Structure-affinity relationships of the affinity of 2-pyrazolyl adenosine analogues for the adenosine A2A receptor

Article abstract of DOI:10.1016/S0960-894X(02)00609-1

The structure-affinity relationships of two novel 2-substituted adenosine series containing a substituted pyrazole attached at the N-1 or C-4 position for the adenosine (ADO) A_2A receptor are described. Compounds in the 2-(N-1-pyrazolyl) adenos

Full text of DOI:10.1016/S0960-894X(02)00609-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2935–2939
Structure–Affinity Relationships of the Affinity of 2-Pyrazolyl
Adenosine Analogues for the Adenosine A2A Receptor
Venkata P. Palle,a,* Elfatih O. Elzein,a,* Scott A. Gothe,c Zhihe Li,b Zhenhai Gao,b
a
Stephanie Meyer,b Brent Blackburna,b andJeff A. Zablocki
aDepartment of Bioorganic Chemistry, CV Therapeutics, 3172 Porter Drive, Palo Alto, CA 94304, USA
bDepartment of Pharmacological Sciences, CV Therapeutics, 3172 Porter Drive, Palo Alto, CA 94304, USA
cTripos, Inc., 601 Gateway Blvd. Suite 720, So. San Francisco, CA, USA
Received15 April 2002; accepted10 July 2002
Abstract—The structure–affinity relationships of two novel 2-substitutedaednosine series containing a substitutedpyrazole
attachedat the N-1 or C-4 position for the adenosine (ADO) A
receptor are described. Compounds in the 2-(N-1-pyrazolyl)
2A
adenosine series IV provided the highest affinity for the ADO A2A receptor as comparedto the 2-(C-4-pyrazolyl) series V. The main
structural differences between the two series point to the N-1 nitrogen of series IV imparting more favorable binding interactions
with the receptor than those of series V.
# 2002 Elsevier Science Ltd. All rights reserved.
Adenosine is an endogenous ligand that has affinity for all
four adenosine receptor subtypes—A1, A2A, A2B, and A3.1
Multiple physiological responses are mediated via local
adenosine production that is dependent on the con-
centration of adenosine producedandthe proximity to the
various adenosine receptor subtypes, since adenosine has
an extremely short half-life in plasma.2 Structurally
modified adenosine analogues provide the ability to
introduce added plasma stability and selective affinity
for a specific receptor subtype.2 Ongoing studies toward
therapeutic intervention with a modified adenosine ana-
logue that selectively activates one receptor subtype with
specific physiological responses are as follows: Ado A1
receptor- negative dromotropic effect (i.e., supraven-
tricular antiarrhythmic agents),3,4 Ado A2A receptor—
coronary vasodilatation (i.e., pharmacological stress
Potent andselective A
agonists that contain a lipo-
2A
philic substituent at the 2-position linkedthrough three
types of functional groups have been described: a pi
system, a polar atom—nitrogen or oxygen, or both a pi
system andpolar atom as illustratedin Figure 1. 1 The 2-
hexynyl 1a11 and2- trans-hexenyl 1b12 adenosine ana-
logues previously described are both potent and selec-
tive A2A agonists (Ia Ki=2.2 nM, Ib Ki=14 nM from ref
12) that are representatives of the pi system class. An
example of a 2-substitutedadenosine analogue with a
polar
linkage
is
foundin
2-[2-
p-methoxy-
phenethyl)amino derivative II that has an IC50 of 23 nM
at the ADO A2A receptor with a 40-foldselectivity over
the A1 receptor with respect to affinity (rat mem-
brane).13 The 2-cyclohexylmethylidene hydrazinoade-
nosine analogue III (WRC-0470, Ki=20 nM) represents
a compoundwith a linkage containing both a pi system
8
agent)5ꢀ7 andanti-inflammatory properties, Ado A2B
receptor—angiogenesis,9 andAdo A receptor—cardio-
andpolar atom, andits side chain is shown in the
Z-
3
protection.10 We describe our initial efforts in preparing
adenosine analogues with high affinity towards the Ado
A2A receptor. Further pharmacological characterization
including selectivity and pharmaceutical use as coronary
vasodilators of selected Ado A2A agonists of this
communication have been described elsewhere.6
conformation (see below).14 Structural elements of
compounds Ib, II, and III are mimickedby design in the
N-pyrazole andC-pyazole class exemplifiedby general
structures IV and V in Figure 1, respectively.15 Briefly,
the N-pyazole class can be construedas a constrained
mimetic of the Z-hydrazone derivitive III (WRC-0470),
andboth classes can be perceivedas constraineddouble
bondmimetics by virtue of their substitution pattern.
Elements of design are discussed below, including the
similarities of compound II with members of the N-
*Corresponding author. Fax: +1-650-858-0390; e-mail: jeff.zablocki@
cut.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00609-1

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V. P. Palle et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2935–2939
Figure 1. Compounds I–III are known selective ADO A2A receptor agonists andgeneral structures IV and V represent the pyrazole series described
herein that incorporate elements of compounds I–III.
Scheme 1.
pyrazole class (3–5). In general, our findings show that
despite a similar substitution pattern on the 5-mem-
beredring heterocycle between series IV and V, the
compounds of N-pyrazole class IV tolerateda larger
variance of substitution including lipophilic and hydro-
philic substituents that retainedaffinity for the ADO
A2A receptor. The compounds of C-pyrazole class tol-
eratedonly selectedlipophilic substituents that retained
affinity for the ADO A2A receptor.
The preparation of the C-pyrazole class V starts with
known 2-stannyl 20,30,50-tris-t-butyldimethylsilyl pro-
tectedadenosine analogue 17 utilizing a Stille coupling
[tetrakis(triphenylphosphine palladium(0) and cuprous
iodide]19,20 with appropriately substituted N-alkyl-4-
iodopyrazoles VII to affordcompounds 18–31 of Table
1 after deprotection with ammonium fluoride (Scheme
2).21 The various N-alkyl substituted4-ioodpyrazole
analogues VII were preparedby alkylating commer-
cially available 4-iodopyrazole with the corresponding
alkyl halide in DMF using sodium hydride as the base.
Amides 33, 34, 35, and 36 were prepareddirectly from
ester 18 through aminolysis with 40% aqueous methyl-
amine, 30% aqueous ethylamine, n-propylamine and
N,N-dimethylamine, respectively (Table 1). Compound
37 was preparedby reduction of the ester of 18 with
sodium borohydride in ethanol.
The N-pyrazole class was preparedby condensation of
1,3-dicarbonyl compounds (e.g., ethoxycarbonyl-
malondialdehyde16) with the known hydrazine
1
(Scheme 1) to affordcompounds 2–8 which are shown in
Table 1.17 The ester 2 was converted directly to amides
9–13 by aminolysis with ammonia, methylamine, di-
methylamine, ethylamine, andpropylamine, respectively
(Table 1). Amides 14–16 (Table 1) were preparedfrom
the acidof ester 2 obtainedthrough basic hydrolysis
(1 N KOH/MeOH/H2O) followedby standardpeptide
coupling (HBTU/HOBT in dichloromethane) with the
Our goal was to discover novel A2A agonists with good
affinity towards the A2A receptor through the structural
modification of adenosine by the addition of a 2-pyr-
azolyl substituent as representedby series IV and V
(Fig. 1). First, we will describe the SAR of the N-pyra-
zole class IV, a series that contains a linkage with both a
pi system anda polar atom. to the 2 position of adeno-
sine. Compounds 6–8 suggest that simple lipophilic
0
20, 30, and5 hydroxyl groups protected as TBDMS
throughout the process (not shown).18
3,4,5 tri- and3,5 id-substituted
N-pyrazolyl 2-sub-
stitutedadenosine derivatives are not well toleratedby
the Ado A2A receptor. For this reason, we therefore
focusedon 4-monosubstitutedN-pyrazolyl derivatives,
and in addition, they are more favorable mimetics of the
extended trans-hexenyl system of compound Ib (Fig. 1).
A comparison of ethyl ester 2, andethyl amide 12, sug-
gest that both ester andamide links at the 4-position of
the N-pyrazolyl can provide favorable affinity. In gen-
eral, the 4-substituted amide derivatives 9–16 that con-
Scheme 2.

V. P. Palle et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2935–2939
2937
Table 1. Binding affinity of N-pyrazole IV andC-pyrazole V for the ADO A2A receptora
1
CompdR
R2
R3
R4
Ki (uM) Rata
Ki (uM) Piga
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
18
19
20
21
22
23
24
25
26
27
–CO2Et
–Ph-4-Cl
–Ph-4-OMe
4-Me-Ph
–n-Propyl
–n-Butyl
H
H
H
H
H
H
0.06ꢁ0.01
0.39ꢁ0.09
0.19ꢁ0.08
0.19 ꢁ0.05
—
1.28ꢁ0.43
1.12ꢁ0.20
0.56ꢁ0.10
0.49ꢁ0.16
>100
H
H
Me
Me
Ph
H
H
H
H
H
H
H
Me
Me
Me
H
H
H
H
H
H
H
—
—
>100
>100
H
–CONH2
0.35ꢁ0.03
0.29ꢁ0.01
0.14ꢁ0.02
0.060ꢁ0.01
0.050ꢁ0.02
0.21ꢁ0.01
0.29ꢁ0.02
0.060ꢁ0.01
>100
1.35ꢁ0.22
1.12ꢁ0.32
0.44ꢁ0.10
0.38ꢁ0.13
0.12ꢁ0.02
0.71ꢁ0.08
0.98ꢁ0.18
0.38ꢁ0.13
>100
–CONHCH3
–CON(CH3)2
–CONHCH2CH3
–CONH CH2CH2CH3
–CONHCH2Ph-4-Cl
–CONHCH2CO2Et
–CONH-c-C5H9
H
H
CH2CO2Me
H
Me
i-Pr
n-Pentyl
n-Penten-4-yl
n-Decyl
Benzyl
2-Phenethyl
3-Phenpropyl
c-Hexylmethyl
2-c-Hexylethyl
3-c-Hexylpropyl
4-t-Bu-PhCH2
CH2CO2H
CH2CONHMe
CH2CONHEt
CH2CONHPr
CH2CON(Me2)
CH2CH2OH
>100
>100
>100
>100
10
3.88ꢁ1.29
2.59ꢁ0.32
0.47ꢁ0.08
6.11ꢁ0.69
1.61ꢁ0.23
2.26ꢁ0.31
1.28ꢁ0.43
0.22ꢁ0.04
1.10ꢁ0.24
2.62ꢁ0.36
5.29ꢁ1.62
>100
2.14ꢁ0.952
1.71ꢁ0.14
>100
1.58ꢁ0.41
5.90ꢁ0.69
1.66ꢁ0.382
0.88ꢁ0.07
3.43ꢁ0.42
6.68ꢁ0.93
2.42
28
29
30
31
32
33
34
35
>100
>100
>100
>100
>100
>100
>100
>100
>100
21ꢁ6
36
37
>100
0.066ꢁ0.01
III WRC-0470
aReceptor binding affinities for the Ado A2A receptors were determined using [3H]ZM241385 as a radioligand as described in Gao et al.6
tain both lipophilic andpolar substituents proviedd
goodaffinity for the Aod A receptor (rat,
instance, the high affinity N-pyrazole analogues 3, 4,
and 5 of Table 1 overlay nicely with the N-linkedana-
logue II of Figure 1 as shown in Figure 2a. Also, as
mentionedabove, the N-pyrazole class IV can be con-
struedas a constrainedanalogue of the Z-conformation
of the 2-cyclohexylmethylidene hydrazinoadenosine
analogue III (Fig. 1).15 An overlay of the high affinity
analogue cyclopentylamide 16 andanalogue III is
shown in Figure 2b.
2A
Ki<350 nM). In stark contrast, the compounds of the
C-pyrazole series V did not tolerate hydrophilic sub-
stitution (see compounds 32–37, Table 1) andretain
affinity for the Ado A2A receptor, even though they
have the same relative position on the 5-memberedring
pyrazole with respect to adenosine attachment as com-
pounds 2, 9, and 15 of the N-pyrazole class. There are
many possible reasons for this observedtrendin binding
affinities between the two classes. We hypothesize that
the the N-linkedpyrazole of series IV may have addi-
tional interactions with the Ado A2A receptor, possibly
through Nꢀ1, that may enhance affinity or possibly
orient the side chain in a different manner than the C-
pyrazole class V. The choice of the 2-N-pyrazolyl series
was not entirely serendipity as structural similarities
with known active Ado A2A agonists were foreseen. For
The compounds of the C-pyrazole series V required
specific lipophilic substituents to have even moderate
affinity for the Ado A2A receptor. This point is exem-
plifiedby the simple N-alkyl analogues 20–22 wherein
the N-methyl analogue 20 was foundto have affinity
greater than 100 mM, andthe isopropyl 21 andpentyl 22
analogues hadmodest affinity (Table 1). We wouldhave
predicted a higher affinity for the pentyl analogue 22

2938
V. P. Palle et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2935–2939
Figure 2. (a) Compound II (purple) andcompound 4 (red, white, and blue). (b) Compound III (purple) andcompound 16 (red, white, and blue).
(c) Compound Ib (purple) andcompound 22 (red, white, and blue). (d) Compound III (purple) andcompound 28 (red, white, and blue).22
basedon its favorable overlay with the high affinity Ado
A2A receptor ligand trans-hexenyl derivative Ib of Fig. 1
(see Fig. 2c). Similarily, N-benzyl 25, N-2-phenethyl 26,
and N-3-phenpropyl 27 analogues hadmodest affinity
for the Ado A2A receptor (Table 1). It is interesting that
the N-cyclohexylmethyl analogue 28 hadthe highest
affinity of this series for the Ado A2A receptor, even
more so than the N-cyclohexylethyl 29 and N-cyclohexyl-
propyl 30 analogues. This fact promptedthe overlay of
N-cyclohexylmethyl analogue 28 with c-hexylmethyl-
hydrazine analogue III in Fig. 2d.
selectivity, >16,460 nM CHO cells versus 1269 nM
HEK-hA2A AdoR6), anda preliminary account of their
pharmacological use as coronary vasodilators has been
described.6
Acknowledgements
We thank Professor Stephen Baker andDeborah Otero
for their help in screening some of the compounds
reportedherein.
The SAR for both N andC-pyrazole series suggest that
in general the combinedheteroatom linker andpi sys-
tem of the N-pyrazole class provides more favorable
affinity for the Ado A2A receptor than the C-pyrazole
class. As notedabove, adenosine itself has a very short
plasma half-life in part due to the conversion to inosine
by adenosine deaminase. All of the compound binding
affinities for the Ado A2A receptor were testedin the
presence of adenosine deaminase, and prolonged incu-
bation of selectedmembers in the competitive binding
did not lead to a decrease in affinity. Selected members
of each class were evaluatedfor their functional selec-
tivity for the Ado A2A receptor andcoronary vasodi-
lating effects in a separate study.6 Compounds 10 (CVT-
3146) and 22 (CVT-3033) were foundto be functionally
selective for the Ado A2A receptor activation over Ado
References and Notes
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A1 andA
foundto have low affinity for human Ado A receptor
receptors (note: both compounds were
2B
3
8. Fozard, J. R.; Ellis, K. M.; Villela Dantas, M. F.; Tigani,
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> >10 mM, andcompound 10 has low affinity at the
human Ado A1 receptor with greater than 13-fold

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10. Liang, J. T.; Jacobson, K. A. Proc. Natl. Acad. Sci. U.S.A.
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The trisilyl derivative (0.4 g, 0.5 mmol) was then suspended
in 1 mL of water andtreatedwith 4 mL 1 N KOH/MeOH. The
mixture was allowedto stir at room temperature for 72 h. The
solvent was removed under reduced pressure and the residue
was dissolved in 5 mL water and acidified to pH 5 using 1 N
HCl. The resulting precipitate was collectedby filtration and
washedwith water andethyl ether to affordthe trisilyl acid 38,
0
which was usedwithout further purification. The 2 ,30,50 tri-
13. Francis, J.; Webb, R.; Ghai, G.; Hutchison, A.; Moskal,
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silyl adenosine acid derivative of 2 (0.14 g, 0.2 mmol) was then
dissolved in 5 mL dichloromethane. To the solution was added
HBTU (0.19 g, 0.4 mmol), HOBt (0.076 g, 4 mmol), N-methyl-
morpholine (0.04 g, 0.4 mmol) andcat. DMAP. The mixture
was allowedto stir at 23 ꢂC for 24 h. The mixture was then
washedwith 10% citric acid, saturatedNaHCO 3, brine and
dried over MgSO4. The solvent was removedandthe residue
was treatedwith 5 mL 0.5 N NH 4F/MeOH.21 The solution was
heatedat reflux for 24 h. The solvent was evaporatedandthe
residue was purified by preparative TLC to afford compounds
14–16.
17. To
a
suspension of 2-hydrazinoadenosine (0.025 g,
19. Kato, K.; Hayakawa, H.; Tanaka, H.; Kumamoto, H.;
Miyasaka, T. Tetrahedron Lett. 1995, 36, 6507.
20. For review, Mitchell, T. N. Synthesis 1992, 803 (Stille).
21. Zhang, W.; Robins, M. J. Tetrahedron Lett. 1992, 33,
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22. Molecular modeling was performed using a Sybyl 6.6
program wherein the torsional angle about the pyrazolyl-
purine bondwas optimizedto a local minima using the
advanced computational module searching at 15 rotational
degrees followed by alignment of the comparator compound
using the Fit Algorithm within Sybyl.
0.08 mmol) in a 1:1 mixture of MeOH/AcOH was added 2-(4-
methyl)malondialdehyde (0.019 g, 0.12 mmol) and the mixture
was heatedat 80 ꢂC for 3 h. The precipitate formedwas col-
lectedby filtration andwashedwith EtOH andether anddried.
18. The acid 38 was synthesizedas follows: The ester 2 (0.50 g,
1.2 mmol) was dissolved in 30 mL DMF. To the solution was
added imidazole (0.68 g, 10 mmol) and TBDMSCl (1.5 g,
10 mmol). The mixture was allowedto stir at 80 ꢂC for 24 h.
The solvent was removedandthe residue was purifiedusing
column chromatography to affordthe trisilyl-protectedderi-
vative of 2.