Synthesis and SAR studies of analogues of 4-(3,3-dimethyl-butyrylamino)-3,5-difluoro-N-thiazol-2-yl-benzamide (Lu AA41063) as adenosine A2A receptor ligands with improved aqueous solubility

Article abstract of DOI:10.1016/j.bmcl.2015.01.062

An adenosine A_2A receptor antagonist may be useful for the treatment of Parkinson's disease. Synthesis and structure-activity studies starting from 4-(3,3-dimethylbutyrylamino)-3,5-difluoro-N-thiazol-2-yl-benzamide (Lu AA41063, 4) led to a novel series of human (h) A_2A receptor antagonists with improved aqueous solubility. Compound 22 was identified as a key representative from the series, displaying submicromolar hA_2A receptor affinity and excellent aqueous solubility. Compound 22 also displayed good in vitro pharmacokinetic properties and is considered a good starting point for further lead optimisation toward hA_2A receptor antagonists with improved druggability properties.

Full text of DOI:10.1016/j.bmcl.2015.01.062

Accepted Manuscript
Synthesis and SAR studies of analogues of 4-(3,3-dimethyl-butyrylamino)-3,5-
difluoro-N-thiazol-2-yl-benzamide (Lu AA41063) as adenosine A2A receptor
ligands with improved aqueous solubility
Gitte Kobberøe Mikkelsen, Morten Langgård, Tenna Juul Schrøder, Mads
Kreilgaard, Erling B. Jørgensen, Guillaume Brandt, Yann Griffon, Ray Boffey,
Benny Bang-Andersen
PII:
S0960-894X(15)00083-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.01.062
BMCL 22402
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 December 2014
26 January 2015
27 January 2015
Please cite this article as: Mikkelsen, G.K., Langgård, M., Schrøder, T.J., Kreilgaard, M., Jørgensen, E.B., Brandt,
G., Griffon, Y., Boffey, R., Bang-Andersen, B., Synthesis and SAR studies of analogues of 4-(3,3-dimethyl-
butyrylamino)-3,5-difluoro-N-thiazol-2-yl-benzamide (Lu AA41063) as adenosine A2A receptor ligands with
improved aqueous solubility, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/
j.bmcl.2015.01.062
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Synthesis and SAR studies of analogues of 4-(3,3-dimethyl-butyrylamino)-3,5-difluoro-
N-thiazol-2-yl-benzamide (Lu AA41063) as adenosine A2A receptor ligands with
improved aqueous solubility
Gitte Kobberøe Mikkelsena, Morten Langgårdb , Tenna Juul Schrøderc, Mads Kreilgaarda,
Erling B. Jørgensend, Guillaume Brandte, Yann Griffone, Ray Boffeye and Benny Bang-
Andersena
c
aDepartments of Discovery Chemistry & DMPK, bComputational Chemistry, Molecular
Pharmacology, and dBiologics & Pharmaceutical Science Department, H. Lundbeck A/S, 9
Ottiliavej, DK-2500 Valby, Denmark; eArgenta Discovery Ltd, 8-9 The Spire Green Centre
Harlow CM19 5TR, UK
* Corresponding author. Gitte Kobberøe Mikkelsen, H. Lundbeck A/S, Ottiliavej 9, 2500
Valby, Denmark. Email: gmi@lundbeck.com.
Abstract:
An adenosine A2A receptor antagonist may be useful for the treatment of Parkinson’s disease.
Synthesis and structure-activity studies starting from (4-(3,3-dimethylbutanamido)-3,5-
difluoro-N-(thiazol-2-yl)benzamide (Lu AA41063, 4) led to a novel series of human (h) A2A
receptor antagonists with improved aqueous solubility. Compound 22 was identified as a key
representative from the series, displaying submicromolar hA2A receptor affinity and excellent
aqueous solubility. Compound 22 also displayed good in vitro pharmacokinetic properties
and is considered a good starting point for further lead optimisation toward hA2A receptor
antagonists with improved druggability properties.
Keywords: hA2A, solubility

The neurotransmitter adenosine interacts with the four seven transmembrane G protein-
coupled receptors A1, A2A, A2B and A3.1 The adenosine receptors are located in several
tissues, including the central nervous system (CNS)2 and adenosine A2A receptors are
predominantly expressed in the dopamine rich areas of the CNS such as the striatum.
Currently, the interactions between A2A and D2 receptors are of great interest in the treatment
for Parkinson´s disease (PD), which involves a decrease in dopamine levels. The A2A
receptors interact tonically and antagonistically with the D2 receptors, causing a decrease in
affinity of the D2 receptors for dopamine upon stimulation. Thus, an A2A receptor antagonist
may be useful as a monotherapy for the treatment of PD. Alternatively, A2A receptor
antagonists may be capable of enhancing the effect of clinically used dopamine receptor
agonists for example by increase the time-period of dopaminergic drug response.3,4
There has been an extensive effort over the past decade to synthesise novel and selective A2A
receptor antagonists,5, 6 and recently istradefylline (KW-6002, 1)7 was launched under the
name Nouriast® as the first antiparkinsonian agent based on A2A receptor antagonism. Other
companies such as Vernalis (i.e. V-81444 (2)) and Roche currently have A2A receptor
antagonists in Phase II clinical trials.
((Figure 1, single-column width))
Figure 1. Structures and human (h) A2A and hA1 receptor binding affinities of istradefylline (KW-6002, 1), V-
81444 (2) from Vernalis, and Lu AA41063 (4) that is derived from general structure 3.9

Common for many A2A receptor antagonists are their planar aromatic structure, which often
results in low aqueous solubility. This has complicated the development of drugs for this
target in general. Several groups have reported different attempts to improve the aqueous
solubility of A2A receptor antagonists.13, 14
We have previously reported a new class of potent and selective human (h) A2A receptor
antagonists of the general structure 3 (Figure 1).9 Among these compounds, Lu AA41063 (4)
and its water soluble prodrug was identified as a highly selective hA2A receptor antagonist
with effect in an in vivo model of PD. However, this series of compounds in general suffer
from poor aqueous solubility, which complicated the solid dose formulation.9 For example
the aqueous solubility of compound 4 is 1 µg/mL at pH 7.4. To avoid the complications of
prodrug formulation we explored the possibilities to significantly improve the aqueous
solubility of compounds based on the general structure of 3.
We set out to investigate the structure-activity and structure-solubility relationships within
general structure 3, in order to identify space that would allow for the introduction of
solubilising groups at positions with only limited effect on hA2A receptor affinity.
From previously disclosed SAR within this series it was known that modifications around the
aminothiazole were not tolerated.9 An in-house homology model of the A2A receptor based
on the published experimental model 1UPE (PDB code) was used to rationalize the
observation. Figure 2 shows compound 4 docked into the later published crystal structure
PDB: 3EML structure. This model confirmed our hypothesis that the aminothiazole is
orientated inwards not allowing substitution and the alkyl chain pointing in the direction of
the extracellular domain.
((Figure 2, single-column width))

Figure 2. Docking of compound 4 docked into the later published crystal structure PDB:
3EML structure
Firstly, taking compounds 5a and 5b ( Figure 3) as starting points for our effort , we
investigated the effect of breaking the planarity of general structure 3 by reducing the
carbonyl groups to methylene linkers. The replacement of the thiazolyl carbonyl moiety in
compound 5a with a methylene spacer (compound 6) resulted in complete loss of hA2A
receptor affinity. Replacement of the other carbonyl group in compound 5b with a methylene
spacer resulted in compound 7 with a substantially weaker hA2A receptor affinity when
compared to compound 5b.
Introducing a nitrogen atom at the 2- or 3-position of the central benzene ring led to
compounds 8 and 9. Compound 9 was inactive at the hA2A receptor, while compound 8 lost a
factor of 10 when compared to 5a. This could be an acceptable starting point if the aqueous
solubility would have increased significantly. However, the aqueous solubility of compound
8 was only 10 µg/mL and this was considered to be insufficient for a starting point for further
optimization. Introduction of a hydroxy group in the neo-pentyl group (compound 10)
resulted in significant loss of hA2A receptor affinity compared to compound 5a,b.
((Figure 3 , double-column width))

O
S
S
S
O
O
N
N
N
N
H
N
H
N
H
O
N
H
N
H
N
H
R
5
7
6
a) R= Me, hA2A Ki = 36 nM
b) R=H, hA2A Ki = 59 nM
hA2A Ki >6000 nM
O
hA2A Ki = 730 nM
O
O
S
S
S
N
O
O
N
N
N
N
N
H
N
H
N
H
O
OH
N
H
N
H
N
H
8
9
10
hA2A Ki = 300nM
% inhibition hA2A @ 1000 nM = 30%
hA2A Ki = 1100 nM
Figure 3. Structures human (h) A2A receptor binding affinities for compounds 5a and 5b and
structural modifications thereof.
Next we turned to examine the effect of introducing a more basic nitrogen atom in the right
hand side amide alkyl chain (Table 1). Previous SAR investigations had taught us that the β-
branching of the alkyl chain was necessary to achieve high affinity for the hA2A receptor.9
Replacing the β-carbon atom in 11 by a nitrogen atom resulted in a complete loss of hA2A
receptor affinity (compound 12). Similarly, replacing one of the carbon atoms in the
cyclopentane ring of 13 by a nitrogen atom led to significant reduction of hA2A receptor
affinity (compounds 14 and 15). The docked pose of 4 (Figure 2) suggests that to reach the
solvent exposed region of the receptor, a longer linking group would be needed between the
core and the polar moieties. Therefore we extended the R2 alkyl group in order to probe if a
basic nitrogen atom could be introduced at a more distant position from the central benzene
ring, using non-β-branched model systems to probe this hypothesis. In short, replacing the
terminal carbon atom in compound 16 with a nitrogen atom resulted in complete loss of
affinity to the hA2A receptor (compound 17), whereas the homologue 18 with a chain of four
carbon atoms between the carbonyl and the basic nitrogen atom displayed a hA2A receptor
affinity of 830 nM. Compounds with even longer carbon chains of five and seven carbon
atoms between the carbonyl and the nitrogen atom showed increasing affinity for the hA2A
receptor (compounds 19 and 20).
Reintroducing a methyl group at the β-position of compound 18 resulted in compound 21,
which showed a 7-fold improvement in hA2A receptor affinity when compared to 18.
Compound 21 showed submicromolar hA2A receptor affinity (Ki = 110 nM). Restricting the

terminal ethyl groups in a morpholine ring resulted in the equipotent compound 22, which
had a good selectivity toward the hA1 receptor (IC50 > 2000 nM).
((Table 1, single-column width))
Table 1. Compound 5a and right hand side analogues.
O
S
R1
N
N
H
R2
N
H

A2A Ki (nM)
or %inhib.a
R1
H
R2
Cpd
O
O
O
5a
36
11 3-Me
12 3-Me
67
N
1%
O
13
14
15
16
17
H
H
H
H
H
67
O
N
6600
>2100
190
N
O
O
O
-18%
N
O
N
18
19
20
H
H
H
830
240
130
O
N
O
N
O
O
N
N
21
22
H
H
110
110
O
aExpressed as Ki values (nM) or percentage displacement of radioligand at 1000 nM test concentration where indicated.
Compound 22 showed no significant inhibition of CYP1A2, CYP2C9, CYP2C19 and
CYP3A4 liver enzymes (IC50 > 40 µM) as well as CYP2D6 (IC50 = 18 µM), and displayed
low in vitro clearance in human liver microsomes (hClint = 0.62 L/kg/h). The aqueous
solubility at pH=7.4 of compound 22 was significantly higher (116 µg/mL) than for Lu
AA41063 (4) (1 µg/mL). Consequently this compound was considered the overall best

compound of the series and a good starting point for further investigations towards druggable
hA2A receptor antagonists.
Compound 5a, 5b, 11, 13 and 16 were synthesised as previously described9, 11
.
((Scheme 1, double-column width))
S
O
S
S
i, ii
iii
O
N
+
N
H
H
N
N
H
N
NH2
N
H
R= NO2
R= NH2
NO2
R
6
O
S
O
S
O
iv
N
N
H
+
N
N
H
H
N
H
NH2
7
O
O
S
O
vi
O
v
O
O
HO
N
N
HO
+
H
X
Cl
X
X
N
H
Y
N
H
Y
NH2
Y
8: X=N, Y=C
9: X=C, Y=N
O
S
O
S
O
vii
OH
O
N
N
H
N
N
H
OH
+
HO
N
H
NH2
10
Scheme 1. Reaction conditions: (i) NaBH(OAc)3, AcOH, 1,2-DCE; (ii) Zn, EtOH, HCl,
40°C; (iii) 3,3-dimethyl-butyryl chloride, pyridine, 0° to r.t.; (iv) NaBH(OAc)3, AcOH, 1,2-
DCE, DMSO; (v) pyridine, 0° to r.t.; (vi) (COCl)2, 2-aminothiazole, pyridine 1,2-DCE, DMF;
(vii) HATU, DIPEA, DMF, r.t.
Compounds 6-10 were synthesised in the following manner (Scheme 1). Reductive amination
of 4-nitro-benzaldehyde with 2-aminothiazole, followed by reduction of the nitro group and
acylation using 3,3-dimethyl-butyryl chloride gave compound 6.
Compound 7 was synthesised by reductive amination of 3-methyl-butyraldehyde with 4-
amino-N-thiazol-2-yl-benzamide.9 Acylation of 5-amino-pyridine-2-carboxylic acid or 6-
amino-nicotinic acid with 3,3-dimethyl-butyryl chloride followed by treatment with
oxalylchloride and subsequent reaction with 2-aminothiazole gave the compounds 8 and 9.
Coupling of 4-amino-N-thiazol-2-yl-benzamide9 with 3-hydroxy-3-methyl-butyric acid gave
compound 10.
((Scheme 2, double-column width))

O
N
O
12:
N
N
O
i
a)
S
N
H
O
S
+
N
H
HO
R1
HN
14:
17:
R1
NH2
N
H
NH2
O
X
20: X=H
21: X=CH3
N
O
O
b)
O
+
ii for R=Cl
iii for R=OH
R
Br
n
O
NH2
vi, vii
n = 1-3
O
O
R= CH3
R= OH
O
Br
N
H
n
O
O
O
iv, v
+
23a: n=1, R=H
23b: n=2, R=H
23c: n=3, R=H
23d: n=1, R=CH3
NH2
O
O
S
N
1
viii
R1
HO
O
O
R
N
H
N
N
H
N
N
H
n
n
17: n=1, R1=H
18: n=2, R1=H
19: n=3, R1=H
O
S
O
viii
O
N
HO
O
N
H
N
N
H
N
N
H
O
O
22
Synthesis of building blocks for 20 and 21:
X
O
X
O
ix
N
HO
O
O
O
X= CH3, H
Scheme 2. Reaction conditions: (i) For compound 12, 14, 16: DMF, HATU, N,N-
diisopropylethylamine, overnight, room temperature followed by treatment with TFA for boc
protected compounds. For compound 20, 21: DMF, EDC, N,N-diisopropylethylamine, 60°C,
3 days (ii) NEt3, DCM, 0°C to room temperature; (iii) DCM, EDCI, DMAP, room
temperature; (iv) AlCl3, DCE (v) CBr4, polystyrene supported PPh3, DCM ; (vi) CH3CN, KI,
K2CO3, diethylamine or morpholine, reflux, overnight; (vii) THF, H2O, LiOH·H2O, room

temperature overnight; or 1M LiOH in methanol, room temperature, overnight; (viii) DCM,
EDCI, DMAP, 2-aminothiazole, room temperature; ix: THF, Et2N, r.t.
Compounds 12, 14 and 17-22 were synthesised in the following manner (Scheme 2).
The basic nitrogen atom in the amide alkyl chain was introduced by different methods.
Compound 12, 14, 17, 20 and 21 were synthesised by coupling of 4-amino-N-thiazol-2-yl-
benzamide10 with carboxylic acids. The boc group was removed from amino acids that
contained a boc group, after the coupling to 4-amino-N-thiazol-2-yl-benzamide.
Intermediate 23a, 23b and 23c were synthesised by acylation of 4-amino-benzoic acid methyl
ester using acid chlorides or by coupling with carboxylic acids. The intermediate 23d was
synthesized by treatment of 4-amino-benzoic acid methyl ester with AlCl3 and 4-methyl-
tetrahydro-pyran-2-one. The formed alcohol was converted to the intermediate bromide 23d
by treatment with CBr4 and polystyrene supported triphenylphosphine in DCM. The
compounds 17-19 and 22 were obtained by alkylation of the intermediates 23a, 23b, 23c and
23d with diethylamine or morpholine followed by hydrolysis of the methyl ester using LiOH
in THF/water, the final coupling with 2-aminothiazole gave the compounds 17-19 and 22.
The building blocks for synthesis of 20 and 21 were synthesised by reaction of glutaric
anhydride or 3-methylglutaric anhydride with diethylamine in THF at room temperature.
In summary, synthesis and structure-activity and -solubility relationship studies of analogues
of Lu AA41063 (4) have resulted in the identification of a series of new adenosine A2A
receptor ligands, including specifically compound 22 that contain a basic nitrogen atom in the
amide alkyl chain of the of 4-carboxamido-N-thiazol-2-yl-benzamide scaffold. Compound 22
showed submicromolar hA2A receptor affinity, good selectivity toward the hA1 receptor, as
well as good in vitro microsomal stability. Furthermore, compound 22 was devoid of any
CYP enzyme inhibition, and it showed significant better aqueous solubility than 4.
Compound 22 is considered a good starting point for further optimisation toward druggable
A2A receptor antagonists.
Acknowlegdement
Thanks to Anette Graven Sams for fruitfull medchem discussions.

Abbreviations
HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
EDC, N-ethyl-N’-dimethylaminopropyl-carbodiimide hydrochloride,
HOBt, 1-hydroxy-benzotriazole
DMAP 4-dimethylaminopyridine
DMF, dimethylformamide
DMSO dimethyl sulfoxide
1,2-DCE 1,2-dichloroethane
AcOH acetic acid
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Table 1. Compound 5a and right hand side analogues.
O
S
R1
N
N
H
R2
N
H
A2A Ki (nM)
or %inhib.a
Cpd
R1
R2
O
H
3-Me
3-Me
H
36
67
5a
11
12
13
14
15
16
17
O
O
O
O
N
1%
67
HN
H
6600
>2100
190
H
N
O
H
O
H
O
O
NH2
H
-18%
N
H
830
18
O
H
H
240
130
19
20
N
O
O
N
N
N
H
110
21
O
H
110
22
O
aExpressed as Ki values (nM) or percentage displacement of radioligand at 1000 nM test concentration where indicated.

Graphical abstract:
O
S
O
S
F
O
N
O
N
N
N
H
H
N
N
H
N
H
O
F
Lu AA41063 (4)
hA2A: Ki = 5.9 nM
aq. solubility @ pH 7.4 = 1g/mL
22
hA2A: Ki = 110 nM
aq. solubility @ pH 7.4 =116 g/mL