Discovery of novel, potent and bioavailable proline-urea based macrocyclic HCV NS3/4A protease inhibitors

A novel series of P3-truncated macrocyclic HCV NS3/4A protease inhibitors containing a P2 proline-urea or carbamate scaffold was synthesized. Very potent inhibitors were obtained through the optimization of the macrocycle size, urea and proline substitution, and bioisosteric replacement of the P1 carboxylic acid moiety. Variation of the lipophilicity by introduction of small lipophilic substituents resulted in improved PK profiles, ultimately leading to compound 13Bh, an extremely potent (K(i)=0.1 nM, EC(50)=4.5 nM) and selective (CC(50) (Huh-7 cells)>50 microM) inhibitor, displaying an excellent PK profile in rats characterized by an oral bioavailability of 54% and a high liver exposure after oral administration.     

Synthetic FP-prostaglandin-induced contraction of rat uterus smooth muscle in vitro

Numerous synthetic FP-class prostaglandin (PG) analogs stimulated the contraction of isolated non-pregnant female rat uterus in a concentration-dependent manner with the following agonist potencies: bimatoprost acid (17-phenyl-trinor PGF(2alpha); EC(50)=0.68+/-0.06 nM)=cloprostenol (EC(50)=0.73+/-0.01 nM)>travoprost acid (EC(50)=1.3+/-0.07 nM)>latanoprost acid (EC(50)=2.7+/-0.08 nM)>PGF(2alpha) (EC(50)=52+/-11 nM)>unoprostone (UF-021; EC(50)=310+/-101 nM)>S-1033 (EC(50)=610+/-4 nM)>bimatoprost (EC(50)=1130+/-173 nM). The FP-receptor antagonist, AL-8810, antagonized the contractile effects of PGF(2alpha) (K(i)=2.9+/-0.2 microM), travoprost acid (K(i)=0.6+/-0.1 microM) and bimatoprost (K(i)=0.2+/-0.02 microM). Agonist and antagonist potencies for rat uterus contraction by these PGs compared well with their potencies for inducing/blocking functional responses in other systems (r=0.83-0.94) except with bovine iris sphincter (r=0.2; p<0.7). In conclusion, the rat uterus contains functionally active FP-receptors whose activation by a variety of free acid and an amide forms of synthetic PGs leads to the contraction of this tissue and which can be pharmacologically blocked by an FP-receptor antagonist, AL-8810.     

Evaluation of the cyclopentane-1,2-dione as a potential bio-isostere of the carboxylic acid functional group

Cycloalkylpolyones hold promise in drug design as carboxylic acid bio-isosteres. To investigate cyclopentane-1,2-diones as potential surrogates of the carboxylic acid functional group, the acidity, tautomerism, and geometry of hydrogen bonding of representative compounds were evaluated. Prototypic derivatives of the known thromboxane A₂ prostanoid (TP) receptor antagonist, 3-(3-(2-((4-chlorophenyl)sulfonamido)-ethyl)phenyl)propanoic acid, in which the carboxylic acid moiety is replaced by the cyclopentane-1,2-dione unit, were synthesized and evaluated as TP receptor antagonists. Cyclopentane-1,2-dione derivative 9 was found to be a potent TP receptor antagonist with an IC₅₀ value comparable to that of the parent carboxylic acid. These results indicate that the cyclopentane-1,2-dione may be a potentially useful carboxylic acid bio-isostere.     

Book Review

1-Aminocyclopropane carboxylic acid (ACPC) competitively inhibited (IC50, 38 +/- 7 nM) [3H]glycine binding to rat forebrain membranes but did not affect [3H]strychnine binding to rat brainstem/spinal cord membranes. Like glycine, ACPC enhanced 3H-labelled (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([3H]MK-801) binding to N-methyl-D-aspartate receptor-coupled cation channels (EC50, 135 +/- 76 nM and 206 +/- 78 nM for ACPC and glycine, respectively) but was approximately 40% less efficacious in this regard. The maximum increase in [3H]MK-801 binding produced by a combination of ACPC and glycine was not different from that elicited by glycine, but both compounds potentiated glutamate-stimulated [3H]MK-801 binding. These findings indicate that ACPC is a potent and selective ligand at the glycine modulatory site associated with the N-methyl-D-aspartate receptor complex.     

Novel selective small molecule agonists for peroxisome proliferator-activated receptor delta (PPARdelta)--synthesis and biological activity

We report the synthesis and biological activity of a new series of small molecule agonists of the human Peroxisome Proliferator-Activated Receptor delta (PPARdelta). Several hits were identified from our original libraries containing lipophilic carboxylic acids. Optimization of these hits by structure-guided design led to 7k (GW501516) and 7l (GW0742), which shows an EC(50) of 1.1 nM against PPARdelta with 1000-fold selectivity over the other human subtypes.     

Bimatoprost (Lumigan((R))) is an agonist at the cloned human ocular FP prostaglandin receptor: real-time FLIPR-based intracellular Ca(2+) mobilization studies

Bimatoprost is the ethyl amide derivative of 17-phenyl-trinor prostaglandin F(2alpha). Here, we show that bimatoprost (K(i)=9250+/-846nM) and bimatoprost free acid (17-phenyl-trinor prostaglandin F(2alpha); K(i)=59+/-6nM) bind to the FP receptor and displace [(3)H]-travoprost acid, a selective FP agonist. Bimatoprost (EC(50)=3070+/-1330nM), Lumigan((R)) (bimatoprost 0.03% ophthalmic solution; EC(50)=1150+/-93nM) and bimatoprost acid (EC(50)=15+/-3nM) mobilized intracellular Ca(2+) ([Ca(2+)](i)) in <5s in HEK-293 cells expressing the cloned human ciliary body FP receptor on a fluorometric imaging plate reader (FLIPR). Furthermore, agonist effects of bimatoprost and bimatoprost acid were blocked by AL-8810 (11beta-fluoro-15-epi-15-indanyl prostaglandin F(2alpha); K(i)=0.7-2.1 MicroM), an FP receptor-selective antagonist. Therefore, the prodrug bimatoprost and its hydrolytic product, bimatoprost free acid, bind to and activate the human ocular FP prostaglandin receptor to mobilize [Ca(2+)](i), thus behaving as FP receptor agonists.     

Structure/function of the human glucocorticoid receptor: tyrosine 735 is important for transactivation.

Ligand-induced activation of the glucocorticoid receptor (GR) is not well understood. The GR ligand-binding domain was modeled, based on homology with the progesterone receptor. Tyrosine 735 interacts with the D ring of dexamethasone, and substitution of D ring functional groups results in partial agonist steroids with reduced ability to direct transactivation. Loss of the Tyr735 hydroxyl group by substitution to phenylalanine (Tyr735Phe) did not reduce ligand binding affinity [dissociation constant (Kd) 4.3 nM compared with Kd 4.6 nM for wild-type] and did not alter transrepression of an nuclear factor-kappaB (NF-kappaB reporter. But, there was a significant 30% reduction in maximal transactivation of a mouse mammary tumor virus (MMTV) reporter, although with an unchanged EC50 (8.6 nM compared with 6 nM). Substitution to a nonaromatic hydrophobic amino acid, valine (Tyr735Val), retained high-affinity ligand binding for dexamethasone (Kd 6 nM compared with 4.6 nM) and did not alter transrepression of NF-kappaB. However, there was a 36% reduction in MMTV activity with a right shift in EC50 (14.8 nM). The change to serine, a small polar amino acid (Tyr735Ser), caused significantly lower affinity for dexamethasone (10.4 nM). Maximal transrepression of NF-kappaB was unaltered, but the IC50 for this effect was increased. Tyr735Ser had a major shift in EC50 (118 nM) for transactivation of an MMTV reporter. Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr735Ser. These data suggest that tyrosine 735 is important for ligand interpretation and transactivation.     

Covalently cyclized agonist and antagonist analogues of bombesin and related peptides.

During a search for possible cyclization points in shortened, potent bombesin agonists and antagonists, it was found that the joining of amino acid residues in positions 6 and 14 by various means resulted in retention of significant binding affinity for rat pancreatic acini and murine Swiss 3T3 cells. In one series of analogues, Cys residues in these positions were used for bridging via a disulfide bond. (D)-C-Q-W-A-V-G-H-L-C-NH2 retained significant binding affinity for rat pancreatic acini cells and was a full amylase releasing agonist (EC50 187 nM). Potency was markedly increased by substituting D-Ala for Gly (EC50 67 nM compared to 10 nM for its linear counterpart) and was decreased by substituting L-Cys for D-Cys in this analogue (EC50 214 nM), thus strongly suggesting stabilization of peptide folding by the D residues. Elimination of the COOH-terminal amino acid produces competitive antagonists in the linear analogues; however, (D)-C-Q-W-A-V-G-H-C-NH2 was devoid of activity. Likewise, cyclization to position 13 with the 14 amino acids intact to give (D)-C-Q-W-A-V-G-H-C-L-NH2 resulted in an almost inactive peptide. On the other hand, as in the linear series, the reduced peptide bond analogue, (D)-C-Q-W-A-V-(D)-A-H-L-psi (CH2NH)-C-NH2, was a receptor antagonist (IC50 5.7 mM), albeit much weaker than the corresponding linear analogues, but with no residual agonist activity. Direct head-to-tail cyclization was also tried. Both cyclo[(D)-F-Q-W-A-V-G-H-L-L] (EC50 346 nM) and the shorter cyclo [Q-W-A-V-G-H-L-L] (EC50 1236 nM) were full agonists. Elimination of the COOH-terminal residue in cyclo[(D)-p-Cl-F-Q-W-A-V-(D)-A-H-L] produced an agonist (EC50 716 nM) rather than an antagonist. These results provide support for the proposal that both bombesin agonists and antagonists adopt a folded conformation at their receptor(s). Furthermore, the retention of appreciable potencies using several cyclization strategies and chain lengths suggests that further optimization of these structures both in terms of potency and ring size is possible. Since these peptides have increased conformational restriction, they should begin to serve as useful substrates for NMR and molecular modeling studies aimed at comparing the obviously subtle differences between agonist and antagonist structures.     

Bioisosteres of 9-carboxymethyl-4-oxo-imidazo[1,2-a]indeno-[1,2-e]pyrazin-2-carboxylic acid derivatives. Progress towards selective, potent in vivo AMPA antagonists with longer durations of action

A novel series of 2- and 9-disubstituted heterocyclic-fused 4-oxo-indeno[1,2-e]pyrazin derivatives was synthesized. One of them, the 9-(1H-tetrazol-5-ylmethyl)-4-oxo-5,10-dihydroimidazo[1,2-a]indeno[1,2-e]pyrazin-2-yl phosphonic acid 4i exhibited a strong and a selective binding affinity for the AMPA receptor (IC50 = 13 nM) and demonstrated potent antagonist activity (IC50 = 6nM) at the ionotropic AMPA receptor. This compound also displayed good anticonvulsant properties against electrically-induced convulsions after ip and iv administration with ED50 values between 0.8 and 1 mg/kg. Furthermore, a strong increase in potency was observed when given iv 3 h before test (ED50 = 3.5 instead of 25.6 mg/kg for the corresponding 9-carboxymethyl-2-carboxylic acid analogue). These data confirmed that there is an advantage in replacing the classical carboxy substituents by their bioisosteres such as tetrazole or phosphonic acid groups.     

Structure-activity relationships of dynorphin analogs substituted in positions 2 and 3

Following up on the observation that the dynorphin analog [Pro(3)]Dyn A(1-11)-NH(2) 2 possesses high affinity and selectivity for the kappa opioid receptor, a number of related peptides were prepared and characterized by radioligand binding and [(35)S]GTPgammaS assays. While incorporation of 2-azetidine carboxylic acid in position 3 led to the equally potent analog 3, the corresponding analog containing piperidine-2-carboxylic acid showed a nearly 90-fold reduction in kappa affinity. Differential preferred bond angles phi in the three building blocks might account for these observations. Compounds 2 and 3 were kappa antagonists with IC(50) values of 380 and 350 nM, respectively. The Sar(3) analog 7 and the Sar(2) analog 8 were kappa agonists, with greater selectivity than Dyn A(1-11)-NH(2) 1. In view of their high kappa affinities (8: K(i) = 1.5 nM; 2: K(i) = 2.4 nM), the new analogs were surprisingly weak kappa agonists or antagonists, e.g., the EC(50) value for the agonist 8 was 280 nM. Different kappa receptor subtypes in binding vs functional assays can not account for these results, since both assays were performed using the same membrane preparation.     

Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).     

Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion Channel Complex by l-Glutamate, Glycine, and Polyamines

In extensively washed rat cortical membranes [3H](+)-5-methyl-10,11-dihydro-5 H-dibenzo [a,d]cyclohepten-5,10-imine ([3H]MK-801) labeled a homogeneous set of sites (Bmax = 1.86 pmol/mg protein) with relatively low affinity (KD = 45 nM). L-Glutamate, glycine, and spermidine produced concentration-dependent increases in specific [3H]MK-801 binding due to a reduction in the KD of the radioligand. In the presence of high concentrations of L-glutamate, glycine, or spermidine, the KD values for [3H]MK-801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK-801 binding affinity as follows: L-glutamate + glycine, KD = 6.2 nM; L-glutamate + spermidine, KD = 2.2 nM; glycine + spermidine, KD = 8.3 nM. High concentrations of spermidine did not inhibit either [3H]glycine orf [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to the N-methyl-D-aspartate (NMDA) receptor complex. The concentration of L-glutamate required to produce half-maximal enhancement (EC50) of [3H]MK-801 binding was reduced from 218 nM to 52 nM in the presence of 30 microM glycine and to 41 nM in the presence of 50 microM spermidine. The EC50 value for glycine enhancement of [3H]MK-801 binding was 184 nM. This was lowered to 47 nM in the presence of L-glutamate and to 59 nM in the presence of spermidine. Spermidine enhanced [3H]MK-801 binding with an EC50 value of 19.4 microM which was significantly reduced by high concentrations of L-glutamate (EC50 = 3.9 microM) or glycine (EC50 = 6.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and SAR requirements of adamantane-colchicine conjugates with both microtubule depolymerizing and tubulin clustering activities

A series of analogues of conjugate 1, combining an adamantane-based paclitaxel (taxol) mimetic with colchicine was synthesized and tested for cytotoxicity in a cell-based assay with the human lung carcinoma cell line A549. The most active compounds (10 EC(50) 2 ± 1.0 nM, 23 EC(50) 6 ± 1.4 nM, 26 EC(50) 5 ± 1.8 nM, 28 EC(50) 11 ± 1.7 nM, 30 EC(50) 4.8 ± 0.5 nM) were found to interfere with the microtubule dynamics in an interesting manner. Treatment of the cells with these compounds promoted disassembly of microtubules followed by the formation of stable tubulin clusters. Structure-activity relationships for the analogues of 23 revealed the sensitivity of both cytotoxicity and tubulin clustering ability to the linker length. The presence of adamantane (or another bulky hydrophobic and non-aromatic moiety) in 23 was found to play an important role in the formation of tubulin clusters. Structural requirements for optimal activity have been partially explained by molecular modeling.     

Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase

Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b) <10 nM; EC(50) (1b)=34 nM) as well as good stability towards human liver microsomes (HLM t(1/2) =59 min).     

Structure-activity studies of new melanocortin peptides containing an aromatic amino acid at the N-terminal position

Cyclic melanotropin peptides, designed with an aromatic amino acid substitution at the N-terminal position of the MT-II-type scaffold, were prepared by solid-phase peptide synthesis and evaluated for their ability to bind to and activate human melanocortin-1, -3, -4, and -5 receptors. The structure-activity studies of these MT-II analogues have identified a selective antagonist at the hMC4R (H-Phe-c[Asp-Pro-d-Nal(2')-Arg-Trp-Gly-Lys]-NH(2), pA(2)=8.7), a selective partial agonist at the hMC4R (H-d-Nal(2')-c[Asp-Pro-d-Phe-Arg-Trp-Gly-Lys]-NH(2), IC(50)=11nM, EC(50)=56nM), and a selective partial agonist at the hMC3R (H-d-Phe-c[Asp-Pro-d-Phe-Arg-Trp-Lys]-NH(2), IC(50)=3.7nM, EC(50)=4.9nM). Aromatic amino acid substitution at the N-terminus in conjuction with the expansion of the 23-membered cyclic lactam MT-II scaffold to a 26-membered scaffold by addition of a Gly residue in position 10 leads to melanotropin peptides with enhanced receptor selectivity.     

Sub-nanomolar hMC1R agonists by end-capping of the melanocortin tetrapeptide His-D-Phe-Arg-Trp-NH(2)

Twenty three derivatives of the core fragment His(6)-D-Phe(7)-Arg(8)-Trp(9)-NH(2) end-capped with carboxylic and sulfonic acids were synthesized and evaluated at human melanocortin receptors (hMC1, hMC3, and hMC4Rs). The SAR within this series allowed us to map the hMCRs near the His(6) binding site and design a superpotent MC1R agonist, LK-184, Ph(CH(2))(3)CO-His-D-Phe-Arg-Trp-NH(2) (19) with EC(50) 0.01 nM (5 nM at MC3 and MC4Rs).     

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.     

Gastrin and CCK-8 induce inositol 1,4,5-trisphosphate formation in rabbit gastric parietal cells

The role of phosphoinositide turnover in the mediation of acid secretion was examined in an enriched preparation of isolated rabbit parietal cells (75%). Both gastrin and CCK-8 (octapeptide of cholecystokinin) stimulated [14C]aminopyrine (AP) uptake by cells (EC50 0.07 +/- 0.03 nM (gastrin) and 0.093 +/- 0.065 nM (CCK-8] and increased [3H]inositol phosphates cellular contents (EC50 0.142 +/- 0.016 nM (gastrin) and 0.116 +/- 0.027 nM (CCK-8] in a parallel fashion. In addition, the EC50 values for both phenomenon were quite similar to the Kd values obtained from binding experiments. HPLC analysis of the different [3H]inositol phosphates produced under gastrin or CCK-8 stimulation showed a 2-fold increase in [3H]Ins(1,4,5)P3 levels within 5 s with a concomitant increase in [3H]Ins(1,4)P2 content within 15 s. A low but significant rise in [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 cellular contents was also observed. No difference between gastrin- and CCK-8-induced inositol phosphates production could be shown. We can conclude that gastrin and CCK-8 display an identical profile of action, suggesting that they stimulate the acid secretory function of parietal cells through the same receptor site coupled to the Ins(1,4,5)P3 production.     

Design and synthesis of 1-(4-benzoylphenyl)imidazole derivatives as new potent 20-HETE synthase inhibitors

Structural modification of the novel 20-HETE synthase inhibitor 1 (IC(50) 310nM) is described. Introduction of a side chain with a carboxylic acid at the terminal position to 1 resulted in increased ability to inhibit human renal microsomal production of 20-HETE (7c: IC(50) 7.9nM), with good selectivity toward CYP2D6 and cyclooxygenases (COX)-1 and -2.