2-Substitution of adenine nucleotide analogues containing a bicyclo[3.1.0]hexane ring system locked in a northern conformation: enhanced potency as P2Y1 receptor antagonists.
Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of P2Y(1) receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y(1) receptor and by using the radiolabeled antagonist [(3)H]2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y(1) receptor was an (N)-methanocarba N(6)-methyl-2-iodo analogue 12, which displayed a K(i) value in competition for binding of [(3)H]5 of 0.79 nM and a K(B) value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y(1) receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y(1) receptors. An enzymatic method of synthesis of the 3',5'-bisphosphate from the corresponding 3'-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-0810, USA.
Hak Sung
HS
Kim
Michihiro
M
Ohno
Bin
B
Xu
Hea Ok
HO
Kim
Yongseok
Y
Choi
Xiao D
XD
Ji
Savitri
S
Maddileti
Victor E
VE
Marquez
T Kendall
TK
Harden
Kenneth A
KA
Jacobson
D000227:Adenine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001667:Binding, Competitive; D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D008968:Molecular Conformation; D058919:Purinergic P2 Receptor Antagonists; D011869:Radioligand Assay; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
C479686:4-(2-iodo-6-methylaminopurin-9-yl)-1-((phosphato)methyl)-2-(phosphato)bicyclo(3.1.0)hexane; D000227:Adenine Nucleotides; C546382:P2RY1 protein, human; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate