Synthesis and pharmacological evaluation of dual acting ligands targeting the adenosine A2A and dopamine D2 receptors for the potential treatment of parkinsons disease

Article abstract of DOI:10.1021/jm501254d

A relatively new strategy in drug discovery is the development of dual acting ligands. These molecules are potentially able to interact at two orthosteric binding sites of a heterodimer simultaneously, possibly resulting in enhanced subtype selectivity, higher affinity, enhanced or modified physiological response, and reduced reliance on multiple drug administration regimens. In this study, we have successfully synthesized a series of classical heterobivalent ligands as well as a series of more integrated and drug-like dual acting molecules, incorporating ropinirole as a dopamine D₂ receptor agonist and ZM 241385 as an adenosine A_2A receptor antagonist. The best compounds of our series maintained the potency of the original pharmacophores at both receptors (adenosine A_2A and dopamine D₂). In addition, the integrated dual acting ligands also showed promising results in preliminary blood-brain barrier permeability tests, whereas the classical heterobivalent ligands are potentially more suited as pharmacological tools.

Full text of DOI:10.1021/jm501254d

Subscriber access provided by University of Sunderland
Article
Synthesis and Pharmacological Evaluation of Dual Acting
Ligands Targeting the Adenosine A2A and Dopamine D2
Receptors for the Potential Treatment of Parkinson’s Disease
Manuela Jorg, Lauren May, Frankie S Mak, Kiew Ching K
Lee, Neil D Miller, Peter J. Scammells, and Ben Capuano
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501254d • Publication Date (Web): 09 Dec 2014
Downloaded from http://pubs.acs.org on December 19, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis and Pharmacological Evaluation of Dual Acting Ligands
Targeting the Adenosine A and Dopamine D Receptors for the Potential
2
A
2
Treatment of Parkinson’s Disease
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
†
‡
║
║
║
Manuela Jörg, Lauren T. May, Frankie S. Mak, Kiew Ching K. Lee, Neil D. Miller, Peter
†
,*
†,*
J. Scammells, Ben Capuano
†
‡
Medicinal Chemistry and Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 381 Royal
║
Parade, Parkville, Victoria 3052, Australia, GSK R&D, Neural Pathways DPU, Neurosciences TAU, 11
Biopolis Way, Helios Bldg #03ꢀ01/02, Singapore 138667, Singapore
ABSTRACT: A relatively new strategy in drug discovery is the development of dual acting
ligands. These molecules are potentially able to interact at two orthosteric binding sites of a
heterodimer simultaneously, possibly resulting in enhanced subtype selectivity, higher
affinity, enhanced or modified physiological response, and reduced reliance on multiple drug
administration regimens. In this study, we have successfully synthesized a series of classical
heterobivalent ligands as well as a series of more integrated and “drugꢀlike” dual acting
2
molecules, incorporating ropinirole as a dopamine D receptor agonist and ZM 241385 as an
adenosine A2A receptor antagonist. The best compounds of our series maintained the potency
of the original pharmacophores at both receptors (adenosine A2A and dopamine D ). In
2
addition, the integrated dual acting ligands also showed promising results in preliminary
bloodꢀbrainꢀbarrier permeability tests, whereas the classical heterobivalent ligands are
potentially more suited as pharmacological tools.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
Parkinson’s disease is a progressive neurodegenerative disorder affecting about 1% of the
1,2
world population over 60 years of age. Mechanistically, current clinical therapeutics almost
exclusively focus on the deficiency in dopamine production in specific parts of the brain
which is associated with the primary symptoms observed in patients with Parkinson’s
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
,4
disease. Dopamine replacement therapies such as the proꢀdrug levodopa or dopamine D
2
receptor (D R) agonists are able to improve the quality of life enormously but are insufficient
2
5ꢀ8
due to side effects and their inability to alter or delay the progression of the disease. Hence,
there is an urgent need for a better understanding of the disease and more effective drugs.
Animal models as well as clinical studies have given evidence that adenosine A2A receptor
(A2AR) antagonists, as monotherapy and in conjunction with levodopa or D
2
R agonists, are
able to amplify the therapeutic effects of levodopa and reduce motor complications such as
9
ꢀ12
dyskinesia, onꢀoff oscillation and wearing off effects in patients with Parkinson’s disease.
Another potential benefit of A2AR antagonists in contrast to current drugs on the market is
their proposed capability of slowing down the degeneration of dopaminergic neurons thereby
1
3,14
delaying the onset and progression of the disease.
Therefore, A2AR antagonists are
considered a promising new drug class that could potentially complement the dopaminergic
drugs currently available for patients suffering from Parkinson’s disease.
Consequently, scientists have investigated the possibility of designing a single molecule
which is able to target the A2AR and D
2
R. A strategy which gained greater attention after the
discovery of A RꢀD R heterodimers is the development of heterobivalent ligands; a
2
A
2
molecule consisting of two different pharmacophores separated through a spacer which is
15,16
linked to each individual drug entity (Fig. 1).
The simultaneous interactions of these
RꢀA2AR heterodimer has the potential to
molecules with both orthosteric binding sites of a D
2
result in enhanced subtype selectivity, higher affinity, enhanced or modified physiological
2
ACS Paragon Plus Environment

Page 3 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
response, and reduced reliance on multiple drug administration regimens. Furthermore, these
heterobivalent systems can be used as pharmacological tools permitting estimations of the
17
distance between the targets and their spatial distribution. Soriano et al. published a series of
heterobivalent ligands (XCCꢀPPHTꢀNH
2
) ranging from 26 to 118 atoms spacer length to
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
target A2ARꢀD R heterodimers. The molecules are constructed of a functionalized xanthineꢀ
2
based adenosine A2AR antagonist (XCC) and a 2ꢀaminoꢀ5ꢀhydroxytetralinꢀbased dopamine
D R agonist (PPHTꢀNH ) component. In this study, higher affinity was observed for the
2
2
heterobivalent ligands compared to the monovalent analog in brain striatum and cells coꢀ
expressing both receptors, whereas in cells expressing just one receptor type no change in
2
affinity was reported. These findings were explained by the existence of A2ARꢀD R
heterodimers in brain striatum and simultaneous binding of the heterobivalent ligands to both
receptors. Even though, such molecules are interesting pharmacological tools, their use as
drugs is potentially limited due to their size.
Consequently, the design of integrated dual acting ligands (also referred to as merged or fused
designed multiple ligands or twin ligands) is a more promising approach for the development
1
8
of drugs for the treatment of CNS drugs such as Parkinson’s disease (Fig. 1). The
advantages of integrated dual acting ligands over heterobivalent ligands are the reduction in
molecular mass, topological polar surface area and clogP, which is equivalent to the
enhancement of the “drugꢀlike” properties including their ability to cross the blood brain
barrier. Dual acting ligands for the treatment of Parkinson’s disease have been reported with
19,20
the aim to target the A2A receptor while inhibiting MAOꢀB.
To the best of our knowledge,
comparatively small “drugꢀlike” molecules targeting the A2A and D receptors simultaneously
2
have not formerly been reported. A related concept is the design of dual acting proꢀdrugs as
21
exemplified by Gogoi et al.; in this instance a molecule consisting of two pharmacophores is
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
required to cross the blood brain barrier, before it dissociates into two entities able to interact
at the A R and D R individually.
2A
2
In summary, both of these concepts provide interesting aspects that are worthwhile
investigating. Heterobivalent ligands are potentially more suited as pharmacological tools to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
further investigate the properties of the A2ARꢀD R heterodimers, whereas integrated dual
acting ligands may find their application in drug discovery as novel drugs for the treatment of
Parkinson’s disease.
Figure 1. (left) Schematic representation of a heterobivalent ligand and integrated dual acting ligand consisting
2
of a D R agonist and an A2AR antagonist. (right) Schematic examples of a heterobivalent ligand binding to a
heterodimer and an integrated dual acting ligand acting at the two orthosteric sites of two different types of
receptor monomers.
22,23
In this project the A R antagonist of choice was ZM 241385 (1) (Fig. 2);
the first A R
2A
2A
antagonist where an Xꢀray structure of an A2AR antagonist in complex with the A2A receptor
24
existed, which allowed good predictions of the binding mode of triazolotriazineꢀbased
analogs. The D R agonist of choice was the established antiparkinsonian drug ropinirole (2)
trade name: Requip) (Fig. 2) due to its simple structure compared to other D R agonist on the
Furthermore, its low molecular weight is beneficial for the design of dual acting
2
(
2
25,26
market.
ligands with more “drugꢀlike” properties. Notably, ropinirole (2) is a nonꢀergot derived D
2
R
4
ACS Paragon Plus Environment

Page 5 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
agonist which has been shown to be superior to ergot derived first generation drugs due to
27,28
reduced sideꢀeffects.
The drug is also available as an extendedꢀrelease formulation
permitting onceꢀdaily administration. Our group has previously published the successful
development of functionalized congeners of each pharmacophore (3ꢀ5), allowing further
extension into the extracellular space, whilst maintaining comparable activity to the parent
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
29ꢀ31
molecules.
For the design of the heterobivalent ligands we have used the functionalized
congeners TCAC (3) and OAC (5) as our starting point, before moving onto more “drugꢀlike”
analogs with reduced molecular mass. The synthesized dual acting ligands were tested in cellꢀ
based assays to determine their inhibitory potency at the A2A receptor, and functional potency
2
as well as the maximum response at the D receptor. In addition, preliminary permeability
testing was performed on selected analogs to predict their ability to cross the blood brain
barrier, which is an essential attribute for a potential CNS drug.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
Figure 2. Chemical structures of the A2AR antagonist ZM 241385 (1) and D R agonist ropinirole (2) and their
respective functionalized carboxylic acid congeners TCAC (3) and OCAC (4), as well as the functionalized
amine congener OAC (5).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SYNTHESIS OF THE DUAL ACTING LIGANDS
We first investigated classical heterobivalent ligands, consisting of the fully furnished
pharmacophores ZM 241385 (1) and ropinirole (2), separated with various lengths and types
of linkers or/and spacers spanning the two moieties (Fig. 3). Secondly, we focused on more
structurally integrated dual acting ligands with desirable physical properties (molecular mass,
polar surface area and clogP) for the development of potential CNS drugs (Fig. 3). This was
achieved by the elimination of the spacer and tyramine moiety, which according to structureꢀ
3
2
24
activity relationship studies and the receptor bound Xꢀray structure are nonꢀessential for
the activity of the A2AR antagonist ZM 241385 (1).
Figure 3. An example of the design principle of the “classical” heterobivalent ligands inꢀcooperating the fully
furnished pharmacophores ZM 241385 (1) and ropinirole (2) and the integrated dual acting ligands without the
tyramine moiety of the A2AR antagonist ZM 241385 (1).
Heterobivalent ligands
We first investigated ester linked heterobivalent ligands (Scheme 1) due to their simplicity
and facile synthetic accessibility. The OAC (5) was added to succinic anhydride (6) in toluene
and stirred at reflux. LCꢀMS indicated the formation of the carboxylic acid intermediate 7, but
this compound was not sufficiently stable and readily converted to the pyrrolidineꢀ2,5ꢀdione
6
ACS Paragon Plus Environment

Page 7 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
byproduct 7a. The addition of ZM 241385 (1) and the appropriate coupling reagents to the in
situ formed intermediate 7 also afforded mainly the pyrrolidineꢀ2,5ꢀdione byproduct 7a and
not the desired heterobivalent ligand. To avoid this ring closure, the order of the reaction steps
was reversed. Consequently, the cyclic anhydride was first added to the A2AR antagonist
ZM 241385 (1) followed by OAC (5). Therefore, ZM 241385 (1) was reacted with succinic
anhydride (6) or glutaric anhydride (9) in the presence of triethylamine in toluene at room
temperature. In the case of succinic anhydride (6), the reaction failed whereas glutaric
anhydride (9) furnished the desired compound 10 albeit in modest yields (28%). The
carboxylic acid 10 was then converted under standard peptide coupling conditions to the
heterobivalent ligand 11. The disadvantage of this approach is the limited range of linker
lengths available (larger cyclic anhydrides are not stable or commercially available) as well as
the metabolic instability of ester bonds. However, this approach provided a relatively straight
forward synthetic route to obtain a first dual acting ligand for preliminary in vitro testing as a
proofꢀofꢀconcept system to evaluate the overall approach.
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
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 1. Synthesis of the esterꢀlinked heterobivalent ligand 11
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) toluene, 24 h, reflux, 13% (7a); (b) TEA, toluene, 29 h, rt, no product observed (8),
8% (10); (c) DIPEA, HCTU, DMF, 3 h, rt, 10%.
2
Next, we designed and synthesized heterobivalent ligands where the two pharmacophores
were connected via a more hydrolytically and metabolically stable amide and ether linkage
(
Scheme 2). Consequently, molecules of this type provide pharmacological tools that are
potentially able to interact simultaneously at both orthosteric binding sites of a D RꢀA2AR
2
heterodimer. Therefore, the functionalized congeners OAC (5) and TCAC (3) as well as a
shorter version of the latter (compound 16) were either connected directly to afford
compounds 17a and 17b or incorporated amino acid spacers of varying lengths to afford
compounds 18aꢀ18c. The formation of the amide bonds was performed using standard
coupling reagents and conditions. It was important to protect the amino acids 12aꢀc to avoid
undesired side products, which simplified the purification step and drastically increased the
8
ACS Paragon Plus Environment

Page 9 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
yield. Initially benzotriazoleꢀbased coupling reagents such as BOP and HCTU were used, but
the byꢀproducts of these coupling reagents were challenging to remove in the final step. The
use of COMU as a coupling reagent furnished higher conversion and simplified the
purification process to remove undesired byꢀproducts associated with the coupling reagent.
In addition to the heterobivalent ligands (17a, 17b and 18a-c), the monovalent analogs with
the protected 12 carbon spacer attached to either the oxindole (14c) or triazolotriazine (19)
congener were synthesized and tested as additional reference compounds.
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
Scheme 2. Synthesis of heterobivalent ligands 17a, 17b and 18a-c incorporating amide bonds and the chemical
a
structure of the triazolotriazine monovalent ligand 19
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Reagents and conditions: (a) Boc anhydride, 1 M NaOH, THF, 3 h, rt, 66% (13a); Boc anhydride, TEA,
methanol, 2 h, 60 °C, 81% (13b) and 68% (13c); (b) COMU, DIPEA, DMF, 1 h, 0 °C followed by 2 h, rt, 31%
14a), 23% (14b) and 41% (14c); (c) TFA, DCM, 2.5ꢀ9 h, rt, 69% (15a), 99% (15b) and 39% (15c); (d) BOP,
(
DIPEA, DMF, 5 h, rt, 7% (17a); HCTU, DIPEA, DMF, 3 h, rt, 24% (17b); (e) COMU, DIPEA, DMF, 1 h, 0 °C
followed by 2ꢀ9 h, rt, 17% (18a), 17% (18b) and 8% (18c).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, we investigated the possibility of incorporating a triazine spacer, which was further
functionalized with a morpholine group to potentially improve the overall solubility of the
heterobivalent ligand 24 (Scheme 3). The triazine linker unit has been incorporated into the
heterobivalent ligands since it is a commonly used motif in drug discovery (i.e. PI3K
3
3ꢀ36
inhibitors, dendrimers).
The starting material cyanuric chloride (20) is an inexpensive and
commercially available compound with diverse reactivity of the three chlorine atoms allowing
selective substitution. Therefore, cyanuric chloride (20) in acetone was added dropwise to a
solution of morpholine (21) and triethylamine in acetone at ꢀ20 °C to afford intermediate 22
in 75% yield, which was used in the next step without further purification. ZM 241385 (1)
was deprotonated with sodium hydride, treated with the triazine spacer 22 and the reaction
mixture stirred at room temperature for two days to obtain compound 23 in 53% yield.
Intermediate 23 was then further reacted with OAC (5) in a sealed microwave tube to furnish
the heterobivalent ligand 24 in 67% yield.
10
ACS Paragon Plus Environment

Page 11 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Scheme 3. Synthesis of triazine spacer containing heterobivalent ligand 24
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
a
Reagents and conditions: (a) TEA, acetone, 15 min, ꢀ20 °C, 75%; (b) NaH 60%, THF, 2 days, rt, 53%; (c)
2 3
K CO , DMF, microwave, 15 min, 100 °C, 67%.
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Integrated dual acting ligands
Unlike the synthesis of the heterobivalent ligands, the more integrated molecules did not
consist of the fully furnished pharmacohore ZM 241385 (1). Therefore, the sulfone
intermediate 28 (which does not contain the tyramine moiety of ZM 241385 (1)) was key to
facilitate the introduction of the 2ꢀ(furanꢀ2ꢀyl)ꢀ5ꢀ(methylsulfonyl)ꢀ[1,2,4]triazolo[1,5ꢀ
a][1,3,5]triazinꢀ7ꢀamine portion of the A2AR antagonist instead of the TCACs 3 or 16. The
synthesis of the integrated molecules 32aꢀc and 29 (Scheme 4) started with commercially
available 4ꢀ(2ꢀhydroxyethyl)indolinꢀ2ꢀone (25), which was activated by conversion into the
tosylated intermediate 26, thereby allowing substitution with either methylamine or
propylamine at reflux to furnish the secondary amines 27a and 27b. Our dual acting ligand 29
was formed by stirring intermediate 27b and sulfone 28 in acetonitrile at room temperature
for 2.5 days. In the design of compound 29 the linker between the two pharmacophores was
fully removed to further reduce the molecular weight. This approach is associated with the
loss of the ionizable nitrogen; as a consequence the pharmacological evaluation of this
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
molecule will further validate the vitality of this feature for activity at the D receptor.
Intermediates 27a and 27b were also utilized for the synthesis of the target compounds 32aꢀc.
Bocꢀprotected amine linkers of different lengths were incorporated into the secondary amine
moieties of compound 27a and 27b to afford analogs 30aꢀc which, after cleavage of the Boc
protecting group, furnished the oxindole amine congeners 31a, 31c and 5. The final step
involved stirring the newly formed congeners 31a, 31c and 5 with sulfone 28 in acetonitrile at
room temperature to afford the target compounds 32a-c in yields of 23ꢀ65%. The dual acting
ligands 32a-c and 29 represent a small set of compounds with varying linker lengths between
the two pharmacophores as well as in the length of the side chain attached to the ionizable
tertiary amine of the ropinirole portion of the molecule.
12
ACS Paragon Plus Environment

Page 13 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Scheme 4. Synthesis of integrated dual acting ligands based on ZM 241385 (1) and ropinirole (2)
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
a
Reagents and conditions: (a) pꢀtoluenesulfonyl chloride, DCM, pyridine, 5ꢀ10 °C, 4 h, 80%; (b) 40% aq.
methylamine solution or neat nꢀpropylamine, reflux, 1.5 h, 57% and 74%, respectively; (c) 1 M NaOH, ethyl
acetate or DCM, rt, 15ꢀ20 min, 54% (27a) and 92% (27b); (d) 28, acetonitrile, rt, 2.5 days, 8%; (e)
2 2 3
Br(CH )n+2NHBoc, K CO , acetonitrile, reflux, 3ꢀ19 h, 46% (30a), 38% (30b) and 48% (30c); (f) TFA, DCM, rt,
1
h, 43% (31a), 84% (5) and 87% (31c); (g) 28, acetonitrile, toluene, rt, 5ꢀ60 h, 23% (32a), 65% (32b) and
3% (32c).
6
Next, we looked at molecules with a cyclic linker unit between the two pharmacophores,
which increases the rigidity of the molecule without interfering with the original
triazolotriazine and oxindole scaffolds of ZM 241385 (1) and ropinirole (2), respectively. The
idea behind this research was based on the fact that molecules with a reduced number of
rotatable bonds tend to show improved penetration of the BBB compared to highly flexible
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
7,38
and branched compounds.
Synthesized were three variations thereof; the aminopiperidine
3
7, piperazine 40a and homopiperazine 40b analog (Scheme 5). The first pathway for the
synthesis of the dual acting ligand 37 commenced with sulfone 28 which was treated with
tertꢀbutyl piperidinꢀ4ꢀylcarbamate in acetonitrile to afford intermediate 33 in 63%. The Boc
protecting group was removed using trifluoroacetic acid followed by alkaline treatment to
furnish intermediate 34 in free base form. The aminopiperidine functionalized triazolotriazine
analogs 34 was then further reacted under alkaline conditions with tosylate 26 in acetonitrile
at reflux, however, no conversion to molecule 37 was observed and mainly starting material
was recovered. Therefore, an alternative pathway starting from tosylate 26 was investigated.
Compound 26 was reacted with tertꢀbutyl piperidinꢀ4ꢀylcarbamate under alkaline conditions
at reflux affording intermediate 35 in 34% yield. Cleavage of the Boc protecting group with
TFA and treatment with 1 M aqueous sodium hydroxide furnished intermediate 36, which
was subsequently converted to the desired dual acting ligand 37 in 75% yield by adding
sulfone 28 in acetonitrile at room temperature.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The second synthetic pathway was used to obtain the dual acting ligands 40a and 40b. The
tosylate 26 was stirred under alkaline conditions with either Bocꢀpiperazine or Bocꢀ
homopiperazine to afford intermediate 38a and 38b, respectively. The Boc group of
compound 38a was removed with trifluoroacetic acid followed by an alkaline treatment to
afford the free amine 39a, which was further reacted with sulfone 28 to furnish the target
compound 40a in 66% yield. Intermediate 39a was extracted into the aqueous layer after
washing with 1 M sodium hydroxide solution, therefore intermediate 39b was isolated as the
trifluoroacetate salt after the cleavage of Boc protecting group by simply evaporating the
residual under reduced pressure. Consequently, the nonꢀnucleophilic base 1,8ꢀ
diazabicycloundecꢀ7ꢀene (DBU) was added to intermediate 39b and sulfone 28 in acetonitrile
at room temperature to afford the target compound 40b in low yield (5%).
14
ACS Paragon Plus Environment

Page 15 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Scheme 5. Synthesis of cyclic integrated dual acting ligands based on ZM 241385 (1) and ropinirole (2)
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
a
Reagents and conditions: (a) tertꢀbutyl 4ꢀaminopiperidineꢀ1ꢀcarboxylate, acetonitrile, rt, 3 h, 63%; (b) TFA,
DCM, rt, 3.5 h; (c) 26, DIPEA, DBU, acetonitrile, 85 °C, 2 days, no product; (d) tertꢀbutyl piperidinꢀ4ꢀ
ylcarbamate, DIPEA, acetonitrile, reflux 6 h, 34%; (e) TFA, DCM, rt, 2 h, 46%; (f) 28, acetonitrile, DMF, rt,
2
2 h, 49%; (g) Bocꢀpiperazine, DIPEA, acetonitrile, reflux, 22 h, 75% (38a); 26, Bocꢀhomopiperazine, DIPEA,
acetonitrile, 23 h, reflux, 74% (38b); (h) TFA, DCM, rt, 5 h, followed 1 M NaOH, 71% (39a); TFA, DCM, rt,
3
.5 h (39b); (i) 28, acetonitrile, toluene, rt, 18 h, 66% (40a); DBU, acetonitrile, rt, 23 h, 5% (40b).
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
PHARMACOLOGICAL RESULTS
2
2AR inhibitory potency and D R functional potency
A
The adenosine A2AR inhibitory potency (IC50) of the dual acting ligands and some selected
monovalent ligands was determined in a cyclic adenosine monophosphate (cAMP) assay,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
whereas the dopamine D R functional activity (EC50) and efficacy (Emax) were determined in
35
a [ S]GTPγS assay (Table 1). The functional assays were used to compare the activity (IC50
and EC ) of the synthesized molecules to each other as well as the parent pharmacophores
50
ZM 241385 (1) and ropinirole (2).
The reference and parent compound ZM 241385 (1) inhibited 5'ꢀNꢀ
ethylcarboxamidoadenosine (NECA) (500 nM) mediated cAMP accumulation with a pIC50
value of 7.48 ± 0.14. The attachment of the longest spacer to the triazolotriazine scaffold
(compound 19) showed a 13ꢀfold decrease in inhibitory potency at the A2A receptor; this
result is in accordance with a study from our group focusing on the biological impact of
30
extensions of selected literature A2AR antagonists. The reference and parent compound
3
5
39
ropinirole (2) has a literature pEC50 value of 6.52 ± 0.02 in a [ S]GTPγS assay. Attachment
of the 12ꢀcarbon spacer to the OAC (5) lead to a 3ꢀfold increase of the functional activity at
29
the D R which again is in accordance with work published earlier. The triazolotriazine
2
based monovalent 1 and 19 ligands didn’t exhibit any detectable activity at the D
likewise the oxindoleꢀbased monovalent ligand 14c did not show any detectable activity at the
2A receptor, as expected.
2
receptor,
A
The fused heterobivalent ligands 11, 17a and 17b (without the incorporation of an amino acid
spacer) showed very comparable activity to the initial pharmacophores. In this small group of
2
analogs only small changes in D functional potency were observed; analog 11 exhibited the
lowest functional potency, being about 1.5ꢀfold less potent than ropinirole (2), whereas 17b
was the most potent analog with 1.5ꢀfold improved functional potency. Similar variations of
16
ACS Paragon Plus Environment

Page 17 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
inhibitory potency were observed at the A2A receptor ranging from 18 nM for molecules 17b
to 63 nM for 17a in comparison to 33 nM for the parent compound 1. The “classical”
heterobivalent ligands 18aꢀ18c in general showed reduced potencies at both receptors in
comparison to analogs 11, 17a and 17b, with 18b being the exception exhibiting an IC50 value
of 41 nM. There is no obvious correlation between linker length and the observed potency
values. However, analog 18c (molecule with the longest spacer) not only showed the lowest
potency at both receptors but also exhibited a significant drop in efficacy of the dopamine
response possibly indicating steric clashes with the receptor.
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
The triazine based analog 24 exhibited a 4ꢀfold decrease in A2AR inhibitory potency
compared to the A2AR antagonist ZM 241385 (1) while maintaining equipotent activity
compared to the D
inhibitory potency to final compound 24, however failed to exhibit any functional potency at
the D receptor. This result was expected as the ligand lacked the D pharmacophore,
2
R agonist ropiniole (2). Intermediate 23 showed comparable A2AR
2
2
therefore confirming that the introduction of the oxindole scaffold is responsible for D
2
agonism of molecule 24.
The nonꢀcyclic integrated dual acting ligands (compounds 32a-c and 29) in general showed a
significant drop (28ꢀ54ꢀfold) in inhibitory potency at the A2A receptor, with the exception of
compound 29 that was equipotent to ZM 241385 (1). The decrease in antagonism of analogs
32aꢀc can potentially be explained by the introduction of an ionizable nitrogen atom at
physiological pH whereas compound 29 does not contain this feature and therefore
maintained antagonism. Conversely, compound 29 didn’t exhibit any detectable activity at the
D receptor, which may be related to the lack of the ionizable nitrogen atom whereas dual
2
acting ligands 32aꢀc exhibited D
2
R functional potencies ranging from 38 to 389 nM.
R functional potency,
Compounds 32b and 32c showed a respectable 3ꢀ and 8ꢀfold gain in D
2
respectively, compared to the parent compound 2. The exchange of the propyl chain of
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
molecule 32b with the methyl group in analog 32a caused a decrease in functional potency at
the D receptor (note 32a was equipotent to ropinirole (2)) which is not unexpected as
2
literature shows that incorporation of a propyl chain in commonly used D
2
agonist on the
The cyclic monovalent (35, 36, 38a, 38b, 39a and 39b)
as well as the dual acting ligands (37, 40a and 40b) did not exhibit any detectable activity at
the D receptor, which is possibly due to the introduction of a constrained ring system not
40ꢀ43
2
market improves their D affinity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
allowing sufficient flexibility for the molecule to interact with the D receptor in a favorable
2
pose. The inhibitory potencies at the A2A receptor of monovalent compounds 33 and 34 as
well as the dual acting ligands 37, 40a and 40b were comparable to the nonꢀcyclic analogs
32aꢀc with compound 40a being the stand out. It is noteworthy, that the physical properties of
the integrated dual acting ligands (29, 32aꢀc, 37, 40a and 40b) with topological polar surface
2
areas and clogPs ranging from 127ꢀ140 Å and 1.65ꢀ4.19, respectively are very respectable
2
considering that ZM 241385 (1) has a polar surface area of 127 Å and clogP of 2.82.
18
ACS Paragon Plus Environment

Page 19 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
Table 1. Reported are the calculated molecular weight (MW), topological polar surface area (tPSA) and partition coefficient (clogP) as well as the inhibitory potency at the
adenosine A2A receptor and the functional potency and efficacy at the dopamine D receptor of the reference compounds, selected monovalent ligands as well as dual acting
2
molecules. Emax values were referenced to quinelorane at 100 ꢁM. All the active compounds showed full inhibition in the cAMP assay. Compounds with Emax less than 20% were
considered as inactive (pXC50 <4).
a
MW
a
2
a
A
2AR inhibitory potency
D
2
R functional potency and efficacy
Compound #
tPSA [
Å
]
clogP
[g/mol]
pIC50 ± SEM
IC50 [nM]
33
pEC50 ± SEM
EC50 [nM]
>100,000
304
Emax [%]
b
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
ZM 241385 (1)
Ropinirole (2)
337.34
260.38
708.82
572.84
652.75
680.80
765.92
822.03
878.14
634.78
535.95
774.87
418.46
447.50
475.55
517.64
400.44
300.33
359.47
259.35
459.51
345.44
359.46
245.32
259.35
445.48
459.51
127
32
2.82
2.49
3.76
5.83
3.91
4.23
3.73
5.37
7.41
6.87
3.71
4.87
3.04
2.02
3.04
4.19
3.00
1.53
1.90
1.10
2.27
1.62
1.95
n.d.
74 ± 1
7.48 ± 0.14
n.d.
7.45 ± 0.06
<4
e
d
n.d.
35
6.52 ± 0.02
6.32 ± 0.25
6.95 ± 0.22
6.83 ± 0.01
6.69 ± 0.10
6.11 ± 0.02
6.30 ± 0.08
5.86 ± 0.11
c
d
11
195
100
178
178
207
207
207
172
168
212
127
140
140
140
137
119
71
58
140
62
62
44
44
131
131
484
114
148
204
785
501
1396
>100,000
>100,000
311
>100,000
389
88
38
n.d.
n.d.
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
79 ± 5
66 ± 0
88 ± 0
95 ± 1
68 ± 0
77 ± 1
38 ± 5
n.d.
n.d.
65 ± 3
n.d.
77 ± 2
89 ± 4
77 ± 0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
d
d
14c
17a
17b
18a
18b
<4
>100,000
63
18
110
41
1072
437
135
120
32
1122
912
1778
1738
8511
n.d.
c
d
7.20 ± 0.10
7.74 ± 0.10
6.96 ± 0.18
7.39 ± 0.13
5.97 ± 0.15
6.36 ± 0.30
6.87 ± 0.07
6.92 ± 0.16
7.49 ± 0.16
5.95 ± 0.04
6.04 ± 0.08
5.75 ± 0.04
5.76 ± 0.07
5.07 ± 0.07
n.d.
n.d.
5.82 ± 0.15
c
d
b
d
b
d
d
d
1
8c
d
d
1
2
2
2
9
3
4
9
<4
b
d
<4
b
d
6.51 ± 0.14
d
d
<4
d
d
3
3
2a
2b
6.41 ± 0.22
c
f
7.06 ± 0.09
d
d
3
2c
7.43 ± 0.09
c
3
3
3
3
3
3
4
5
6
7
n.d.
n.d.
c
d
<4
d
n.d.
1514
n.d.
<4
d
d
<4
d
38a
38b
39a
39b
40a
40b
n.d.
n.d.
n.d.
n.d.
<4
d
n.d.
n.d.
<4
d
ꢀ0.19
0.09
1.65
1.98
<4
d
n.d.
59
1479
<4
c
d
7.23 ± 0.12
5.83 ± 0.13
<4
n.d.
n.d.
d
d
<4
a
b
c
The physical properties were calculated with the software ChemSketch from ACD Lab; Data represent the mean ± SEM of three experiments performed in duplicate; Data
represent the mean ± SEM of four separate experiments performed in duplicate; Data represent the mean ± SEM of two experiments performed in duplicate; Literature value
(Ghosh et al.) , ropinirole (2) was not tested in this assay but the potency of the reference compound quinelorane (pEC50 of 7.35 ± 0.14 (EC50 = 45 nM)) is consistent with the
literature thereby permitting subsequent comparisons of test compounds to ropinirole (2); Data represent the mean ± SEM of five separate experiments performed in duplicate;
d
e
3
9
4
4
f
n.d. = not determined.
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Prediction of in vivo CNS penetration
Selected analogs were tested in a brain:blood partition ratio (Kbb) assay using blood and brain
tissue from a Winstar Han Rat. Selection criteria were based on the inclusion of analogs with
different levels of integration (MW ranging from 775 to 445 g/mol), structural diversity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
cyclic and nonꢀcyclic analog), the availability of sufficient quantities for testing (>5 mg) and
the requirement of being in solid from. The percent fraction unbound in blood and brain tissue
was determined which indicates the percentage amount of unbound drug available to reach the
site of action (results Table 2). Compounds that exhibited <1% fraction unbound were
considered to be very highly protein bound. Further determined was the in vitro brain:blood
45ꢀ47
partition ratio (Kbb) which is a prediction of in vivo CNS penetration (results Table 2).
As
a general rule compounds with a Kbb value >1 have a greater likelihood to be CNS penetrant,
due to their higher affinity for binding to brain tissue over blood components provided the
compound have a high level of membrane permeability and is not a substrate of an active
4
5ꢀ47
efflux transporter.
The literature A2AR antagonist ZM 241385 (1) showed a Kbb value of 0.76 and moderate
protein binding. The D R agonist ropinirole (2) was not included in the testing, but given it is
2
an established D R agonist for the treatment of Parkinson’s disease permits that the molecule
2
is able to cross the bloodꢀbrain barrier. Dual acting ligand 24, although having unfavorable
2
physical properties (MW: 775 g/mol, tPSA: 212 Å , clogP: 4.87), exhibited a Kbb value of
3
.65 this value potentially is driving a higher level of CNS penetrance based on the protein
binding equilibrium. Clinically used CNS drugs, for example diazepam, exhibit similar Kbb
values to compound 24, therefore indicating a realistic change of compound 24 being CNS
48
accessible. Unfortunately, compound 24 is highly protein bound implying that only a limited
amount of the free drug would reach the site of action when orally administered. The higher
integrated “drugꢀlike” dual acting ligands 32b exhibited a Kbb value of 1.29 which
corresponds to higher affinity for binding to brain tissue than blood components.
20
ACS Paragon Plus Environment

Page 21 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound 32b also exhibited low protein binding suggesting a sufficient amount of unbound
drug is able to reach the site of action in an in vivo model. The cyclic integrated dual acting
2
ligand 40a exhibited promising Kbb data, but its low potency at the D receptor makes it an
undesirable compound for further investigations. In summary, all the designed dual acting
ligands that were tested (24, 32b and 40a) exhibited promising Kbb values even though their
physical properties are at the upper limit (or above for compound 24) considering common
guidelines for the design of CNS drugs. Additional studies, for example a Madin Darby
canine kidney ꢀ MDR1 gene assay, will be necessary to further assess both membrane
permeability and Pꢀglycoprotein (Pꢀgp) liability of the presented compounds. Furthermore,
the initial in vitro permeability data will need to be confirmed by in vivo brain uptake studies
in rodents.
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
Table 2. Percent fraction unbound (Fu) in blood and brain tissue and the in vitro brain: blood partition ratio
(Kbb) of selected analogs
a
a
Compound #
Species / Matrix
Wistar Han Rat blood
% Undiluted Fu Mean Value ± SD Kbb
6.47 ± 0.45
ZM 241385 (1)
0.76
3.64
1.29
1.07
Wistar Han Rat brain tissues
Wistar Han Rat blood
8.49 ± 0.23
2.22 ± 0.086
0.61 ± 0.034
19.76 ± 3.16
15.33 ± 1.01
6.05 ± 0.18
5.63 ± 0.22
24
Wistar Han Rat brain tissues
Wistar Han Rat blood
32b
40a
Wistar Han Rat brain tissues
Wistar Han Rat blood
Wistar Han Rat brain tissues
a
The experiments have been performed in triplicate.
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CONCLUSION
Herein, we report the successful synthesis of a series of compounds ranging from classical
heterobivalent ligands to integrated dual acting molecules based on the chemical structure of
2
the D R agonist ropinirole (1) and the A2AR antagonist ZM 241385 (2) (Fig. 4). The most
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
promising dual acting ligands maintained the potencies of the original pharmacophores at
both receptors (adenosine A2A and dopamine D ). The results presented in this study for a set
2
of dual acting ligands able to act at two distinct GPCRs represents a remarkable achievement
considering the challenges associated with designing and synthesizing compounds to act at
just one receptor system.
Synthesis:
MW (g/mol):
tPSA:
19 steps
878 - 766
207 - 212
7.4 - 3.7
7.4 - 6.0
6.3 - 5.9
15 steps
709 - 652
195 - 178
4.2 - 3.8
7.7 - 6.9
6.8 - 6.3
10 - 12 steps
518 - 448
140
cLogP:
4.2 - 2.0
6.0 - 5.8
7.4 - 6.4
pIC50 (A2AR)
2
pEC50 (D R)
Figure 4. Overview of some of the most promising dual acting ligands analogs including chemical structures,
physical structures as well as the inhibitory and functional potencies at the adenosine A2A and dopamine D
receptors, respectively.
2
The “classical” heterobivalent ligands (18aꢀ18c) exhibited very respectable potencies at both
receptors and therefore, are useful pharmacological tools to probe the aforementioned
receptor systems as monomers or potentially heterodimers, however, they are labor intensive
to synthesize and have unfavorable physical properties for a potential CNS drug.
Heterobivalent ligand 18c, containing the longest spacer in our series, exhibited a substantial
22
ACS Paragon Plus Environment

Page 23 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
drop in the inhibitory potency at the A2A receptor and efficacy at the D receptor. It remains
2
open if heterobivalent ligands incorporating longer spacers would continue this trend or
2
potentially recover the inhibitory potency at the A2A receptor and efficacy at the D receptor.
The removal of the spacer reduced the number of synthetic steps and the molecular weight
slightly while improving the functional potency at both receptors to some extent. Unlike the
classical heterobivalent ligands (18aꢀ18c), the analogs without a spacer (11, 17a and 17b) not
only maintained the equivalent inhibitory potency of ZM 241385 (1) at the A2A receptor but
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
2
also the functional activity of ropinirole (2) at the D receptor. The active nonꢀcyclic
integrated dual acting ligands (32aꢀc) were relatively more straightforward to synthesize from
commercially available starting materials mainly due to the fact that the triazolotriazine
portion was introduced via the sulfone intermediate 28. As a result, the more labor intensive
synthesis of a TCAC (compound 3 or 16) compared to sulfone 28 was circumvented.
2
Compounds 32aꢀc exhibited EC50 values at the D receptor ranging from equivalent to a 8ꢀ
fold increased functional potency in comparison to ropinirole while the inhibitory potency at
the A2A receptor was still respectable but approximately 1.5 orders of magnitude weaker than
ZM 241385 (1). Compounds 32aꢀc have significantly improved physical properties, but are
admittedly still at the upper limit for a potential CNS drug. Nevertheless, it should be pointed
49
out that there are CNS drugs with similar properties on the market. More importantly, initial
permeability testing of selected analogs exhibited promising Kbb values indicating that these
molecules might be able to cross the bloodꢀbrain barrier. Compound 24 is a very interesting
molecule since it is highly potent at the A2A and D receptors (pIC 6.92 and pEC50 6.51,
2
50
respectively). It should be noted that, with a molecular weight of 775 g/mol, topological polar
2
surface area of 212 Å and a partition coefficient of 4.87, compound 24 does not display ideal
physical properties however exhibited a promising Kbb value of 3.65.
To our knowledge, these are the first “drugꢀlike” dual acting ligands successfully targeting the
50
adenosine A2A and dopamine D receptors. These dual acting ligands with relatively low
2
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
molecular weight are potential lead compounds in drug discovery. An important question that
remains is if these dual acting molecules provide a benefit to patients with Parkinson’s disease
over the single administration of D
to compare the dual acting ligands 11, 24, 32a and the D
exhibit comparable functional potency and efficacy at the D
2
R agonists in the clinic. Therefore, it would be interesting
R agonist ropinirole (2), which all
receptor, but differ in their
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
inhibitory potency at the adenosine A2A receptor, in an appropriate animal model. A study of
this type would also provide an insight into what inhibitory potency is needed at the A2A
receptor to observe a beneficial therapeutic effect in an animal model. Consequently, the
integrated analogs that exhibit a lower pIC50 value compared to ZM 241385 (1) are potentially
as beneficial for the treatment of Parkinson’s disease as molecules that have better inhibitory
potency at the A2A receptor (compound 24 and 11). Future studies would include the
determination of the subtype selectivity for the different adenosine and dopamine receptors,
allowing a comparison of the selectivity profile of the presented dual acting ligands with the
monovalent pharmacophores. This information can potentially lead to a better understanding
of the properties of the different subtypes as well as improve the design of more selective D
and A R agonists and antagonists, respectively.
2
R
2
A
24
ACS Paragon Plus Environment

Page 25 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EXPERIMENTAL SECTION
General Information All reactions were stirred magnetically in ovenꢀdried glassware.
Anhydrous solvents were transferred via ovenꢀdried syringe or cannula. Technical grade
solvents used for extraction and column chromatography were distilled prior to use. Absolute
solvents were used without further purification. The ropinirole hydrochloride salt was
purchased from Betapharma Shanghai Co., Ltd. and 4ꢀ(2ꢀhydroxyethyl)indolinꢀ2ꢀone was
purchased from China Langchem Inc. and tertꢀbutyl (6ꢀbromohexyl)carbamate from Astatech
Inc. All the other reagents were purchased from Merck, TCI Chemicals, Aldrich or AK
Scientific in the highest available grade and used without further purification. Analytical thin
layer chromatography (TLC) plates from Merck were used for reaction control (silica gel 60
on aluminum sheets). Silica gel 60 (Fluka) was used for silica gel flash chromatography.
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
1
Proton nuclear magnetic resonance ( H NMR) spectra and carbonꢀ13 nuclear magnetic
1
3
resonance ( C NMR) spectra were recorded on Bruker spectrometers Avance 400 (400 MHz
1
13
for H NMR and 101 MHz for C NMR) at ambient temperature in the solvents indicated and
1
3
referenced to tetramethylsilane (TMS). C NMR spectra were routinely run with broadband
decoupling. Distortionless enhancement by polarization transfer (DEPT) experiments were
1
3
routinely used for C NMR spectra. Chemical shifts (δ) are reported in parts per million
(ppm). Coupling constants (J) are reported in Hertz (Hz). The following abbreviations are
used: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).
Characterization of known compounds has been in accordance to literature and references
have been provided for relevant compounds in the Experimental Section and Supporting
Information. High resolution mass spectra (HRMS) were obtained on a Waters LCT Premier
XE (TOF) spectrometer fitted with an electrospray ion source. Mass signals are given in mass
units per charge (m/z). The fragments and intensities are written in brackets. Liquid
Chromatography Mass Spectra (LCMS) were measured on either two instruments. An Agilent
6100 Series Single Quad LC/MS, Agilent 1200 Series HPLC. (Pump: 1200 Series G1311A
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Quaternary pump, Autosampler: 1200 Series G1329A Thermostatted Autosampler, Detector:
1200 Series G1314B Variable Wavelength Detector). Gradient takes 4 minutes to get to 100%
acetonitrile; maintain for 3 minutes and a further 3 minutes to get back to the original 5%
acetonitrile. An Agilent 1290 Infinity (Agilent, Palo Alto, CA); Ionisation mode: Electrospray
Ionisation. Chromatographic separation was performed using an Agilent Zorbax SBꢀC18
Rapid Resolution HT 2.1 × 50 mm, 1.8 ꢁm column (Agilent Technologies, Palo Alto, CA)
using an acetonitrile gradient (5% to 100%) over 3.5 min at 0.5 mL/min. Solvent A =
Aqueous 0.1% Formic Acid; Solvent B = Acetonitrile/0.1% Formic Acid. Preparative HPLC
was performed on a Agilent 1260 infinity coupled with binary prep pump and Agilent 1260
FCꢀPS fraction collector. It operates on Agilent OpenLAB CDS Rev C.01.04 software. The
column was an Alltima C85u 22 mm × 250 mm. Analytical HPLC was performed on an
Agilent 1260 Infinity. It was operated on Agilent OpenLAB CDS Rev C.01.04 software. The
column used was a Poroshell 120 SBꢀC18 4.6 × 100 mm, 2.7 ꢀm. All the final compounds
showed a single peak at the designated retention time and are at least 95% pure.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All studies were conducted in accordance with the GSK Policy on the Care, Welfare and
Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use
Committee either at GSK or by the ethical review process at the institution where the work
was performed.
NMR analysis: Compound which include a triazolotriazine moiety show doubling of some
1
13
signals in the H and C NMR spectra due to dimer formation and/ or hindered rotation. For
details refer to the extensive NMR study of the adenosine A2AR antagonist ZM 241385 (1) by
23
our group that was previously published.
26
ACS Paragon Plus Environment

Page 27 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis
-(4-(2-((7-Amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)
phenoxy)-5-oxopentanoic acid (10)
ꢀ(2ꢀ((7ꢀAminoꢀ2ꢀ(furanꢀ2ꢀyl)ꢀ[1,2,4]triazolo[1,5ꢀa][1,3,5]triazinꢀ5ꢀyl)amino)ethyl)phenol
ZM 241385) (1) (50.0 mg, 148 ꢁmol) and glutaric anhydride (9) (18.6 mg, 163 ꢁmol) was
suspended in toluene (4 mL). The reaction mixture was stirred at reflux for 18 h before TEA
100 ꢁL, 717 ꢁmol) was added. Another 5 equivalent of glutaric anhydride were added in
5
4
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
(
(
portions while the reaction mixture was stirred for another 29 h at rt. The reaction mixture
was then partitioned between ethyl acetate, toluene and brine. The organic layer was washed
with water and dried with anhydrous sodium sulfate, filtered and the solvent was removed
under reduced pressure. The crude material was purified by column chromatography (CH Cl
2
2
→
CH Cl : CH OH 9:1) to afford the title compound 10 (19 mg, 28%) as a white solid.
2
2
3
1
H NMR (d
6
ꢀDMSO) δ 12.17 (br s, 1H), 8.30 (2 × br s, ratio 1:6, 2H), 7.87 (m, 1H), 7.52 (2 ×
br t, J = 5.6 Hz, 1H), 7.29 (m, 2H), 7.11 – 6.99 (m, 3H), 6.68 (m, 1H), 3.49 (m, 2H), 2.87 (m,
1
3
2
H), 2.61 (t, J = 7.4 Hz, 2H), 2.34 (t, J = 7.2 Hz, 2H), 1.85 (m, 2H). Some of the C signal
13
show up twice, the signal with lower intensity is marked with an asterisk (*) C NMR (d ꢀ
6
DMSO) δ 174.0 (C), 171.5 (C), 161.1 (C), 159.2 (C), 155.8 (C), 150.0 (C), 148.7 (C), 146.2
(C), 144.6 (CH), 137.1 (C), 129.6 (CH), 121.6 (CH), 111.9 (CH), 111.6 (CH), 42.7 (CH
2
*),
42.1 (CH
2
), 34.5 (CH *), 34.0 (CH ), 32.7 (CH ), 32.6 (CH ), 19.8 (CH ). LCMS: m/z (ESI
2 2 2 2 2
+
2
0 V) 452.2 (MH , 100).
4-(2-((7-Amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenyl
5-oxo-5-((3-((2-(2-oxoindolin-4-yl)ethyl)(propyl)amino)propyl)amino)pentanoate (11)
ꢀ(2ꢀ((3ꢀAminopropyl)(propyl)amino)ethyl)indolinꢀ2ꢀone (5) (12.2 mg, 44.3 µmol) and 5ꢀ(4ꢀ
2ꢀ((7ꢀaminoꢀ2ꢀ(furanꢀ2ꢀyl)ꢀ[1,2,4]triazolo[1,5ꢀa][1,3,5]triazinꢀ5ꢀyl)amino)ethyl)phenoxy)ꢀ5ꢀ
4
(
oxopentanoic acid (10) (20.0 mg, 44.3 µmol) were dissolved DMF (4 mL) and DIPEA
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(11.6 ꢁL,
benzo[d][1,2,3]triazoleꢀ1ꢀium 3ꢀoxide hexafluorophosphate (V) (HCTU) (18.3 mg,
4.3 ꢁmol) were added and the reaction mixture was stirred at rt for 3 h. The volatile
66.5
µmol)
and
1ꢀ(bis(dimethylamino)methylene)ꢀ5ꢀchloroꢀ1Hꢀ
4
components were removed under reduced pressure. The residue was reꢀdissolved in ethyl
acetate and washed with 1 M aqueous potassium carbonate solution. The organic layer was
dried with anhydrous sodium sulfate, filtered and evaporated to dryness. The crude material
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was purified by column chromatography (ethyl acetate → ethyl acetate: CH OH 1:1) to afford
3
1
the title compound 11 (3 mg, 10%) as a purple oil. H NMR (d
6
ꢀDMSO) δ 10.35 (br s, 1H),
8
.60 – 7.95 (2 × br s, ratio 1:6, 2H), 7.85 (m, 1H), 7.83 (t, J = 5.5 Hz, 1H), 7.62 (2 × br t, ratio
1:2, J = 5.7 Hz, 1H), 7.27 (m, 2H), 7.11 – 7.01 (m, 4H), 6.76 (m, 1H), 6.68 (m, 1H), 6.64 (m,
H), 3.49 (m, 2H), 3.43 (s, 2H), 3.06 (m, 2H), 2.86 (m, 2H), 2.58 (s, 4H), 2.54 (m, 2H), 2.44
1
(m, 2H), 2.37 (m, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.84 (m, 2H), 1.52 (m, 2H), 1.37 (m, 2H),
ꢀ
0
.82 (t, J = 7.4 Hz, 3H). LCMS: m/z (ESI 20 V) 707.4 (MH , 100). HPLC: t
R
7.83 min, >95%
+
(214 nm), >97% (254 nm). HRMS (C37
H
44
N O
10 5
): Calcd. 709.3574 [M+H] , Found
709.3558.
4-((tert-Butoxycarbonyl)amino)butanoic acid (13a)
4ꢀAminobutanoic acid (12a) (1.00 g, 9.70 mmol) was dissolved in THF (15 mL) and 1 M
aqueous sodium hydroxide solution (10 mL) and diꢀtertꢀbutyl dicarbonate (2.75 g,
2.61 mmol) were added at 0 °C before the reaction mixture was allowed to warm up to rt and
1
stirred for another 3 h. The solvent was then evaporated under reduced pressure. The residue
was taken up in water and extracted with diethyl ether. The aqueous phase was acidified with
1 M aqueous hydrochloric acid solution and quickly extracted with diethyl ether. The
combined organic layers were dried with anhydrous sodium sulfate, filtered and the solvent
2 2
was removed under reduced pressure. Purification by column chromatography (CH Cl →
1
CH Cl : CH OH 95:5) gave the title compound 13a (1.30 g, 66%) as a colorless oil. H NMR
2
2
3
28
ACS Paragon Plus Environment

Page 29 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(CDCl ) δ 10.00 (br s, 1H), 4.75 (br s, 1H), 3.19 (m, 2H), 2.40 (t, J = 7.2 Hz, 2H), 1.83 (p, J =
3
13
7
.0 Hz, 2H), 1.45 (s, 9H). C NMR (CDCl ) δ 178.4 (C), 156.6 (C), 79.7 (C), 40.0 (CH ),
3 2
2 3 2
31.3 (CH ), 28.3 (CH ), 25.1 (CH ).
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
8
-((tert-Butoxycarbonyl)amino)octanoic acid (13b)
8ꢀAminooctanoic acid (12b) (292 mg, 1.83 mmol) was suspended in CH OH (25 mL). TEA
3
(
365 ꢁL, 2.75 mmol) and a solution of diꢀtertꢀbutyl dicarbonate (800 mg, 3.67 mmol) in
CH OH (5 mL) were added and the reaction mixture was heated up to 60 °C and stirred for
h before the solvent was removed under reduced pressure. The residue was taken up in
CHCl and washed quickly with 1 M aqueous hydrochloric acid solution. The organic phase
3
2
3
was dried with anhydrous sodium sulfate, filtered and the solvent was removed under reduced
pressure. Purification by column chromatography (CH Cl → CH Cl : CH OH 9:1) gave the
2
2
2
2
3
1
title compound 13b (383 mg, 81%) as a white solid; mp: 82ꢀ85 °C. H NMR (CDCl
3
) δ 4.56
(s, 1H), 3.10 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.62 (m, 2H), 1.46 (m, 11H), 1.32 (m, 6H).
1
2-((tert-Butoxycarbonyl)amino)dodecanoic acid (13c)
1
2ꢀAminododecanoic acid (12c) (860 mg, 4.00 mmol) was suspended in CH OH (40 mL).
3
TEA (796 ꢁL, 5.99 mmol) and a solution of diꢀtertꢀbutyl dicarbonate (1.74 g, 7.99 mmol) in
CH OH (10 mL) were added. The reaction mixture was heated up to 60 °C and stirred for
.5 h before the solvent was removed under reduced pressure. The residue was taken up in
3
1
CHCl and extracted quickly with 1 M aqueous hydrochloric acid solution. The organic phase
3
was dried with anhydrous sodium sulfate, filtered and the solvent was removed under reduced
pressure. Purification by column chromatography (CH
2 2 2 2 3
Cl → CH Cl : CH OH 95:5) gave the
1
title compound 13c (860 mg, 68%) as a white solid; mp: 59ꢀ61 °C. H NMR (CDCl
3
) δ 4.53
(s, 1H), 3.10 (m, 2H), 2.34 (t, J = 7.4 Hz, 2H), 1.68 – 1.56 (m, 2H), 1.44 (m, 11H), 1.27 (m,
1
4H).
29
ACS Paragon Plus Environment