Exploring distal regions of the A3 adenosine receptor binding site: Sterically constrained N6-(2-phenylethyl)adenosine derivatives as potent ligands

Article abstract of DOI:10.1016/j.bmc.2004.02.037

We synthesized phenyl ring-substituted analogues of N⁶-(1S,2R)- (2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A₃AR with a K_i value of 0.63nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA₃AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A₃AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A₃AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A₃AR binding. Other related N⁶-substituted adenosine derivatives were included for comparison. Although the N ⁶-(2-phenyl-1-cyclopropyl) derivatives were full A₃AR agonists, several other derivatives had greatly reduced efficacy. N ⁶-Cyclopropyladenosine was an A₃AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N⁶-(2,2-Diphenylethyl)adenosine was an A ₃AR antagonist, and either adding a bond between the two phenyl rings (N⁶-9-fluorenylmethyl) or shortening the ethyl moiety (N ⁶-diphenylmethyl) restored efficacy. A QSAR study of the N ⁶ region provided a model that was complementary to the putative A₃AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A₃AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.

Full text of DOI:10.1016/j.bmc.2004.02.037

Bioorganic & Medicinal Chemistry 12 (2004) 2021–2034
Exploring distal regions of the A3 adenosine receptor binding site:
sterically constrained N6-(2-phenylethyl)adenosine derivatives as
potent ligands
Susanna Tchilibon, Soo-Kyung Kim, Zhan-Guo Gao, Brian A. Harris,
Joshua B. Blaustein, Ariel S. Gross, Heng T. Duong, Neli Melman
and Kenneth A. Jacobson*
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes & Digestive & Kidney Diseases,
National Institutes of Health (NIH), DHHS, Bethesda, MD 20892-0810, USA
Received 13 December 2003; revised 11 February 2004; accepted 28 February 2004
Abstract—We synthesized phenyl ring-substituted analogues of N6-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly
potent in binding to the human A3AR with a Ki value of 0.63 nM. The effects of these structural changes on affinity at human and rat
adenosine receptors and on intrinsic efficacy at the hA3AR were measured. A 3-nitrophenyl analogue was resolved chromato-
graphically into pure diastereomers, which displayed 10-fold stereoselectivity in A3AR binding in favor of the 1S,2R isomer. A
molecular model defined a hydrophobic region (Phe168) in the putative A3AR binding site around the phenyl moiety. A hetero-
aromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A3AR binding. Other related N6-
substituted adenosine derivatives were included for comparison. Although the N6-(2-phenyl-1-cyclopropyl) derivatives were full
A3AR agonists, several other derivatives had greatly reduced efficacy. N6-Cyclopropyladenosine was an A3AR antagonist, and
adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N6-(2,2-Diphenylethyl)adenosine
was an A3AR antagonist, and either adding a bond between the two phenyl rings (N6-9-fluorenylmethyl) or shortening the ethyl
moiety (N6-diphenylmethyl) restored efficacy. A QSAR study of the N6 region provided a model that was complementary to the
putative A3AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A3AR agonists and related
nucleoside antagonists was explored through both empirical and theoretical approaches.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
We have studied the microscopic interactions of ligands
with the A3AR and other members of the AR family
from the perspectives of both ligand modification and
structure–function aspects of the receptors.6–9 Extensive
mutagenesis studies and molecular modeling based on a
high-resolution template of rhodopsin have implicated
TM (transmembrane domain) regions 3, 6, and 7 in the
coordination of adenosine agonists and a putative
rotation of TM6 in the activation of the A2A and
A3ARs.
The adenosine receptors (ARs) consist of four subtypes
(A1, A2A, A2B, and A3) and represent a physiologically
important family of G protein-coupled receptors.1 AR
agonists are current targets for the development of
therapeutic agents for a variety of diseases, including
agents with neuroprotective, antiseizure, anti-inflam-
matory, anti-ischemic, and cardioprotective effects.2–4
A3AR agonists are also potentially useful for the treat-
ment of cancer. In certain tumor cells, a cytostatic effect
of the A3AR agonist appears to be related to its
downstream activation of the Wnt pathway.5
Adenosine is a nonselective AR agonist that is rapidly
degraded in circulation. Its clinical use for treatment of
supraventricular tachycardia is predicated on a short
duration of action. However, other foreseeable appli-
cations of synthetic adenosine agonists would require
greater stability in vivo.10 An early AR agonist showing
improved stability toward adenosine deaminase was the
nonselective agonist 2-chloroadenosine 1, which also
served as the first stable adenosine receptor radioligand.11
Keywords: Nucleoside; Agonist; Molecular modeling; GPCR; Purine
receptor.
* Corresponding author. Tel.: +301-496-9024; fax: +301-480-8422;
e-mail: kajacobs@helix.nih.gov
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.037

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S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
The 2-chloro modification has since been incorporated
in many potent AR agonists, including the 50-uronamide
derivative Cl-IB-MECA 2, the first highly selective
A3AR agonist. The intrinsic efficacy of various adeno-
sine derivatives as A3AR agonists was studied, leading
to the characterization of structure–activity relation-
ships (SARs) for efficacy, which are distinct from those
of affinity.6;7 The ability of a tightly binding adenosine
derivative to activate the A3AR is highly dependent on
ligand conformation. The flexibility of the ribose moiety
has been established as a required feature for A3AR
agonists. Steric constraint of the ribose moiety, for
example, the introduction of a bridged carbocyclic ring
system, tends to reduce intrinsic efficacy of the adeno-
sine derivatives at the human A3AR (hA3AR). These
effects are overridden by the presence of a flexible 50-
uronamide group.
2. Results
2.1. Chemical synthesis
Most of the analogues of 3 were prepared as diastereo-
meric mixtures (Schemes 1–3). The phenylcyclopropyl
amines 41a–n were prepared from the corresponding
trans-cinnamic acid derivatives 37.12 With the exception
of the 3-cyano-cinnamic acid (37k), which was obtained
by a Knovenagel condensation between 3-cyano-benz-
aldehyde and malonic acid, the other acids were com-
mercially available.
€
After esterification with methanol in the presence of
H2SO4, compounds 38a–n were cyclopropanated with
diazomethane in the presence of catalytic Pd(OAc)2.
This is a very high-yield reaction with the only incon-
venience being that it must be monitored by NMR,
because the starting material and product were undis-
tinguishable by TLC. Hydrolysis of the esters 39a–n
gave the desired trans-arylcyclopropanecarboxylic acids
40a–n. The primary amines 41a–n were obtained by a
Curtius degradation, in a one-pot reaction with diphenyl
phosphorazidate, with the exception of 44 and 45
(Scheme 2). For these two compounds a two-step reac-
tion with sodium azide was preferred, because in this
way the resulting acyl azides were transformed into the
2-(trimethylsilyl)ethyl carbamate, which could be
hydrolyzed with tetrabutylammonium fluoride without
affecting the acetamido moiety. A diphenylcyclopropyl
intermediate, the carboxylic acid 47, was prepared by
the method reported (Scheme 3).21 In this case the
cyclopropanation of 3-phenyl-cinnamic acid was
unsuccessful, probably because of the steric hindrance.
Once obtained, the amines 41, 44a–n, 45, and 48 reacted
with 6-chloropurine riboside to give the desired adeno-
sine analogues 6–15 and 19–25. Compound 18 was
obtained by simple hydrogenation on Pd/C of the nitro
derivative 15.
Highly selective A3AR agonists thus far reported, for
(2-chloro-N6-(3-iodobenzyl)-50-N-methyl-
example,
2
carboxamidoadenosine), all contain multiple substitu-
tions of the adenosine molecule.1 A recent study by Gao
et al.7 identified the singly substituted adenosine deriv-
ative compound 3 (N6-(1S,2R)-(2-phenyl-1-cycloprop-
yl)adenosine), with a Ki value in binding to the hA3AR
of 0.63 nM (38-fold more potent than the 1R,2S isomer
4), as a lead for the development of N6 derivatives of
adenosine with high hA3AR affinity. The present study
aimed to identify analogues of 3 and other N6 deriva-
tives of adenosine that are selective, while maintaining
the remainder of the molecule unchanged, and to cate-
gorize structural features of the N6 substituents that
affect the intrinsic efficacy (Chart 1).
I
NHCH2
N
NH2
One of the analogues, containing a 3-nitro group (15),
was resolved into pure diastereomers with HPLC, using
the chiral column Chiralpak AD. The assignment of
absolute configuration was done by analogy to the
unsubstituted phenylcyclopropyl derivatives as standards.7
In the condition used for the chiral separation, the
pure N6-[(1R,2S)-2-phenyl-1-cyclopropyl]adenosine (4)
showed a retention time of 10.1 min and N6-[(1S,2R)-2-
phenyl-1-cyclopropyl]adenosine (3) of 13.3 min. By
analogy, in the resolution of the racemic 15, the first
eluting peak at the retention time of 20.8 min was
assigned as the N6-[(1R,2S)-2-(3-nitrophenyl)-1-cyclopro-
pyl]adenosine (17) and the later one at 31.5 min as N6-
[(1S,2R)-2-(3-nitrophenyl)-1-cyclopropyl]adenosine (16).
N
N
N
N
Cl
Cl
HO
N
N
O
N
O
CH3NH
O
HO
OH
HO
OH
1
2
H
2R
1S
H
NH
N
N
N
N
O
HO
3. Biological activity
3
HO
OH
The analogues 5–25 of N6-(trans-2-phenyl-1-cycloprop-
yl)adenosine (Table 1) generally bound to the hA3AR in
the low nanomolar range. Selectivity was high when
Chart 1. The structure of the various adenosine agonists studied at the
A3AR.

S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
2023
O
O
CH2N2, Pd(OAc)2
0°C
MeOH, H2SO4
reflux
OCH3
OH
R
R
38 a - n
37 a R = 2-Me, b R = 3-Me,
c R = 4-Me
f R = 4-Cl
i R = 3-OCF3
l R = 3-OMe
d R = 2-Cl,
g R = 3-F,
e R = 3-Cl,
h R = 3, 5-di-F,
j R = 3-NO2, k R = 3-CN,
m R = 3-CF3, n R = 3,5-di-CF3,
O
O
1) DPPA, (Et)3N, t-BuOH,
80°C, 48h
NaOH, MeOH
OCH3
OH
2) 1N HCl, 100°C,
overnight
R
R
40 a - n
39 a - n
NH
6-chloropurine riboside
EtOH, (Et)3N, 800C
N
N
NH2
N
N
R
HO
R
O
41 a - n
HO
OH
6 R = 2-Me, 7 R = 3-Me,
9 R = 2-Cl, 10 R = 3-Cl,
8 R = 4-Me
11 R = 4-Cl
12 R = 3-F, 13 R = 3, 5-di-F, 14 R = 3-OCF3
15 R = 3-NO2, 20 R = 3-CN,
22 R = 3-CF3, 23 R = 3,5-di-CF3,
21 R = 3-OMe
Scheme 1. General synthetic route used to prepare N6-(2-phenyl-1-cyclopropyl)adenosine analogues.
1) ethyl chloroformate, Et3N,
COOH
NH2
1) C6H6, CuSO4
NaN3, dry acetone
R
R
+
2) 100ºC in toluene, 2h
COOH
O
CH2
2) 2N NaOH, MeOH
N2CH
OC2H5
3) 2-(trimethylsilyl)ethanol,
60ºC, 16h.
4) TBAF 1M, 50ºC, 24 h
42, 43
44, 45
47
46
NH
1) DPPA, (Et)3N, t-BuOH
80 C, 48h
NH2
6-chloropurine riboside
°
R
N
N
6-chloropurine riboside,
HO
EtOH, (Et) N, 80 C
°
3
N
2) 1N HCl, 100 C
°
N
EtOH, (Et)3N, 80ºC.
O
48
HO
OH
NH
19, 25
42, 44, 19 R =
43, 45, 25 R =
N
N
NHAc
HO
N
N
O
24
HO
OH
S
Scheme 3. Synthetic route used to prepare N6-(2,2-diphenyl-1-cyclo-
propyl)adenosine 24.
Scheme 2. Synthetic route used to prepare compounds 19 and 25 via a
trimethylsilylethyl intermediate.
compared with the A2AARs, but only moderate, at most,
compared with the A1ARs. The diastereomeric mixture
of N6-(trans-2-phenyl-1-cyclopropyl)adenosine 5 was
prepared for comparison with the phenyl-substituted
analogues. Compound 5 bound to the hA3AR with a Ki
value of 0.86 nM. Its affinity at the rat A3AR (rA3AR)
was 460-fold lower than at the human receptor. Sub-
stitution of the 2-phenyl ring at the 3-position was

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S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
Table 1. Binding affinities of adenosine derivatives at human and rat A1, A2AARs and A3ARs and maximal agonist effects at the hA3AR expressed in CHO
cells.a The adenosine derivatives are substituted at the N6 position as indicated. Compounds 1 and 2 are additionally substituted at the 2-position with chloro.
All compounds except 2 are simple 9-b-
N6 substitution
D-riboside derivatives
#
Ki (nM)
hA1ARa
Maximum
effect (%)d
rA2AARb
hA2AARb
hA3AR, h or (r)c
rA1ARa
trans-N6-(2-Phenyl-1-cyclopropyl) analogues
3
4
5
(1S,2R)-2-Phenyl-1-cPr
(1R,2S)-2-Phenyl-1-cPr
2-Phenyl-1-cPr
11.8 2.4e
15.2 3.2e
10.4 1.9
560 232e
3040 490e
2980 310
30.1 6.1
15.6 1.7
124 30
2250 430
2340 330
2530 720
0.63 0.17e
(358 33)e
24.1 10.9e
(694 157)e
0.86 0.09
(399 28)e
12.9 3.9
1.59 0.50
1.96 0.14
6.0 1.4
117
87
9
4
5
4
101
98
6
2-(2-Methylphenyl)-1-cPr
2-(3-Methylphenyl)-1-cPr
2-(4-Methylphenyl)-1-cPr
2-(2-Chlorophenyl)-1-cPr
2-(3-Chlorophenyl)-1-cPr
18.3 3.3
17.3 2.9
20.0 1.9
11.8 1.5
10.3 1.1
1080 250
2500 550
2760 970
1430 330
349 56
116 19
12.1 2.7
28.0 3.4
28.4 13.7
24.9 2.1
4480 460
970 340
3130 710
1820 170
1520 540
7
107 11
8
99
104
101
7
6
3
9
10
0.98 0.27
(642 153)
1.63 0.26
7.6 1.4
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2-(4-Chlorophenyl)-1-cPr
9.70 2.73
13.6 1.7
18.8 1.8
36.3 9.9
15.4 2.8
13.6 3.2
13.2 4.6
46.9 7.8
21.5 9.4
57.4 5.8
15.8 1.4
15.6 1.6
46.1 6.7
22.6 8.7
5.65 1.97
1430 540
1410 750
4060 1330
1360 230
1060 260
79.5 41.0
35.1 8.3
39.7 4.0
146 63
4120 890
2930 330
3270 700
7110 930
2260 270
>10,000
103
7
2-(3-Fluorophenyl)-1-cPr
110 12
2-(3,5-Difluorophenyl)-1-cPr
2-(3-Trifluoromethyloxyphenyl)-1-cPr
2-(3-Nitrophenyl)-1-cPr
34.7 3.1
77.3 21.3
18.8 5.2
11.2 2.7
116 13
99
97
8
6
5
4
7
7
9
7
2
5
8
4
7
47.5 0.5
83.9 8.2
55.4 7.8
131 52
100
108
64
2-(3-Nitrophenyl)-1-cPr, B (1S,2R)
2-(3-Nitrophenyl)-1-cPr, A (1R,2S)
2-(3-Aminophenyl)-1-cPr
1320 370
>10,000
8040 1380
9.0 3.3
103
100
99
2-(3-Acetamidophenyl)-1-cPr
2-(3-Cyanophenyl)-1-cPr
32.6 7.5
45.2 3.3
29.1 5.9
104 30
2130 700
2480 780
2830 340
2370 780
7660 1040
579 186
2120 670
6.02 0.74
22.5 7.5
2.8 0.3
2450 610
795 193
459 97
2-(3-Methoxyphenyl)-1-cPr
2-[3-(Trifluoromethyl)phenyl]-1-cPr
2-[3,5-Di(trifluoromethyl)phenyl]-1-cPr
2,2-Diphenyl-1-cPr
103
101
98
1.9 0.2
869 351
54.6 17.6
40.7 2.4
71 15
387 37
91 14
100
99
2-(3-Thienyl)-1-cPr
31.6 8.2
2.88 0.90
Other analogues
CADO
1
6.7 1.0f
63e
7.5 1.4
630 220
87 24
(1890 900)g
1.4 0.3h (0.33)e
41.3 5.3e
(120 20)g
2.1 0.4
100 7e
100
2
3-Iodobenzyl, Cl-IB-MECA
Benzyl
820e
175 20e
470e
285e
1240 320h
77.8 6.5
5360 2470h
2180 670
26
55
3
27
2-Phenylethyl
24.0 8.8f
521 90
12.9 2.1
676 39
84
5
(240 58)g
8.7 0.9e (158)g
68 12e (920)g
9.1 0.3
28
29
30
(R)-1-Phenyl-2-propyl, (R)-PIA
(S)-1-Phenyl-2-propyl, (S)-PIA
(R)-2-Phenyl-1-propyl
1.2 0.1f
49.3f
1.4 0.1e
124f
1820f
2.04i
75i
859i
7780i
102 6e
97 3e
319 114
4.0 1.3
325 85
99
4
(202 20)
31
32
33
34
(S)-2-Phenyl-1-propyl
Cyclopropyl
3.5 0.3e
14.7 1.7
44.1 1.7
112 49
459 162
3490 260
75.4 14.9
4.4g
26.6 6.8
6.9 2.4
1120 260
7860 550
510 49
38.8 4.1
98
6
(276 20)
100 33
0j
(1950 120)
3.9 0.7
2,2-Diphenylethyl
49.9 16.2
168 29e
0j
(538 202)
106 22e
2-(3,5-Dimethoxyphenyl)-2-(2-meth-
ylphenyl)ethyl, DPMA
Diphenylmethyl
153 26e
0e
(3570 1700)g
3340 360
0.91 0.38
35
36
208 36
2490 420
33.4 24.4
490 242
14.0 4.0
>10, 000
145 26
87
99
6
6
9-Fluorenylmethyl
9.41 3.11
a Binding experiments at rat brain and recombinant human A1ARs used [3H]R-PIA (2.0 nM) as radioligand, unless noted.
b Binding experiments at rat brain and recombinant human A2AARs used [3H]CGS21680 (15 nM) as radioligand, unless noted.
c All A3AR binding experiments were performed using adherent CHO cells stably transfected with cDNA encoding the human or rat A3 receptor. [125I]I-AB-
MECA was used as radioligand.
d Data of hA3AR at 10 lM. Functional assay consisting of inhibition of forskolin-stimulated adenylyl cyclase. The value for compound 2 is the standard for
100% efficacy.
e Data from Gao et al.7
f Data from Daly et al.14
g Data from van Galen et al.23
h Data from Jacobson et al.24
i Data from Klotz et al.25 Radioligands used were [3H]CPX and [3H]NECA at human A1 and A2A ARs, respectively.
j When the stock DMSO solution was subjected to repeated freeze-thaw cycles, the observed efficacy was affected. Values given are for freshly prepared
solutions.

S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
2025
favorable for hA3AR affinity. Thus, among the methyl-
substituted analogues 6–8, the most potent in A3AR
binding was the 3-methyl analogue 7. Similarly, within a
series of chloro-substituted analogues 9–11, the order of
potency in binding was 3-Cl > 4-Cl > 2-Cl. The 3-chloro
analogue 10 bound to the hA3AR with a Ki value of
0.98 nM, and the affinity at the rA3AR was 660-fold
lower. Thus, there were dramatic species differences in
affinity, and A3AR selectivity of 10 was present when
comparing human but not rat ARs. At rARs, com-
pound 10 was moderately selective for the A1AR.
N6-cyclopropyl analogue 32. Compound 32, however,
was not highly potent in binding to the hA3AR.
A functional assay of A3AR-mediated inhibition of
forskolin-stimulated adenylyl cyclase showed marked
effects of certain N6 substitution on intrinsic efficacy.
The prototypical A3AR agonist Cl-IB-MECA 2 is con-
sidered a full agonist in most studies,1;6 although in
calcium responses in monocyte-derived dendritic cells it
appeared to be a partial agonist.18 The efficacy of the
analogues was reported as a single percentage value
(relative to the full agonist 2) at a nucleoside concen-
tration of 10 lM. The efficacy at the hA3AR of the N6-
(2-phenyl-1-cyclopropyl) analogues 3–25 was generally
nearly full (i.e., 100%). In a full concentration-response
experiment (Fig. 1), compound 10 was found to be a full
agonist at the hA3AR in the inhibition of adenylate
cyclase. The functional potency of 10 was comparable to
that of 2. The functional EC50 values for both were
ꢀ3 nM, which is in agreement with the Ki values in
binding of ꢀ1 nM. In contrast, N6-cyclopropyladeno-
sine 32 appeared to be an antagonist, with lack of effi-
cacy at 10 lM, at a concentration 100 times greater than
its Ki value in binding at the hA3AR. A N6-(2,2-diphen-
ylethyl) analogue 33 also proved to be an antagonist. A
Schild analysis of inhibition by 33 of the effects of an
A3AR agonist at the hA3AR was carried out (Fig. 2); it
demonstrated that the KB value for this competitive
antagonist was 5.0 nM. This was consistent with the
previously observed antagonist properties of the closely
related DPMA 34 at the hA3AR.7 Shortening the ethyl
moiety by one carbon, that is to obtain 35, or con-
straining the two rings with a biaryl bond, that is 36,
largely restored efficacy, although the A3AR affinity of
these two compounds varied greatly. The 9-fluor-
enylmethyl analogue 36 was as potent at the hA3AR as
the most potent N6-(2-phenyl-1-cyclopropyl) analogues
in this study. Nevertheless, 36 displayed only minimal
selectivity in comparison to the A1AR.
The 3-fluoro analogue 12 was 8-fold less potent than 10
at the hA3AR. 3,5-Difluoro or 3-trifluoromethyl
oxy substitution, in 13 and 14, respectively, further
lowered the affinity. The 3-nitro analogue 15 displayed
A3AR selectivity of 120-fold compared with the
hA2AAR. The two isomers of this diastereomeric mix-
ture, 16 and 17, differed 10-fold in A3AR affinity. The
more potent 1S,2R analogue 16 was >900-fold selective
for the hA3 compared with hA2AAR. The corresponding
3-amino analogue 18 was 9-fold weaker than 15 at
the hA3AR. This 3-amino derivative also displayed
decreased affinity at the human and rat A2A ARs. The 3-
acetamido derivative 19 was more potent than the cor-
responding 3-amino derivative at all subtypes. Another
electron-withdrawing group, 3-cyano in compound 20,
did not provide high affinity, but an electron-donating
group, 3-methoxy in 21, resulted in high A3AR affinity.
The analogue 22, which contains an electron-with-
drawing 3-trifluoromethyl group, bound to the hA3AR
with high affinity. However, two 3-trifluoromethyl
groups in 23 greatly reduced the affinity at the A3AR.
The 2,2-diphenylethyl substituted analogue 24 was not
highly potent at the A3AR and was generally nonselec-
tive. However, a heteroaromatic group could be
substituted in the place of the phenyl moiety of com-
pound 5; thus, a 2-(3-thienyl) analogue 25 displayed
high A3AR affinity.
For comparison, a number of previously reported ana-
logues were studied in the same assays. The N6-benzyl
group has been widely explored in the design of A3AR
agonists, such as Cl-IB-MECA 2. The combination of
N6-(3-iodobenzyl) and 50-uronamido groups in 2 greatly
enhanced both the A3AR potency and the selectivity in
comparison to 1. However, the simple 2-H benzyl
derivative 26 was only 2-fold more potent at the hA3AR
than 1. The next higher homologue, the 2-phenylethyl
derivative 27, was of greatly increased affinity and
selectivity at the human (but not rat) A3AR. Branching
of the alkyl groups in N6-substituted adenosines has
been well studied at the A1AR and A2AAR.13–16 At the
hA3AR, a moderate degree of stereoselectivity of bind-
ing was observed for introduction of a methyl group in
the R configuration at either the 1-(28 compared with
29) or 2-(30 compared with 31) position.
100
80
60
40
Cl-IB-MECA
Compd. 10
20
0
-11
-10
-9
-8
-7
-6
-5
Conc [log M]
Although the cyclopropyl group of the N6-(2-phenyl-1-
cyclopropyl) analogues 3–25 was introduced for steric
constraint, intended to freeze the biologically preferred
conformation, these analogues could also be considered
hybrids of the N6-phenylethyl analogue 27 and the
Figure 1. Effects of Cl-IB-MECA and 10 on cAMP production in
CHO cells stably expressing the hA3AR. The cAMP level corre-
sponding to 100% (10 lM forskolin) was 220 30 pmol mLꢁ1. The
EC50 value for compound 10 was 3.7 0.6 nM. Results are from a
single representative experiment, which was carried out in triplicate.

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S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
2
1
0
-9
-8
-7
-6
-5
log [Compd. 33], M
Figure 2. Antagonism by compound 33 of the inhibition of cyclic
AMP production elicited by Cl-IB-MECA in CHO cells stably trans-
fected with the hA3AR. The experiment was performed in the presence
of 10 lM rolipram and 3 units/mL adenosine deaminase. Forskolin
(10 lM) was used to stimulate cyclic AMP levels. The level of cAMP
corresponding to 100% was 220 30 pmol mLꢁ1. The KB value for
compound 33 was calculated to be 5.0 nM.
The effect of selected compounds, at a single concen-
tration of 10 lM, on stimulation of adenylyl cyclase via
the hA2BAR in stably transfected CHO (Chinese ham-
ster ovary) cells was examined. 50-N-Ethyluronamido-
Figure 3. The complex of the A3AR with two regioisomers in the
putative binding site of the hA3AR model. (A) (1S,2R)-2-phenyl-1-
cyclopropyl-adenosine
3 and (B) (1R,2S)-2-phenyl-1-cyclopropyl-
adenosine
(NECA)
stimulated
cyclic
AMP
adenosine 4. All ligands are displayed as ball and stick models, and the
side chains of hA3AR are shown as stick models. The H-bonding
between ligand and hA3AR is displayed in yellow. The A3AR is repre-
sented by a tube model with a different color for each TM domain
(TM3 in yellow, TM4 in green, TM5 in cyano, TM6 in blue, TM7 in
purple).
accumulation in a concentration-dependent manner
corresponding to an EC50 of 140 19 nM (n ¼ 4). The
maximum stimulation level of NECA at 10 lM was
expressed as 100%. Most compounds tested at 10 lM
had only a small effect (<50%) on cyclic AMP accu-
mulation. The ranges of percentage stimulation were 30–
50% (3, 6–10, and 15), 15–30% (5, 11, 12, 17, 19, 25, and
35), <15% (13, 14, 16, 18, 20–23, and 31). Compounds
that stimulated to >50% of the NECA response were 4
(60 2%), 24 (78 2%), 30 (56 3%), 34 (6 3 8%), and
36 (96 2%).
F168 (EL2), S181 (5.42), M177 (5.38), V178 (5.39), F182
(5.43), F239 (6,44), W243 (6.48), L246 (6.51), S247
(6.52), N250 (6.55), I268 (7.39), S271 (7.42), H272
(7.43), and N274 (7.45). For comparison, the less potent
isomer 4 was docked in a similar manner.
4. Molecular modeling
The purine ring of 3 was located in a hydrophobic
pocket defined by L91 (3.33) and L246 (6.51). The amine
of the N6 substituent in proximity to N250 (6.55) was H-
bonded with the hydroxyl group of S247 (6.52), and the
purine N3 atom formed an H-bond with the side chain
of Q167 (EL2). The 20-OH group of the ribose ring was
involved in H-bonding with the backbone carbonyl
group of I268 (7.39), and the 30-OH group formed H-
bonds with the carbonyl group of the backbone of S271
(7.42) and the side chain of H272 (7.43), consistent with
our A3 neoceptor model.31 The 50-hydroxyl group also
formed an H-bond with T94 (3.36). The phenyl moiety
showed an additional hydrophobic interaction with
F168 (EL2). Compared with the Cl-IB-MECA complex,
in the case of N6-phenylcyclo propyladenosine, the
phenyl ring showed a better p–p stacking interaction
with the aromatic ring of F168 (EL2). The distances
between the centers of the N6-phenyl ring and the F168
As the first step in modeling the environment within the
hA3AR surrounding the distal portion of the N6-binding
region, the agonist template molecule N6-(1S,2R)-(2-
phenyl-1-cyclopropyl) adenosine 3 was docked in a
rhodopsin-based homology model of the receptor.8 We
used the previously reported Cl-IB-MECA/hA3AR
complex as the starting geometry for the position of the
ribose moiety of 3, followed by a systematic conforma-
tional search varying the rotatable bonds of the N6
substitutent, that is t0 (C5–C6–N6–Ccp) and t1 (C6–N6–
Ccp–Ccp) angles. The constraint of the cyclopropyl ring
fixed the t2 (N6–Ccp–Ccp–Car) torsion angle. The ener-
getically optimized result of docking 3 in the hA3AR is
shown in Figure 3. Mutagenesis results were consistent
with molecular modeling that featured direct interaction
of nucleosides with TMs 3, 6, 7, and EL2 (the second
extracellular loop).6;8;30 Residues that were within 5 A
proximity to 3 in this putative binding site were L91
(3.33), T94 (3.36), H95 (3.37), N150 (EL2), Q167 (EL2),
aromatic ring were 3.69, 4.55, and 4.21 A, for com-
pounds 3, 4, and Cl-IBMECA, respectively. Thus, the
1S,2R isomer 3 showed a better p–p stacking interaction
ꢀ
ꢀ

S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
2027
than 4 in the distal region of the binding site of the N6
substituent, consistent with its higher measured binding
affinity.
cally favorable for binding to the hA3AR, and this
would be consistent with the experimental results.
Following the initial receptor docking of the set of N6-
modified adenosine derivatives, 3D-QSAR (quantitative
structure activity relationship) studies were performed
with CoMFA (comparative molecular field analysis)34;35
and CoMSIA (comparative molecular similarity indices
analysis).36 Two distinct models for the training set in
3D-QSAR, 1 and 2 as defined in the experimental sec-
tion,33 were derived by superimposition of the com-
plexes in which the ligands were docked in the putative
binding site of the hA3AR. The results were compared
according to the two models (Table 2). PLS (partial least
squares) analysis of model 1 generated in the CoMFA
3D-QSAR model displayed a modest q2 value of 0.44
and an r2 value of 0.98 for 27 compounds only after
removing five compounds (1, 10, 15, 18, and 29). These
compounds were detected as outliers from the residual
plot of the leave-one-out cross-validation. Model 2, with
a similar 3D orientation to the docking study, generated
a better 3D-CoMFA result than did model 1. This
model displayed a q2 value of 0.57 and an r2 value of
0.97, for 29 out of 32 compounds. Fewer compounds
(13, 21, and 32) were outliers in model 2 than in model 1.
The docked conformation of 3 displayed a preference
for t0, t1, and t2 angles of approximately )110ꢁ, )80ꢁ,
and 140ꢁ, respectively, in the docking complex of 4 the
angles of t0, t1, and t2 were )130ꢁ, 80ꢁ, and )140ꢁ. Thus,
the preferred t1 and t2 angles differed between the two
diastereomers. The 3-hA3AR complex showed ꢀ5 kcal
lower energy than that of the agonist 4, correlating with
the binding affinity. The binding affinity of 3 was ꢀ40-
fold higher than that of 4. The relative stability of the
hA3AR complex with the nitrophenyl diastereomer N6-
(1S,2R)-(2-phenyl-1-cyclopropyl) adenosine 16 and its
less potent 1R,2S diastereomer 17 was also checked. The
receptor complex of 16 showed ꢀ1.4 kcal lower energy
than the complex of 17, consistent with the stereoselec-
tivity of binding at the hA3AR. Thus, the docking study
of the hA3AR suggested that the 1S,2R diastereomer
might show more favorable enthalpy of binding to the
hA3AR compared with the 1R,2S isomer, thus increas-
ing the binding affinity.
The adenosine analogues with substitution of the phenyl
ring of the N6 substituent displayed a more limited
rotational freedom of this ring when docked in the
hA3AR.32 The 1S,2R diastereoisomers were assumed to
be generally more potent and therefore were used in the
docking. The phenyl substituents tended to be directed
toward EL2, especially residue N150. However, the
docked complexes of 15 and 18 showed different loca-
tions of the nitro and amino groups of the phenyl ring,
that is directed toward TM4 and TM5 rather than EL2,
because of more favorable nonbonding van der Waals
interactions, while keeping the same preference of t0, t1,
and t2 angles as for other phenyl substitutions.
CoMSIA methods with additional hydrophobic and H-
bonding fields37 have been shown to be of comparable
statistical significance to traditional CoMFA models,
but with somewhat more easily interpreted isocontour
surface maps. Here, CoMSIA performed better than
CoMFA. The better statistical result of CoMSIA was
attributed to the large contributions of hydrophobic and
H-bonding interactions (Table 2). In addition, the con-
formational diversity, using model 1 for 3D-QSAR, did
not affect any statistical parameters of CoMSIA,
whereas CoMFA was very sensitive to the conformation
used for the model. Both CoMSIA models required two
different outliers to reach the predictable model with a q2
value of >0.4. The result of the CoMSIA model was a q2
value of 0.53 for model 1 and a q2 value of 0.47 for
model 2 with the same r2 value of 0.96 for both models.
All ligands in Table 1 were subjected to similar receptor
docking, and binding energies were calculated. The rel-
ative binding energies for analogues substituted on the
phenyl moiety at the ortho, meta, and para positions
were 4.0, 0, and 1.4 kcal for a Cl atom, and 1.3, 0, and
0.3 kcal for a methyl group. For both substituents,
receptor docking confirmed that the meta position was
the most and the ortho position was the least energeti-
The superimposition of the CoMFA/CoMSIA map onto
the binding site of the receptor was interpretable with
respect to the SAR, because all conformations of the
Table 2. The result of CoMFA and CoMSIA 3D-QSAR for two models
Statistics
CoMFA
Model 2
0.57
29
CoMSIA
Model 1
Model 1
Model 2
q2
0.44
27
0.53
30
0.47
30
Number of compounds
Number of components
r2
5
6
6
6
0.98
0.13
198
0.97
0.16
0.96
0.18
99
0.96
0.19
87
SEEa
F
124
Contribution (%)
Steric
59.70
40.30
60.80
39.20
10.10
30.60
28.30
24.80
6.20
10.70
38.90
31.20
15.10
4.20
Electrostatic
Hydrophobic
H-bond donor
H-bond acceptor
a Standard error of estimate.

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S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
two models used for 3D-QSAR analyses were obtained
from the docking complex without any modifications,
such as RMS fitting and database alignment with a
template. The CoMFA and CoMSIA maps of the two
models showed similar contour maps for steric, elec-
trostatic, hydrophobic, and H-bonding fields, except for
the contours representing regions of hydrophilicity and
disfavored steric bulk (data not shown). The best con-
tour maps for each property through the correlation
with the binding site environment were the steric and the
electrostatic maps from the CoMFA model 2 and the
hydrophobic and H-bonding maps from the CoMSIA
model 1 (Fig. 4). The projection of the CoMFA/CoM-
SIA contour maps onto the binding site, which was
validated by the experimental results, displayed a good
complementarity. As shown in Figure 4B, blue regions,
indicating that an electropositive group would increase
the affinity, were located near the meta position of the
phenyl group (V141 (4.56), N150 (EL2)) and sur-
rounding the adenine ring (N250 (6.55), Q167 (EL2),
T94 (3.36)). The interpretation of the existence of two
blue contour maps at the meta position of the phenyl
ring was that most of the electron-withdrawing groups
with a negative charge did not improve the binding
affinity compared with the unsubstituted compound 3. A
red CoMFA contour favoring a negatively charged
group at the para position of the N6-phenyl ring binding
site was in proximity to S181 (5.42). For the steric
CoMFA map in Figure 4A, a large green contour
indicating tolerance of steric bulk around the N6-phenyl
group matched well with the hydrophobic binding site
surrounded by F168 (EL2), M177 (5.38), and F182
(5.43). The green contour of CoMFA also coincided
closely with the contour region of CoMSIA favoring
hydrophobicity. Model 1 with the conformational
diversity showed more regions of disfavored steric bulk
in CoMFA and favored hydrophilic groups in CoMSIA
than did model 2 (Fig. 4C). One of the hydrophilic
contours from CoMSIA overlapped well with the less
bulky contours from CoMFA. The H-bonding field map
(Fig. 4D) was very useful because of the importance of
H-bonding of adenosine analogues in binding to the
hA3AR, especially at the ribose-binding position and at
the exocyclic amine of the adenine moiety. Because the
Figure 4. CoMFA stddev * coeff contour plots for model 2 (Top) and CoMSIA stddev * coeff contour plots for model 1 (bottom). (A) Predicted
effects of structural modification on the binding affinity of 3 as docked in the hA3AR: green contours indicate sterically favored regions; yellow
contours indicate sterically disfavored regions. (B) Blue contours define a region where increased positive charge will result in increased affinity, and
red contours define a region where increased negative charge will be favorable. (C) To enhance the binding affinity of hA3AR, yellow and white areas
define hydrophobic and hydrophilic preferences, respectively. (D) The H-bond donor field contour display regions where H-bond acceptors on the
ligand are predicted to enhance (cyan) and disfavor (purple) binding. For the H-bond acceptor field, H-bond donors on the ligand are predicted to
enhance (magenta) and disfavor (red) binding.

S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
2029
ribose ring was the core moiety, H-bonding fields were
detected only near the N6 and 50-position. An H-bond-
ing donor contour around the N6 position was located at
the side chain of S247 (6.52), and an H-bond acceptor
contour around the N6 position was directed toward the
side chain of N250 (6.55). Thus, the steric, electrostatic,
hydrophobic, and H-bonding contour maps were sup-
ported by the docking complex, with each contour map
matched well with its surrounding amino acids with
hydrophobic, hydrophilic, and H-bonding properties in
the putative binding site of hA3AR.
is highly dependent on the nature of the N6 substituent.
A new series of high-affinity A3AR agonists and related
nucleoside antagonists was explored, and ligand docking
in a molecular model of the hA3AR defines a hydro-
phobic region for interaction with the N6-(2-phenyl-
ethyl) moiety. These findings for the N6 region may be
combined synthetically with other structural modifica-
tions to enhance the pharmacological profile for either
A3AR selectivity or mixed A1/A3AR selectivity. Agon-
ists of mixed A1/A3AR selectivity may be useful for
treating cardiac ischemia.22 To investigate the structural
basis for the differences in affinity between species and
the striking variation in intrinsic efficacy will require the
use of receptor mutagenesis.6;8
5. Discussion
CoMFA/CoMSIA 3D-QSAR and docking studies were
conducted on a series of potent, conformationally con-
strained A3AR agonists. The CoMFA/CoMSIA maps
from the 3D-QSAR study and the putative binding site,
based on both experimental results and a docking study,
were integrated to propose a binding mode for hA3AR
agonists. The superimposition of the contour map from
the CoMFA/CoMSIA study and the hA3AR docking
complex validated each other for the predictability of
the ligand-based method as a 3D-QSAR and receptor-
based approach through a homology modeling and
docking study.
6. Conclusion
A new series of high-affinity A3AR agonists was
explored. The adenosine derivatives are mainly sterically
constrained analogues of N6-(2-phenylethyl)adenosine,
found previously to display high affinity at the hA3AR.
The affinity and selectivity of these nucleosides is highly
dependent on the species examined and on the substi-
tution of a distal aryl substitution. A molecular model
defines a hydrophobic region, which includes Phe168 of
EL2, in the putative A3AR binding site around the distal
phenyl moiety. Upon probing of the SAR in this series,
several novel nucleoside antagonists of the A3AR were
identified.
The molecular modeling studies of the A3AR6 indicate
that required flexibility of the ribose moiety and a
movement of TM6 were correlated with receptor acti-
vation. Similarly, at the A2AAR a rotation of a con-
served Trp of TM6 has been proposed to be involved in
activation.8 We have assembled this series of adenosine
derivatives to apply a similar analysis of SAR to the N6
region.
7. Experimental
7.1. Chemistry
The present study focused on derivatives of N6-(2-
phenylethyl)adenosine, in which various degrees of
rigidity (a cyclopropane ring) and steric bulk (e.g., me-
thyl groups of 28–31) were included. The strikingly high
affinity of the N6-(1S,2R)-2-phenyl-1-cyclopropyl ana-
logue 3 was further explored through phenyl ring sub-
stitution. None of the diastereomeric analogues proved
to be more potent and selective than 3, although mod-
erate selectivity (22) and high affinity at the hA3AR (7,
8, 10, 11, 21, 22, and 25) were achieved. The consistently
greater affinity of these analogues at human compared
with rat A3ARs was also characteristic of the unsubsti-
tuted compounds 3 and 4 and the parent N6-(2-phen-
ylethyl)adenosine 27.
7.1.1. Materials and instrumentation. Reagents and sol-
vents were purchased from Sigma–Aldrich (St. Louis,
MO). H NMR spectra were obtained with a Varian
1
Gemini 300 spectrometer using CDCl3 as a solvent. The
chemical shifts are expressed as ppm downfield from
TMS. High-resolution FAB mass spectrometry was
performed with a JEOL SX102 spectrometer using 6 kV
Xe atoms. The chiral separation was done with a Hewlet
Packard 1090 HPLC system using a Chiralpak AD
column at an isocratic method with methanol as a
mobile phase, with flow rate 1mL/min. Peaks were
detected by UV absorption with a diode array detector.
All final compounds were analyzed by LC/MS showing
more than 96% purity. TLC analysis was carried out on
aluminum sheets precoated with silica gel F254 (0.2 mm)
from Aldrich.
Curiously, although the 2-phenyl-1-cyclopropyl ana-
logues tended to be full agonists, several related deriv-
atives had greatly reduced efficacy. The N6-cyclopropyl
derivative 32 was an A3AR antagonist; adding one or
two phenyl rings at the 2-position restored efficacy. The
N6-(2,2-diphenylethyl) derivative was
a
somewhat
7.2. General procedure for the synthesis of compounds
6–18 and 20–23
selective A3AR antagonist, but either adding a bond
between the two phenyl rings (N6-9-fluorenylmethyl) or
shortening the ethyl moiety (N6-diphenylmethyl)
restored efficacy. Thus, extending earlier findings,6;7 the
ability of an adenosine derivative to activate the A3AR
7.2.1. Methyl trans-3-(3-Chlorophenyl)propenoate (38e).
Concentrated H2SO4 (0.1 mL) was added to a solution
of trans-3-(3-chlorophenyl) propenoic acid (0.2 g,

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S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
1.09 mmol) in MeOH (5 mL). The solution was heated at
reflux overnight. After cooling the solution, the acid was
neutralized with saturated aqueous Na2CO3. The
aqueous solution was extracted with ether (3 · 50 mL).
The organic phase was washed with brine, dried over
Na2SO4, filtered, and concentrated. The residue was
purified by flash chromatography on silica gel (ether/
overnight. Then the mixture was cooled and concen-
trated. The solution was washed with ether (10 mL · 3),
and the aqueous phase was alkalinized with 10% aque-
ous K2CO3 until pH 10. The mixture was extracted with
EtOAc (10 mL · 3). The combined organic phases were
dried over K2CO3, filtered, and concentrated. The resi-
due was purified by PTLC (chloroform/methanol 9:1) to
1
1
petroleum ether 1:9), to give 38e as a white solid. H
give 41e. H NMR (CDCl3, 300 MHz) d 7.19–7.08 (m,
NMR (CDCl3, 300,MHz) d 7.63 (d, J ¼ 16:0 Hz 1H),
7.51 (br s, 1H), 7.34–7.26 (m, 2H), 6.43 (d, J ¼ 16:0 Hz
1H), 3.81 (s, 3H).
2H), 6.98–6.96 (m, 1H), 6.91–6.88 (m, 1H), 2.56–2.52
(m, 1H), 1.86–1.81 (m, 1H), 1.80–1.48 (br s, 2H), 1.11–
1.04 (m, 1H), 1.00–0.94 (m, 1H).
7.2.2. trans-2-(3-Chlorophenyl)-cyclopropanecarboxylic
acid methyl ester (39e). Diazomethane was generated
with a diazomethane-generating glassware kit (Aldrich).
A solution of N-methyl-N-nitroso-4-toluenesulfonamide
(Diazald, 2.23 g, 10.4 mmol) in ether (24 mL) was added
dropwise to a mixture of KOH (1.75 g, 31.2 mmol) in
H2O (18 mL), ether (4 mL), and 2-(2-ethoxyethoxy)eth-
anol (18 mL) kept at 70 ꢁC. The ethereal solution of
diazomethane was continuously distilled into a stirred
solution of 38a (206 mg, 1.05 mmol) and Pd(OAc)2
(1.16 mg, 0.00052) in CH2Cl2/ether (28/10 mL) kept at
0 ꢁC. The rate of distillation was controlled to match the
rate of addition. After the addition of Diazald was
complete, the solution of 38a was stirred at rt for 30 min.
The excess of diazomethane was destroyed with acetic
acid. The resulting mixture was washed with a saturated
solution of NaHCO3, brine, dried over Na2SO4, filtered,
and concentrated. NMR confirmed the resulting prod-
uct to be pure 39e, to be used for the next reaction
without further purification. 1H NMR (CDCl3,
300 MHz) d 7.21–7.18 (m, 2H), 7.08–7.07 (m, 1H), 7.00–
6.97 (m, 1H), 3.72 (s, 3H), 2.53–2.46 (m, 1H), 1.93–1.87
(m, 1H), 1.64–1.59 (m, 1H), 1.34–1.29 (m, 1H).
7.2.5. trans-2-(3-Chlorophenyl)-cyclopropyladenosine (10).
Compound 41e (12 mg, 0.07 mmol) and 6-chloropurine
riboside (25.8 mg, 0.09) were placed in a sealed tube with
absolute ethanol (1 mL) and triethylamine (10 lL) and
heated at 80 ꢁC for 8 h. The solution was evaporated and
the residue dissolved in chloroform (10 mL) and washed
with H2O (10 mL · 3). The organic phase was dried over
Na2SO4, filtered, and concentrated. The residue was
purified by PTLC (chloroform/methanol 9:1) to give
1
pure 10. H NMR (CDCl3, 300 MHz) d 8.23–8.21 (m,
1H), 7.77–7.76 (m, 1H), 7.22–7.08 (m, 4H), 6.48 (br s,
1H), 6.16 (br s, 1H), 5.79 (d, J ¼ 7:2 Hz 1H), 5.05 (t,
J ¼ 5:7 Hz 1H), 4.46 (d, J ¼ 4:5 Hz 1H), 4.34 (s, 1H),
3.94 (d, J ¼ 13:2 Hz 1H), 3.78–3.75 (m, 1H), 3.18 (br s,
1H), 2.25–2.06 (m, 1H), 1.73–1.51 (m, 1H), 1.41–1.25
(m, 2H). HR-MS: calculated for C19H21O4N5Cl
418.1282 found 418.1282.
7.2.6. N6-[trans-2-(2-Methylphenyl)cyclopropyl]adenosine
(6). Yield 45%. 1H NMR (CDCl3, 300 MHz) d 8.18–8.15
(m, 1H), 7.76 (s, 1H), 7.18–7.14 (m, 4H), 6.72–6.65 (m,
1H), 6.36–6.31 (m, 1H), 5.79 (d, J ¼ 7:2 Hz 1H), 5.02 (br
s, 1H), 4.43 (d, J ¼ 4:5 Hz 1H), 4.32 (s, 1H), 3.94 (d,
J ¼ 13:2 Hz 1H), 3.75–3.70 (m, 1H), 3.22 (br s, 1H), 2.40
(d, J ¼ 3 Hz, 3H), 2.18–2.13 (m, 1H), 1.36–1.23 (m, 4H).
MS (APCI) m=z ¼ 398 (Mþ1).
7.2.3. trans-2-(3-Chlorophenyl)-cyclopropanecarboxylic
acid (40e). Aqueous NaOH (2 M, 3 mL) was added to
a solution of 39e (200 mg, 1.13 mmol) in MeOH (2 mL),
and the mixture was stirred for 2 h at rt. The mixture
was concentrated and H2O (20 mL) was added. The
aqueous phase was washed with ether, acidified with
3 M aqueous HCl, and extracted with ether (20 mL · 3).
The combined organic phases were dried over Na2SO4,
7.2.7. N6-[trans-2-(3-Methylphenyl)cyclopropyl]adenosine
1
(7). Yield 49%. H NMR (CDCl3, 300 MHz) d 8.19 (s,
1H), 7.74 (m, 1H), 7.22–7.15 (m, 1H), 7.05–6.95 (m, 3H),
6.29 (br s, 1H), 5.78–5.72 (m, 1H), 5.04 (m, 1H), 4.45 (m,
1H), 4.32 (s, 1H), 3.92 (d, J ¼ 13:2 Hz 1H), 3.73 (d,
J ¼ 13:2 Hz 1H), 3.17 (s, 1H), 2.33 (s, 3H), 2.09 (m, 1H),
1.37–1.24 (m, 2H). MS (APCI) m=z ¼ 398 (Mþ1).
1
filtered, and concentrated to give pure 40e. H NMR
(CDCl3, 300 MHz) d 7.23–7.20 (m, 2H), 7.10–7.08 (m,
1H), 7.02–6.98 (m, 1H), 2.61–2.54 (m, 1H), 1.93–1.89
(m, 1H), 1.71–1.65 (m, 1H), 1.44–1.37 (m, 1H).
7.2.8. N6-[trans-2-(4-Methylphenyl)cyclopropyl]adenosine
1
7.2.4. trans-2-(3-Chlorophenyl)-cyclopropylamine (41e).
A mixture of 40e (105 mg, 0.72 mmol) in dry t-BuOH
(1.3 mL), diphenylphosporazidate (170 lL, 0.8 mmol),
and triethylamine (105 lL, 1 mmol) was stirred at 90 ꢁC
under nitrogen atmosphere for 48 h. The solution was
concentrated and poured into 10% aqueous Na2CO3
(20 mL) and extracted with ether (10 mL · 3). The
combined organic phases were dried over Na2SO4, fil-
tered, and concentrated. The resulting t-butyl carbamate
was dissolved in MeOH (3 mL), and 1 M aqueous HCl
(5 mL) was added. The solution was maintained at reflux
(8). Yield 47%. H NMR (CDCl3, 300 MHz) d 8.32 (s,
1H), 7.78 (s, 1H), 7.11 (m, 2H), 6.17 (br s, 1H), 5.79 (d,
J ¼ 7:8 Hz 1H), 5.05 (m, 1H), 4.50 (d, J ¼ 3:8 Hz 1H),
4.35 (s, 1H), 3.96 (d, J ¼ 13:2 Hz 1H), 3.75 (d,
J ¼ 13:2 Hz 1H), 3.49 (s, 1H), 3.20 (m, 1H), 2.84 (br s,
1H), 2.33 (s, 3H), 2.16 (m, 1H), 1.29–1.25 (m, 2H). MS
(APCI) m=z ¼ 398 (Mþ1).
7.2.9. N6-[trans-2-(2-Chlorophenyl)cyclopropyl]adenosine
(9). Yield 40%. 1H NMR (CDCl3, 300 MHz) d 8.17–8.16

S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
2031
(m, 1H) 7.76 (s, 1H), 7.78–7.77 (m, 1H), 7.38–7.35 (m,
1H), 7.21–7.11 (m, 4H), 6.72–6.65 (br m, 1H), 6.48–6.31
(br m, 1H), 5.79–5.78 (m, 1H), 5.01 (br s, 1H), 4.45 (br s,
1H), 4.32 (s, 1H), 3.94 (d, J ¼ 13:2 Hz 1H), 3.75–3.70
(m, 1H), 3.52 (br s, 1H), 3.15(br s, 1H), 2.48 (m, 1H),
1.77 (br s, 1H), 1.43–1.23 (m, 4H). MS (APCI)
m=z ¼ 418 (Mþ1).
7.2.16.
N6-[trans-2-(3-Acetamidophenyl)cyclopropyl]-
adenosine (19). Yield 39%. 1H NMR (CD3OD,
300 MHz) d 8.26 (br s, 2H), 7.43–7.36 (m, 2H), 7.25–7.19
(m, 1H), 6.97–6.95 (m, 1H), 5.97 (d, J ¼ 6:6, 1H), 4.36–
4.31 (m, 1H), 4.17–4.14 (m, 1H), 3.92–3.87 (m, 2H),
3.25–3.07 (m, 1H), 2.17–2.04 (m, 4H), 1.38–1.23 (m,
2H). MS (APCI) m=z ¼ 441 (Mþ1).
7.2.10. N6-[trans-2-(4-Chlorophenyl)cyclopropyl]adeno-
sine (11). Yield 42%. 1H NMR (CDCl3, 300 MHz) d
8.35 (s, 1H), 7.80 (s, 1H), 7.32–7.15 (m, 4H), 6.27 (br m,
1H), 6.09 (br s, 1H), 5.80 (d, J ¼ 7:8 Hz 1H), 5.06 (br s,
1H), 4.51 (d, J ¼ 3:8 Hz 1H), 4.35 (s, 1H), 3.96 (d,
J ¼ 13:2 Hz 1H), 3.78–3.73 (m, 1H), 3.25 (br s, 1H), 2.74
(br s, 1H), 2.14 (m, 1H), 1.33–1.25 (m, 2H). MS (APCI)
m=z ¼ 418 (Mþ1).
7.2.17. N6-[trans-2-(2-Cyanophenyl)cyclopropyl]adeno-
sine (20). Yield 45%. (CDCl3, 300 MHz) d 8.27 (s, 1H)
7.83 (s, 1H), 7.33–7.30 (m, 1H), 7.29–7.20 (m, 3H), 6.19–
6.03 (m, 2H), 5.79 (d, J ¼ 7:2 Hz 1H), 5.05 (m, 1H), 4.48
(m, 1H), 4.34 (s, 1H), 3.94 (d, J ¼ 13:2 Hz 1H), 3.81–
3.75 (m, 1H), 3.22 (m, 1H), 2.95–2.86 (m, 1H), 2.12–2.08
(m, 1H), 1.48–1.25 (m, 2H). MS (APCI) m=z ¼ 408
(Mþ1).
7.2.11. N6-[trans-2-(3-Fluorophenyl)cyclopropyl]adeno-
sine (12). Yield 35%. 1H NMR (CDCl3, 300 MHz) d
8.25 (s, 1H), 8.15 (s, 1H) 7.86–7.24 (m, 4H), 7.07 (br s,
1H), 7.02 (br s, 1H), 5.79 (d, J ¼ 7:8 Hz 1H), 5.15 (m,
1H), 4.52–4.30 (m, 2H), 3.95–3.72 (m, 2H), 2.93–2.85
(m, 1H), 2.35 (br s, 1H), 1.43–1.20 (m, 2H). MS (APCI)
m=z ¼ 402 (Mþ1).
7.2.18. N6-[trans-2-(3-Methoxyphenyl)cyclopropyl]adeno-
sine (21). Yield 38%. 1H NMR (CDCl3, 300 MHz) d 8.24
(s, 1H), 7.77 (br s, 1H), 6.85–6.72 (m, 3H), 6.25 (br s,
1H), 5.79 (br s, 1H), 5.05 (m, 1H), 4.48–4.34 (m, 2H),
3.96–3.72 (m, 6H), 3.20 (br s, 1H), 2.17 (m, 1H), 1.50–
1.38 (m, 2H). MS (APCI) m=z ¼ 414 (Mþ1).
7.2.19. N6-[trans-2-(3-Trifluoromethylphenyl)cycloprop-
yl]adenosine (22). Yield 30%. 1H NMR (CDCl3,
300 MHz) d 8.34 (s, 1H), 7.8 1 (br s, 1H), 7.52–7.40 (m,
4H), 6.30 (br s, 1H), 6.12 (br s, 1H), 5.81 (d, J ¼ 6:9,
1H), 5.07 (m, 1H), 4.48 (m, 1H), 4.35 (s, 1H), 3.97 (m,
1H), 3.81–3.75 (m, 1H), 3.25 (br s, 1H), 2.25 (m, 1H),
1.51–1.38 (m, 2H). MS (APCI) m=z ¼ 452 (Mþ1).
7.2.12. N6-[trans-2-(3,5-Difluorophenyl)cyclopropyl]adeno-
sine (13). Yield 40%. 1H NMR (CDCl3, 300 MHz) d 8.35
(s, 1H) 7.81 (s, 1H), 6.78–6.66 (m, 3H), 6.09 (br s, 1H),
5.80 (d, J ¼ 7:8 Hz 1H), 5.07 (br s, 1H), 4.50 (br s, 1H),
4.35 (s, 1H), 5.96 (d, J ¼ 12 Hz, 1H), 3.76 (d, J ¼ 12 Hz,
1H), 3.21 (br s, 1H), 2.77 (br s, 1H), 2.17 (m, 1H), 1.35–
1.15 (m, 2H). MS (APCI) m=z ¼ 420 (Mþ1).
7.2.20. N6-[trans-2-[3,5-Di(trifluoromethyl)phenyl]cyclo-
propyl]adenosine (23). Yield 32%. 1H NMR (CDCl3,
300 MHz) d 8.37 (s, 1H), 8.21 (s, 1H) 7.86–7.34 (m, 3H),
7.07 (br s, 1H), 6.99 (br s, 1H), 5.79 (d, J ¼ 7:8 Hz 1H),
5.07 (m, 1H), 4.51–4.34 (m, 2H), 3.97–3.70 (m, 2H),
2.92–2.89 (m, 1H), 2.32 (br s, 1H), 1.31–1.25 (m, 2H).
MS (APCI) m=z ¼ 520 (Mþ1).
7.2.13.
N6-[trans-2-(3-Trifuoromethyloxyphenyl)cyclo-
propyl]adenosine (14). Yield 30%. 1H NMR (CDCl3,
300 MHz) d 8.35 (s, 1H), 8.18 (s, 1H) 7.96–7.34 (m, 4H),
7.07 (br s, 1H), 7.02 (br s, 1H), 5.79 (d, J ¼ 7:8 Hz 1H),
5.05 (m, 1H), 4.51–4.34 (m, 2H), 3.97–3.70 (m, 2H),
2.92–2.85 (m, 1H), 2.32 (br s, 1H), 1.41–1.25 (m, 2H).
MS (APCI) m=z ¼ 468 (Mþ1).
7.2.21. N6-(2,2-Diphenylcyclopropyl)adenosine (24).
Yield 52%. 1H NMR (CDCl3, 300 MHz) d 8.31–8.24
(m, 2H), 7.51–7.13 (m, 10H), 6.48 (br s, 1H), 5.89 (br s,
1H), 5.61 (d, J ¼ 7:2 Hz 1H), 4.91–4.87 (m 1H), 4.34 (d,
J ¼ 4:5 Hz 1H), 4.24 (m, 1H), 3.90–3.83 (m, 2H), 3.70–
3.63 (m, 1H), 1.78–1.45 (m, 2H). MS (APCI) m=z ¼ 460
(Mþ1).
7.2.14. N6-[trans-2-(3-Nitrophenyl)cyclopropyl]adenosine
(15). Yield 42%. 1H NMR (CDCl3, 300 MHz) d 8.26–8.1
6 (m, 1H), 8.15 (br s, 1H), 8.07 (d, J ¼ 8:4 Hz, 1H), 7.82
(s, 1H), 7.60–7.57 (m, 1H), 7.50–7.39 (m, 4H), 5.85 (d,
J ¼ 6:3 1H), 5.02 (t, J ¼ 6:5 Hz, 1H), 4.47 (br s, 1H),
4.35 (s, 1H), 3.97 (d, J ¼ 13:2 Hz 1H), 3.78–3.73 (m,
1H), 3.17 (br s, 1H), 2.25 (m, 1H), 1.50–1.41 (m, 2H).
MS (APCI) m=z ¼ 429 (Mþ1).
7.2.22. N6-[2-(3-Thienyl)cyclopropyl]adenosine (25).
Yield 45%. 1H NMR (CDCl3, 300 MHz) d 8.32 (s,
1H), 7.80 (s, 1H) 7.02–7.01 (m, 2H), 6.13 (br s, 1H), 5.81
(d, J ¼ 7:5 Hz 1H), 5.06 (m, 1H), 4.50 (d, J ¼ 4:5 Hz
1H), 4.35 (s, 1H), 3.96 (d, J ¼ 12:8 Hz, 1H), 3.76 (d,
J ¼ 12:8 Hz, 1H), 3.22–3.13 (m, 1H), 2.82 (br s, 1H),
2.22–1.18 (m.1H), 1.31–1.20 (m, 2H). MS (APCI)
m=z ¼ 390 (Mþ1).
7.2.15. N6-[trans-2-(3-Aminophenyl)cyclopropyl]adeno-
sine (18). Yield 35%. H NMR (CD3OD, 300 MHz) d
8.26 (s, 2H), 7.02 (t, J ¼ 7:8 Hz, 1H), 6.56 (t, J ¼ 7:8 Hz,
3H), 5.96 (d, J ¼ 6:6, 1H), 4.76–4.72 (m, 1H), 4.33–4.31
(m, 1H), 4.17–4.16 (m, 1H), 3.91–3.71 (m, 2H), 2.15–
2.07 (m, 1H), 1.35–1.22 (m, 2H). MS (APCI) m=z ¼ 399
(Mþ1).
1

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S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
1
7.2.23. N6-(Cyclopropyl)adenosine (32). Yield 65%. H
NMR (CDCl3, 300 MHz) d 8.25 (s, 1H), 7.77 (s, 1H)
6.46 (d, J ¼ 11:4 Hz, 1H), 6.03 (br s, 1H), 5.79 (d,
J ¼ 7:5 Hz 1H), 5.06 (m, 1H), 4.48 (d, J ¼ 4:5 Hz 1H),
4.34 (s, 1H), 3.94 (d, J ¼ 10:8 Hz, 1H), 3.12–2.93 (m,
2H), 0.93 (d, J ¼ 6:9, 2H), 0.66 (s, 1H). MS (APCI)
m=z ¼ 308 (Mþ1).
nol (11.5 mL) was added, and the mixture was refluxed
for 6 h. The solution was concentrated in vacuo, and
H2O (20 mL) was added. The aqueous mixture was
heated to 90 ꢁC and filtered and allowed to cool overnight.
The crystals were filtered and redissolved in hot water,
and the solution was filtered. The filtrate was acidified
with 10% aqueous HCl, and the resulting precipitate was
separated and identified as pure 47. H NMR (CDCl3,
300 MHz) d 7.38–7.35 (m, 2H), 7.27–7.12 (m, 8), 2.52–
2.47 (m, 1H), 2.08–2.05 (m, 1H), 1.62–1.57 (m, 1H).
1
7.2.24.
N6-[2,2-Di-(phenylethyl)cyclopropyl]adenosine
1
(33). Yield 60%. H NMR (CDCl3, 300 MHz) d 8.13
(s, 1H), 7.59 (s, 1H) 7.32–7.17 (m, 10H), 6.01 (br s, 1H),
5.68 (d, J ¼ 7:2 Hz 1H), 4.92 (t, J ¼ 6:5 Hz 1H), 4.36–
4.26 (m, 4H), 4.09 (br s, 1H), 3.90–3.86 (d, J ¼ 12:9 Hz
1H), 3.73–3.66 (m, 2H). MS (APCI) m=z ¼ 448 (Mþ1).
8. Pharmacology
8.1. Materials
7.2.25.
trans-2-(3-Acetamidophenyl)-cyclopropylamine
[3H]R-PIA and [125I]I-AB-MECA were from Amersham
(44). Ethylchloroformate (0.035 mL, 0.35 mmol) was
added to a solution of 42 (50 mg, 0.25 mmol) and tri-
ethylamine (0.04 mL, 0.3 mmol) in dry acetone at
)10 ꢁC. The solution was stirred at the same tempera-
ture for 2 h, and a solution of NaN3 (25 mg, 0.38 mmol)
in H2O (0.5 mL) was added. The stirring was interrupted
after 1 h, and additional H2O (5 mL) was added. The
solution was concentrated and extracted with EtOAc
(20 mL · 3). The combined organic layers were dried
over Na2SO4, filtered, and concentrated. The residue
was dissolved in toluene (50 mL), and the solution was
concentrated by half to eliminate traces of H2O. The
solution was heated at 90 ꢁC for 2 h while observing N2
evolution. Then the toluene was evaporated, and the
resulting isocyanate was dissolved in dry 2-(trimethyl-
silyl)ethanol (2 mL) and the solution was heated to 60 ꢁC
overnight. The solution was cooled and concentrated.
The residue was purified by flash chromatography on
silica gel (ether/petroleum ether 1:9), to give the pure
carbamate. The carbamate was treated with tetrabuty-
lammonium fluoride (1 M solution in THF 0.28 mL,
0.28 mmol) at 50 ꢁC for 24 h. The solution was cooled,
and H2O (5 mL) was added, and the resulting mixture
was stirred for 20 min. Then it was concentrated, and the
aqueous solution was acidified with 1 M HCl and
washed with ether (20 mL · 3), and the aqueous phase
was alkalinized with 10% aqueous Na2CO3. The mixture
was extracted with EtOAc (10 mL · 3). The combined
organic phases were dried over K2CO3, filtered, and
concentrated. The residue was purified by PTLC (chlo-
roform/methanol 9:1) to give 44. 1H NMR (CDCl3,
300 MHz) d 8.18–8.01 (m, 1H), 7.33–7.28 (m, 1H), 7.17–
7.12 (m, 1H), 6.73–6.71 (m, 1H), 3.30–3.24 (m, 1H), 2.54
(br s, 1H), 2.18 (s, 3H), 1.87–1.83 (m, 1H), 1.61 (br s,
2H), 1.44–1.35 (m, 2H).
Pharmacia
Biotech
(Piscataway,
NJ),
and
[3H]CGS21680 was from Perkin–Elmer. (Boston, MA).
Adenosine deaminase was obtained from Sigma (St.
Louis, MO). All other compounds were obtained from
standard commercial sources and were of analytical
grade.
8.2. Biological assays
The procedures for [3H]R-PIA and [3H]CGS21680
binding to A1 and A2A receptors, respectively, were as
previously described.7 Briefly, membranes (10–20 lg of
protein) were incubated with radioligand and the com-
peting adenosine derivative in duplicate, together with
increasing concentrations of the competing compounds,
in a final volume of 0.4 mL TrisÆHCl buffer (50 mM,
pH 7.4) at 25 ꢁC for 60 min. Binding reactions were
terminated by filtration through Whatman GF/B glass–
fiber filters under reduced pressure with a MT-24 cell
harvester (Gaithersburg, MD). Filters were washed
three times with ice-cold buffer and placed in scintilla-
tion vials with 5 mL scintillation fluid, and bound
radioactivity was determined by using a liquid scintil-
lation counter. Functional assays of adenylyl cyclase
(either stimulation via the hA2B AR or inhibition via the
hA3AR) stably transfected CHO cells was carried out as
previously described.26
8.3. Statistical analysis
Binding and functional parameters were estimated with
GraphPAD Prism software (GraphPAD, San Diego,
CA). IC50 values obtained from competition curves were
converted to Ki values with the Cheng–Prusoff equa-
tion.19 Data were expressed as mean standard error.
7.2.26. 2,2-Diphenylcyclopropanecarboxylic acid (47).
1,1-Diphenylethylene (1.02 g, 5.67 mmol), dry Cu2SO4
(58.3 mg, 0.36 mmol), and dry benzene (2 mL) were put
in a three-necked flask equipped with two condensers
and a dropping funnel. The solution was stirred and
heated at 75 ꢁC for 6 h. Ethyldiazoacetate (1.3 mL) was
added dropwise over 1 h. The mixture was cooled to rt
and stirred overnight. NaOH (1.45 g, 36 mmol) in etha-
8.4. Molecular modeling
All calculations were performed on a Silicon Graphics
Octane workstation (300 MHz, MIPS R12000 (IP30)
processor, Mountain View, CA). All ligand structures

S. Tchilibon et al. / Bioorg. Med. Chem. 12 (2004) 2021–2034
2033
were constructed using the Sketch Molecule of SYBYL
6.9.20 A conformational search of compounds 3 and 4 to
be docked was performed by random search for all
rotatable bonds. The options of random search were
3,000 iteration, 3 kcal energy cutoffs, and chirality
checking. In all cases, MMFF force field27 and charge
were applied using distance-dependent dielectric con-
stants and conjugate gradient method until the gradient
CoMSIA descriptors were derived from the same lattice
box used for the CoMFA calculations. For the calcul-
ation of CoMSIA similarity indices, five different simi-
larity fields including steric, electrostatic, hydrophobic,
H-bond donor, and H-bond acceptor were calculated at
the regularly spaced grid points with a common probe
ꢀ
atom with radius of 1 A, charge, hydrophobicity, and H-
bonding properties of þ1. A Gaussian function with the
default value (0.3) of the attenuation factor, a, was used
for the distance dependence between the molecule and
the probe atoms. The steric CoMSIA fields were from
the internally coded parameters of the van der Waals
table in SYBYL program. The electrostatic fields were
calculated from the atomic partial charge of MMFF94.
The hydrophobic fields were derived from atom-based
values based on the research of Viswanadhan et al.38
The H-bond donor and acceptor fields were obtained by
a rule-based method from experimental values, creating
dummy atoms at donor and acceptor sites like extension
points of DISCO (distance comparisons).37
ꢀ
reached to 0.05 kcal/mol/A. After clustering the low-
energy conformers from the result of the conformational
search, the representative conformers from all groups
were reoptimized by semiempirical molecular orbital
calculations with the PM3 method in the MOPAC 6.0
package.28
A hA3AR model (PDB code: 1o74) constructed by
homology to the high-resolution X-ray structure of
bovine rhodopsin17 was used for the docking study. The
(1S,2R)-isomers of all ligands in Table 1 were docked
within the hA3AR model. The atom types were manu-
ally assigned with the Amber all atom force field29 and
their charges were calculated before docking. The
starting geometry of ligand conformation was chosen
from the hA3AR complex model with Cl-IB-MECA,
which was already validated by point mutation.6 The
ribose-binding position of this series was fixed by an
atom-by-atom fitting method for the carbon atoms of
the ribose ring. Only N6-binding regions were variously
positioned in the putative binding cavity, rotating the
flexible bonds of N6 substituents, t0 and t1 angles. Sev-
eral conformations without any steric bump were
selected for further optimization. The initial structures
of all complexes were optimized with the Amber force
field with a fixed dielectric constant of 4.0 and termi-
The PLS regression analyses39 were used to derive a
linear relationship. The predictive value of the models
was evaluated first by SAMPLS (sample distance partial
least squares)40 and then by leave-one-out cross-valida-
tion, with a 2 kcal/mol column filtering with a column
scaling of CoMFA standard. The outlier points whose
target values were badly predicted in the residual plot
from the cross-validation analyses were omitted to get
the predictable model with a sufficiently high q2 value
(>0.4). For the conventional r2 value, final noncross-
validation with the number of components to the opti-
mum value from the cross-validation analysis was per-
formed. CoMFA and CoMSIA stdev * coeff contour
maps were generated by a default value of contribution.
nating gradient of 0.1 kcal molꢁ1 ꢁ1. Binding energy
A
ꢀ
was calculated by the following equation: binding
E¼complex E)(receptor Eþligand E). These energies
are not rigorous thermodynamic quantities but can only
be used to compare the relative stabilities of the com-
plexes. Consequently, these interaction energy values
cannot be used to calculate binding affinities because
changes in entropy and solvation effects are not taken
into account. In addition, nonbonding van der Waals
and electrostatic energies were calculated.
Acknowledgements
Dr. Neli Melman thanks the Cystic Fibrosis Foundation
for financial support. We thank Dr. Tom Spande and
Dr. Victor Livengood (NIDDK) for mass spectral
measurements.
For the training set of 3D-QSAR, two models were
generated. In model 1, a series of energetically favorable,
bound conformations from the docking complex were
selected and aligned in the 3D Cartesian space, and
similar conformations for the t0, t1, and t2 binding
preference were used for model 2. The Ki values of
hA3AR for the training set were converted to pKi
(ꢁ log Ki) values as dependent variables in the CoMFA
and CoMSIA. To derive the CoMFA and CoMSIA
descriptors as independent variables, a 3D cubic lattice
References and notes
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