Purification of combinatorial libraries.

In the early days of combinatorial chemistry, much attention focused on preparation of large libraries for lead discovery. Recently, though, the focus has shifted toward smaller, more focused libraries for lead optimization. These focused libraries generally consist of individual discrete compounds. Biological assay requirements often require compounds of high purity, thus development of automated high throughput purification methods has received new attention in the past several years. This paper covers automated high throughput purification methods that have been applied to libraries of discrete compounds. Literature published through February 1998 is included. Purification methods discussed include extraction methods, scavenger methods, solid phase extraction, and preparative HPLC.

High throughput analysis and purification in support of automated parallel synthesis.

Rapid reverse-phase analytical and preparative HPLC methods have been developed for application to parallel synthesis libraries. Gradient methods, short columns, and high flow rates allow analysis of over 300 compounds per day on a single system, or purification of up to 200 compounds per day on a single preparative system. Hardware and software modifications allow continuous unattended use for maximum efficiency and throughput.

Development of highly potent inhibitors of Ras farnesyltransferase possessing cellular and in vivo activity.

Analogs of CVFM (a known nonsubstrate farnesyltransferase (FT) inhibitor derived from a CA1A2X sequence where C is cysteine, A is an aliphatic residue, and X is any residue) were prepared where phenylalanine was replaced by (Z)-dehydrophenylalanine, 2-aminoindan-2-carboxylate, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Tic), and indoline-2-carboxylate. The greatest improvement in FT inhibitory potency was observed for the Tic derivative (IC50 = 1 nM); however, this compound was ineffective in blocking oncogenic Ras-induced transformation of NIH-3T3 fibroblast cells. A compound was prepared in which both the Cys-Val methyleneamine isostere and the Tic replacement were incorporated. This derivative inhibited FT with an IC50 of 0.6 nM and inhibited anchorage-independent growth of stably transformed NIH-3T3 fibroblast cells by 50% at 5 microM. Replacing the A1 side chain of this derivative with a tert-butyl group and replacing the X position with glutamine led to a derivative with an IC50 of 2.8 nM and an EC50 of 0.19 microM, a 26-fold improvement over (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-valyl]-1,2,3,4- tetrahydro-3-isoquinolinyl]carbonyl]-L-methionine. This derivative, (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-tert-leucyl]-1,2,3,4 - tetrahydro-3-isoquinolinyl]-carbonyl]-L-glutamine, was evaluated in vivo along with (S*,R*)-N-[[2-[N-(2-amino-3- mercaptopropyl)-L-tert-leucyl]-1,2,3,4-tetrahydro-3- isoquinolinyl]carbonyl]-L-methionine methyl ester for antitumor activity in an athymic mouse model implanted ip with H-ras-transformed rat-1 tumor cells. When administered by injection twice a day at 45 mg/kg for 11 consecutive days, both compounds showed prolonged survival time (T/C = 142-145%), thus demonstrating efficacy against ras oncogene-containing tumors in vivo.

Phosphinyl acid-based bisubstrate analog inhibitors of Ras farnesyl protein transferase.

The rational design, synthesis, and biological activity of phosphonyl- and phosphinyl-linked bisubstrate analog inhibitors of the enzyme Ras farnesyl protein transferase (FPT) are described. The design strategy for these bisubstrate inhibitors involved connection of the critical binding components of the two substrates of FPT (ras protein and farnesyl pyrophosphate, FPP) through a phosphonyl- or phosphinyl-bearing linker. Compound 14, the first example in this series, was found to be a potent FPT inhibitor (I50 = 60 nM). A further 15-fold enhancement in activity was observed upon replacement of the VLS tripeptide sequence in 14 with VVM (15, I50 = 6 nM). The phosphinic acid analog 16 (I50 = 6 nM) was equiactive to phosphonic acid 15. Compounds 14-16 afforded 1000-fold selectivity for FPT against the closely related enzyme geranylgeranyl protein transferase type I, GGT-I [14, I50(GGT-I) = 59 microM; 15 I50(GGT-I) = 10 microM; 16 I50(GGT-I) = 21 microM]. Methyl and POM ester prodrugs 17-19 were prepared and evaluated in whole cell assays and appear to block ras-induced cell transformation, as well as colony formation in soft agar. A distinctive feature of this novel class of potent and selective bisubstrate FPT inhibitors is that they are non-sulfhydryl in nature.

Quinoxaline N-oxide containing potent angiotensin II receptor antagonists: synthesis, biological properties, and structure-activity relationships.

A series of novel quinoxaline heterocycle containing angiotensin II receptor antagonist analogs were prepared. This heterocycle was coupled to the biphenyl moiety via an oxygen atom linker instead of a carbon atom. Many of these analogs exhibit very potent activity and long duration of effect. Interestingly, the N-oxide quinoxaline analog was more potent than the nonoxidized quinoxaline as in the comparison of compounds 5 vs 30. In order to improve oral activity, the carboxylic acid function of these compounds was converted to the double ester. This change did result in an improvement in oral activity as represented by compound 44.

Pharmacology of novel imidazole alcohol inhibitors of primate renin.

SQ 30,774 and SQ 31,844 are representatives of a novel class of renin inhibitors, the imidazole alcohols. These compounds, which contain an imidazole ring as part of their active site binding group are potent in vitro inhibitors of primate renin, but not rat, hog of dog renin. In conscious, sodium-depleted cynomolgus monkeys both compounds produced a dose-related inhibition of plasma renin activity (PRA) at doses ranging between 0.001 and 1.0 mumol/kg, intravenously, and total inhibition was observed after the highest dose. However, a reduction in blood pressure was observed only after an intravenous dose of 10 mumol/kg or when the compounds were administered by infusion. In sodium-replete monkeys, SQ 30,774 inhibited the rise in arterial pressure and PRA following administration of exogenous monkey renin. When the compounds were administered orally at 50 mumol/kg, only SQ 31,844 significantly inhibited PRA (80%). It is concluded that representatives of the imidazole alcohol class of renin inhibitors are potent inhibitors of renin in vitro and inhibit PRA and lower arterial pressure in vivo.

Ketomethylureas. A new class of angiotensin converting enzyme inhibitors.

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term "ketomethylureas", is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE with I50 values in the nM range.

Cyclic (hydroxyphosphinyl)acyl dipeptides: a new class of angiotensin converting enzyme inhibitors.

Conformationally constrained phenylbutyl(hydroxyphosphinyl)acyl dipeptides are potent inhibitors of angiotensin converting enzyme. The activity enhancement obtained by introducing conformational constraint into these molecules is greater than for related sulfhydryl and carboxyl analogs. The results are interpreted in terms of a binding model which optimally positions both zinc binding and hydrophobic groups for active site binding.

Design of conformationally constrained angiotensin-converting enzyme inhibitors.

Modification of alanyl proline by introduction of both zinc coordinating and S1 subsite binding interactions affords potent new carboxy- and mercapto-acyl dipeptide angiotensin-converting enzyme (ACE) inhibitors. Design of these inhibitors was guided by an extension of the hypothetical ACE active site model originally used to derive captopril. Significant increases in ACE inhibitory activity were observed by introduction of conformation constraint into acyclic acyl dipeptides, thus further defining the three dimensional structure of the ACE active site.

Ketomethyldipeptides I. A new class of angiotensin converting enzyme inhibitors.

The design rationale for a new series of angiotensin-converting enzyme (ACE) inhibitors which incorporate a ketone substituent into a peptide backbone is described. Molecular regions which were expected to mimic the binding of an N-acyl tripeptide substrate at secondary binding sites S1 and S1' were systematically varied in order to study the specificity of inhibitor binding and optimize inhibition against ACE. The most effective ketomethyldipeptides inhibit ACE in the 10(-9) M range.

Ketomethyldipeptides II. Effect of modifications of the alpha-aminoketone portion on inhibition of angiotensin converting enzyme.

Results of an investigation aimed at identifying the consequences of chemical modifications of the alpha-aminoketone moiety of ketomethyldipeptides on angiotensin converting enzyme (ACE) inhibition are reported. These studies lead to the conclusion that within this series, the optimal structural backbone formulation for inhibition of ACE is represented by 1. Introduction of a Sar-Pro C-terminal dipeptide in this system, in contrast to other inhibitor classes, is compatible with potent inhibitory activity. Other structure-activity relationships for ketomethyldipeptides and related derivatives are presented, and speculations on possible modes of binding of these inhibitors to ACE, and on the question of ketone rehybridization are offered.