Discovery of imidazo[1,5-a]pyrido[3,2-e]pyrazines as a new class of phosphodiesterase 10A inhibitiors

Article abstract of DOI:10.1021/jm1002793

Novel imidazo[1,5-a]pyrido[3,2-e]pyrazines have been synthesized and characterized as both potent and selective phosphodiesterase 10A (PDE10A) inhibitors. For in vitro characterization, inhibition of PDE10A mediated cAMP hydrolysis was used and a QSAR mod

Full text of DOI:10.1021/jm1002793

J. Med. Chem. 2010, 53, 4399–4411 4399
DOI: 10.1021/jm1002793
Discovery of Imidazo[1,5-a]pyrido[3,2-e]pyrazines as a New Class of Phosphodiesterase 10A
Inhibitiors†
Norbert Hofgen,*,‡ Hans Stange,‡ Rudolf Schindler,‡ Hans-Joachim Lankau,‡ Christian Grunwald,‡ Barbara Langen,‡
€
Ute Egerland,‡ Peter Tremmel,‡ Menelas N. Pangalos,§ Karen L. Marquis,§ Thorsten Hage,‡ Boyd L. Harrison,§
Michael S. Malamas,§ Nicholas J. Brandon,§ and Thomas Kronbach‡
§
‡Biotie Therapies GmbH, Meissner Strasse 191, 01445 Radebeul, Germany, and Pfizer Neuroscience Princeton, 865 Ridge Road,
Monmouth Junction, New Jersey 08852
Received March 3, 2010
Novel imidazo[1,5-a]pyrido[3,2-e]pyrazines have been synthesized and characterized as both potent and
selective phosphodiesterase 10A (PDE10A) inhibitors. For in vitro characterization, inhibition of
PDE10A mediated cAMP hydrolysis was used and a QSAR model was established to analyze
substitution effects. The outcome of this analysis was complemented by the crystal structure of PDE10A
in complex with compound 49. Qualitatively new interactions between inhibitor and binding site were
found, contrasting with previously published crystal structures of papaverine-like inhibitors. In
accordance with the known antipsychotic potential of PDE10A inhibitors, MK-801 induced stereotypy
and hyperactivity in rats were reversed by selected compounds. Thus, a promising compound class has
been identified for the treatment of schizophrenia that could circumvent side effects connected with
current therapies.
Chart 1
Introduction
Phosphodiesterases (PDEsa) are a family of enzymes that
inactivate cyclic nucleotides cAMP and cGMP via hydrolyza-
tion.1 Because cAMP and cGMP are important secondary
messengers in the signal cascade of G-protein-coupled receptors,
PDEs are involved in a broad spectrum of physiological me-
chanisms which contribute to the homeostasis of the organism.2
In mammals, 11 PDE isoenzyme families have been identi-
fied so far,3 encoded by 21 genes. The PDE families differ in
their substrate specificity for cyclic nucleotides, regulatory
mechanisms, and sensitivity to inhibitors.1 Moreover, they are
differentially expressed at the tissue, cellular, and subcellular
level.4 These differences resultin a diverse array of roles forthe
PDEs across a range of physiological functions.
PDE10Ais a dual substratePDE, primarily expressedinthe
brain, specifically in the nucleus accumbens and the caudate
putamen. Areas with moderate expression levels include thala-
mus, hippocampus, frontal cortex, and olfactory tubercle.5-7
Each of these brain areas is thought to be critical in the patho-
physiology of schizophrenia.8
The antipsychotic activity of PDE10A inhibitors was first
identified using a prototype PDE inhibitor 1-(3,4-dimethoxy-
benzyl)-6,7-dimethoxyisoquinoline 1 (papaverine, Chart 1), a
potent (IC50 = 76 nM) and selective PDE10A inhibitor.9,10
Kostowski et al.11 showed that papaverine reduces apomor-
phine-induced stereotypies in a rat model of psychosis and
increases haloperidol-induced catalepsy in rats while concur-
rently reducing brain dopamine concentration. Papaverine also
increased striatal extracellular cAMP and cGMP levels10 but
had no effect on cGMP levels in PDE10A KO mice, suggesting
that the effects of papaverine were PDE10A-mediated. In
addition, papaverine demonstrated efficacy in a broad range
of psychosis models including “conditioned avoidance re-
sponding” as well as reversal of PCP and D-amphetamine
induced hyperactivity. In a separate study, papaverine im-
proved executive function in a rat model,12 suggesting that
the PDE10A mechanism may have a broader therapeutic
profile and will provide an approach for treating cognitive
deficits of the disease as well as the positive symptoms.
†Coordinates of the PDE10/compound 49 complex have been depo-
sited in the Protein Data Bank. PDB ID is 3LXG.
*To whom correspondence should be addressed. Phone: (þ49)
351 40 43 1301. Fax: (þ49) 351 40 43 2169. E-mail: norbert.hoefgen@
biotie.com.
a Abbreviations: cAMP, cyclic adenosine monophosphate; cGMP,
cyclic guanine monophosphate; FWA, Free-Wilson analysis; IC50
concentration of inhibitor leading to 50% inhibition; MED, mini-
mal effective dose; PDE, cyclic nucleotide phosphodiesterase; pIC50
,
,
-log(IC50 [M]); (Q)SAR, (quantitative) structure-activity relationship;
SEM, standard error of the mean.
r
2010 American Chemical Society
Published on Web 05/07/2010
pubs.acs.org/jmc

4400 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ho€fgen et al.
Papaverine was used as starting point for a number of
lead optimization programs accompanied by pharmacophore
modeling and cocrystallization studies. 6,7-Dimethoxy-4-[8-
(4-methyl-piperazine-1-sulfonyl)-3,4-dihydro-1H-isoquinolin-
2-yl]-quinazoline 2 from Pfizer (Chart 1) was one of the first
examples used for X-ray studies of PDE10A complexes.13
Structure-based drug design permitted identification of a
number of high affinity inhibitors of PDE10A. However, lack
of isoenzyme selectivity toward PDE3 remains an issue in
many cases.
The introduction of biaryl pyrazoles as a new type of
PDE10A inhibitors, structurally distinct from papaverine,
generated a series of potent PDE10A inhibitors with im-
proved selectivity over other PDEs. Currently, the most
advanced example is 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyra-
zol-3-yl)-phenoxymethyl]-quinoline 3 (PF-2545920/MP-10,
Chart 1), which is undergoing phase II clinical studies.14
Other PDE10A inhibitors, not listed here, have recently
been reviewed elsewhere.15,16
[3,2-e]pyrazines 47-59 and 62 were synthesized by the
reaction of II with Grignard reagents (Scheme 5).
For synthesis of 4-trifluoromethyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine 61, this approach was modified by first
replacing chlorine by iodine and subsequently treating with
trifluoromethyl-tris-methylsilane (Scheme 6).
An alternative synthetic route was used for preparation of
4-alkyl-imidazo[1,5-a]pyrido[3,2-e]pyrazines 60, 63-64
(Scheme 7). 4-Amino-2-(4-methyl-2-propyl-imidazolyl)-6-
methoxy-pyridine was treated with carboxylic acid anhy-
drides to form the corresponding amides, which were cyclo-
condensed at higher temperatures.
Inhibition of PDE10 Activity: SAR and QSAR. Inhibitory
potencies of our compounds were tested using in vitro
inhibition of human recombinant PDE10A catalyzed cAMP
hydrolysis. The results are compiled in Table 1.
The impact of R1 on target affinity was investigated in a
number of subseries of derivatives. The replacement of the
hydrogen in compound 4 with an aliphatic chain yielded a
significant improvement in affinity (compounds 5, 6, and 8).
The n-propyl group seems to be the optimal, confirmed with
compounds including 11, 18, 25, 39, and 49. Similar groups
like isobutyl and cyclohexyl are also well tolerated. In
contrast, phenyl-substituted chains, e.g. phenylethyl (com-
pound 59), may be too large at this position, resulting in a
slight drop of affinity. R2 was identified to be a critical
position for good affinity. Here only small groups (hydro-
gen, methyl) can be used. A large variety of substituents was
investigated for R3. Again, small substituents are preferred.
Hydrogen (compound 46), cyano (compounds 25 to 27), and
methyl groups were frequently used to get highly affine
derivatives. Among the larger substituents generally redu-
cing target affinity, the methylsulfonylamino group seems to
be an exception (compound 39). Finally, only a narrow range
of substituents was tested at position R4. No major effects on
the IC50 values were found.
Our initial SAR findings were complemented by a QSAR
study to obtain a comprehensive overview of relationships
between substituent patterns and biological activity and to
differentiate between significant and nonsignificant effects.
A Free-Wilson analysis18 (FWA) as modified by Fujita and
Ban19 was applied. The most frequent R groups were used as
standards resulting in a structure that is equal to 49. Two
significant models M1 and M2 were derived. M1: n = 61, r =
0.990, s = 0.242, F = 13.13, p < 0.001; M2: n = 27, r =
0.979, s = 0.242, F = 22.83, p < 0.001. M1 is based on all
available compounds. For M2, only the subset of com-
pounds with nonunique substituents was included, i.e., with
substituents that were used at a specific position in two or
more compounds. Model M2 excludes known statistical
problems associated with unique substituents.20 However,
M1 may give hints as to which unique substituents may
potentially be advantageous. Results of the FWA are com-
piled in Table 2. Listed coefficients are numerical contribu-
tions of the standard-substituted compound and the
different substituents to the -log(IC50) value. Positive and
negative coefficients indicate an increase in or decrease of
inhibition, respectively, compared to standard substitution.
Numerical results are presented in detail in the Supporting
Information.
In the course of our studies to identify isoenzyme selective
inhibitors of PDE10A, we discovered the imidazo[1,5-a]-
pyrido[3,2-e]pyrazines as a new compound class. Random
screening yielded first experimental indications as to the suit-
ability of these compounds. On the basis of this screen, com-
pound 4 (Chart 1) was selected for structure optimization.
Results and Discussion
Chemistry. The synthetic procedures of imidazo[1,5-a]-
pyrido[3,2-e]pyrazines (Table 1) are outlined in Schemes
1-7. Key starting points are imidazo[1,5-a]pyrido[3,2-e]-
pyrazinones (I), synthesized according to the method of
Norris et al.17 Treatment of I with POCl3 produced the
corresponding 4-chloro-imidazo[1,5-a]pyrido[3,2-e]pyrazines
(II). The chlorine atom can be replaced via classical nucleo-
philic substitution reactions in the presence of potassium
hydroxide (Scheme1). Nucleophiles used here were alcohols
for synthesis of 4-14, mercaptanes for derivatives 15-18, and
amines for preparation of 22-24. Under similar conditions,
potassium cyanide was used to prepare nitriles 25-27.
Stepwise oxidation of 4-methylmercapto-imidazo[1,5-a]-
pyrido[3,2-e]pyrazines (III) with 3-chloroperoxybenzoic acid
produced the corresponding sulfines and sulfones 19-21
(Scheme 2). Individual products were separated by prepara-
tive HPLC.
Treatment of II with ammonia or methylamine formed 4-
amino-imidazo[1,5-a]pyrido[3,2-e]pyrazines (IV), which upon
reaction with anhydrides or carboxylic acid chlorides produced
carboxylic acid amides 28-30 (Scheme 3). To investigate the
role of the remaining hydrogen at the amide group, a second
acylation was carried out to synthesize compound 33.
4-Alkoxycarbonylamino-imidazo[1,5-a]pyrido[3,2-e]pyra-
zines 31, 32, and 34 were prepared using chloroformic acid
esters for acylation. Ureas 35-37 were synthesized by react-
ing IV with carbonyldiimidazole and amines or ammonia.
For comparison with carboxylic acid amides, a series of
corresponding sulfonamides 38-45 were prepared from IV
by treatment with sulfonic acid chlorides or anhydrides in an
aprotic solvent (Scheme 4). Depending on the structure of
the 4-amino-imidazo[1,5-a]pyrido[3,2-e]pyrazine and the
amount of sulfonic acid derivative, either one or two (43)
sulfonations were obtained.
Starting from the global result of the FWA, the excellent
correlation coefficients indicate that assumptions under-
lying this QSAR method are met with regard to derivatives
with nonunique substituents: There is no indication of
Hydrogenation of II displaced the chlorine atom exempli-
fied here with compound 46. 4-Alkyl-imidazo[1,5-a]pyrido-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4401
Table 1. Substituent Pattern and Measured Inhibition of PDE10A Activity
compd
R1
R2
R3
R4
IC50/INHa
SEMb
196
17.2
3.38
4
H
CH3
CH3
CH3
CH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OC3H7
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
2270
270
5
6
C2H5
C3H7
C3H7
C2H5
C2H5
C3H7
C2H5
C3H7
C3H7
H
63.0
92.2
24.8
149
7
9.44
2.98
8
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
9
17.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
1
OCH(CH3)2
OCH(CH3)2
cyclopentyloxy
OC2H4C6H5
OC3H6C6H5
SCH3
731
391
54.1
11.2
25.4%
383
962
0.96%
31.7
125
4.31%
288
51.5
21.0%
2390
617
CH3
SCH3
SCH3
SCH3
C2H5
C3H7
C2H5
C3H7
C3H7
C2H5
C3H7
C3H7
C3H7
C2H5
209
284
26.8
27.3
S(=O)CH3
S(=O)CH3
S(=O)2CH3
NHCH3
89.6
23.3
482
2.75
1.43
59.8
NHCH3
N(CH3)2
246
146
34.6
16.7
1.6
CN
CN
10.9
36.4
9.62
175
5.3
1.41
cyclohexyl
C2H5
C3H7
CN
NHC(dO)CH3
NHC(dO)CH3
NHC(dO)C2H5
NHC(dO)OCH3
NHC(dO)OC2H5
N(C(dO)OCH3)2
N(CH3)C(dO)OCH3
NHC(dO)NH2
NHC(dO)NHCH3
NHC(dO)NHCH(CH3)2
NHS(=O)2CH3
NHS(=O)2CH3
NHS(=O)2C2H5
NHS(=O)2C3H7
NHS(=O)2CH(CH3)2
N(S(=O)2CH3)2
NHS(=O)2CF3
N(CH3)S(=O)2CH3
H
39
61.4
472
C3H7
C3H7
1050
22.9
206
169
1.97
20.5
8.21
54.2
7.82
C3H7
C3H7
81.5
486
C3H7
C3H7
55.4
535
381
C3H7
C3H7
47.3
49.1
C2H5
C3H7
64.7
7.16
39.2
617
3.96
0.74
2.01
C3H7
C3H7
10.7
15.6
1.07
6.77
89
C3H7
C3H7
132
15.3
53.4
1195
33.9
81.2
15.7
7.28
25.1
32.5
8.32
13.2
51.3
223
C3H7
C3H7
C3H7
CH3
4.69
3.46
0.44
0.6
CH3
CH3
C2H5
C3H7
CH3
CH3
C3H7
C3H7
1.91
3.16
0.47
0.82
2.28
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C3H7
C3H7
CH3
CH3
CH3
OCHF2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
C5H11
C6H13
cyclohexyl
CH2CH(CH3)2
CH2C6H5
C2H4C6H5
C3H7
CH3
CH3
26.8
2.84
1.43
21.4
116
0.355
0.23
2.17
CH3
CH3
CH3
CHF2
13.1
0.7
7.33
45.8
12.4
285
C3H7
C3H7
CF3
C2H5
2.12
1.15
C3H7
C3H7
C(dO)OCH3
C(dO)OC2H5
CH3
CH3
35.7
5.7
326
56.9
1.34
4.4
0.19
3
a Means of IC50 [nmol/L] or, in case of 12 and 15, percent inhibition at a concentration of 1000 nM; n = 4. b Standard error of the mean

4402 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ho€fgen et al.
Scheme 1
Scheme 2
smaller (hydrogen) and larger (ester) groups. A large spec-
trum of substituents was applied at position R3. No substit-
uents offering significant improvement on the standard
methyl were found. Smaller (hydrogen) and most larger and
polar substituents decrease the inhibitory potential. How-
ever, two comparatively large sulfonamide groups do not
change IC50 values significantly. It can thus be hypothesized
that there is, in this position, space for bulkier groups and
specific interactions such as hydrogen bonding. At position
R4, only four groups were tested. No significant differences
were found between polar methoxy and difluoromethoxy
Scheme 4
interactions between the four substituents, and the implicitly
used simple alignment of the three-ring systems appears to be
correct. From the alignment, we conclude that at least this
subset of compounds will have a similar binding position
compared to 49 for which the PDE10 cocrystal structure was
solved. Docking calculations were used to further check this
assumption (see below). Regarding the four substituents,
standard substitution is generally favorable with regard to
IC50. At position R1, inhibition may be enhanced by use of
isobutyl (unique substituent) instead of propyl. Smaller as
well as the majority of larger substituents decrease inhibition
but cyclohexyl and benzyl do not change the IC50 signifi-
cantly, indicating a voluminous subpocket. Only a small
number of substituents were used at position R2. Among
these, the standard methyl is optimal in comparison to both
Scheme 3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4403
Table 2. Free-Wilson Analysis Results: Substituent Coefficients
Scheme 5
Pos.
substituent
coefficient Ca
CS
standard-substituted compound
8.168b
R1
CH2CH(CH3)2
cyclohexyl
(0.677)c
nsd
e
C3H7
C2H5
0
-0.369
(ns)
CH2C6H5
C5H11
CH3
(-0.878)
-1.047
(-1.232)
(-1.516)
-1.593
C2H4C6H5
C6H13
H
R2
CH3
H
0
-0.526
(-1.623)
(-1.681)
C(dO)OCH3
C(dO)OC2H5
Scheme 6
R3
CH3
CHF2
0
(ns)
C2H5
NHS(=O)2CH3
CN
(ns)
ns
-0.332
(ns)
N(S(=O)2CH3)2
NHC(dO)OCH3
S(=O)2CH3
OCH3
(-0.528)
(-0.535)
-0.65
H
(-0.698)
(-0.761)
(-0.829)
(-0.896)
(-0.912)
(-0.972)
(-1.079)
-1.186
(-1.289)
(-1.332)
-1.432
-1.442
(-1.482)
-1.521
-1.712
(-1.749)
(-1.751)
(-1.855)
(-1.896)
(-1.958)
(-2.151)
(-2.189)
(-2.245)
(-2.267)
NHS(=O)2C2H5
CF3
NHS(=O)2CF3
NHC(dO)NH2
OC3H7
Scheme 7
N(C(dO)OCH3)2
S(=O)CH3
NHS(=O)2CH(CH3)2
N(CH3)2
SCH3
NHC(dO)CH3
NHC(dO)OC2H5
NHCH3
OCH(CH3)2
NHC(dO)NHCH(CH3)2
OC2H4C6H5
N(CH3)C(dO)OCH3
NHC(dO)NHCH3
NHS(=O)2C3H7
OC3H6C6H5
NHC(dO)C2H5
N(CH3)S(=O)2CH3
cyclopentyloxy
groups and nonpolar methyl. The small hydrogen atom
however decreases inhibitory potency.
X-ray. Crystal structure determination of the potent com-
pound 49 in complex with PDE10A (rat) was used to comple-
ment data obtained from SAR studies regarding binding
posture and key interactions of the invariant core structure
and binding site residues. The structure of a papaverine-like
inhibitor in complex with PDE10A is known21 and displays
some similarities with inhibitors of other PDE’s, e.g. rolipram
at PDE4D.22 Recently, the structure of PDE10A in complex
with papaverine was published.23 In contrast to this data,
qualitatively new interactions were to be expected with our
compounds due to the lack of a dimethoxy moiety and the new
core structure.
Binding interactions of compound 49 are summarized
schematically in Figure 2, and the binding pocket is depicted
in Figure 3. 49 interacts with Gln716 via a hydrogen bond to
the pyrazine nitrogen. This glutamine residue is conserved in
all PDE isoforms and known to play a crucial role in binding
natural PDE substrates cGMP and cAMP.24,25 It is also
involved in hydrogen bonding interactions with most known
R4
OCH3
CH3
0
(ns)
OCHF2
H
(ns)
(-0.68)
a Coefficients C from multiple linear regression analysis. pIC50
=
-log(IC50) = CCS þ CR1 þ CR2 þ CR3 þ CR4
.
b Related coefficients of
models M1 and M2 are identical. c Results for unique substituents are
stated in parentheses. d ns: not significantly different from zero: p > 0.1.
e Standard substituent bold.
PDE inhibitors. Additional water-mediated hydrogen bonds
between the imidazole nitrogen and residues Tyr514 and
Asp664 further anchor this inhibitor within the binding site.
This hydrogen bond network is not known from other
PDE10A inhibitors.26 The binding pose is further stabilized

4404 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ho€fgen et al.
Figure 1. Effect of PDE10 inhibitors on MK-801 induced hyperactivity and stereotyped sniffing in female rats. Data are shown as mean (
SEM. Significantly different from MK-801 stimulated control (Cs): * p < 0.05, ** p < 0.01, *** p < 0.001. Significantly different from saline
control (Co): ### p < 0.001.
Figure 2. Ligplot38 representation of hydrophobic and hydrogen bond interactions of compound 49 and the amino acid residues involved in
the PDE10 binding pocket.
by π-stacking of the fused ring system with Phe719 and
hydrophobic contacts with Leu665, Ser667, Val668, Ile682,
Phe686, and Met703. Phe719, Phe686, and Ile682 form a cleft
known as a “hydrophobic clamp”.27 On the basis of the
crystal structure, some aspects of the experimentally observed
SAR can be rationalized. At positions R1 (propyl) and R4
(methoxy), no interactions were detected (Figure 2), indicat-
ing room for larger substituents. The oxygen of R4 is not
involved inpolar interactions or hydrogen bonding. Methylat
position R2 has the optimal size to occupy a small subpocket.
The R2 substituent is involved in hydrophobic interactions
with a couple of amino acid residues. As we found in our SAR
studies, there is no space to accommodate larger groups. In
contrast, a methyl group at the R3 position points toward a
rather large subpocket. There are hydrophobic contacts with
Ile682 and Val668, but there is space for bulkier groups which
could be involved in additional interactions. The methylsul-
fonamide substituent of 39 is one such example.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4405
Figure 3. Co-crystal structure of PDE10 and 49 (orange C atoms): depiction of the shape of the binding pocket (clipped in z direction), key
amino acids (blue-gray C atoms), metal ions (spheres), and a water involved in binding. H bonds are symbolized by yellow broken lines.
Table 3. Reversal of MK-801 Induced Hyperactivity and Stereotyped Sniffing
MEDa hyperact (mg/kg po)
compd
F hyperactb
MEDa sniffing (mg/kg po)
F sniffingb
IC50 (nM)c
25
26
7.5
15
5
34.813
14.764
20.180
13.684
17.360
29.580
16.533
5
10
5
47.893
58.876
43.586
29.536
23.693
94.475
16.279
10.9
36.4
22.9
7.16
33.9
7.28
n.i.d
31
39
5
2.5
10
2.5
20
46
49
30
5
clozapine
20
a Minimal effective dose that significantly reduced MK-801-induced hyperactivity or stereotyped sniffing, respectively (p < 0.05). b p < 0.001, n = 6.
c Inhibition of PDE10 activity from Table 2. d ni: no inhibition at PDE10.
Table 4. Selectivity of 39 and 49 towards PDE Isoforms
IC50a [nM]
To test our initial hypothesis of a similar binding mode of
our compounds, 4-64 were docked into the crystal structure.
It was found that most derivatives can adopt a binding
PDE
39
49
position like 49, even those with rather big R3 substituents
(sulfonamides, ureas, urethanes). However, the extended R3
substituents of 12, 13, 14, and 33 on the one hand and R2 ester
groups of 63 and 64 on the other hand do not seem to fit into
the respective subpockets;only alternative binding poses
were calculated. Interestingly, there is no total loss of inhibi-
tory potency despite a likely changed binding mode. Thus,
these derivatives might be used as starting points for new
PDE10 inhibitors. Because the four R3 and two R2 substitu-
ents causing potentially changed binding poses are unique in
the present set of derivatives, SAR (either qualitative or
QSAR like FWA) did not allow determination if reduced
inhibitionwas duetounfavorablelocalinteractionorchanged
binding mode. Recalculation of FWA model M1 without 12,
13, 14, 33, 63, and 64 yielded identical regression coefficients
for all other substituents and nearly unchanged statistics.
Pharmacology. A representative subset of six high affinity
PDE10A inhibitors was selected for investigation of the anti-
psychotic potential of imidazo[1,5-a]pyrido[3,2-e]pyrazines
in vivo. To enable the characterization of the antipsychotic
activity, we utilized a MK-801 induced hyperactivity reversal
model.28 MK-801 (0.1 mg/kg ip) significantly increased
horizontal activity (t test: p < 0.001) and induced stereo-
1B
2A
3A
4A
5A
6
>1000
∼1000
>5000
239 (25)
3700 (491)
∼5000
1260 (125)
>1000
561 (77)
>1000
>1000
919 (81)
>5000
7B
8A
9A
10A
11A
3100 (491)
>5000
>5000
>1000
>1000
7.16 (0.74)
68.6 (3.9)
7.28 (0.60)
779 (107)
a SEM of IC50 data stated in parentheses if IC50 values could be
determined. Otherwise, IC50 values are related to the maximum con-
centration used in the screen.
typed sniffing (t test: p < 0.001) in female rats. These beha-
viors were reversed significantly by 8-chloro-11-(4-methyl-1-
piperazinyl)-5H-dibenzo(b,e)(1,4)diazepine (clozapine), an
atypical antipsychotic.29 The selected PDE10A inhibitors also
reversed hyperactivity and stereotyped sniffing in a dose depen-
dent and significant manner. Results are shown in Table 3 and
Figure 1.
The minimal effective dose (MED) of clozapine for both end
points is 20 mg/kg; the MEDs of the novel PDE10A inhibitors

4406 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ho€fgen et al.
are, with one exception, below this value (Table 3). Further, a
qualitative correlation can be seen between MEDs and PDE10
inhibition data. Among the six compounds studied, 39 and 49
are most effective regarding both enzyme inhibition and re-
versal of MK-801 induced behavioral symptoms. Both com-
pounds were characterized regarding their selectivity profiles.
They were found to be highly specific PDE10A inhibitors with
selectivities >100 to most of the isoenzymes: Table 4.
4,8-Dimethoxy-3-methyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
(4). Yield 72%; mp 152-154 °C; C12H12N4O2; MW 244.25. 1H
NMR (300 MHz, CDCL3) δ 8.55 (s, 1 H), 7.73 (d, J = 9 Hz,
1 H), 6.71 (d, J = 9 Hz, 1 H), 4.03 (s, 3 H), 3.95 (s, 3 H), 2.60 (s,
3 H). 13C NMR (300 MHz, CDCL3) δ 160.72, 154.61, 137.96,
136.66, 133.95, 128.57, 124.12, 115.36, 109.97, 53.90, 53.39, 14.33.
4,8-Dimethoxy-1,3-dimethyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
(5). See Supporting Information for details.
4,8-Dimethoxy-1-ethyl-3-methyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (6). See Supporting Information for details.
4,8-Dimethoxy-1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
(7). See Supporting Information for details.
Conclusions
A series of novel imidazo[1,5-a]pyrido[3,2-e]pyrazines has
been identified. These compounds were characterized to be
potent and selective PDE10A inhibitors structurally distinct
from inhibitor families published to date. Structural optimi-
zation was supported by a QSAR model established to
analyze substitution effects. The optimal combination of
substituents was realized with compound 49. The outcome
of this analysis was complemented by the crystal structure of
PDE10Aincomplexwithcompound49. Qualitatively distinct
interactions between inhibitor and binding site were found
contrasting with previously published crystal structures of
papaverine-like inhibitors. In addition to the common hydro-
gen bond to Gln716, a water mediated interaction between the
imidazole nitrogenand Tyr514 and Asp664 was detected. This
hydrogen bond network was not known from other types of
PDE10A inhibitors. In accordance with the known antipsy-
chotic potential of PDE10A inhibitors, MK-801 induced
stereotypies and hyperactivity in ratswere reversed by selected
compounds. The most effective derivatives are compounds 39
and 49, with MED’s of 2.5 and 5 mg/kg po, respectively.
Compound 49 is favorablewith regard toits selectivitytoward
all other PDE’s. Thus, a promising compound class for the
treatment of schizophrenia has been found.
4,8-Dimethoxy-3-methyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (8). See Supporting Information for details.
1-Ethyl-8-methoxy-3-methyl-4-propyloxy-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (9). See Supporting Information for details.
1-Ethyl-8-methoxy-3-methyl-4-isopropyloxy-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (10). See Supporting Information for
details.
4-Isopropyloxy-8-methoxy-3-methyl-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (11). See Supporting Information for details.
1-Ethyl-8-methoxy-3-methyl-4-cyclopentyloxy-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (12). See Supporting Information for
details.
8-Methoxy-3-methyl-4-phenethyloxy-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (13). See Supporting Information for
details.
8-Methoxy-3-methyl-4-(3-phenyl-propoxy)-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (14). See Supporting Information
for details.
Synthesis of 4-Methylsulfanyl-imidazo[1,5-a]pyrido[3,2-e]pyra-
zines 15-18. General Procedure. First, 5 mmol of 4-chloro-
imidazo[1,5-a]pyrido[3,2-e]pyrazine were dissolved in 25 mL of
N,N-dimethylformamide, then 20 mmol of sodium thiomethylate
were added. The mixture was heated up to 60 °C for 4 h. The
solvent was removed, and the residue was treated with 20 mL
of water and 20 mL of dichloromethane. The organic layer
was separated. The aqueous layer was extracted with 20 mL of
dichloromethane. The unified organic layers were washed with
two times 20 mL of water. The solvent was removed completely.
The residue was purified by column chromatography.
8-Methoxy-3-methyl-4-methylsulfanyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (15). Yield 69%; mp 185-187 °C; C12H12N4OS;
MW 260.32. 1H NMR (300 MHz, CDCL3) δ 8.60 (s, 1 H), 7.86
(d, J = 9 Hz, 1 H), 6.75 (d, J = 9 Hz, 1 H), 3.99 (s, 3 H), 2.74 (s,
3 H), 2.62 (s, 3 H). 13C NMR (300 MHz, CDCL3) δ 160.99,
152.92, 138.47, 136.26, 134.11, 127.71, 125.18, 121.15, 110.32,
54.08, 15.75, 12.11.
8-Methoxy-1,3-dimethyl-4-methylsulfanyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (16). See Supporting Information for details.
1-Ethyl-8-methoxy-3-methyl-4-methylsulfanyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (17). See Supporting Information for
details.
8-Methoxy-3-methyl-4-methylsulfanyl-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (18). See Supporting Information for
details.
Experimental Section
Chemistry. Column chromatography was performed on silica
gel eluting with 5% MeOH in CH2Cl2. Melting points were
determined on a Boetius melting point apparatus PHMK 05 and
are uncorrected. All substances were analyzed with an Agilent
1100 series HPLC/MSD system. Purities were ascertained using
the area percentage method on the UV trace recorded at a
wavelength of 254 nm and found to be g95%. Proton (1H
NMR) and carbon (13C NMR) nuclear magnetic resonance
spectra were recorded on a Bruker ARX 300 NMR spectro-
meter. Chemical shifts (δ) are in parts per million (ppm) relative
to Si(CH3)4, and coupling constants (J) are in hertz. The NMR
solvent used was either CDCl3 or DMSO. Analytical and
spectroscopic data are listed here only for key compounds. A
comprehensive list including all compounds can be found in
Supporting Information.
Key Intermediates. The synthesis of the used imidazo[1,5-a]-
pyrido[3,2-e]pyrazinones I is described by Norris et al.17 The
synthesis of the used 4-chloro-imidazo[1,5-a]pyrido[3,2-e]pyrazines
II is described by Chen et al.30
Synthesis of 4-Methylsulfinyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazines 19-20. General Procedure. First, 2 mmol of 4-
methylsulfanyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine were dis-
solved in 40 mL of dichloromethane. At 0-5 °C, about 0.8 g of
3-chloroperoxybenzoic acid were added in small portions.
The mixture was stirred for 2 h at 5 °C. At room temperature,
the reaction solution was washed first with 2 ꢀ 30 mL
saturated NaHCO3 solution and then with 2 ꢀ 30 mL water.
The solvent was removed from the isolated organic layer. The
residue was purified by column chromatography.
1-Ethyl-8-methoxy-3-methyl-4-methylsulfinyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (19). Yield 67%; mp 189-192 °C; C14H16-
N4O2S; MW 304.37. 1H NMR (300 MHz, CDCL3) 8.23 (d, J =
9 Hz, 1 H), 6.89 (d, J = 9 Hz, 1 H), 4.05 (s, 3 H), 3.66 (q, J =
Synthesis of 4-Alkoxy-imidazo[1,5-a]pyrido[3,2-e]pyrazines
4-14. General Procedure. First, 5 mmol of 4-chloro-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine were dissolved in a mixture of
15 mL of dichloromethane and 15 mL of the correspond-
ing alcohol, then 1 g of solid KOH was added. The reaction
mixture was heated up to reflux for 7 h. At room temperature,
30 mL of water were added. The organic layer was separated.
The aqueous layer was extracted with 20 mL of dichloro-
methane. The unified organic layers were washed with two
times 20 mL of water. The solvent was removed completely.
The residue was purified by column chromatography.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4407
7 Hz, 2 H), 2.94 (s, 3 H), 2.72 (s, 3 H), 1.45 (t, J = 7 Hz, 3 H). 13
NMR (300 MHz, CDCL3) δ 162.64, 156.66, 147.80, 140.63,
137.67, 134.88, 125.24, 120.11, 110.54, 54.42, 39.13, 24.32,
16.28, 13.20.
8-Methoxy-3-methyl-4-methylsulfinyl-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (20). See Supporting Information for
details.
8-Methoxy-3-methyl-4-methylsulfonyl-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (21). First, 2 mmol of 8-methoxy-4-methyl-
sulfanyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine (18) were
dissolved in 40 mL of dichloromethane. At 0-5 °C, about 1.58 g
of 3-chloroperoxybenzoic acid were added in small portions. The
mixture was stirred for 2 h at 25 °C. At room temperature, the
reaction solution was washed first with 2 ꢀ 30 mL of saturated
NaHCO3 solution and then with 2 ꢀ 30 mL of water. The solvent
was removed from the isolated organic layer. The residue was
purified by column chromatography.
C
30 mmol of 4-chloro-imidazo[1,5-a]pyrido[3,2-e]pyrazine and
200 mL of an aqueous solution of NH3 (32%) were stirred in an
autoclave at 125-130 °C for 8 h. The reaction mixture was
diluted with 200 mL of water. The corresponding 4-amino-
imidazo[1,5-a]pyrido[3,2-e]pyrazine precipitated. The crude in-
termediate was filtered off and washed with 50 mL of water.
Finally, it was stirred with 30 mL of dichloromethane for 15 min
at room temperature. After filtration, the intermediate was dried
at room temperature.
First, 3 mmol of the corresponding 4-amino-imidazo[1,5-
a]pyrido[3,2-e]pyrazine and 10 mL of acetic acid anhydride or
propionic acid anhydride were stirred at 100 °C for 2 h. At room
temperature, the mixture was diluted with 100 mL of ice-water.
Solid sodium hydrogencarbonate is added until a pH value of
6.0 was achieved. The precipitated product was filtered off and
washed with 50 mL of water. The product was purified by
column chromatography.
Yield 59%; mp 43-46 °C; C15H18N4O3S; MW 334.4. 1H
NMR (300 MHz, CDCL3) 7.95 (d, J = 9 Hz, 1 H), 6.83 (d, J = 9
Hz, 1 H), 4.02 (s, 3 H), 3.57 (t, J = 8 Hz, 2 H), 3.40 (s, 3 H), 2.79
(s, 3 H), 1.85 (m, 2 H), 0.99 (t, J = 7 Hz, 3 H). 13C NMR (300
MHz, CDCL3) δ 163.10, 151.86, 146.58, 140.34, 137.94, 137.00,
123.04, 117.01, 110.67, 54.53, 39.50, 32.60, 22.20, 16.27, 13.70.
Synthesis of 4-Alkylamino-imidazo[1,5-a]pyrido[3,2-e]pyrazines
22-24. General Procedure. First, 7 mmol of 4-chloro-imidazo[1,5-
a]pyrido[3,2-e]pyrazine and 25 mL of an aqueous solution of
methylamine or dimethylamine (40%) were stirred in an autoclave
at 125-130 °C for 5 h. At room temperature, the mixture was
extracted with 2 ꢀ 50 mL of dichloromethane. The organic
solution was washed with 2 ꢀ 50 mL of water. The solvent was
removed from the isolated organic layer. The residue was purified
by column chromatography.
4-Acetylamino-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (28). Yield 90%; mp 199-202 °C; C15H17N5O2;
MW 299.33. 1H NMR (300 MHz, DMSO) δ 10.26 (s, 1 H), 8.01 (d,
J = 9 Hz, 1 H), 6.95 (d, J = 9 Hz, 1 H), 3.99 (s, 3 H), 3.54 (q, J = 7
Hz, 2 H), 2.44 (s, 3 H), 2.13 (s, 3 H), 1.36 (t, J = 7 Hz, 3 H). 13
C
NMR (300 MHz, DMSO) δ 170.44, 160.65, 145.40, 143.96, 139.28,
136.33, 134.49, 124.71, 119.83, 109.51, 54.01, 23.53, 23.03, 14.42,
12.82, 12.44.
4-Acetylamino-8-Methoxy-3-methyl-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (29). See Supporting Information for
details.
8-Methoxy-3-methyl-4-propionylamino-1-propyl-imidazo[1,5-
a]pyrido[3,2-e]pyrazine (30). See Supporting Information for
details.
Synthesis of 4-Alkoxycarbonylamino-imidazo[1,5-a]pyrido-
[3,2-e]pyrazines 31-34. General Procedure. First, 30 mmol of
4-chloro-imidazo[1,5-a]pyrido[3,2-e]pyrazine and 200 mL of an
aqueous solution of NH3 (32%) were stirred in an autoclave at
125-130 °C for 8 h, then the reaction mixture was diluted with
200 mL of water. The corresponding 4-amino-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine precipitated. The crude intermediate was filtered off
andwashedwith50mLofwater. Finally, itwasstirredwith30mLof
dichloromethane for 15 min at room temperature. After filtration,
the intermediate was dried at room temperature.
First, 3 mmol of the corresponding 4-amino-imidazo[1,5-
a]pyrido[3,2-e]pyrazine were stirred into a mixture of 20 mL of
dichloromethane, 5 mL of methanol, and 1 mL of triethylamine.
At 0 °C, a solution of 3 mmol of chloroformic acid ester in 10 mL
of dichloromethane was added slowly. The reaction mixture was
stirred for 2 h at 0 °C, then the solution was heated up to reflux
for 10 h. At room temperature, the solution was washed with
30 mL of a saturated aqueous solution of sodium hydrogencar-
bonate and with 30 mL of water. The solvent was removed. The
product was purified by column chromatography.
1-Ethyl-8-methoxy-3-methyl-4-methylamino-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (22). Yield 88%; mp 172-174 °C;
C
14H17N5O; MW 271.32. 1H NMR (300 MHz, DMSO) δ
7.72 (d, J = 9 Hz, 1 H), 6.76 (d, J = 9 Hz, 1 H), 6.37 (q, J =
5 Hz, 1 H), 3.90 (s, 3 H), 3.46 (q, J = 8 Hz, 2 H), 2.94 (d, J =
5 Hz, 3 H), 2.56 (s, 3 H), 1.32 (t, J = 8 Hz, 3 H). 13C NMR (300
MHz, DMSO) δ 157.20, 149.22, 144.89, 136.71, 134.12, 131.43,
126.96, 116.05, 108.54, 53.46, 28.00, 23.58, 15.12, 12.78.
8-Methoxy-3-methyl-4-methylamino-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (23). See Supporting Information for
details.
4-Dimethylamino-8-methoxy-3-methyl-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (24). See Supporting Information for details.
Synthesis of 4-Cyano-imidazo[1,5-a]pyrido[3,2-e]pyrazines
25-27. General Procedure. First, 10 mmol of 4-chloro-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine were stirred into a solution of 32 g
of ethoxycarbonyldifluoromethyl magnesia chloride in 100 mL
of tetrahydrofuran. The mixture was stirred and heated up to
reflux for 10 h. The solvent was removed. About 15 mL of N,N-
dimethylformamide and 2 g of KCN were added. This reaction
mixture was heated up to reflux for 5 h. After this time, 100 mL
of toluene were added. After a stirring period of 10 min, the
organic layer was separated and washed with 3 ꢀ 50 mL of
water. The solvent was removed from the isolated organic layer.
The residue was purified by column chromatography.
4-Cyano-8-methoxy-3-methyl-1-propyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (25). Yield 70%; mp 178-180 °C; C15H15N5O;
MW 281.32. 1H NMR (300 MHz, CDCL3) δ 8.21 (d, J = 9 Hz,
1 H), 7.10 (d, J = 9 Hz, 1 H), 4.10 (s, 3 H), 3.86 (t, J = 8 Hz, 2
H), 2.02 (m, 2 H), 1.07 (t, J = 7 Hz, 3 H). 13C NMR (300 MHz,
CDCL3) δ 164.33, 144.06, 141.72, 136.43, 130.17, 126.43,
126.38, 121.05, 113.98, 113.96 55.23, 30.13. 22.42, 13.61, 11.03.
4-Cyano-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (26). See Supporting Information for details.
4-Cyano-1-cyclohexyl-8-methoxy-3-methyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (27). See Supporting Information for details.
Synthesis of 4-Acetylamino- and 4-Propionylamino-imidazo-
[1,5-a]pyrido[3,2-e]pyrazines 28-30. General Procedure. First,
8-Methoxy-4-methoxycarbonylamino-3-methyl-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (31). Yield 76%; mp 127-130 °C;
1
C16H19N5O3; MW 329.36. H NMR (300 MHz, DMSO) δ 9.88
(s,1H), 8.00(d, J=9Hz, 1H), 6.94(d,J= 9 Hz, 1 H), 3.98 (s, 3 H),
3.69 (s, 3 H), 3.49 (t, J =8 Hz, 2H), 2.46(s, 3H), 1.82(m, 2H), 0.97
(t, J = 7 Hz, 3 H). 13C NMR (300 MHz, DMSO) δ 160.58, 154.73,
144.41, 143.33, 139.35, 136.24, 134.44, 124.77, 119.61, 109.62, 54.01,
52.22, 31.88, 21.40,14.45, 13.65.
4-Ethoxycarbonylamino-8-methoxy-3-methyl-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (32). See Supporting Information for
details.
4-(N,N-Bis-ethoxycarbonyl-amino)-8-methoxy-3-methyl-1-pro-
pyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine (33). In this case, 6 mmol
of chloroformic acid ethyl ester were used in the last step.
8-Methoxy-4-(methoxycarbonyl-methyl-amino)-3-methyl-1-
propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine (34). 8-Methoxy-3-
methyl-4-methylamino-1-propyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (23) was used as the corresponding 4-amino-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine.

4408 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ho€fgen et al.
Synthesis of 4-Ureido-imidazo[1,5-a]pyrido[3,2-e]pyrazines
35-37. General Procedure. First, 30 mmol of 4-chloro-8-meth-
oxy-3-methyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine and
200 mL of an aqueous solution of NH3 (32%) were stirred in an
autoclave at 125-130 °C for 8 h, then the reaction mixture was
diluted with 200 mL of water. The reaction product 4-amino-8-
methoxy-3-methyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
precipitated. The crude intermediate was filtered off and washed
with 50 mL of water. Finally, it was stirred with 30 mL of di-
chloromethane for 15 min at room temperature. After filtration,
the intermediate was dried at room temperature.
First, 2 mmol of 4-amino-8-methoxy-3-methyl-1-propyl-
imidazo[1,5-a]pyrido[3,2-e]pyrazine were stirred into 20 mL of
tetrahydrofuran at room temperature, then 6 mmol of carbo-
nyldimidazole were added. The reaction mixture was heated up
to reflux for 3 h. Then 20 mmol of NH3 (aqueous solution, 32%)
or methylamine (aqueous solution, 40%) or isopropylamine
were added. Again, the mixture was heated up to reflux for 1 h.
The solvent was removed. The residue was treated with 30 mL of
water. The crude product precipitated. It was filtered off and
washed with 50 mL of water. The product was purified by
column chromatography.
8-Methoxy-3-methyl-1-propyl-4-ureido-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (35). Yield 61%; mp 185-187 °C; C15H18N6O2;
MW 314.35. 1H NMR (300 MHz, CDCL3) δ 9.35 (bs, 1 H), 7.73
(d, J = 9 Hz, 1 H), 7.22 (bs, 1 H), 6.77 (d, J = 9 Hz, 1 H), 5.63
(bs, 1 H), 3.99 (s, 3 H), 3.54 (t, J = 8 Hz, 2 H), 2.75 (s, 3 H), 1.88
(m, 2 H), 1.01 (t, J = 7 Hz, 3 H). 13C NMR (300 MHz, CDCL3)
δ 160.28, 154.97, 146.22, 144.36, 137.58, 135.49, 132.88, 124.24,
115.56, 109.94, 54.14, 32.59, 22.15, 15.63, 13.84.
4-(N-Methyl-ureido)-8-methoxy-3-methyl-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (36). See Supporting Information
for details.
4-(N-Isopropyl-ureido)-8-methoxy-3-methyl-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (37). See Supporting Information for
details.
Synthesis of 4-Sulfonylamino-imidazo[1,5-a]pyrido[3,2-e]-
pyrazines 38-45. General Procedure. First, 30 mmol of
4-chloro-imidazo[1,5-a]pyrido[3,2-e]pyrazine and 200 mL of
an aqueous solution of NH3 (32%) were stirred in an auto-
clave at 125-130 °C for 8 h, then the reaction mixture was
diluted with 200 mL of water. The corresponding 4-amino-
imidazo[1,5-a]pyrido[3,2-e]pyrazine precipitated. The crude
intermediate was filtered off and washed with 50 mL of water.
Finally, it was stirred with 30 mL of dichloromethane for
15 min at room temperature. After filtration, the intermediate
was dried at room temperature.
A mixture of 30 mmol of the corresponding 4-amino-
imidazo[1,5-a]pyrido[3,2-e]pyrazine with 50 mmol of an alkyl-
sulfonic acid anhydride and 350 mL of toluol was heated up to
reflux for 1 h. Then 15 mL triethylamine were added at about
70 °C. Again, the reaction mixture was stirred and heated to
reflux for 1 h. The reaction mixture was treated with 100 mL of
water. The precipitated crude product was filtered off and
washed with 3 ꢀ 80 mL of water. The product was purified by
recrystallization from toluol.
8-Methoxy-3-methyl-4-propylsulfonylamino-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (41). See Supporting Information for
details.
4-Isopropylsulfonylamino-8-methoxy-3-methyl-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (42). See Supporting Information for
details.
4-(N,N-Bis-methylsulfonyl)-8-methoxy-3-methyl-1-propyl-
imidazo[1,5-a]pyrido[3,2-e]pyrazine (43). In this case, 80 mmol
of methylsulfonic acid anhydride were used in the last step.
8-Methoxy-3-methyl-1-propyl-4-trifluoromethylsulfonylamino-
imidazo[1,5-a]pyrido[3,2-e]pyrazine (44). See Supporting Infor-
mation for details.
8-Methoxy-3-methyl-4-(N-methylsulfonyl-N-methyl-amino)-
1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine (45). 8-Methoxy-
3-methyl-4-methylamino-1-propyl-imidazo[1,5-a]pyrido[3,2-e]
pyrazine (23) was used as the corresponding 4-amino -imidazo-
[1,5-a]pyrido[3,2-e]pyrazine.
8-Methoxy-3-methyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (46). First, 3 mmol of 4-chloro-8-methoxy-3-methyl-1-
propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine were suspended in
50 mL of ethanol, then about 1 mL of triethylamine and 1 g of
palladium catalyst were added. This suspension was stirred in an
autoclave. Hydrogen was pressed in up to 20 bar pressure. The
mixture was stirred for 4 h at 30 °C. After filtration, the solvent was
removed from the clear solution. The residue was dissolved in
100 mL of dichloromethane. The solution was washed with 50 mL
of water. The solvent was removed again. The product was purified
by column chromatography.
1
Yield 66%; mp 128-130 °C; C14H16N4O; MW 256.31. H
NMR (300 MHz, DMSO) δ 8.78 (s, 1H), 8.10 (d, J = 9 Hz, 1 H),
6.96 (d, J = 9 Hz, 1 H), 3.99 (s, 3 H), 3.48 (t, J = 8 Hz, 2 H), 1.82
(m, 2 H), 0.97 (t, J = 7 Hz, 3 H). 13C NMR (300 MHz, DMSO) δ
160.78, 143.81, 143.39, 140.23, 136.67, 134.38, 125.93, 123.17,
109.15, 54.01, 31.77, 21.44, 13.64, 12.35.
Synthesis of 4-Alkyl-imidazo[1,5-a]pyrido[3,2-e]pyrazines 47-
59. General Procedure. First, 10 mmol of 4-chloro-imidazo[1,5-
a]pyrido[3,2-e]pyrazine were suspended in 150 mL of tetrahy-
drofuran, then 30 mL of a solution of alkyl magnesium bromide
in tetrahydrofuran (3M) were added. The mixture was stirred
for 4 h at room temperature. After filtration, the solvent was
removed from the clear solution. The product was purified by
column chromatography.
8-Methoxy-1,3,4-trimethyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
(47). See Supporting Information for details.
3,4-Dimethyl-1-ethyl-8-methoxy-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (48). See Supporting Information for details.
3,4-Dimethyl-8-methoxy-1-propyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (49). Yield 87%; mp 91-93 °C; C15H18N4O; MW
1
270.33. H NMR (300 MHz, CDCL3) δ 7.87 (d, J = 9 Hz, 1
H), 6.73 (d, J = 9 Hz, 1 H), 3.96 (s, 3 H), 3.51 (t, J = 8 Hz, 2 H),
2.66 (s, 3 H), 2.65 (s, 3 H), 1.84 (m, 2 H), 0.98 (t, J = 7 Hz, 3 H).
13C NMR (300 MHz, CDCL3) δ 160.81, 152.32, 144.46, 139.11,
136.96, 134.63, 126.08, 123.14, 109.42, 54.02, 32.57, 23.57, 22.22,
15.76, 13.80.
8-Methoxy-4-methyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
(50). See Supporting Information for details.
1-Ethyl-8-methoxy-3-methyl-4-methylsulfonylamino-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (38). See Supporting Information
for details.
3,4-Dimethyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine (51).
See Supporting Information for details.
1-Propyl-3,4,8-trimethyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
(52). See Supporting Information for details.
8-Methoxy-3-methyl-4-methylsulfonylamino-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (39). Yield 92%; mp 244-246 °C;
C15H19N5O3S; MW 349.41. 1H NMR (300 MHz, DMSO) δ
10.74 (s, 1 H), 8.16 (d, J = 9 Hz, 1 H), 6.80 (d, J = 9 Hz, 1 H), 3.87
(s, 3 H), 3.32 (t, J = 8 Hz, 2 H), 3.10 (s, 3 H), 2.54 (s, 3 H), 1.75 (m,
2 H), 0.94 (t, J = 7 Hz, 3 H). 13C NMR (300 MHz, DMSO) δ
158.42, 146.76, 146.15, 142.59, 132.23, 130.47, 116.62, 108.55,
53.60, 42.22, 31.66, 20.60, 14.96, 13.21.
8-Difluoromethoxy-3,4-dimethyl-1-propyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (53). See Supporting Information for details.
3,4-Dimethyl-8-methoxy-1-pentyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (54). See Supporting Information for details.
1-Hexyl-8-methoxy-3,4-dimethyl-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (55). See Supporting Information for details.
1-Cyclohexyl-3,4-dimethyl-8-methoxy-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (56). See Supporting Information for details.
3,4-Dimethoxy-1-isobutyl-8-methoxy-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (57). See Supporting Information for details.
4-Ethylsulfonylamino-8-methoxy-3-methyl-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (40). See Supporting Information
for details.

Article
1-Benzyl-3,4-dimethyl-8-methoxy-imidazo[1,5-a]pyrido[3,2-e]-
pyrazine (58). See Supporting Information for details.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4409
The organic layer was separated. The aqueous layer was washed
three times with 10 mL of ethyl acetate. The combined organic
layers were dried with MgSO4. The solvent was removed by
evaporation. The crude product was purified by column chro-
matography.
8-Methoxy-3,4-dimethyl-1-phenethyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (59). See Supporting Information for details.
4-Difluoromethyl-8-methoxy-3-methyl-1-propyl-imidazo[1,5-a]-
pyrido[3,2-e]pyrazine (60). To a solution of 100 mmol of 4-methyl-
2-propyl-imidazole in 500 mL of dimethylformamide 120 mmol of
KOH were added at 0 °C. The mixture was stirred for 10 min, then
100 mmol of 2-chloro-6-methoxy-3-nitropyridine were added to
the mixture. The resulting solution was stirred at room tempera-
ture for 2 h. The solvent was removed under vacuum. The residue
was diluted with water and extracted with 3 ꢀ 100 mL of ethyl
acetate. The organic layer was washed with water and dried
with magnesium sulfate. The solvent was evaporated under
vacuum. The residue was purified by column chromatography to
provide pure 6-methoxy-2-(4-methyl-2-propyl-imidazol-1-yl)-3-
nitropyridine.
90 mmol of 6-methoxy-2-(4-methyl-2-propyl-imidazol-1-yl)-
3-nitropyridine and 5 mmol of the palladium catalyst were
stirred in 250 mL of tetrahydrofurane and 250 mL of methanol,
then 500 mmol of ammonium formiate were added in small
portions. This mixture was stirred at room temperature for 15
min and at 50 °C for 1 h. Filtration at room temperature yielded
a clear solution. The solvent was removed. The reaction product
3-amino-6-methoxy-2-(4-methyl-2-propyl-imidazol-1-yl)-pyr-
idine precipitated.
1
Yield 58%; mp 107-110 °C; C15H15F3N4O; MW 324.3. H
NMR (300 MHz, DMSO) δ 8.22 (d, J = 9 Hz, 1 H), 7.03 (d, J =
9 Hz, 1 H), 4.01 (s, 3 H), 3.51 (t, J = 8 Hz, 2 H), 2.51 (q, J = 1.5
Hz, 3 H), 1.83 (m, 2 H), 0.98 (t, J = 7 Hz, 3 H). 13C NMR (300
MHz, DMSO) δ 162.64, 145.31, 140.95, 138.51 (q, J = 36 Hz),
137.33, 133.86, 132.22, 120.30 (q, J = 275 Hz), 118.30, 110.56,
54.44, 32.02, 21.23, 14.69 (q, J = 4 Hz), 13.60.
4-Ethyl-8-methoxy-3-methyl-1-propyl-imidazo[1,5-a]pyrido-
[3,2-e]pyrazine (62). This compound was synthesized accord-
ing to the general procedure for compounds 47-59 using ethyl
magnesium bromide.
Yield 67%; mp 78-81 °C; C16H20N4O; MW 284.36. 1H
NMR (300 MHz, CDCL3) δ 7.85 (d, J = 9 Hz, 1 H), 6.68 (d,
J = 9 Hz, 1 H), 3.92 (s, 3 H), 3.47 (t, J = 8 Hz, 2 H), 2.91 (q, J =
7 Hz, 2 H), 2.61 (s, 3 H), 1.81 (m, 2 H), 1.28 (t, J = 7 Hz, 3 H),
0.96 (t, J = 7 Hz, 3 H). 13C NMR (300 MHz, CDCL3) δ 160.67,
156.78, 144.37, 139.10, 136.75, 133.62, 126.04, 122.52, 109.24,
53.92, 32.59, 29.03, 22.15, 15.75, 13.76, 12.12.
8-Methoxy-3-methoxycarbonyl-4-methyl-1-propyl-imidazo-
[1,5-a]pyrido[3,2-e]pyrazine (63). This compound was synthe-
sized according to the procedure for compound 60. 2-Propyl-
imidazole-4-yl carboxylic acid methyl ester was used instead of
4-methyl-2-propyl-imidazole in the first step and acetic acid
anhydride instead of difluoroacetic acid anhydride in the third
step.
80 mmol of 3-amino-6-methoxy-2-(4-methyl-2-propyl-imida-
zol-1-yl)-pyridine was solved in 15 mL of acetic acid and
4 mL of difluoroacetic acid anhydride. The reaction mixture
was stirred at room temperature for 1 h. A mixture of ice-water
and ammonia was added. N-[6-Methoxy-2-(4-methyl-2-propyl-
1
Yield 73%; mp 181-182 °C; C16H18N4O3; MW 314.34. H
NMR (300 MHz, DMSO) δ 8.22 (d, J = 9 Hz, 1 H), 7.10 (d, J =
9 Hz, 1 H), 4.04 (s, 3 H), 3.88 (s, 3 H), 3.61 (t, J = 8 Hz, 2 H), 2.84
(s, 3 H), 1.87 (m, 2 H), 1.00 (t, J = 7 Hz, 3 H).
imidazol-1-yl)-pyridin-3-yl]-difluoroacetamide
precipitated
slowly. The product was filtered off and dried at 40 °C under
vacuum.
3-Ethoxycarbonyl-8-methoxy-4-methyl-1-propyl-imidazo[1,5-
a]pyrido[3,2-e]pyrazine (64). This compound was synthesized
according to the procedure for compound 60. 2-Propyl-imida-
zole-4-yl carboxylic acid ethyl ester was used instead of 4-meth-
yl-2-propyl-imidazole in the first step and acetic acid anhydride
instead of difluoroacetic acid anhydride in the third step.
Yield 71%; mp 158-159.5 °C; C17H20N4O3; MW 328.37. 1H
NMR (300 MHz, DMSO) δ 8.22 (d, J = 9 Hz, 1 H), 7.11 (d, J =
9 Hz, 1 H), 4.36 (q, J = 7 Hz, 2 H),4.04 (s, 3 H), 3.62 (t, J = 7 Hz,
2 H) 2.83 (s, 3 H), 1.87 (m, 2 H), 1.35 (t, J = 7 Hz, 3 H), 1.00 (t,
J = 7 Hz, 3 H). 13C NMR (300 MHz, DMSO) δ 160.19, 150.83,
144.78, 139.87, 135.34, 126.44, 125.72, 110.90, 60.58, 54.16,
32.29, 24.75, 21.22, 14.04, 13.49.
Screening. Inhibition of recombinant PDE10A (baculovirus/
SF21 system). The DNA of PDE10A1 (AB 020593, 2340 bp) was
synthesized and cloned into vector pCR4.TOPO (Entelechon
GmbH, Regensburg, Germany). The gene was then inserted into
a baculovirus vector, ligated with the baculovirus DNA, and the
enzyme protein expressed in SF21 cells. To isolate the enzyme, cells
were first harvested by centrifugation at 500 g followed by resus-
pension in 50 mM Tris-HCl/1 mM EDTA/250 mM sucrose buffer,
pH = 7.4. (Sigma, Deisenhofen, Germany; Merck, Darmstadt,
Germany) and lysis by sonication (3 ꢀ 15 s, Labsonic U, Fa. Braun,
Degersheim, Switzerland, level setting “high”). Cytosolic PDE10A
was obtained by centrifugation at 48000g for 1 h. The supernatant
containing the active enzyme was stored at -70 °C.
50 mmol of N-[6-Methoxy-2-(4-methyl-2-propyl-imidazol-1-
yl)-pyridin-3-yl]-difluoroacetamide were stirred in 30 mL of
POCL3, then 3 g of P2O5 were added. The reaction mixture
was placed into an autoclave and heated up to 180 °C for 11 h,
then it was pured into a mixture of ice-water and ammonia. The
crude product precipitated. It was filtered off and purified by
column chromatography.
Yield 88%; mp 114-117 °C; C15H16F2N4O; MW 306.31. 1H
NMR (300 MHz, DMSO) δ 8.19 (d, J = 9 Hz, 1 H), 7.10 (t, J =
54 Hz, 1 H), 7.02 (d, J = 9 Hz, 1 H), 4.01 (s, 3 H), 3.52 (t, J = 8
Hz, 2 H), 2.57 (s, 3 H), 1.83 (m, 2 H), 0.98 (t, J = 7 Hz, 3 H). 13
C
NMR (300 MHz, DMSO) δ 162.08, 144.96, 144.64 (t, J = 27
Hz), 140.64, 137.10, 134.09, 123.99, 119.30, 114.09 (t, J = 244
Hz), 110.22, 54.30, 31.98, 21.32, 14.99, 13.61.
8-Methoxy-3-methyl-1-propyl-4-trifluoromethyl-imidazo[1,5-
a]pyrido[3,2-e]pyrazine (61). First, 10 mmol of 4-chloro-8-meth-
oxy-3-methyl-1-propyl-imidazo[1,5-a]pyrido[3,2-e]pyrazine
and 15 mmol of HI (aqueous solution, 57%) were stirred at 0 °C
for 3 h, then the reaction mixture was poured in 200 mL of an
aqueous solution of NaHCO3. To the stirred mixture, Na2S2O5,
200 mL of water, and 200 mL of ethyl acetate were added. The
organic layer was separated. The aqueous layer was washed
twice with 50 mL of ethyl acetate. The combined organic layers
were dried with MgSO4. The solvent was removed by evapora-
tion. The crude intermediate is used in the next step without
further purification.
A mixture of 0.75 g KF and 2.5 g CuI was stirred and heated
under vacuum until a homogeneous green color of the mixture
was obtained. The reaction was cooled to room temperature,
and the same procedure was done three times. After cooling to
room temperature, 20 mL of NMP and 1.7 mL of CF3Si(CH3)3
were added under nitrogen. The reaction mixture was stirred at
50 °C for 45 min. It was cooled to room temperature, and the
previous prepared intermediate was added under nitrogen. The
reaction mixture was stirred at 50 °C for 20 h. The mixture was
cooled to room temperature, and aqueous ammonia was added.
PDE activity was determined applying a one-step procedure
in microtiter plates. The reaction mixture contained 50 mM
Tris-HCl/5 mM MgCl2 buffer (pH = 7.4. Sigma, Deisenhofen,
Germany; Merck, Darmstadt, Germany), 0.1 μM [3H] cAMP
(Amersham, Buckinghamshire, UK), and the enzyme in a total
volume of 100 μL. Nonspecific activity was determined in the
absence of enzyme. The reaction was initiated by addition of the
substrate solution and carried out at 37 °C for 30 min. Enzy-
matic activity was stopped by addition of 25 μL Ysi-SPA-beads
(Amersham-Pharmacia). After 1 h, the mixture was quantified

4410 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ho€fgen et al.
in a liquid scintillation counter for microtiter plates (Microbeta
Trilux). Pipetting of the incubation mixture was routinely per-
formed using the Biomek 2000 (Beckman). The optimal amount
of enzyme to use per assay was determined for each enzyme
preparationbatchprior tousageincompoundtesting. IC50 values
were determined using the Hill plot, two-parameter model.
MK-801-Induced Psychosis in Rats. Animals: Female Wistar
rats (Crl: (WI) BR from Charles River, Germany) weighing
160-180 g were used. They were housed in groups of five under
standard conditions on a 12 h light/dark cycle (lights on at 06:00
h) with ad libitum access to water and food (ssniff M/R 15,
Acknowledgment. We thank Proteros Biostructures
GmbH (Martinsried) for solving the crystal structure of the
PDE10A/compound 49 complex. The presented investiga-
tions were funded by EFRE grants from the EU and by grants
from the Free State of Saxony (project no. 12525).
Supporting Information Available: Analytical and spectro-
scopic results for compounds 4-64, QSAR details, detailed
pharmacological results, and X-ray crystallographic data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
€
Spezialdiaten GmbH, Soest/Westfalen, Germany). Experiments
were approved by the Committee for Animal Care and Usage of
the Federal State of Saxony and carried out in accordance with
German animal protection laws.
Experimental procedure: Behavior induced by the NMDA
antagonist MK-801 is generally accepted as a rat model of
psychosis. MK-801 induced stereotypies and hyperactivity in
rats after intraperitoneal administration.28
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Stereotyped sniffing was scored by the experimenter every 5
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28
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ꢀ
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100 - P
IC50E ¼ ½Iꢁ ꢀ
ð1Þ
P
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˚
including residues within a radius of 18 A. The number of
docking runs was set to 30 for each compound and “early
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an essential role for binding of 49 (Figure 3) was retained.
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bond to Gln716.
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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4411
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