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B. ALLOSTERIC MODULATION ON THE ADENOSINE RECEPTOR

B. ALLOSTERIC MODULATION ON THE ADENOSINE RECEPTOR

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Figure 4 Dose–response curves for the potentiation by brucine of ACh wholecell M1 muscarinic receptor responses. (A) Brucine (10À4 M) enhanced the

potency of ACh to increase cAMP accumulation in M1 CHO cells by 2.6-fold. (B)

Brucine (100 AM) produced a 3.0-fold increase in ACh potency in Ca2+ response

to ACh. (From Ref. 9.)



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 5 (A) In a dose-dependent manner, N-chloromethyl brucine (CMB)

enhanced the field-stimulated contractions of isolated guinea pig ileum strips.

The contractions were inhibited by atropine (30 nM). (B) Histogram of the

percentage enhancement of contraction produced in four independent experiments of the type illustrated in A. (From Ref. 9.)



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



receptors offers both opportunities and drawbacks for therapeutic intervention [38,41]. For example, A1 adenosine agonists, through their interaction with adenosine A1 receptors on fat cells, are able to reduce free fatty

acid levels in the blood. Since this effect sensitizes insulin’s action [42]. It

may be a very useful feature in non-insulin-dependent diabetes mellitus

(type II diabetes). However, serious side effects occur by the concomitant

bradycardia and drop in mean arterial pressure due to interference with

cardiovascular adenosine receptors [43]. Various strategies have been followed to circumvent all or some of these problems, such as the development of partial agonists for that purpose [44–48]. It was shown that some

of these compounds were virtually ‘‘silent’’ on the heart, while keeping a

pronounced, full effect on adipose tissue [49].

On the other hand, among the effects of receptor-bound adenosine

is the ability to protect organs, including the heart and brain, from

ischemic injury [50–52]. The formation of extracellular adenosine as a

breakdown product of ATP is a local phenomenon, induced by a tissue

at risk (e.g., under hypoxic or anoxic conditions: heart failure, stroke,

etc.). As a consequence, compounds that would increase adenosine’s

concentration, and thus its tissue-protective effect, might have a better

therapeutic profile than the agonists described earlier. Marketed nucleoside transport blockers such as dipyridamole and dilazep have already

proven this concept by inhibiting the intracellular uptake of extracellular

adenosine, and thereby effectively increasing its concentration outside

the cell [53,54].

Another interesting approach is to enhance adenosine’s action

locally by means of an allosteric enhancer. In 1990, Bruns and coworkers reported on various 2-amino-3-benzoylthiophene derivatives

capable of enhancing the binding and activity of reference A1 receptor

agonists, such as N6-cyclopentyladenosine (CPA) [14,55]. One of these

‘‘allosteric modulators,’’ PD81,723, or (2-amino-4,5-dimethyl-trienyl)

[3-(trifluoromethyl) phenyl]methanone (Fig. 6), has been investigated

pharmacologically in greater detail by various independent research

groups [56–61].

The modulator PD81,723 enhances two- to threefold the binding

and function of agonists such as CPA, R-PIA, or NECA to adenosine

A1 receptors [62]. As shown in Figure 7, in displacement experiments of

[3H]DPCPX from the human adenosine A1 receptor (wild type), the binding curve of CPA in the presence of PD81,723 is shifted leftward; it seems

that CPA binds more efficiently, since lower concentrations of this agonist are needed to displace the same concentration of radioligand. This



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 6 Structure of PD81,723, (2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) phenyl]methanone and adenosine A1 agonists/antagonists.



Figure 7 Displacement of 0.4 nM [3H]DPCPX by various concentrations of CPA

from human wild-type (CHO A1) and mutant (CHO A1-mutT277A) adenosine A1

receptors in the absence (n) or presence (5) of PD81,723 (10 AM).



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



‘‘enhanced’’ activity of CPA is also maintained in second messenger

assays, where, for example, lower concentrations of CPA (in the presence of PD81,723) are needed for the inhibition of forskolin-stimulated

cAMP production in cells bearing adenosine A1 receptors (Fig. 8). It is

known that PD81,723 slows down the kinetics (dissociation) of 3H-labeled

agonists such as [3H]CHA or [3H]CCPA from the receptor as shown in

Figure 9; the half-life of 17 min for the dissociation of CCPA alone from

the rat A1 receptor is increased to 25 min in the presence of 10 AM

PD81,723 [63]. It is postulated that this compound binds to an allosteric

site on the adenosine A1 receptor—which, unlike the muscarinic one, is

not yet so well defined—while at somewhat higher concentration it binds

to the ligand binding site exhibiting antagonistic action. It is presumed

that via its allosteric activity PD81,723 increases the proportion of adenosine receptors in the ‘‘active’’ (R*) conformation that has a high affinity

for agonists and low for antagonists and inverse agonists (Fig. 2). Not

only are these effects selective for the A1 receptor, but they disappear

upon a mutation of the receptor at the proposed agonist binding site [62].

Threonine at position 277 on the A1 receptor is considered to interact with

ribose ring of agonists, since changing it to alanine greatly decreases the

affinity for agonists but not for antagonists. This mutation also eliminated

the activity of PD81,723 (Fig. 7), which no longer can increase the already



Figure 8 Forskolin-stimulated cAMP production of CHO A1 cells after addition

of CPA in the absence (n) or presence (5) of 10 AM PD81,723.



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 9 Dissociation of agonist [3H]CCPA from rat brain A1 receptors in the

presence (5) or absence (n) of 10 AM PD81,723.



low affinity of agonists [64–67]. This indicates that an intact agonist binding site of the receptor is required for PD81,723 to exert its allosteric

action [62].

Recently we developed a series of novel PD81,723 analogues,

some of which appear to be superior to PD81,723 in their enhancing

activity [68,69]. The synthesis of these derivatives is relatively straightforward, as shown in Figure 10 [68–72]. The 4,5-dimethyl group and

the benzoyl moiety were targets for further modifications, leading to

series of 4,5-dialkyl (1a–g), of tetrahydrobenzo (1h–u) and of tetrahydropyridine (3a–g) derivatives (Fig. 10, Tables 1 and 2). These derivatives were evaluated both as allosteric enhancers of agonist binding to

the rat adenosine A1 receptor and as antagonists on this receptor.

Among them, a number of compounds, in particular 1b, 2e, 1j, 1n,

and 1u (Fig. 11, Table 1), proved to be superior to the reference

compound (PD81,723) in both enhancing activity and diminished antagonistic behavior [68].

Some structure–activity relationships of a further developed R4, R5

alkyl/cycloalkyl series (2a–o, Fig. 10, Table 1) were also investigated.

This study [69] revealed structural features that favored allosteric

enhancing activity, such as benzoyl lipophilic substitution and thiophene

4-alkyl substitution, while other features, such as thiophene 5-bulky

substitution, favored antagonistic properties. Upon further analysis, a



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 10 Scheme of synthesis of PD81,723 analogues. Reagents and

conditions: i, DMF; S8, Et3N, RT (or EtOH, S8, Et2NH, 50jC); ii, C6H6, h-alanine,

HOAc; iii, EtOH, S8, Et2NH; iv, BzCl, CH2Cl2, Et3N.



Table 1 Structure and Enhancing/Antagonistic Activity of 2-Amino-3-benzoylthiophenes

Analogues 1a–u and 2a–o



Compound

PD81,723

1a

1b

1c



R0



R4



R5



Enhancement (%)a



Antagonism (%)b



3-CF3



CH3



CH3



100



39 (F4)



CH3

CH3

CH3



CH3

CH3

CH3



8 (F5)

80 (F19)

93 (F32)



H

3-Cl

4-Cl



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



14 (F3)

19 (F4)

41 (F6)



Table 1 Continued

Compound

PD81,723

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

1q

1r

1s

1t

1u

2a

2b

2c

2d

2e

2f

2g

2h

2i

2j

2k

2l

2m

2n

2o



R0



R4



R5



Enhancement (%)a



Antagonism (%)b



3-CF3



CH3



CH3



100



39 (F4)



H

3-CF3

3-Cl

4-Cl

H

2-Cl

3-CF3

3-Cl

3-I

4-CF3

4-Cl

4-Br

4-I

4-NO2

4-CH3

4-CO2CH3

4-CO2H

3,4-Cl

3-CF3

3-Cl

H

3-CF3

3-Cl

3-Cl

H

3-Cl

3-CF3

H

H

H

3,4-Cl

4-tBu

4-tBu



CH2CH3

CH3

CH2CH3

CH3

CH2CH3

CH3

CH3

CH2CH3

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

—(CH2)4—

H

CH3CH2CH2

H

CH3CH2CH2

H

CH3CH2CH2

H

C5H9

H

C5H9

H

C6H11

H

C6H5

H

C6H5

H

C6H5

H

(CH3)2CHCH2

CH3

CH3CH2

CH3CH2

CH3CH2CH2

CH3

CH3

CH3

CH3

—(CH2)4—



31

112

30

97

47

73

122

93

113

131

123

128

155

34

137

44

29

151

88

67

0

99

52

57

21

38

42

À7

13

69

116

125

137



(F4)

(F10)

(F7)

(F25)

(F4)

(F19)

(F19)

(F6)

(F18)

(F11)

(F15)

(F18)

(F21)

(F22)

(F21)

(F9)

(F3)

(F24)

(F8)

(F18)

(F30)

(F25)

(F12)

(F2)

(F5)

(F6)

(F7)

(F14)

(F17)

(F19)

(F7)

(F24)

(F10)



13

5

22

20

35

35

32

51

66

57

40

42

64

19

30

29

35

52

54

50

49

64

64

75

80

58

47

27

17

50

47

40



(F3)

(F11)

(F2)

(F12)

(F6)

(F3)

(F8)

(F5)

(F1)

(F4)

(F5)

(F4)

(F8)

(F2)

(F3)

(n=1)

nd

(F4)

(F3)

(F5)

(F7)

(F2)

(F1)

(F3)

(F2)

(F1)

(F3)

(F5)

(F7)

(F6)

(F1)

(F2)

(F4)



Enhancing activity (at 10 AM of test compound) is expressed as percentage of decrease (FSEM) in

[3H]CCPA dissociation over control (0%) and that of PD81,723 (100%, n = 3).

b

Antagonistic activity is expressed as percentage of displacement (FSEM) of 0.4 nM of [3H]DPCPX by 10

AM of test compound. nd: not determined.

a



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Table 2 Structure and Enhancing/Antagonistic Activity of 2-Amino-3-benzoyl4,5,6,7-tetrahydrothieno [2,3-c]pyridines 3a–g and 4



Compound

3a

3b

3c

3d

3e

3f

3g

4

Theophylline



R0



R1



H

H

H

H

4-Cl

4-Cl

3,4-Cl





H

3-Cl

4-Cl

3,4-Cl

H

3,4-Cl

H





Enhancement (%)a

53

106

69

57

132

106

174

14

15



(F37)

(F27)

(F23)

(F36)

(F21)

(F31)

(F37)

(F27)

(F7)



Antagonism (%)b

67

80

52

4

60

46

51

72

56



(F5)

(F1)

(F2)

(F2)

(F0)

(F2)

(F0)

(F2)

(F5)



Enhancing activity is expressed as percentage of decrease (FSEM) in [3H]CCPA

dissociation over control (0%) and that of PD81,723 (100%, n = 3).

b

Antagonistic activity is expressed as percentage of displacement (FSEM) of 0.4 nM of

[3H]DPCPX by 10 AM of test compound.

a



significant correlation was found between antagonistic activity and hydrophobic fragment constants (k values) [73] of substituent R5 (Fig. 12),

in contrast to a negative correlation with those of R 4. Finally, comparison of low energy conformations (Fig. 13) of some of the 2-amino-3benzoylthiophene derivatives (PD81,723 and 2f ) with known adenosine

A1 receptor antagonists (theophylline and 8-cyclohexyltheophylline)

indicated that thiophene 5-substituents (R5 ) may interact with the same

lipophilic domain of the adenosine A1 receptor that accommodates 8substituents of xanthine antagonists. The separation of the two activities, antagonism and allosteric enhancement, is ultimately necessary for

the development of more potent and selective allosteric enhancers for the

adenosine A1 receptor with potential therapeutic applications.



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 11 Concentration–effect curves for derivative 1u and PD81,723.

Enhancement of 100% is expressing the maximum decrease in [3H]CCPA

dissociation by the highest concentration of 1u.



Figure 12 Correlation of lipophilicity parameter (k) for substituent R 5 of compounds 1a,d–f,h, j, k, and 2a–o with their antagonistic activity. ***p < 0.0001.



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 13 Structure and low-energy conformation with van der Waals surface of

(a) theophylline; (b) CHT; (c) PD81,723; and (d) 2f.



IV. CONCLUSION

The possibility of allosterically modulating receptors offers novel pharmacological means of ‘‘fine-tuning’’ receptor function. Further clarification is

required with respect to whether such modulated receptors are a general

feature of all or only of a subset of GPCRs and whether endogenous agents

regulate via this mechanism receptor function in vivo. Finally, elucidation

of the molecular mechanisms of the allosteric interactions will provide

useful insights for the therapeutic exploitation of this phenomenon in the

design and development of appropriate modulatory drugs.

Abbreviations

ACh

cAMP

[3H]CCPA

CHO

CH3CN



Acetylcholine

Cyclic-3V,5V-adenosine monophosphate

[3H]-2-Chloro-N6-cyclopentyladenosine

Chinese hamster ovary

Acetonitrile



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



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