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.     

beta-Secretase inhibitors: modification at the P4 position and improvement of inhibitory activity in cultured cells

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-570 and KMI-684 in which we replaced carboxylic acid groups at the P(1)(') position of KMI-420 and KMI-429, respectively, with tetrazole derivatives as carboxylic acid bioisosteres. These modifications improved significantly BACE1 inhibitory activity and chemical stability. In this study, the acidic tetrazole ring of the P(4) position of KMI-420 and KMI-570, respectively, was replaced with various hydrogen bond acceptor groups. We found BACE1 inhibitor KMI-574 that exhibited potent inhibitory activity in cultured cells as well as in vitro enzymatic assay.     

Design and synthesis of highly active Alzheimer's beta-secretase (BACE1) inhibitors, KMI-420 and KMI-429, with enhanced chemical stability

Recently, we reported potent and small-sized BACE1 inhibitors KMI-358 and KMI-370 in which the Glu residue is replaced by a beta-N-oxalyl-DAP (l-alpha,beta-diaminopropionyl) residue at the P(4) position. The beta-N-oxalyl-DAP group is important for enhancing BACE1 inhibitory activity, but these inhibitors isomerized to alpha-N-oxalyl-DAP derivatives in solvents. Hence, we used a tetrazole moiety as a bioisostere of the free carboxylic acid of the oxalyl group. KMI-420 and KMI-429, containing a tetrazole ring, showed improved stability and potent enzyme inhibitory activity.     

Novel non-peptidic and small-sized BACE1 inhibitors

Recently, we reported substrate-based beta-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (approximately 1.2 nM IC(50)). In order to improve in vivo enzymatic stability and permeability across the blood-brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P(2) position were reported from other research groups. We selected isophthalic-type aromatic residues at the P(2) position and an HMC isostere at the P(1) position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P(2) position.     

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.     

KMI-358 and KMI-370, highly potent and small-sized BACE1 inhibitors containing phenylnorstatine

Recently, we reported a novel substrate-based octapeptide BACE1 inhibitor KMI-008 containing hydroxymethylcarbonyl (HMC) isostere as a transition-state mimic. Using KMI-008 as a lead compound, a small-sized and highly potent BACE1 inhibitor KMI-370 (IC(50)=3.4 nM) was designed and synthesized.     

Novel BACE1 inhibitors with a non-acidic heterocycle at the P1′ position

We have reported potent peptidic and non-peptidic BACE1 inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. However, our potent inhibitors possess a tetrazole ring at the P₁′ position. It is desirable that central nervous system (CNS) drugs do not possess an acidic moiety. In this study, we synthesized non-acidic BACE1 inhibitors with heterocyclic derivatives at the P₁′ position. KMI-1764 (27) exhibited potent inhibitory activity (IC₅₀ = 27 nM). Interestingly, these non-acidic inhibitors tended to follow the quantitative structure–activity relationship (QSAR) equation and interacted with BACE1-Arg235 in the binding model.     

Discovery of potent and selective succinyl hydroxamate inhibitors of matrix metalloprotease-3 (stromelysin-1)

Structure activity relationships are described for a series of succinyl hydroxamic acids 4a-o as potent and selective inhibitors of matrix metalloprotease-3 (stromelysin-1). Optimisation of P1' and P3' groups gave compound 4j (MMP-3 IC50=5.9nM) which was >140-fold less potent against MMP-1 (IC50=51,000nM), MMP-2 (IC50=1790nM), MMP-9 (IC50=840nM) and MMP-14 (IC50=1900nM).     

Potent P1' biphenylmethyl substituted aggrecanase inhibitors.

A series of cis-1(S)2(R)-amino-2-indanol based compounds with a biphenylmethyl group at the P1' position was found to be potent aggrecanase inhibitors. Both compounds 2j and 2n possessed very high aggrecanase affinity (IC(50)=1.5nM), and showed excellent selectivity over MMP-1 and MMP-9, with moderate selectivity against MMP-2.     

A new class of orally active glycol renin inhibitors containing phenyllactic acid at P3

We prepared a new series of renin inhibitors based on dipeptide glycols, replacing the P4-P3 subsites with an O-(N-morpholinocarbonyl)-3-L-phenyllactic acid residue. This modification proved bioisosteric with Boc-L-phenylalanine, giving rise to highly potent human renin inhibitors (1-5 nM), e.g., SC-46944 (IC50 = 5 nM). Moreover, this change produced compounds that are orally efficacious in reducing plasma renin activity in salt-depleted marmosets.     

Synthesis and SAR of bis-statine based peptides as BACE 1 inhibitors

A new series of bis-statine based peptidomimetic inhibitors of human beta-secretase (BACE 1) was developed by structure-based modification of the three regions to the initial lead 3: an N-terminus, a central bis-statine core, and a C-terminus. Introduction of a 4-aminomethylbenzoic acid on the C-terminus resulted in a potent BACE 1 inhibitor with an IC50 value of 21 nM. The general requirements for the optimal substrate-enzyme interaction are disclosed herein.     

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).     

Structure–activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P2 position

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aβ production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood–brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P₂ position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.     

Rational design and synthesis of selective BACE-1 inhibitors

An effective approach for enhancing the selectivity of beta-site amyloid precursor protein cleaving enzyme (BACE 1) inhibitors is developed based on the unique features of the S1' pocket of the enzyme. A series of low molecular weight (<600) compounds were synthesized with different moieties at the P1' position. The selectivity of BACE 1 inhibitors versus cathepsin D and renin was enhanced 120-fold by replacing the hydrophobic propyl group with a hydrophilic propionic acid group.     

Bidentate peptides: highly potent new inhibitors of enkephalin degrading enzymes

Three series of bidentates bearing an hydroxamic or an N-Acyl-N-hydroxy amino group on structures related to Phe-Gly or Phe-Ala exhibit strong inhibitory potency against purified enkephalinase with IC50 values in the 4 to 15 nM range. As with thiol-containing inhibitors, such as thiorphan, the most active compounds are those in which a methylene spacer separates the benzyl P1' moiety from the Zn coordinating residue. Formation of a bidentate complex with the metal enzyme is clearly demonstrated by a loss of potency of three order of magnitude following the removal of one component of the bidentate group. All the compounds studied are unable to interact with angiotensin converting enzyme (IC50 greater than 10,000 nM). Moreover, compounds of the general formula HONHCO-CH2-CH(CH2 phi)-CONH-CH(R)-COOH belonging to the most active series of enkephalinase blockers (IC50 approximately 4 nM) behave also as highly potent and competitive inhibitors (IC50 approximately 10 nM) of a Tyr-Gly releasing dipeptidylaminopeptidase purified from rat brain. The pure steroisomer [(R)-3-(N-hydroxy)carboxamido-2-benzylpropanoyl]-L-alanine designated kelatorphan, exhibits also a relatively good inhibitory potency against aminopeptidases (IC50 approximately 10 microM) and can be considered as the first virtually complete inhibitor of enkephalin metabolism. This very interesting property of inhibiting all three enzymes of enkephalin metabolism could enhance the required selectivity for a possible clinical use of these inhibitors as new analgesic and psychoactive drugs.     

Synthesis and biological evaluation of hydroxamate-Based inhibitors of glutamate carboxypeptidase II

A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1' residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxycarbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC(50) value of 220nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure-activity relationships of GCP II inhibition.     

Phosphinic acid-based MMP-13 inhibitors that spare MMP-1 and MMP-3

Phosphinic acid-based inhibitors of MMP-13 have been investigated with the aim of identifying potent inhibitors with high selectivity versus MMP-1. Independent variation of the substituents on a P(1)' phenethyl group and a P(2) benzyl group improved potencies in both cases around 3-fold over the unsubstituted parent. Combining improved P(1)' and P(2) groups into a single molecule gave an inhibitor with a 4.5 nM IC(50) against MMP-13 and which is 270-fold selective over MMP-1.     

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.     

P3 cap modified Phe*-Ala series BACE inhibitors

With the aim of reducing molecular weight and adjusting log D value of BACE inhibitors to more favorable range for BBB penetration and better bioavailability, we synthesized and evaluated several series of P3 cap modified BACE inhibitors obtained via replacement of the P3NHBoc moiety as seen in 3 with other polar functional groups such as amino, hydroxyl and fluorine. Several promising inhibitors emerging from this P3 cap SAR study (e.g., 15 and 19) demonstrated good enzyme inhibitory potencies (BACE-1 IC(50) <50 nM) and whole cell activities (IC(50) approximately 1 microM).     

Synthesis and inhibitory activity of acyl-peptidyl-pyrrolidine derivatives toward post-proline cleaving enzyme; a study of subsite specificity.

Several pyrrolidine derivatives have been synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain. Almost all the compounds tested in this study inhibited the activity of both enzymes at low IC50 values (from nM to microM) but a specificity difference was observed with alkylacyl-peptidyl-pyrrolidine derivatives which strongly inhibited only the bacterial enzyme. The most effective inhibitors have a proline residue on their P2 sites and a substituted or unsubstituted phenoxybutyryl moiety on their P3 sites. Thus phenoxybutyryl-prolyl-pyrrolidine is the most effective partial structure of the inhibitors. The best inhibitors found were: 4-(4-benzylphenoxy)butyryl-prolyl-pyrrolidine for bacterial enzyme (IC50 1.4 nM) and 4-phenylbutyryl-thioprolyl-pyrrolidine for bovine brain enzyme (IC50 67 nM). In the passive avoidance test, using amnesic rats experimentally induced with scopolamine, the pyrrolidine derivatives which had potent inhibitory activity toward post-proline cleaving enzymes also showed strong anti-amnesic activities at doses of 1-5 mg/kg, i.p.