Cyclic sulfamide HIV-1 protease inhibitors, with sidechains spanning from P2/P2' to P1/P1'

Previous studies of HIV protease inhibitors have shown that it is possible to elongate the P1/P1' sidechains to reach the S3/S3' binding sites. By analogy, we expected that it would be possible to design inhibitors reaching between the S1/S1' and S2/S2' binding sites. Molecular modeling suggested that this could be achieved with appropriate ortho-substitution of the P2/P2' benzyl groups in our cyclic sulfamide inhibitors. Four different spacer groups were investigated. The compounds were smoothly prepared from tartaric acid in five steps and exhibit low to moderate activity, the most potent inhibitor possessing a Ki value of 0.53 microM.     

Structure-guided design and synthesis of P1' position 1-phenylcycloalkylamine-derived pentapeptidic BACE1 inhibitors

Previously, we reported potent pentapeptidic BACE1 inhibitors with the hydroxymethylcarbonyl isostere as a substrate transition-state mimic. To improve the in vitro potency, we further reported pentapeptidic inhibitors with carboxylic acid bioisosteres at the P(4) and P1' positions. In the current study, we screened new P1' position 1-phenylcycloalkylamine analogs to find non-acidic inhibitors that possess double-digit nanomolar range IC(50) values. An extensive structure-activity relationship study was performed with various amine derivatives at the P1' position. The most potent inhibitor of this pentapeptide series, KMI-1830, possessing 1-phenylcyclopentylamine at the P1' position had an IC(50) value of 11.6 nM against BACE1 in vitro enzymatic assay.     

Novel spirocyclic pyrrolidones as P2/P1 mimetics in potent inhibitors of HIV-1 protease

We have developed concise and efficient syntheses of novel spirocyclic pyrrolidones 1-3, which involve the alkylation of pyrrolidone precursor 13 with 1,5-dibromopentane, 16 and 15, followed by an in situ lactamization. Conjugates of 1 and 2 with P1'/P2' hydroxy-indanolamine moiety resulted in novel and potent inhibitors of HIV-1 protease 25 and 26, suggesting that 1 and 2 are novel P2/P1 HIV-PI mimetics.     

BACE1 inhibitors: optimization by replacing the P1' residue with non-acidic moiety

Recently, we reported potent BACE1 inhibitors KMI-429, -684, and -574 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays, especially, KMI-429 was confirmed to significantly inhibit Abeta production in vivo. However, acidic moieties at the P(4) and P(1)' positions of KMI-compounds were thought to be unfavorable for membrane permeability across the blood-brain barrier. Herein, we replaced acidic moieties at the P(4) position with other hydrogen bond acceptor groups, and these inhibitors exhibited improved BACE1 inhibitory activities in cultured cells. In this study, we replaced the acidic moieties at the P(1)' position with non-acidic and low molecular sized moieties.     

Symmetric fluoro-substituted diol-based HIV protease inhibitors. Ortho-fluorinated and meta-fluorinated P1/P1'-benzyloxy side groups significantly improve the antiviral activity and preserve binding efficacy.

HIV-1 protease is a pivotal enzyme in the later stages of the viral life cycle which is responsible for the processing and maturation of the virus particle into an infectious virion. As such, HIV-1 protease has become an important target for the treatment of AIDS, and efficient drugs have been developed. However, negative side effects and fast emerging resistance to the current drugs have necessitated the development of novel chemical entities in order to exploit different pharmacokinetic properties as well as new interaction patterns. We have used X-ray crystallography to decipher the structure-activity relationship of fluoro-substitution as a strategy to improve the antiviral activity and the protease inhibition of C2-symmetric diol-based inhibitors. In total we present six protease-inhibitor complexes at 1.8-2.3 A resolution, which have been structurally characterized with respect to their antiviral and inhibitory activities, in order to evaluate the effects of different fluoro-substitutions. These C2-symmetric inhibitors comprise mono- and difluoro-substituted benzyloxy side groups in P1/P1' and indanoleamine side groups in P2/P2'. The ortho- and meta-fluorinated P1/P1'-benzyloxy side groups proved to have the most cytopathogenic effects compared with the nonsubstituted analog and related C2-symmetric diol-based inhibitors. The different fluoro-substitutions are well accommodated in the protease S1/S1' subsites, as observed by an increase in favorable Van der Waals contacts and surface area buried by the inhibitors. These data will be used in the development of potent inhibitors with different pharmacokinetic profiles towards resistant protease mutants.     

Increased antiviral activity of cyclic urea HIV protease inhibitors by modifying the P1/P1' substituents.

A series of alkyl substituted P1/P1' analogs was prepared in an attempt to increase translation of the 3-aminoindazole class of HIV protease inhibitors. Increasing the lipophilicity of the P1/P1' residues dramatically improved translation of enzyme activity to antiviral activity in the whole cell assay.     

Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.     

Inhibitory serpins from rye grain with glutamine as P1 and P2 residues in the reactive center

Six of seven serpins detected in grains of rye (Secale cereale) were purified and characterized. The amino acid sequence close to the blocked N-terminus, the reactive center loop sequence and the second order association rate constant (k(a)') for irreversible complex formation with chymotrypsin were determined for each serpin. Three of four serpins containing the unusual reactive center P2-P1' QQ/S and one with P2-P1' PQ/M were equally efficient inhibitors of chymotrypsin (k(a)' approximately 10(5) M(-1) s(-1)). One serpin with P2-P1' PY/M was a faster inhibitor (k(a)' approximately 10(6) M(-1) s(-1)). Similar but differently organized glutamine-rich reactive centers were recently found in grain serpins cloned from wheat [Ostergaard et al. (2000) J. Biol. Chem. 275, 33272] but not from barley. The prolamin storage proteins of cereal grains contain similar sequences in their glutamine-rich repeats. A possible adaption of hypervariable serpin reactive centers late in Triticeae cereal evolution as defence against insects feeding on cereal grains is discussed.     

Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors.

HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 A resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the delta-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.     

P1 and P3 optimization of novel bicycloproline P2 bearing tetrapeptidyl alpha-ketoamide based HCV protease inhibitors

With the aim of discovering potent and selective HCV protease inhibitors, we synthesized and evaluated a series of 1a based tetrapeptidyl ketoamides with additional modification(s) at P1', P1, and P3 positions. As a result of this effort, we found that replacement of the P3 valine with tert-leucine resulted in the discovery of a series of inhibitors (e.g., 3a, 3c, and 4c) endowed with improved enzyme and/or cellular activity relative to 1a. When dosed to F-344 rats orally at 50mg/kg, 3a achieved 2.5x higher liver and plasma exposure in comparison to that detected with 1a.     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

P1' oxadiazole protease inhibitors with excellent activity against native and protease inhibitor-resistant HIV-1

HIV-1 protease inhibitors (PI's) bearing 1,3,4-oxadiazoles at the P1' position were prepared by a novel method involving the diastereoselective installation of a carboxylic acid and conversion to the P1' heterocycle. The compounds are picomolar inhibitors of native HIV-1 protease, with most of the compounds maintaining excellent antiviral activity against a panel of PI-resistant strains.     

Potent and selective P2-P3 ketoamide inhibitors of cathepsin K with good pharmacokinetic properties via favorable P1', P1, and/or P3 substitutions

A series of ketoamides were synthesized and evaluated for inhibitory activity against cathepsin K. Exploration of the interactions between achiral P(2) substituents and the cysteine protease based on molecular modelling suggestions resulted in potent cathepsin K inhibitors that demonstrated high selectivity versus cathepsins B, H, and L. Subsequent modifications of the P(3), P(1), and P(1') moieties afforded orally bioavailable inhibitors.     

Phe*-Ala-based pentapeptide mimetics are BACE inhibitors: P2 and P3 SAR

We describe herein the syntheses and evaluation of a series of C-termini pyridyl containing Phe*-Ala-based BACE inhibitors (5-19). In conjunction with four fixed residues at the P1 (Phe), P1' (Ala), P2' (Val), and P2' cap (Pyr.), rather detailed SAR modifications at P2 and P3 positions were pursued. The promising inhibitors emerging from this SAR investigation, 12 and 17 demonstrated very good enzyme potency (IC(50)=45 nM) and cellular activity (IC(50)=0.4 microM).     

Design and synthesis of potent beta-secretase (BACE1) inhibitors with P1' carboxylic acid bioisosteres

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-420 and KMI-429 in which we replaced the Glu residue at the P4 position of KMI-260 and KMI-360, respectively, with a 1H-tetrazole-5-carbonyl DAP (L-alpha,beta-diaminopropionic acid) residue. At the P1' position, these compounds contain one or two carboxylic acid groups, which are unfavorable for crossing the blood-brain barrier. Herein, we report BACE1 inhibitors with P1' carboxylic acid bioisosteres in order to develop practical anti-Alzheimer's disease drugs. Among them, tetrazole ring-containing compounds, KMI-570 (IC50=4.8 nM) and KMI-684 (IC50=1.2 nM), exhibited significantly potent BACE1 inhibitory activities.     

Synthesis and antiviral activity of P1' arylsulfonamide azacyclic urea HIV protease inhibitors

A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P1' were synthesized utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more potent inhibitors. The effects of substituting the P1' benzenesulfonyl group on antiviral activity and protein binding are described.     

Design and synthesis of HIV-1 protease inhibitors. Novel tetrahydrofuran P2/P2'-groups interacting with Asp29/30 of the HIV-1 protease. Determination of binding from X-ray crystal structure of inhibitor protease complex

A series of HIV-1 protease inhibitors having new tetrahydrofuran P2/P2' groups have been synthesised and tested for protease inhibition and antiviral activity. Six novel 4-aminotetrahydrofuran derivatives were prepared starting from commercially available isopropylidene-alpha-D-xylofuranose yielding six symmetrical and six unsymmetrical inhibitors. Promising sub nanomolar HIV-1 protease inhibitory activities were obtained. The X-ray crystal structure of the most potent inhibitor (23, K(i) 0.25 nM) co-crystallised with HIV-1 protease is discussed and the binding compared with inhibitors 1a and 1b.     

A synthetic method for diversification of the P1' substituent in phosphinic dipeptides as a tool for exploration of the specificity of the S1' binding pockets of leucine aminopeptidases

A novel, general, and versatile method of diversification of the P1' position in phosphinic pseudodipeptides, presumable inhibitors of proteolytic enzymes, was elaborated. The procedure was based on parallel derivatization of the amino group in the suitably protected phosphinate building blocks with appropriate alkyl and aryl halides. This synthetic strategy represents an original approach to phosphinic dipeptide chemistry. Its usefulness was confirmed by obtaining a series of P1' modified phosphinic dipeptides, inhibitors of cytosolic leucine aminopeptidase, through computer-aided design basing on the structure of homophenylalanyl-phenylalanine analogue (hPheP[CH(2)]Phe) bound in the enzyme active site as a lead structure. In this approach novel interactions between inhibitor P1' fragment and the S1' region of the enzyme, particularly hydrogen bonding involving Asn330 and Asp332 enzyme residues, were predicted. The details of the design, synthesis, and activity evaluation toward cytosolic leucine aminopeptidase and aminopeptidase N are discussed. Although the potency of the lead compound has not been improved, marked selectivity of the synthesized inhibitors toward both studied enzymes was observed.     

Introduction of Pro and its analogues in the conserved P1' position of trypsin inhibitor SFTI-1 retains its inhibitory activity

A number of monocyclic SFTI-1 analogues modified in the conserved inhibitor P1' position by Pro, its L-hydroxyproline (Hyp) derivative as well as mimetics with different ring size were synthesized by the solid-phase method. Replacement of Ser6 by Pro, Hyp, and a four-member ring, L-azetidine-2-carboxylic acid (Aze), retained trypsin or chymotrypsin inhibitory activity. The determined association equilibrium constants of these analogues with a cognate enzyme were about two orders of magnitude lower than those obtained for ones with conserved Ser6. In all analogues, with the exception of one, [Phe5,Aze6]SFTI-1, the P1-P1' reactive site remained intact. The results provide first evidence that the conserved Ser in the P1' position of Bowman-Birk inhibitors can be successfully replaced by an amino acid with a secondary amine group.