|[Molecules: 8] [Related articles/posters: 043 114 050 032 115 ]|
Combinatorial chemistry methods have unleashed a powerful strategy for diversity-based discovery of new leads.1 Convergent, resin-based parallel synthesis of discrete chemical libraries is a focused adaptation of combinatorial chemistry that provides an attractive lead development tool for the refinement of biological activity.2 Herein, we report on the solid-phase parallel synthesis of analogues of a prototypical fibrinogen receptor (GPIIb/IIIa) antagonist lead, RWJ-50042 (1). This approach resulted in a marked compression of the drug lead-to-clinical-candidate timeline vis-a-vis traditional solution-phase techniques.
RWJ-50042 is an orally active antagonist of the platelet fibrinogen receptor (binding IC50 = 0.0045 uM; sustained ex vivo inhibition of collagen-induced platelet aggregation at 10 mg kg-1 in dogs), which was discovered by using the solution structure of the C-terminal g-chain of fibrinogen as a basis for drug design.3-7 This substituted nipecotic acid modelled the b-turn structure contained within the KQADG sequence of the g-chain (residues 406-410). Given the competitive environment surrounding this type of antithrombotic therapy,8 we pursued synthetic methodology to expedite the study of RWJ-50042 analogues.
Scheme 1 Strategy for preparation of a "virtual" library of 288 variants of RWJ-50042 (6 x 4 x 12)
Isonipecotic acid and five- and seven-membered-ring variants of nipecotic acid were chosen to study scaffold size/conformation change of the "central ring" in relation to the other two components. While 288 variants (6 x 4 x 12) were targeted in principle for synthesis, a concurrent refinement process was implemented to select relatively optimal components for subsequent analogue synthesis/evaluation. This process of using a "virtual" compound library discards inferior components to avoid the unnecessary synthesis and bioassay of weakly active agents.
Resin-based preparation of the 3-phenyl-3-aminopropionic acid analogue (15) of RWJ-50042 typifies our strategy of convergent, high-throughput synthesis. N-attachment of 2-chlorotrityl chloride resin to allyl 4-piperidinepropanoate furnished "N-protected" intermediate A. Allyl ester removal under mild, reproducible palladium(0) conditions and then DIC-mediated coupling to allyl piperidinepropanoate afforded pseudo-dipeptide B. Starting at the N-terminus allows one to accrue large quantities of resin-bound intermediate B in a common reaction vessel. Once B was saponified, the resin was split for coupling with twelve readily available b-amino esters, leading to final products. For modifications at the tertiary amide, analogous urethane and urea couplings at the N-nipecotyl position were performed by using standard p-nitrophenylchloroformate conditions9 with the appropriate resin-bound primary alcohol or primary amine (e.g., 2 and 3). Solution-phase coupling of CBZ-4-piperidineethanesulfonyl chloride with ethyl nipecotate gave the corresponding sulfonamide intermediate (e.g., 4).10 Intermediate B was saponified and coupled to methyl 3-phenyl-3-aminopropionate to render C. To isolate a variety of carboxylic acid targets from readily available methyl or ethyl b-amino esters, an organic solution method of KOSiMe3/THF saponification was adapted to intermediates such as C. This method allows the resin to swell suitably to complete ester cleavage when basic aqueous conditions fail. Potassium carboxylates were then acidified with dilute AcOH and cleaved with CF3CO2H to give products, exemplified by 3-phenyl derivative 15.
A representative synthesis that uses the asymmetric Michael addition as the key step is shown for methylenedioxybenzene derivative 25 (Scheme 4). Conjugate addition of (R)-(+)-1-phenylethylamine to ethyl 3,4-methylenedioxybenzeneacrylate proceeds with >90% diastereomeric excess. Hydrogenolysis of the a-methylbenzyl group and HBTU-mediated coupling with Boc-(R)-(-)-nipecotic acid15 gives a Boc-nipecotamide intermediate which is then N-terminally deprotected and coupled iteratively with Boc-piperidinepropanoic acid. Purifications were performed on fully protected coupling products via chromatography on silica gel. Nipecotamide 25, for instance, was isolated in 17% overall yield.
This parallel synthesis methodology rapidly produced greater than a dozen analogues of RWJ-50042, which were targeted for enantiospecific scale-up synthesis and subsequent in vivo evaluation (some are shown in Table 2). From an in vitro standpoint, some of the more promising compounds were the doubly racemic 3-(3,4-methylenedioxyphenyl) and 3-(3-quinoline) congeners. Indeed, enantiospecific synthesis of these fibrinogen receptor antagonists afforded the most potent compounds in this series (3S-enantiomers 25 and 28). 3-Substituted b-aminopropionic acid analogues with the R absolute configuration are only weakly active.
To address potential oral bioavailability limitations of our amino acid-like antagonists, compound 25 was "capped" at the N- or C-terminus. N-Methylpiperidine 26 and ethyl ester 27 exhibit inferior in vitro and in vivo characteristics relative to 25, however. The power of parallel synthesis played a decisive role in overcoming this bioavailability hurdle. Due to the ample selection of potent, solid-phase synthesis-derived compounds, analogues were identified with useful systemic availability (15-20%) without the need for prodrug modifications (oral canine studies).
Table 1 Inhibition of human platelet aggregation and fibrinogen binding by RWJ-50042 analogues (uM)
Pl. Agg. Bndg Pl. Aggr. Bndg
# Y Z IC50* IC50+ # n IC50* IC50+
1 COCH2 CH 0.66 0.005 1 1 0.66 0.005
2 COO CH 4.7 0.027 7 0 7.0 0.004
3 CONH CH 6.7 0.016 8 2 7.6 0.013
4 SO2CH2 CH 11.0 0.025 9 1 >25 (isonipec.) >25
5 COCH2 CH 2.4 0.006 10 1 0.34 (3R) 0.005
(N-Me-piperidine) 11 1 2.93 (3S) 0.004
6 COCH2 N >25 >25
Pl. Agg. Bndg Pl. Agg. Bndg
# X IC50* IC50+ # X IC50* IC50+
1 b-Ala 0.66 0.005 17 2-Me-b-Ala >25 >25
12 N-Me-b-Ala 27.5 0.20 18 2-OH-b-Ala 0.85 0.005
13 3-Me-b-Ala 2.0 0.003 19 4-oxo-nipecotic acid >25 0.33
14 3-Bui-b-Ala 4.1 0.0025 20 3-NH-c-C6H10-CO2H >25 0.20
15 3-Ph-b-Ala 3.6 0.003 21 NH(CH2)2SO3H 10.8 0.18
16 L-Asp-OMe 1.1 0.003 22 NH(CH2)2-5-tetrazole 25.5 1.39
* Thrombin-induced gel-filtered platelet aggregation (uM, n = 3).3
+ Inhibition of biotinylated fibrinogen binding to immobilized GPIIb/IIIa (uM, n = 2).3
Table 2 In vitro data for three-substituted b-amino acid GPIIb/IIIa antagonists
Fg Binding+ Human GFP*
# R1 R2 R3 IC50 (uM) IC50 (uM)
10 H H H 0.005 0.34
23 H C C-Ph H 0.0002 0.080
24 H C C-But H 0.021 0.079
25 H 3,4-methylenedioxy-Ph H 0.0005 0.028
26 Me 3,4-methylenedioxy-Ph H 0.0002 0.84
27 H 3,4-methylenedioxy-Ph Et 0.046 36
28 H 3-quinoline H 0.0002 0.019
29 H 2-thiophene H 0.0002 0.090
Xemlofiban (SC-54684) 0.0006 0.31
* Thrombin-induced gel-filtered platelet aggregation (n = 3).3
+ Inhibition of biotinylated fibrinogen binding to immobilized GPIIb/IIIa (n = 2).3
Table 3 Canine ex vivo data for four GPIIb/IIIa antagonists
Dog PRP* Dog ex vivo platelet aggregation+
Compd IC50 (uM) PO Dose Duration
10 0.41 3 mg kg-1 120 min
24 0.45 10 mg kg-1 90 min
25 0.015 3 mg kg-1 >180 min
Xemlofiban 1.20 3 mg kg-1 >180 min
* Collagen-induced platelet-rich plasma aggregation (n = 3).
+ Oral dose required to inhibit collagen-induced canine ex vivo platelet aggregation at least 50% (3 dogs).