2,6-Diaryl-4-acylaminopyrimidines as potent and selective adenosine A2A antagonists with improved solubility and metabolic stability

Article abstract of DOI:10.1016/j.bmcl.2008.09.048

In this report, the strategy and outcome of expanding SAR exploration to improve solubility and metabolic stability are discussed. Compound 35 exhibited excellent potency, selectivity over A₁ and improved solubility of >4 mg/mL at pH 8.0. In addition, compound 35 had good metabolic stability with a scaled intrinsic clearance of 3 mL/min/kg (HLM) and demonstrated efficacy in the haloperidol induced catalepsy model.

Full text of DOI:10.1016/j.bmcl.2008.09.048

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5402–5405
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,6-Diaryl-4-acylaminopyrimidines as potent and selective adenosine
A2A antagonists with improved solubility and metabolic stability
a
a
a
a
a
Manisha Moorjani a, , Zhiyong Luo , Emily Lin , Binh G. Vong , Yongsheng Chen , Xiaohu Zhang ,
Jaimie K. Rueter a, Raymond S. Gross a, Marion C. Lanier a, John E. Tellew a, John P. Williams a,
Sandra M. Lechner b, Siobhan Malany c, Mark Santos c, María I. Crespo d, José-Luis Díaz d,
John Saunders a, Deborah H. Slee a
*
a Department of Medicinal Chemistry, Neurocrine Biosciences, 12780 El Camino Real, San Diego, CA 92130, USA
b Department of Neuroscience, Neurocrine Biosciences, 12780 El Camino Real, San Diego, CA 92130, USA
c Departments of Pharmacology and Lead Discovery, Neurocrine Biosciences, 12780 El Camino Real, San Diego, CA 92130, USA
d Almirall Research Center, Almirall, Ctra. Laureà Miró, 408-410, E-08980 St. Feliu de Llobregat, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2008
Revised 11 September 2008
Accepted 11 September 2008
Available online 14 September 2008
In this report, the strategy and outcome of expanding SAR exploration to improve solubility and meta-
bolic stability are discussed. Compound 35 exhibited excellent potency, selectivity over A1 and improved
solubility of >4 mg/mL at pH 8.0. In addition, compound 35 had good metabolic stability with a scaled
intrinsic clearance of 3 mL/min/kg (HLM) and demonstrated efficacy in the haloperidol induced catalepsy
model.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
A2A antagonists
Adenosine receptor
Parkinson’s disease
Adenosine is considered to be one of the human body’s most
important neuromodulators, in both the central and peripheral
nervous systems.1 The effects of this purine nucleoside are modu-
lated via four receptor subtypes: A1, A2A, A2B and A3.2 These four
subtypes belong to the family of seven trans-membrane G-protein
coupled receptors (GPCRs).3 Adenosine A2A receptors are highly
distributed in the central nervous system and are found in abun-
dance in the basal ganglia, a region of the brain associated with
motor function.4 A number of A2A receptor antagonists have been
shown to improve motor disabilities in animal models of Parkin-
son’s disease.5 Parkinson’s disease is a debilitating motor disorder
arising from the progressive degeneration of dopaminergic neu-
rons in the nigrostriatal pathway.6 Unfortunately, current dopa-
mine replacement therapies for Parkinson’s disease suffer from
poor long term control and undesirable side effects, namely dyski-
nesia (involuntary movements). A number of companies have pro-
gressed A2A antagonists into clinical trials including KW-6002
(istradefylline) from Kyowa Hakko Kogyo, which showed efficacy
in alleviating symptoms of the disease in Phase II clinical trials.7
In addition, Schering-Plough has progressed SCH 420814 into
Phase II clinical trials (Fig. 1).8
Previously, we reported on a series of non-xanthine based A2A
antagonists which incorporated a pyrimidine core. A number of
compounds from our initial exploration, including compound 1,
showed good in vivo efficacy in rat models of Parkinson’s disease.8
However, as this series of compounds contained an unsubstituted
furan ring, we sought to replace this heterocycle (Fig. 2).9 It has
been well documented that unsubstituted furan rings can be
metabolized to form reactive intermediates, which in turn can
form protein adducts leading to liver toxicity.10 A number of het-
erocycles were surveyed in the context of the right hand side
methyl piperazine. Although compound 2 was less active than
the starting lead 1, replacement of the furan with a dimethyl pyr-
azole was tolerated and significantly decreased binding against the
A1 receptor. In addition, we found that by removing the bulky right
hand side methyl piperazine (3), the potency against the A2A recep-
tor was increased. We hoped to further benefit from these findings
by utilizing the dimethyl pyrazole as a furan replacement and
eliminating the need for a bulky right hand side substituent. In
an effort to further increase potency, an SAR exploration was
undertaken to replace the left hand side heterocycle with previ-
ously unexplored substituted phenyl groups.
Compounds 8–35 were prepared from intermediate 49 accord-
ing to the general synthesis outlined in Scheme 1. Compounds 8–
22 were prepared in one step by Suzuki coupling with suitable
* Corresponding author.
E-mail address: mmoorjani@neurocrine.com (M. Moorjani).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.048

M. Moorjani et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5402–5405
5403
O
N
H
N
H
NH2
Ar1
N
O
Cl
O
N
N
O
N
N
N
N
N
N
N
N
O
O
N
N
N
O
O
(a)
O
N
N
N
N
N
N
KW 6002
SCH 420814
4
8-22
Figure 1. Examples of A2A antagonists in clinical development.
(a)
X
X
commercially available boronic acids. For compounds 23–30 and
33–35, intermediate 4 was first reacted with either 3-hydrox-
yphenylboronic acid (23–30) or 3-fluoro-5-hydroxyphenylboronic
acid (33–35) by Suzuki coupling to yield intermediates 22 and
19, respectively. The resulting intermediates were reacted with
alcohols via a Mitsunobu reaction to yield final compounds. Like-
wise, compounds 31–32 were prepared by coupling intermediate
4 with 3-(hydroxymethyl)phenylboronic acid, followed by Mitsun-
obu reaction with the appropriate alcohol in a similar fashion to
compounds 23–30 and 33–35.
H
N
H
N
HO
(b)
O
R2
N
N
N
O
N
N
N
O
N
N
X = H 22
X = F 19
X = H (23-30)
F (33-35)
Scheme 1. Reagents and conditions: (a) Ar1B(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane,
100 °C, 12 h, 60–90%; (b) R2OH, DEAD, PPh3, THF, rt, 12 h, 70–85%.
Replacement of the left hand side heterocycle with a simple
phenyl group (8) showed modest A2A activity but poor selectivity
over the A1 receptor (Ki of 87 nM and A1/A2A of 7). However,
encouraged that some potency remained, we further explored var-
ious substituents. By the addition of a methoxy group (9–11), po-
tency and selectivity were greatly increased. In particular,
substitution in the ortho (9) and meta (10) positions gave very po-
tent compounds with Kis of 2 and 1 nM, respectively, and selectiv-
ity of ꢀ70-fold over A1. A further boost in potency came upon the
addition of another methoxy substituent, as in the case of 3,5-
dimethoxy phenyl (18). Compound 18 not only showed an increase
in potency (Ki of 0.2 nM) but the A1 selectivity was 111-fold. In
addition to having very good potency and selectivity, compound
18 was potent in an A2A functional assay (cAMP IC50 of 29 nM).11
Due to the promising in vitro profile, compound 18 was selected
for in vivo efficacy evaluation. The haloperidol induced catalepsy
(HIC) model in rat was used as the primary assay to screen com-
pounds for efficacy.12 Compound 18 showed good efficacy in the
HIC model with a minimum efficacious dose of 1 mg/kg p.o., how-
ever, it was determined to have poor aqueous solubility (<0.1 mg/
mL at pH 8.0).13 In addition, upon further profiling, compound 18
showed time dependant inhibition of CYP3A4. Compound 21 also
showed promising potency and selectivity with a Ki of 3 nM and
selectivity of 164-fold over A1. Unfortunately, compound 21 also
exhibited poor aqueous solubility of <0.1 mg/mL at pH 8.0. From
this initial survey, we determined that meta substitution off of
the phenyl ring was preferred. Also, the incorporation of a hydro-
gen bond acceptor, as in the case of the 3-methoxyphenyl and
3,5-dimethoxyphenyl, increased A2A potency. However, poor solu-
bility of the most promising compounds, and the inhibition of a
major CYP enzyme for compound 18, prevented further develop-
ment. As such, an SAR exploration was initiated to improve solubil-
ity while maintaining potency at A2A. The idea was to introduce a
basic center off of the phenyl group, while maintaining the pre-
ferred meta substitution pattern and a hydrogen bond acceptor at
that site, in the form of an oxygen atom (see Table 2).
By replacing the methoxy group with an N-dimethylamino eth-
oxy (23), we obtained a potent and selective compound. Solubility
testing at pH 8.0 revealed that incorporation of an amine did in fact
increase the solubility significantly to >4 mg/mL. To reduce the
number of rotatable bonds in an attempt to improve drug like
properties,14 cyclized analogues of compound 23 were made,
exploring ring size and substitution off of the basic nitrogen. The
methyl pyrrolidine compound 24, showed an increase in A2A po-
tency and selectivity over A1 as compared to the straight chain ana-
logue 23. Extending the substitution off of the pyrrolidine to ethyl
(25) and isopropyl (26) resulted in similar potency as the methyl
version (24) but a slight loss of selectivity from 191-fold over A1
to 167-fold was observed. As the six membered piperidine com-
pounds did not significantly increase potency or selectivity (27),
the single enantiomers of compound 24 were made. The S enantio-
mer (30) showed slightly better potency than the R enantiomer
(29) with a Ki of 2 nM versus 4 nM. A more significant improve-
ment was seen in the selectivity over A1 as the S isomer was
320-fold selective, double the selectivity of the R isomer. Further
in vitro profiling of compound 24 revealed similar human func-
tional activity to compound 18 (cAMP IC50 of 42 nM). Unfortu-
nately, this compound showed poor metabolic stability in human
liver microsomes with
a scaled intrinsic clearance value of
54 mL/min/kg.15 As this series proved to have good potency and
selectivity while greatly improving the solubility (compound 24
>4 mg/mL at pH 8.0), we turned to improving the poor metabolic
stability. From our initial survey, we determined that fluorine
atoms were well tolerated off of the phenyl ring (19–21). The addi-
tion of an electron withdrawing group such as a fluorine atom may
help to stabilize the electronic rich phenyl group, thereby improv-
ing the metabolic stability (see Table 3).
The 3-fluoro-5-(N-methyl-3-pyrrolidol) compound 33, showed
good potency with a Ki of 1 nM and selectivity of 135-fold over
A1. The addition of a 3-fluorine atom did not adversely affect the
potency or the selectivity when compared to compound 24. The
S
H
H
N
N
H
N
N
N
N
N
N
N
N
N
N
N
O
O
N
N
N
N
O
N
N
O
O
N
2
1
3
Ki (nM) hA2A
Ki (nM) hA1
27
1700
Ki (nM) hA2A
Ki (nM) hA1
0.6
9.2
Ki (nM) hA2A
Ki (nM) hA
3
270
Figure 2. Potent A2A antagonists

5404
M. Moorjani et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5402–5405
Table 1
Table 2
Binding affinities of 8–21 towards the human A2A receptor and selectivity over the
Binding affinities of 22–32 towards the human A2A receptor and selectivity over the
human A1 receptor
human A1 receptor
H
R
N
N
R
N
N
N
O
N
N
N
O
N
N
a
Compound
R
Ki (nM) hA2A
hA1/hA2A
7
a
Compound
R
Ki (nM) hA2A
hA1/hA2A
74
8
87
22
OH
2
O
O
23
24
25
26
27
5
2
3
3
6
134
191
168
167
146
9
2
1
71
72
N
N
O
O
O
10
N
O
11
12
17
7
44
59
N
O
N
O
CN
13
2
10
N
28
29
30
31
1
203
145
320
99
O
NC
N
4
O
O
14
15
2
2
50
40
N
N
2
CF3
67
O
O
N
32
73
102
16
17
18
19
8
43
a
See footnotes of Table 1.
19
0.2
2
46
Table 3
Binding affinities of 33–35 towards the human A2A receptor, selectivity over the
human A1 receptor and intrinsic clearance
O
111
54
F
O
H
OH
N
R
N
N
N
O
F
N
F
20
4
3
64
a
b
Compound
R
Ki (nM) hA2A
hA1/hA2A
CLint (HLM)
F
F
(mL/min/kg)
N
N
N
21
164
33
34
1
2
1
135
125
218
3
O
O
O
O
3
3
a
Displacement of specific [3H]-DPCPX binding at human A1 receptors expressed
in HEK293 cells. Displacement of specific [3H]-ZM241385 binding at human A2A
expressed in HEK293. Data are expressed as geometric means of at least three runs
with a standard deviation less than or equal to 20%.17
35
a
See footnotes of Table 1.
See note 15.
b
racemic compound was followed up with the single isomers. As
seen with the des-fluoro analogues, both the R (34) and S (35) iso-
mers had similar A2A potency to the racemic compound. However,
a significant difference in selectivity over A1 was noted with the R
isomer being 125-fold selective and the S over 200-fold selective.
The more promising compound 35 was further profiled in vitro
and found to have better functional activity than compound 18
with a cAMP IC50 of 15 nM. In addition, compound 35 showed

M. Moorjani et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5402–5405
5405
7. (a) Hockemeyer, J.; Burbiel, J. C.; Muller, C. E. J. Org. Chem. 2004, 69, 3308; (b)
Kyowa Hakko Kogyo company website: http://www.kyowa.co.jp/eng/netext/
ero60307.htm.
8. Slee, D. H.; Zhang, X.; Moorjani, M.; Lin, E.; Lanier, M. C.; Chen, Y.; Rueter, J. K.;
Lechner, S. M.; Markison, S.; Malany, S.; Joswig, T.; Santos, M.; Gross, R. S.;
Williams, J. P.; Castro-Palomino, J. C.; Crespo, M. I.; Prat, M.; Gual, S.; Díaz, J. L.;
Jalali, K.; Sai, Y.; Zuo, Z.; Yang, C.; Wen, J.; O’Brien, Z.; Saunders, J. J. Med. Chem.
2008, 51, 400.
9. Slee, D. H.; Chen, Y.; Zhang, X.; Moorjani, M.; Lanier, M. C.; Lin, E.; Rueter, J. K.;
Williams, J. P.; Lechner, S. M.; Markison, S.; Malany, S.; Santos, M.; Gross, R. S.;
Jalali, K.; Sai, Y.; Zuo, Z.; Castro-Palomino, J. C.; Crespo, M. I.; Prat, M.; Gual, S.;
Díaz, J. L.; Saunders, J. J. Med. Chem. 2008, 51, 1719.
10. (a) Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh-Bakht, S. C.; Obach, R. S.; O’Donnell,
J. P. Chem. Res. Toxicol. 2002, 15, 269; (b) Evans, D. C.; Watt, A. P.; Nicoll-Griffith,
D. A.; Baillie, T. A. Chem. Res. Toxicol. 2004, 17, 3.
11. General experimental details for this assay may be found in the following
reference Selkirk, J. V.; Nottebaum, L. M.; Ford, I. C.; Santos, M.; Malany, S.;
Foster, A. C.; Lechner, S. M. J. Biomol. Screen. 2006, 11, 351.
12. General experimental details for this assay may be found in the following
reference Slee, D. H.; Zhang, X.; Moorjani, M.; Lin, E.; Lanier, M. C.; Chen, Y.;
Rueter, J. K.; Lechner, S. M.; Markison, S.; Malany, S.; Joswig, T.; Santos, M.;
Gross, R. S.; Williams, J. P.; Castro-Palomino, J. C.; Crespo, M. I.; Prat, M.; Gual,
S.; Díaz, J. L.; Jalali, K.; Sai, Y.; Zuo, Z.; Yang, C.; Wen, J.; O’Brien, Z.; Saunders, J. J.
Med. Chem. 2008, 51, 400.
excellent solubility of >4 mg/mL at pH 8.0. Furthermore, by the
introduction of a fluorine atom on the electron rich phenyl ring,
the metabolic stability in human liver microsomes was signifi-
cantly increased from 54 mL/min/kg (scaled intrinsic clearance)
for compound 24 to 3 mL/min/kg for compound 35. In addition,
compound 35 was not a potent inhibitor of the major CYP enzymes
(CYP3A4 and 2D6).16 Due to the promising in vitro profile, good sol-
ubility and improved metabolic stability, compound 35 was as-
sayed in the rat efficacy model (HIC), and demonstrated
significant effect at 30 mg/kg p.o.
a
In summary, we have found potent and selective A2A antago-
nists which show efficacy in the haloperidol induced catalepsy
model. By introducing small basic phenyl substituents, we greatly
improved solubility from <0.1 mg/mL at pH 8.0 to >4 mg/mL. In
addition, a significant increase in metabolic stability was achieved
while maintaining a promising in vitro profile. We have identified a
potent, soluble, and metabolically stable series with the best com-
pound showing significant efficacy in the HIC model at 30 mg/kg.
13. To determine solubility of compounds, approximately 1 mg of sample was
weighed into a 15 mL Falcon Centrifuge tube, and the weight recorded to
Acknowledgment
0.001 mg. Assay medium, (200 lL, Phosphate Buffer Solution pH 8.0) was
added and the sample sonicated for 10 min, then shaken overnight. The sample
was then centrifuged and the supernatant was analyzed by HPLC to determine
the concentration of sample in solution. The concentration in solution was then
calculated based on a standard curve generated from known dilutions of
authentic sample.
We are indebted to Shawn Ayube, Chris DeVore, Paddi Ekhlassi,
and John Harman for their analytical support.
References and notes
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Connors, P. J.; Rowbottom, M. W.; Chen, Y.; Struthers, R. S.; Xie, Q.; Saunders, J.;
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16. Inhibition assays were carried out using microsomes isolated from transfected
cells expressing only CYP3A4, and in the presence of the fluorescent substrate
BFC. Ketoconazole was used as a positive control. The CYP2D6 assay was
carried out in the presence of the fluorescent substrate, AMMC. Quinidine was
used as a positive control. All compounds described with an IC50 < 30
assayed in 2 or 3 experiments.
lM were
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17. On each assay plate, a standard antagonist of comparable affinity to those
being tested was included as a control for plate-to-plate variability. Overall Ki
values were highly reproducible, the standard deviations were less than or
equal to 20%. All compounds reported were assayed in 3–6 independent
experiments.