JOURNAL OF
MEDICINAL CHEMISTRY 0 Copyright 1994 by the American Chemical Society
Volume 37, Number 5
March 4, 1994
Communications to the Editor Discovery of New Non-Phospholipid Inhibitors of the Secretory Phospholipases A2 Haydn G. Beaton,+Colin Bennion,t Stephen Connolly,t Anthony R. Cook,? Nigel P. Gensmante1,t Catherine Hallam,$ Kim Hardy,? Barbara Hitchin,$ Clive G. Jackson,s and David H. Robinson**t Departments of Medicinal Chemistry, Physical Chemistry, and Biochemistry, Fisons plc, Pharmaceutical Division, Research and Development Laboratories, Bakewell Road, Loughborough, Leicestershire LE1 1 ORH, U.K. Received November 8, 1993
Phospholipases A2 (PLA2s)l catalyze the hydrolysis of fatty acids from the sn-2 position of aggregated glycerophospholipids. The first PLAz enzymes to be characterized were the closely related, low molecular weight secretory proteins (sPLAns), isolated from such sources as mammalian pancreas (designated as group I sPLA2), snake venom (groups I and I1 sPLA2), bee venom (group I11 sPLA2), and human platelets (group I1 sPLA2). More recently, however, a quite different, high molecular weight cytosolic PLA2 was describede2 This enzyme (cPLA2) selectively cleaves arachidonic acid from membrane phospholipids and is considered to catalyze the rate-limiting step in the cellular generation of biologically active eicosanoid products. Due to this pivotal role in the formation of a variety of proinflammatory lipid mediators, both the mammalian group I1 sPLAz3and cPLAz4 have been widely implicated in the initiation and exacerbation of the inflammatory response in animals. In attempt to discover novel antiinflammatory agents, considerable effort has been expended in the design of potential inhibitors of both of these enzyme classes, but to date, no bona fide selective inhibitor has been reported to be undergoing clinical de~elopment.~ The properties of these enzymes and the associated reasons for the slow progress in inhibitor design have been discussed in a number of recent reviews.l In a previous publication,6 we described the design and synthesis of some phospholipid substrate analogue int
Department of Medicinal Chemistry.
t Department of Physical Chemistry. 8
Department of Biochemistry.
0022-2623/94/1837-0557$04.50/0
hibitors of pancreatic sPLA2 and defined the binding interactions in the enzyme-inhibitor complex using NMR and molecular modeling techniques. The usual ambiguities associated with in vitro screening of these enzymes were resolved using parallel assays with both monomerically dispersed and aggregated substrate systems. Phosphocholine 1 was one of the more potent compounds we described, but due to its physical properties and metabolic instability represented a poor drug candidate. In this communication we describe the discovery of a novel series of highly potent, non-phospholipid inhibitors of both the porcine pancreatic and human group I1 sPLAz enzymes. Inhibitor Design and Structure-Activity Relationships. Pancreatic (Group I) PLA2. During our earlier enzyme-inhibitor interaction studies? we concluded that one of the phosphate oxygen atoms of compounds such as 1 forms an ionic interaction with the Ca2+ ion at the active site of pancreatic PLA2 while another accepts a hydrogen bond from the OH of mobile residue, Tyr-69. Furthermore, we observed that replacement of the quaternary N atom of l with an OH group produced a phosphoglycol derivative with enhanced activity over the phosphocholine. These findings led us to speculate that it may be possible to achieve similar strength of binding with a simple anionic group in place of the phosphocholine moiety present in the parent inhibitor. Biochemical screening (Table 1)of a newly synthesized series of carboxylate analogues of 1 showed the foregoing hypothesis to be correct. Inspection of the activities of compounds 2a-d indicates that direct replacement of the phosphocholine moiety by carboxylate (compound 2b) produces an inhibitor with comparable potency to 1. Homologue 2c was similarly active, whereas 2a and 2d with shorter and longer methylene chains, respectively, were less active. In an attempt to reduce the overall inhibitor lipophilicity, the hexadecanoyl chain of 2b was replaced by a decanoyl moiety to give 3, a molecule with somewhat reduced activity. However, as was previously observed6 in the phosphocholine series, we found that the even less lipophilic trans-dec-3-enoyl analogue (4) displayed a similar level of potency to the longer chain compound (2b). A combination of 2-D NMR and molecular modeling techniques indicated that the binding interactions between 0 1994 American Chemical Society
Communications to the Editor
558 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 5 Table 1. In Vitro Activity of Substrate Analogue Inhibitors of PLA2 compd
structure
panc PLA2 monob 0.30 f 0.05 (15)
2a: n = 0 2b n = l 2c: n = 2 2d n = 3
6a: n = 1 6b n = 2
9a: n = 1 9b: n = 2
ICw ( r M P panc PLA2 DOCC
human platelet PLA&
30 (4)
NT
15 (2) 0.19 ( 5 ) 0.20 (2) 1.3 (2) 1.4 (2)
>30 (2) 7.3 (3) 4.5 (2) 19 (2) 15 (2)
NT NT NT NT NT
0.30 (3)
8.5 (2)
NT
0.095 (2)
2.2 (2)
NT
0.023 (2) 0.016 (2)
0.79 f 0.06 (10) 0.38 (2)
NT
>250 (2)
>loo (2)
12 (3)
0.030 (2)
0.12 (2)
3.8 (2)
NT
NT
0.015 (2)
0.030 (4)
0.065 (2) 0.021 0.004 (33)
1.85 (2)
a Mean of at least two separate results (number of individual results shown in parentheses); data for small sets (individual results
