´╗┐
Molecular Recognition Section, Division of Intramural Research : NIDDK : National Institutes of Health
NavigationINDEX

Medicinal Chemistry

This page describes the current research in our lab to better understand the molecular mechanisms involved in receptor-ligand interactions.

For this purpose we use

This particular page contains data from the following paper:

  • van Rhee, A.M.; Jiang, J.L.; Melman, N.; Olah, M.E.; Stiles, G.L.; Jacobson, K.A.
    Interaction of 1,4-dihydropyridine and pyridine derivatives with adenosine receptors: selectivity for A3 receptors.
    J. Med. Chem., 1996, 39: 2980-2989.

in which the concept of "privileged structures" is used to convert a highly potent, and moderately A3 selective (vs either A1 or A2A receptors, but not calcium channels), calcium channel blocker into a potent and very selective A3 antagonist (vs any other binding site tested).

Introduction

The 1,4-dihydropyridines (DHPs) have been developed extensively as potent blockers and activators of L-type calcium channels. A number of these channel blockers, such as nifedipine (Figure 1, 7) and nicardipine (Figure 1, 10), are used therapeutically in the treatment of cardiovascular disorders, especially hypertension and coronary heart disease.

DHPs appear to be "privileged structures" in medicinal chemistry and pharmacology, i.e. they display affinity for many diverse binding sites. This adaptability of DHPs has been utilized to optimize affinity in binding to alpha character1a-adrenergic receptors (e.g., the antagonist SNAP 5089, Figure 1), and to platelet activating factor (PAF) receptors (Figure 1, 3). Thus, by careful structural modification, it has been possible to select for affinity at sites other than Ca2+-channels.

Studies of the structure-activity relationship of xanthines at A3 adenosine receptors have thus far failed to identify principles of achieving receptor subtype selectivity. In the current study, we aim to show that the affinity of DHPs can be optimized selectively for adenosine receptors versus L-type calcium channels and for A3 versus A1 and A2A adenosine receptors.



Results & Discussion

Structure-activity relationship (SAR; Table 1) analysis at adenosine receptors indicated that sterically bulky groups are tolerated at the 4-, 5-, and 6-positions. Compound 3 is a potent PAF-acether antagonist and not a calcium channel ligand, thus there is an uncoupling of the SAR in this series for interaction with both PAF and adenosine receptors on the one hand and L-type calcium channels on the other hand.

In a series of methyl (1), n-propyl (4), and even larger alkyl substituents (5) at the 4-position it was demonstrated that steric bulk at this position is tolerated in adenosine receptor binding, favored at the A3 subtype, and not detrimental at the A1 subtype.

In general, the affinity and receptor subtype selectivity of 4-aryl analogues was highly dependent on the substituents of the phenyl ring. Compound 14, bearing an ortho-trifluoromethyl group behaved similarly to the ortho-nitro substituted compound 8, and was 2-fold selective for A1 vs A3 receptors. The piperonal derivative 17 had a generally increased affinity at A1 and A3 adenosine receptor subtypes, but did not display subtype selectivity.

The relatively high affinity of a 4-aryl group larger than phenyl (17), an arylalkyl group (18), and dehydro analogues thereof (19, 22) further indicated a bulk tolerance at this position.

The 4-trans-beta character-styryl derivative, 19, was particularly potent and selective at human A3 receptors. Nicardipine, 10, differing from nitrendipine, 8, in the presence of a sterically bulky ester group at the 5-position, displayed a moderate enhancement of affinity at human A3 receptors of 2.6-fold, while at rat A1 and A2A receptors affinity was diminished 2-3 fold.


Table 1: Affinities of dihydropyridines at A1, A2A, and A3 receptors in Ki (ÁM) ▒ sem.a-e
compound R3 R4 R5 rA1a rA2Ab hA3c
1
MRS1036
Me CH3 CO2CH2CH3 32.6 ▒ 6.3 46.1 ▒ 6.8 32.3 ▒ 5.1
2
MRS1059
Me CH3 CO2CH2Ph 6.45 ▒ 1.47 9.72 ▒ 0.63 2.78 ▒ 0.89
3
Et CH3 CO2(CH2)2SPh 6.50 ▒ 0.47 7.10 ▒ 2.46 5.56 ▒ 1.36
4
MRS1043
Me (CH2)2CH3 CO2CH2CH3 8.17 ▒ 1.58 11.5 ▒ 3.8 6.51 ▒ 0.74
5
MRS1055
Me CH2CHCH3(CH2)2-
CH=C(CH3)2 (R,S)
CO2CH2CH3 9.10 ▒ 2.90 23.1 ▒ 8.6 7.90 ▒ 0.88
6
MRS1044
Me Ph CO2CH2CH3 11.0 ▒ 1.6 2.74 ▒ 0.85 12.0 ▒ 3.3
7
nifedipine
Me 2-NO2-Ph CO2CH3 2.89 ▒ 0.23 18.2 ▒ 2.51 8.29 ▒ 2.41
8
nitrendipine
Me 3-NO2-Ph CO2CH2CH3 8.96 ▒ 2.06 23.0 ▒ 3.7 8.30 ▒ 1.41
9
MRS1098
Et 3-NO2-Ph CO2CH2CH3 3.34 ▒ 2.17 18.2 ▒ 7.9 2.51 ▒ 0.15
10
nicardipine
Me 3-NO2-Ph CO2CH2CH2N(CH3)CH2Ph 19.6 ▒ 1.9 63.8 ▒ 4.2 3.25 ▒ 0.26
11
nimodipine
iPr 3-NO2-Ph CO2CH2CH2OCH3 20.1 ▒ 1.7 44.3 ▒ 14.4 8.47 ▒ 2.75
12
R-niguldipine
Me 3-NO2-Ph scientific formula 41.3 ▒ 3.5 d (10-4) 1.90 ▒ 0.40
13
S-niguldipine
Me 3-NO2-Ph scientific formula d (10-4) d (10-4) 2.80 ▒ 0.35
14
MRS1050
Me 2-CF3-Ph CO2CH2CH3 6.68 ▒ 2.37 20.7 ▒ 2.8 11.6 ▒ 1.7
15
R-BayK8644
Me 2-CF3-Ph NO2 0.785 ▒ 0.113 35.1 ▒ 10.1 2.77 ▒ 0.34
16
S-BayK8644
Me 2-CF3-Ph NO2 6.66 ▒ 1.89 86.3 ▒ 23.4 23.5 ▒ 0.6
17
MRS1054
Me 3,4-OCH2O-Ph CO2CH2CH3 3.66 ▒ 0.61 5.27 ▒ 1.97 4.58 ▒ 1.11
18
MRS1096
Me CH2CH2Ph CO2CH2CH3 8.81 ▒ 0.92 6.71 ▒ 2.06 2.30 ▒ 0.70
19
MRS1045
Me trans CH=CH-Ph CO2CH2CH3 16.1 ▒ 0.5 49.3 ▒ 12.5 0.670 ▒ 0.195
20e
MRS1081
Me CH3 CO2CH2CH3 10.8 ▒ 3.52 38.0 ▒ 10.6 47.1 ▒ 10.8
21e
MRS1073
Et CH3 CO2CH2CH3 25.9 ▒ 7.3 35.9 ▒ 15.3 7.24 ▒ 2.13
22e
MRS1097
Et trans CH=CH=Ph CO2CH2CH3 5.93 ▒ 0.27 4.77 ▒ 0.29 0.108 ▒ 0.012

a) Displacement of specific [3H]PIA binding in rat brain membranes.
b) Displacement of specific [3H]CGS 21680 binding in rat striatal membranes.
c) Displacement of specific [125I]AB-MECA binding at human A3 receptors expressed in HEK cells, in membranes.
d) Displacement of < 10 % of specific binding at the indicated concentration in M.
e) R6 = Me, except 20: R6 = butyl; 21 & 22: R6 = phenyl.


Affinities of enantiomeric pairs (12,13 & 15,16) indicated a general preference for the R- over the S- enantiomer at all of the receptor subtypes. This is in contrast to the affinity at L-type calcium channels and at alpha character1a adrenoceptors at which the S(+)-enantiomer is preferred.

At the 6-position, n-butyl substitution (20) was tolerated, and phenyl substitution (21) enhanced A3 adenosine receptor binding (4.5-fold vs 1), and lead to slight subtype selectivity.

Combination of 6-phenyl and 4-trans-beta character-styryl substituents greatly enhanced both affinity and selectivity. Consequently, the novel compound MRS1097 (22) displayed an affinity of 108 ▒ 12 nM at cloned human A3 adenosine receptors, was 55-fold selective for A3 vs A1 receptors, and 44-fold selective for A3 vs A2A receptors.


Table 2: Specifity for L-type Calcium Channels.a
compound
Ki (rCa2+)
Ratio of Ki (rCa2+)/Ki (hA3)
12
0.0597 ▒ 0.0001
0.031
18
0.910 ▒ 0.207
0.40
19
0.694 ▒ 0.165
1.04
21
17 ▒ 5 % (10-4)
> 14
22
< 10 % (10-4)
> 1000

a) Inhibition of specific [3H]isradipine binding at L-type calcium channels in rat brain membranes expressed in ÁM as Ki ▒ sem or percent displacement of specific binding at the indicated concentration (M), and the selectivity ratio versus affinity at cloned human A3 receptors.


Moreover , MRS1097 (22) was 1000-fold selective for A3 adenosine receptors vs L-type Ca2+-channels (Table 2), displaced less than 10 % of total [3H]NBI binding from the Na+- independent adenosine transporter in rat forebrain at a concentration of 10-4 M (data not shown), and was able to reverse N6-(3- iodobenzyl)adenosine-5'-N-methyluronamide induced inhibition of forskolin- stimulated adenylyl cyclase in CHO cells transfected with rat A3 adenosine receptors (Figure 2).



In addition to the aforementioned assays, the affinity of MRS1097 (22) was evaluated in a battery of radioligand binding assays (NovaScreen®, Div. of Oceanix Biosciences, Hanover, MD) at a concentration of 10-5 M (Table 3).


Table 3: Binding Sites with Negligible Affinity for MRS1097 (22)a
binding site subtype
amino acid receptors GABAA (muscimol)
GABAB (baclofen)
NMDA (kainate)
NMDA (quisqualate)
NMDA (phencyclidine)
glycine (strychnine sensitive
glycine (strychnine insensitive)
biogenic amine receptors alpha character1,2
beta character
D1,2
H1,2
5-HT1,2,3
M1,2,3
nicotinic
central benzodiazepine receptors (RO 151788)
ion channels N-type calcium
chloride
low conductance potassium
opioid receptors sigma character
peptide receptors AT1
CCKB
neuropeptide Y
neurotensin
somatostatin
ANF-1
EGF
C5a complement
second messengers sites forskolin
phorbol ester
inositol trisphosphate
uptake transporters adenosine
choline
dopamine
noradrenaline
serotonin

a) Displacement of < 25 % of the specific ligand from the relevant binding site.


Conclusions

  • In the present study, we have demonstrated that structural modification and careful examination of the SAR of DHPs resulted in the development of one of the first A3 adenosine receptor-selective non-xanthine ligands (patent pending).
  • MRS1097 (22) displayed a submicromolar affinity for human A3 adenosine receptors in a radioligand binding assay (Ki = 0.108 ▒ 0.012 ÁM).
  • In addition, we have shown that DHPs can be effective in attenuating the IB-MECA elicited inhibition of adenylyl cyclase in CHO cells expressing the cloned rat A3 adenosine receptor.
  • MRS1097 (22) was 55-fold selective versus A1 receptors, 44-fold selective versus A2A receptors, and over 1000-fold selective versus L-type Ca2+-channels.
MRS Home Page
Staff
Selected Publications
Adenosine Receptors
P2 Receptors
Medicinal Chemistry
Computational Chemistry
The Purine Club
GPCRs
Links

Chief: Dr. Kenneth Jacobson
The Molecular Recognition Section (MRS) is in the Laboratory of Bioorganic Chemistry at the National Institute of Diabetes and Digestive and Kidney Diseases which is part of the National Institutes of Health, Bethesda, MD, USA. General inquiries may be addressed to NIDDK, NIH, Building 31, Room 9A04, 31 Center Drive, MSC 2560, Bethesda, MD 20892-2560, USA.
Privacy || Copyright, Disclaimers and Access Restrictions || Accessibility

National Institute of Diabetes, Digestive & Kidney Diseases U. S. National Institutes of Health U.S. Department of Health & Human Services USA.gov Logo - link to the U.S. government's official web portal
National Institutes of Health