Optimization of 1H-tetrazole-1-alkanenitriles as potent orally bioavailable growth hormone secretagogues.

1H-tetrazole-1-alkanenitrile SR-9g exhibits a >10-fold in vivo potency enhancement over the lead nitrile 1 and has acceptable oral bioavailability in rats and dogs. An enantiospecific synthesis of 1H-tetrazole-1-alkanenitrile nitriles 9 has been developed.

Design and synthesis of tetrazole-based growth hormone secretagogue: the SAR studies of the O-benzyl serine side chain.

The structure-activity relationship of the O-benzyl serine side chain was investigated based on the tetrazole-based growth hormone secretagogue BMS-317180 (2). The ortho position of the benzyl moiety was found to be favorable for introduction of substituents. A series of ortho-substituted compounds were synthesized with improved in-vitro and in-vivo activity. Among them, the biphenyl compound 2p shows twofold improvement in potency compared to its parent compound BMS-317180 (2).

Discovery of a tetrazole-based growth hormone secretagogue: 4-(hydroxybutyl)carbamic acid 2-{5-[1-(2-amino-2-methylpropionylamino)-2- benzyloxyethyl]tetrazol-1-yl}ethyl ester (BMS-317180).

A tetrazole-based peptidomimetic 2 (BMS-317180) was discovered as a human growth hormone secretagogue (GHS). Compound 2 is a potent, novel, orally effective GHS that shows an excellent safety profile in preclinical studies. The compound was advanced into clinical development.

Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay.

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates.

An automated high throughput process, termed the MetFast assay, is described to assess in vitro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphate-mediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-UV/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision-induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions, generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.

Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor (hPXR) and correlation to known CYP3A4 drug interactions.

The human transcription factor pregnane X receptor (hPXR) is a key regulator of enzyme expression, especially cytochrome P450 3A4 (CYP3A4). Due to the prominence of CYP3A4 in the elimination of many drugs, the development of high throughput in vitro models to predict the effect of drugs on CYP3A4 expression have increased. To better interpret and predict potential drug-drug interactions due to CYP3A4 enzyme induction, we evaluated 170 xenobiotics in a hPXR transactivation assay and compared these results to known clinical drug-drug interactions. Of the 170 xenobiotics tested, 54% of them demonstrated some level of hPXR transactivation. By taking into consideration cell culture conditions (solubility, cytotoxicity, appropriate drug concentration in media), as well as in vivo pharmacokinetics (therapeutic plasma C(max), distribution, route of administration, dosing regimen, liver exposure, potential to inhibit CYP3A4), the risk potential of CYP3A4 enzyme induction for most compounds reduced dramatically. By employing this overall interpretation strategy, the final percentage of compounds predicted to significantly induce CYP3A4 reduced to 5%, all of which are known to cause drug-drug interactions. Also, this is the first report that identifies several potent compounds that have the ability to transactivate hPXR that previously have not been identified, such as terbinafine, diclofenac, sildenafil, glimepiride, montelukast, and ticlopidine.

Dual angiotensin II and endothelin A receptor antagonists: synthesis of 2'-substituted N-3-isoxazolyl biphenylsulfonamides with improved potency and pharmacokinetics.

In a previous report we demonstrated that merging together key structural elements present in an AT(1) receptor antagonist (1, irbesartan) with key structural elements in a biphenylsulfonamide ET(A) receptor antagonist (2) followed by additional optimization provided compound 3 as a dual-action receptor antagonist (DARA), which potently blocked both AT(1) and ET(A) receptors. Described herein are our efforts directed toward improving both the pharmacokinetic profile as well as the AT(1) and ET(A) receptor potency of 3. Our efforts centered on modifying the 2'-side chain of 3 and examining the isoxazolylsulfonamide moiety in 3. This effort resulted in the discovery of 7 as a highly potent second-generation DARA. Compound 7 also showed substantially improved pharmacokinetic properties compared to 3. In rats, DARA 7 reduced blood pressure elevations caused by intravenous infusion of Ang II or big ET-1 to a greater extent and with longer duration than DARA 3 or AT(1) or ET(A) receptor antagonists alone. Compound 7 clearly demonstrated superiority over irbesartan (an AT(1) receptor antagonist) in the normal SHR model of hypertension in a dose-dependent manner, demonstrating the synergy of AT(1) and ET(A) receptor blockade in a single molecule.

Discovery of N-isoxazolyl biphenylsulfonamides as potent dual angiotensin II and endothelin A receptor antagonists.

The ET(A) receptor antagonist (2) (N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)-[1,1'-biphenyl]-2-sulfonamide, BMS-193884) shares the same biphenyl core as a large number of AT(1) receptor antagonists, including irbesartan (3). Thus, it was hypothesized that merging the structural elements of 2 with those of the biphenyl AT(1) antagonists (e.g., irbesartan) would yield a compound with dual activity for both receptors. This strategy led to the design, synthesis, and discovery of (15) (4'-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(3,4-dimethyl-5-isoxazolyl)-2'-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl]-[1,1'-biphenyl]-2-sulfonamide, BMS-248360) as a potent and orally active dual antagonist of both AT(1) and ET(A) receptors. Compound 15 represents a new approach to treating hypertension.

Correlation of high-throughput pregnane X receptor (PXR) transactivation and binding assays.

Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z' > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays. EC(50) values of compounds in the transactivation assay correlated reasonably well with their IC(50) values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonist-dependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules.

Discovery, synthesis, and structure-activity studies of tetrazole based growth hormone secretagogues.

A novel class of Growth Hormone Secretagogues (GHS), based on a tetrazole template, has been discovered. In vitro SAR and in vivo potency within this new class of GHS are described. The tetrazole 9q exhibits good oral bioavailability in rats and dogs as well as efficacy following an oral 10 mg/kg dose in dogs. Solution and solid phase protocols for the synthesis of tetrazole based GHS have been developed.

Tetrahydroisoquinoline 1-carboxamides as growth hormone secretagogues.

Several novel series of tetrahydroisoquinoline 1-carboxamides were prepared and shown to be potent growth hormone (GH) secretagogues. Among them, carbamate 12a-E2 displays excellent in vivo activity by increasing plasma GH 10-fold in an anesthetized IV rat model.

Novel dual action AT1 and ETA receptor antagonists reduce blood pressure in experimental hypertension.

Angiotensin II and endothelin-1 activate their respective AT(1) and ET(A) receptors on vascular smooth muscle cells, producing vasoconstriction, and both peptides are implicated in the pathogenesis of essential hypertension. Angiotensin II potentiates the production of endothelin, and conversely endothelin augments the synthesis of angiotensin II. Both AT(1) and ET(A) receptor antagonists lower blood pressure in hypertensive patients; thus, a combination AT(1)/ET(A) receptor antagonist may have greater efficacy and broader utility compared with each drug alone. By rational drug design a biphenyl ET(A) receptor blocker was modified to acquire AT(1) receptor antagonism. These compounds (C and D) decreased Sar-Ile-Angiotensin II binding to AT(1) receptors and endothelin-1 binding to ET(A) receptors, and compound C inhibited angiotensin II- and endothelin-1-mediated Ca(2+) transients. In rats compounds C and D reduced blood pressure elevations caused by intravenous infusion of angiotensin II or big endothelin-1. Compound C decreased blood pressure in Na(+)-depleted spontaneously hypertensive rats and in rats with mineralocorticoid hypertension. Compound D was more efficacious than AT(1) receptor antagonists at reducing blood pressure in spontaneously hypertensive rats, and its superiority was likely due to its partial blockade of ET(A) receptors. Therefore compounds C and D are novel agents for treating a broad spectrum of patients with essential hypertension and other cardiovascular diseases.