show
enzymatic activity in in vitro binding study. The co-crystal structures of inhibitors
bound to PDE10A provided detailed information about different binding modes.
Besides the conserved hydrogen bonds between Gln716 and small molecules, the
binding site also contained several subpockets: a selectivity pocket, a hydrophobic
6
clamp, a ribose region and a large exo-binding region. The small molecular inhibitors
occupying more than one of above-mentioned regions in the binding site usually
exhibited potent binding affinity.
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Our
previous
chemistry
offered
an
efficient
route
to
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1
8-aminoimidazo[1,2-a]pyrazines (Structure A in Figure 2). Replacement of NHR
with morpholine ring (Structure B in Figure 2) yielded a structure resembling closely
the PDE10A inhibitors by Bartolome-Nebreda et al (Compound C). These compounds
contained the same imidazo[1,2-a]pyrazine scaffold and showed a unique binding
1
mode that the N of the imidazole made an interaction with the backbone NH of
Gln716 and the morpholine group extended into a small PDE10A specific
2
0-21
subpocket.
Based on this binding mode, the purine skeleton was also considered
as a suitable scaffold.
Thus, above-mentioned two types of compounds were screened against PDE10A
enzymatic assay, and showed several submicromolar PDE10A inhibitors, which were
further optimized to two compounds with potent enzymatic and in vivo activities and

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2
good metabolic stability. The new compounds could be useful for further
antipsychotic drug development and the details of the study were reported herein.
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Figure 2. Design of hit compounds
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Chemistry
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Compound 4 was prepared by the methods we published previously. Commercially
available 2-Amino-3-chloropyrazine was reacted with morpholine to give compound
2 which underwent Groebke–Blackburn–Bienaymé reaction and deprotection to yield
intermediate compound 4. Compound 4 was coupled with phenylacetic acids and the
carbonyl groups were reduced to yield 10-14. Compounds 19-20 were obtained by
Suzuki reaction from 17-18 that were prepared as described above. Compounds 23-46
were obtained from intermediate 4 through nucleophilic substitution and Suzuki
reaction.
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Scheme 1

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2
o
Reagents
and
conditions:
a)
morpholine,
DIEA,
120-130
C,
12h;
b)
o
3
4
5
6
7
8
Yb(OTf) ,2-isocyano-2,4,4-trimet-hylpentane, CH CHO, MeOH, 65 C, 2h; (c) HCl/EtOAc,
3
3
o
MeOH, 25 C, 8h; d) phenylacetic acids, HOBt, EDC, TEA, CH Cl , rt, 8h; e) BH -THF complex,
2
2
3
o
THF, 80 C, 1h; f) dioxane/water or DMF, Pd (dba) or Pd(OAc) , x-phos, K CO or Cs CO , boric
2
3
2
2
3
2
3
o
acids or boronic acid pinacol esters, 80 C, overnight; g) DMF, benzyl bromides, 8h; h)
o
dioxane/water, Pd (dba) , x-phos, K CO , boric acids or boronic acid pinacol esters, 100 C,
2
3
2
3
overnight.

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0
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The synthesis route of purine series was shown in scheme 2. 6-chloro-9H-purine (47)
was reacted with morpholine and then with benzyl bromides to give compounds 49-58.
6-Chloro-4,5-diaminopyrimidine and ethyl orthopropionate were condensed to
generate compound 61, from which compound 63 was obtained by the same route of
49-58 described above. Compounds 67-68 were achieved with the same methods as
63 using trimethyl orthoacetate to replace ethyl orthopropionate. Suzuki reaction with
67-68 provided compounds 69-80. Compound 81 was also obtained with the methods
of 63. Then, it was hydrolyzed with LiOH and coupled with aniline to give 83.
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1
Scheme 2

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o
Reagents and conditions: a) morpholine, 80 C overnight; b) benzyl bromides, DMF, Cs CO , rt,
2
3
o
3
4
5
6
overnight; c) TEA, acetonitrile, 120 C, overnight; d) benzyl bromides, DMF, K CO , rt, overnight;
2 3
o
o
e) boric acids, 2M Na CO , Pd(PPh ) , DME, 100 C, overnight; f) LiOH, MeOH/H O(3:1), 60 C,
2
3
3 4
2
5h; g) aniline, EDC, DMAP, DMF, rt, overnight.
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Results and Discussion
Hit identification
Based on our multi-component reaction and reported PDE10A inhibitors, we designed
and synthesized fourteen compounds of morpholine substituted
aminoimidazo[1,2-a]pyrazines as shown in Table 1 for preliminary evaluation. Since
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1

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5
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0
there was an amino group left on the imidazo[1,2-a]pyrazine ring using our synthesis,
modifications were made on it at the first stage. From the PDE10A enzymatic assay,
these compounds all showed moderate inhibition at concentration 5 μM. In particular,
the amides (5-9) displayed lower activities than their reduction products (10-14). To
further explore the SAR, compounds 17-20 with different substitutions on the phenyl
ring were synthesized. 2,4-Bis-substituted compounds 17 and 19 showed better
inhibitory activities than that of 3,4-bis-substituted compounds 18 and 20. And large
substitution like phenyl group was also well-tolerated (19, 20). These indicated that 2-
and 4-position might be the direction for further optimization.
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Table 1. Inhibition of Compound 5-14，17-20 on PDE10A
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2
a
Compd.
R
Inhibition%@5μM
5
6
7
8
51%
44%
50%
24%
9
47%

1
0
1
74%
82%
83%
78%
1
1
1
2
3
9
8%
14
17
18
19
20
IC = 447 nM
5
0
9
2%
IC = 243 nM
5
0
81%
9
3%
IC = 336 nM
5
0
80%
1
2
3
4
5
6
7
8
9
0
1
a: All assay data are reported as the average of at least two measurements.
To predict the binding conformation of this series of compounds, we selected
compound 17 as representative to dock into the binding site of PDE10A catalytic
domain. Initially, the crystal structure of PDE10A bound with an analogue of
compound C (PDB ID: 4BBX) was downloaded from PDB data bank, and prepared
with Maestro program of Schrödinger software by adding the hydrogen atoms and
assigning the bond order to the complex. Then the Glide program implemented in
Schrödinger software package was adopted to make the grid file and perform the SP
docking with default parameters. According to the predicted binding conformation,
the parts of imidazo[1,2-a]pyrazine and morpholine fit well into the binding site of
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1

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5
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PDE10A structure and the N had a potential hydrogen bond with Gln716. The part of
phenylethylamine pointed to a different direction from the ligand in original crystal
structure, making van der Waals interactions with residues Ser561, Met581 and
Leu625. These results showed that it was a proper design for modification on our
scaffold to achieve PDE10A inhibition activities.
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Figure 3. Superimposition of compound 17 (Red) with PDE10A crystal structure
(PDB ID: 4BBX, Green).
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SAR of Aminoimidazo[1,2-a]pyrazines
Further SAR investigation on phenyl ring was carried out. From the docking results
we predicted that the phenyl ring pointed outward of the binding site, offering the
possibility to introduce larger substitutions. An additional aryl group was attached to
the 2- or 4-position of the phenyl ring. However, such modification would increase

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2
the molecular weight significantly. To compensate this unwanted effect, the
modification was carried out on benzylamine skeleton in Table 2, instead of on
phenylethylamine skeleton in Table 1. As shown in Table 2, compounds with aryl
substitutions at 4-position of the phenyl ring generally showed better inhibitory
activities than those substituted at 2-position. Further, analyzing the substitution
pattern on the distal phenyl ring indicated that compounds with methoxyl group (39,
42) showed stronger inhibition than other substituents such as methyl (35), tert-butyl
(45) and chlorine atom (46). Heterocycles such as pyridyl (37, 38), isoxazolyl (40)
and pyrazolyl (41) generally showed better activities than substituted phenyl rings.
However all these compounds did not exhibit satisfactory inhibition against PDE10A.
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Table 2. Inhibition of Compound 21-46 on PDE10A
1
3
Inhibition IC50
Inhibition
IC50
nM
--
Compd.
2-R
Compd.
4-R
a
a
%
@5μM
nM
%@5μM
2
2
1
3
Br
73%
57%
--
22
35
Br
77%
74%
--
--
2
2
4
5
61%
76%
--
--
36
37
79%
90%
--
529

2
6
7
70%
60%
--
--
38
39
91%
87%
391
--
2
2
2
8
9
68%
64%
--
--
40
41
90%
88%
518
--
3
3
3
0
1
2
51%
63%
75%
--
--
--
42
43
44
92%
54%
55%
452
--
--
3
3
3
4
39%
87%
--
--
45
46
38%
77%
--
--
1
2
a: All assay data are reported as the average of at least two measurements.
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0
SAR of Purines
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According to the solved crystal structure (PDB entry: 4BBX) the N on imidazole ring
(see Figure 2 for the numbering) interacted with the protein backbone and was
12
essential to the binding activities. Thus, besides the imidazo[1,2-a]pyrazine scaffold,
other eligible structures were also considered. The structures of cAMP and cGMP
both contain the purine ring, and moreover, the first-generation PDE inhibitors known
as pan-PDE inhibitors including theophylline and xanthine also contained the purine
moiety. Together with the SAR of aminoimidazo[1,2-a]pyrazines, we decided to
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4
combine the purine and the morpholine ring to form a new series of PDE10A
inhibitors.
Table 3. Inhibition of Compound 49-58 on PDE10A
5
a
Compd.
R
Inhibition%@5μM
IC nM
5
0
4
5
9
0
13%
--
20%
--
5
5
5
1
2
3
11%
66%
70%
--
--
--
5
4
85%
--
5
5
5
6
90%
530
107
100%
5
5
7
8
94%
97%
103
75

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3
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7
a: All assay data are reported as the average of at least two measurements.
As shown in Table 3, if substitution R was phenylethyl group the compounds (49, 50)
showed weak inhibition on PDE10A; while if substitution R was benzyl group the
compounds (52-58) had an obvious increase of the inhibitory activities from lower
than 20% up to 100 % at 5 μM. This implied that the length between the purine and
phenyl group was an important factor for binding. Large groups replacing the phenyl
group could improve the activity, as demonstrated by compounds 55 and 56. And
biphenyls (57 and 58) exhibited better potency over phenyls, which confirmed that
para-position of phenyl group was important for the binding. Taking together, these
pointed the direction for following optimization. Another worrisome issue associated
with purine scaffold was that the hydrogen atom on C8 of the purine ring was
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2
2
commonly considered to be metabolically unstable. Therefore we prepared
compounds with substitutions at this position to potentially block the metabolism and
also assessed their binding affinities.
Table 4. Inhibition of Compound 63, 69 on PDE10A
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8
a
Compd.
R
Inhibition%@5μM
IC nM
50
5
7
H
94
103

6
6
3
9
Et
97
99
168
113
Me
1
2
a: All assay data are reported as the average of at least two measurements.
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5
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As indicated in Table 4, the compound 69 with a methyl group had equivalent activity
to compound 57. If substitution R was the ethyl group, the activity of the compound
decreased slightly (63). Based on the aforementioned SAR information, we chose
8-methyl-purine as the scaffold for further optimization.
Table 5. Inhibition of Compound 67-81, 83 on PDE10A
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a
Compd.
R
-
Inhibition%@5μM
IC nM
5
0
6
6
6
7
7
7
7
8
9
0
1
2
87
93
99
94
91
93
--
Br
327
118
62
168
258
7
3
98
38

74
75
76
77
96
97
98
97
116
61
56
76
7
7
8
9
96
96
68
119
8
8
8
0
1
3
98
90
96
60
--
130
1
2
a: All assay data are reported as the average of at least two measurements.
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4
5
6
7
8
9
0
1
2
To reassess the obtained SAR information, we prepared two compounds (67 and 68)
and found that 4-Br indeed showed higher activity than 2-Br. Then, as shown in Table
5, further optimization was focused on the 4-position to synthesize derivatives with
the aryl substitution on the phenyl ring. From the enzymatic assay, the bi-aryl
compounds obviously improved the inhibition on PDE10A and half of the tested
compounds showed IC value lower than 100 nM. For compounds with substituted
5
0
phenyl as R group (71-73), their inhibition activities did not show large differences
(within 4 fold) when comparing with compound 70. The 2-Cl phenyl (73) was the
most potent compound in this series with the IC value of 38 nM. Compounds with
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1
5
0
heterocyclic rings as R group (74-80) showed similar activities and their IC values
5
0

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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
ranged from 56-119 nM. Even the amide (83) or ester (81) compounds showed good
activities. And these observations were consistent with our prediction that the
investigated part (R group) might be located at the solvent-accessible range of the
binding site, and therefore various moieties were well-tolerated. Finally, compounds
42, 58, 73, 76, 77 and 78 were selected for further evaluation based on their inhibitory
activities and structural diversity.
Profiling Lead Compounds
Metabolic stability is an important concern for developing successful drugs, as it will
enable the molecules to prolong the effects at the functional sites in the body. We
tested compounds 42, 58, 73, 76, 77 and 78 on mouse and human liver microsomes
respectively (MLM and HLM in Table 6), and all compounds showed good stability
with reasonable half-life. In addition to the liver microsome assay, five cytochrome
P450 enzymes commonly metabolizing exogenous chemicals were used to test the
direct inhibition of these six compounds. The evaluation showed that these
compounds had less than 50% inhibitory activity at concentration 10 μM in direct
inhibition tests against selected five CYPs. The time-dependant inhibition test also
clarified that the compounds had no covalent interaction issue with the five CYPs.
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1
1
2
2
Table 6. Metabolic stability in mouse and human liver microsomes of compounds
42, 58, 73, 76, 77 and 78
a
b
Compd.
HLM
MLM
DI (%) / TDI

t (min) t1/2(min)
3A4
2D6
2C9
1A2
29/ NI
12/20
15/NI
34/30
12/10
21/NI
2C19
NI/ NI
44/NI
65/NI
21/70
NI/90
22/80
1/2
c
4
5
7
7
7
7
2
8
3
6
7
8
18.24
22.35
43.31
18.24
49.5
12.83
49.5
17.77
23.9
69.3
99
42/ NI
24/ NI 41/ NI
NI/NI
9/50
6/NI
22/NI
6/60
30/NI
52/10
18/10
NI/NI
10/40
12/NI
22/20
40/NI
NI/30
7/20
231
1
2
3
a. DI = direct inhibition. If the value is less than 50 that means the compound has no obvious
inhibition on CYPs.
b. TDI = TDI: time-dependent inhibition of CYPs. If the value is less than 200 that means the
4
5
6
compound has no obvious inhibition on CYPs.
c: NI = no inhibition
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8
9
0
1
2
3
4
5
PDE family contains eleven members that distribute in different tissues and regulate
corresponding physiological functions. Since inhibition on different subtypes of PDEs
may cause adverse effects, target selectivity is an important issue to be checked when
developing the PDE inhibitors. Based on the structural diversity, compounds 42, 73
and 77 were subjected to selectivity assessment. All these three compounds showed
weak inhibition on PDE2, PDE4 and PDE5 (representing PDEs that hydrolyze both
cAMP and cGMP, cAMP only and cGMP only, respectively).
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1
1
1
1
1
Table 7. Inhibition on PDEs of compounds 42, 73 and 77

Inhibition%@1μM
PDE10A
IC (nM)
Compd.
PDE2A1
PDE4A1A
PDE5A1
50
4
7
7
2
3
7
5
29
4
14
6
-3
5
452
38
0
-3
76
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
It was reported that PDE10A inhibitors might increase the functionality of striatal
2
3
medium spiny neurons and produce antipsychotic-like effects in rodents.
Compounds 73 and 77 were submitted to locomotor and prepulse inhibition (PPI)
tests to evaluate their in vivo activities. As shown in Figure 4, pretreatment with 73
and 77 significantly attenuated PCP-stimulated locomotor activity. A significant
decrease in total travel distance was observed in rats with 10 mg/kg dosage, however,
the high dose (50 mg/kg) showed no activities. The results did not show a
dose-dependent response where higher doses caused higher reductions in PCP
locomotion until the maximal effect was achieved. The effect of compounds 73 and
77 on sensorimotor gating in rat was assessed by measuring the disruption of PPI,
which have been widely used to evaluate the antipsychotic potential for drug
development. As shown in Figure 5, pretreatment with 10 mg/kg dose of compound
77 significantly attenuated PPI disruption induced by 5 mg/kg PCP at least at one
prepulse level (two-way ANOVA analysis followed by Tukey's test). It was noted that
10 mg/kg and 50 mg/kg 73 and 50 mg/kg 77 did not rescue the PCP-induced PPI
deficit, indicating that the limited dose range for compound 73 to elicit effect on PPI.
1
1
1
1
1
1
1
1

1
2
3
4
5
6
7
It was not surprised since the optimal concentration of cAMP/cGMP was important to
maintain the normal neuronal function and over-inhibition of PDE could induce the
over-shoot of intracellular cAMP/cGMP that was potentially harmful to cellular
function. This was in agreement with our previous observation in which we found low
dose of MP-10, a well-known PDE10A inhibitor, attenuated morphine-induced
2
4-26
conditioned place preference but not with high doses.
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9
Figure 4. Compounds 73 and 77 inhibit PCP-stimulated locomotor activity in rats. Drugs
were administered (p.o.) 30 min prior to 5 mg/kg PCP (phencyclidine) challenge (i.p.). The
locomotor activity was measured for a 1.5 h duration after PCP administration and the total
traveled distance was expressed as mean ± SEM. Statistical analysis showed 10mg/kg 73 and 77
significantly attenuated hyperlocomotor activity induced by PCP.
1
1
1
1
1
0
1
2
3
4

1
2
Figure 5. Effects of compounds 73 and 77 in prepulse inhibition tests. Drugs were pretreated
(p.o.) 30 min prior to 5mg/kg PCP (i.p.) and the test was performed 5-10 min after PCP injection.
Two-way ANOVA analysis with Tukey’s test indicated that 73 (10mg/kg) significantly prevented
PPI disruption induced by PCP.
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7
8
9
0
1
2
3
4
5
6
7
Conclusion
Inspired by a series of PDE10A inhibitors reported by Bartolome-Nebreda et al, two
series were designed, synthesized, and evaluated. The purine-based compounds
showed good inhibitory activities, among which compounds 73 and 77 also showed
good metabolic stability and PDE subtype selectivity. Following several recent reports
that suggested dual cAMP and cGMP phosphodiesterase 10A inhibitors might present
a novel mechanism to treat schizophrenia, we demonstrated that compounds 73 and
77 elicited potent antipsychotic effects as evidenced by attenuating the acute
PCP-induced hyperlocomotive activity. However, higher dose of compound 73 and 77
failed to produce the effects. This indicating that optimal therapeutic window for
PDE10A inhibitor was very important in order to achieve the expected efficacy.
1
1
1
1
1
1
1
1

1
2
Further research is now carried out.

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0
1
2
Experiment and Methods
Chemistry
Reagents (chemicals) were purchased from Alfa-Aesar (Karlsruhe, Germany), Acros
(Geel, Belgium), Aldrich (St. Louis, MO, USA), Adamas-beta (Shanghai, China) and
Shanghai Chemical Reagent Company (Shanghai, China) and were used without
further purification. Analytical thin-layer chromatography was performed on HSGF
1
254 (150–200 mm thickness; Yantai Huiyou Company, Yantai, Shandong, China). H
NMR (300 MHz or 400 MHz) spectra were recorded on a Varian Mercury-300 or 400
High Performance Digital FT-NMR with TMS as an internal standard. HPLC analysis
was performed using a Gilson HPLC system with UV detection at 214 and 254 nm.
LC–MS spectra were obtained on an LCQ Deca XP ion trap mass spectrometer
(Thermo-Finnigan, San Jose, CA, USA). Accurate mass measurements were carried
out on a Q-TOF ultima Globe mass spectrometer (Micromass, Manchester, UK).
Synthesis
1
1
1
1
1
1
1
1
1
1
2
2
2
3-Morpholinopyrazin-2-amine (2). 3-Chloropyrazin-2-amine (10 g, 77 mmol) was stirred with
o
morpholine (40 mL, 463 mmol) at 120 C for 12h. The mixture was diluted with ethyl acetate,
washed with water and brine. The organic layer was dried with Na SO and concentrated. The
2
4
crude product was purified by silica gel (ethyl acetate/ petroleum 1:1) to afford 12.83 g product as
1
white solid (yield 92%). H NMR (400 MHz, CDCl ) δ 7.71 (d, J=2.9 Hz, 1H), 7.67 (d, J=2.9 Hz,
3
1
3
1H), 4.72 (brs, 2H), 3.90-3.82 (m, 4H), 3.22-3.15 (m, 4H). C NMR (100 MHz, CDCl ) δ 144.00,
3
+
+
141.78, 131.43, 127.06, 62.17, 43.83. HRMS (ESI ) calcd for C H ON H 181.1084, found
8
12
4
181.1082.

1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
2-Methyl-8-morpholino-N-(2,4,4-trimethylpentan-2-yl)imidazo[1,2-a]pyrazin-3-amine (3). To
the solution of compound 2 (9 g, 50 mmol) in 30 mL methanol was added 1,1,3,3-tetramethylbutyl
Isocyanide (17.76 mL,100 mmol), CH CHO (14 mL,250 mmol) and Yb(OTf) (6.2 g,10 mmol).
3
3
The mixture was stirred at 65 °C for 2h, cooled to room temperature and concentrated. The residue
was diluted with ethyl acetate and washed with brine. The organic layer was dried with Na SO4
2
and concentrated. The residue was purified by silica gel (ethyl acetate/ petroleum 1:4) to afford
1
compound 3 14.85g as brown oil (yield 86%). H NMR (400 MHz, CDCl ) δ 7.58 (d, J=4.6 Hz,
3
1H), 7.34 (d, J=4.5 Hz, 1H), 4.23-4.17 (m, 4H), 3.93-3.88 (m, 4H), 2.40 (s, 3H), 1.67 (s, 2H), 1.18
13
1
1
1
1
1
1
1
1
1
1
2
2
2
(s, 6H), 1.12 (s, 9H). C NMR (100 MHz, CDCl ) δ 149.10, 136.01, 130.16, 126.30, 125.30,
3
109.32, 67.00, 60.00, 56.74, 46.86, 31.88, 31.74, 31.60, 31.42, 29.29, 29.03, 14.20. HRMS (ESI+)
+
calcd for C H ON H 346.2601, found 346.2599.
9
31
5
2-Methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-amine (4). Compound 3 (7.94 g, 22.98 mmol)
was dissolved in 50 mL methanol and added 4 M HCl in EtOAc (30 mL). The mixture was stirred
at room temperature for 8h and then concentrated. The residue was neutralized by sat.NaHCO3
and extracted with ethyl acetate. The organic layer was washed with brine, dried with Na SO and
2
4
concentrated. The crude product was purified by silica gel (CH Cl / MeOH 10:1) to obtain
2
2
1
compound 4 5.26 g as pale brown solid (yield 98%). H NMR (400 MHz, CDCl ) δ 7.38-7.34 (m,
3
+
2H), 4.23- 4.18 (m, 4H), 3.90-3.85 (m, 4H), 3.08 (brs, 2H), 2.37 (s, 3H). HRMS (ESI ) calcd for
+
C H ON H 234.1349, found 234.1346.
11
15
5

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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
General procedure A for preparing compound 5-9. The corresponding phenylacetic acids
(2.5eq.), EDC (2 eq.) and HOBt (2 eq.) were dissolved in dichloromethane. The mixture was
stirred at room temperature for 15 minutes and then compound 4 (1 eq.) and triethylamine (6 eq.)
were added. After stirred at room temperature for 8h, the reaction became turbid. The mixture was
filtered and the precipitate was washed with dichloromethane. The solid was dried under vacuum
to get the products.
N-(2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-yl)-2-phenylacetamide (5). White solid,
1
yield 90%. H NMR (400 MHz, DMSO-d ) δ 10.09 (s, 1H), 7.40-7.34 (m, 5H), 7.31-7.26 (m, 2H),
6
1
1
1
1
1
1
1
1
1
1
2
2
2
4.13 (t, J=4.7 Hz, 4H), 3.76 (s, 2H), 3.73 (t, J=4.8 Hz, 4H), 2.19 (s, 3H). MS (ESI): m/z 352
(M+H) +.
2-(Benzo[d][1,3]dioxol-5-yl)-N-(2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-yl)acetamide
1
(6). White solid, yield 81%. H NMR (400 MHz, DMSO-d ) δ 10.00 (s, 1H), 7.35 (d, J=4.5 Hz,
6
1H), 7.29 (d, J=4.5 Hz, 1H), 6.95 (d, J=1.6 Hz, 1H), 6.90 (d, J=7.9 Hz, 1H), 6.84 (dd, J= 8.0, 1.7
Hz, 1H), 6.00 (s, 2H), 4.12 (t, J=4.8 Hz, 4H), 3.74- 3.70 (m, 4H), 3.66 (s, 2H), 2.18 (s, 3H). MS
+
(ESI): m/z 396 (M+H) .
2-(3,4-Dimethoxyphenyl)-N-(2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-yl)acetamide
1
(7). White solid, yield 88%. H NMR (400 MHz, DMSO-d ) δ 10.03 (s, 1H), 7.35 (d, J=4.5 Hz,
6
1H), 7.28 (d, J=4.5 Hz, 1H), 7.01 (d, J=1.9 Hz, 1H), 6.96-6.88 (m, 2H), 4.13 (t, J=4.8 Hz, 4H),
3.76 (d, J=6.3 Hz, 6H), 3.73 (t, J=4.7 Hz, 4H), 3.67 (s, 2H), 2.19 (s, 3H). MS (ESI): m/z 412

+
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
(M+H) .
N-(2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-yl)-2-(naphthalen-2-yl)acetamide
(8).
1
White solid, yield 85%. H NMR (400 MHz, DMSO-d ) δ 10.16 (s, 1H), 7.95-7.88 (m, 4H),
6
7.59-7.48 (m, 3H), 7.37-7.32 (m, 2H), 4.13 (t, J=4.8 Hz, 4H), 3.95 (s, 2H), 3.73 (dd, J=5.5, 4.0 Hz,
+
4H), 2.20 (s, 3H). MS (ESI): m/z 402 (M+H) .
2-(2,6-Dichlorophenyl)-N-(2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-yl)acetamide (9).
1
White solid, yield 77%. H NMR (400 MHz, DMSO-d ) δ 10.70 (s, 1H), 7.51 (d, J=8.1 Hz, 2H),
6
1
1
1
1
1
1
1
1
1
1
2
2
2
7.42 (d, J=4.4 Hz, 1H), 7.40-7.31 (m, 2H), 4.20 (s, 2H), 4.13 (t, J=4.8 Hz, 4H), 3.73 (t, J=4.8 Hz,
+
4H), 2.24 (s, 3H). MS (ESI): m/z 420 (M+H) .
General procedure B for preparing compound 10-14. Compound 5-9 (1 eq.) were dissolved in
dry tetrahydrofuran and added 1M BH -THF (6 eq.) by dropwise under N protection. The mixture
3
2
o
was heated at 80 C for 0.5-1h. The solvent was removed by vacuum. Methanol 5 mL was add to
o
the residue and refluxed at 80 C for another 0.5h. The solvent was removed again. The residue
was dissolved with ethyl acetate and washed with sat.NaHCO3 and brine. The organic layer was
dried with Na SO and concentrated. The crude products were purified by silica gel (ethyl acetate/
2
4
petroleum) to afford the corresponding reduction products 10-14.
2-Methyl-8-morpholino-N-phenethylimidazo[1,2-a]pyrazin-3-amine (10). Pale yellow oil,
1
yield 68%. H NMR (400 MHz, CDCl ) δ 7.38-7.30 (m, 3H), 7.30-7.23 (m, 4H), 4.22 (t, J=4.8 Hz,
3

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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
4H), 3.92-3.86 (m, 4H), 3.30 (t, J=7.0 Hz, 2H), 2.89 (t, J=7.0 Hz, 2H), 2.33 (s, 3H). MS (ESI):
+
m/z 338 (M+H) .
N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-amine
1
(11). Pale yellow oil, yield 79%. H NMR (400 MHz, CDCl ) δ 7.33 (d, J = 4.5 Hz, 1H), 7.30 (d,
3
J=4.6 Hz, 1H), 6.79 (d, J=7.8 Hz, 1H), 6.74-6.72 (m, 1H), 6.69 (dd, J=7.9, 1.7 Hz, 1H), 5.97 (s,
2H), 4.22 (t, J=4.8 Hz, 4H), 3.89 (t, J=4.8 Hz, 4H), 3.24 (t, J=7.0 Hz, 2H), 2.79 (t, J=7.0 Hz, 2H),
+
2.34 (s, 3H). MS (ESI): m/z 382 (M+H) .
1
1
1
1
1
1
1
1
1
1
2
2
2
N-(3,4-dimethoxyphenethyl)-2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-amine
(12).
1
Pale yellow oil, yield 72%. H NMR (400 MHz, CDCl ) δ 7.33 (d, J=4.5 Hz, 1H), 7.29 (d, J=4.6
3
Hz, 2H), 6.85 (d, J=8.1 Hz, 1H), 6.79 (dd, J=8.1, 1.9 Hz, 1H), 6.75 (d, J=1.9 Hz, 1H), 4.22 (t,
J=4.8 Hz, 4H), 3.90-3.87 (m, 10H), 3.27 (t, J=7.0 Hz, 2H), 2.83 (t, J=7.0 Hz, 2H), 2.34 (s, 3H).
+
MS (ESI): m/z 398 (M+H) .
2-Methyl-8-morpholino-N-(2-(naphthalen-2-yl)ethyl)imidazo[1,2-a]pyrazin-3-amine
(13).
1
Pale yellow oil, yield 64%. H NMR (400 MHz, CDCl ) δ 7.83 (m, 3H), 7.69 (d, J=1.6 Hz, 1H),
3
7.49 (tt, J=6.9, 5.1 Hz, 2H), 7.38 (dd, J=8.4, 1.7 Hz, 1H), 7.30 (s, 2H), 4.21 (t, J=4.8 Hz, 4H),
3.91-3.86 (m, 4H), 3.40 (t, J=7.0 Hz, 2H), 3.05 (t, J=7.0 Hz, 2H), 2.33 (s, 3H). MS (ESI): m/z 388
+
(M+H) .
N-(2,4-dichlorophenethyl)-2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-amine (14). Pale

1
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8
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0
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7
8
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2
yellow oil, yield 77%. H NMR (400 MHz, CDCl ) δ 7.34-7.30 (m, 4H), 7.13 (t, J=8.0 Hz, 1H),
3
4.25-4.19 (m, 4H), 3.92-3.87 (m, 4H), 3.30-3.19 (m, 4H), 2.40 (s, 3H). MS (ESI): m/z 406 (M+H)
+
.
Compound 15-16 were prepared using general procedure A.
2-(2-Bromo-4-methoxyphenyl)-N-(2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-yl)acetam
1
ide (15). White solid, yield 82%. H NMR (400 MHz, DMSO-d ) δ 10.07 (s, 1H), 7.43-7.36 (m,
6
3H), 7.22 (d, J=2.6 Hz, 1H), 6.97 (dd, J=8.5, 2.7 Hz, 1H), 4.15-4.11 (m, 4H), 3.89 (s, 2H), 3.78 (s,
+
+
1
1
1
1
1
1
1
1
1
1
2
2
2
3H), 3.75-3.71 (m, 4H), 2.24 (s, 3H). HRMS (ESI ) calcd for C H O N BrH 460.0979, found
20 22 3 5
460.0977.
2-(3-Bromo-4-methoxyphenyl)-N-(2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-yl)acetam
1
ide (16). White solid, yield 88%. H NMR (400 MHz, DMSO-d ) δ 7.61 (d, J =2.1 Hz, 1H),
6
7.38-7.31 (m, 3H), 7.11 (d, J=8.5 Hz, 1H), 4.13 (t, J=4.8 Hz, 4H), 3.85 (s, 3H), 3.77-3.69 (m, 6H),
+
2.19 (s, 3H). MS (ESI): m/z 460 (M+H) .
Compound 17-18 were prepared using general procedure B.
N-(2-bromo-4-methoxyphenethyl)-2-methyl-8-morpholinoimidazo[1,2-a]pyrazin-3-amine
1
(17). Pale brown solid, yield 81%. H NMR (400 MHz, CDCl ) δ 7.35-7.30 (m, 2H), 7.16-7.12 (m,
3
2H), 6.83 (dd, J=8.5, 2.7 Hz, 1H), 4.26-4.18 (m, 4H), 3.92-3.86 (m, 4H), 3.81 (s, 3H), 3.25 (dd,