A nonpeptidyl growth hormone secretagogue.

A nonpeptidyl secretagogue for growth hormone of the structure 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5 -yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamid e (L-692,429) has been identified. L-692,429 synergizes with the natural growth hormone secretagogue growth hormone-releasing hormone and acts through an alternative signal transduction pathway. The mechanism of action of L-692,429 and studies with peptidyl and nonpeptidyl antagonists suggest that this molecule is a mimic of the growth hormone-releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6). L-692,429 is an example of a nonpeptidyl specific secretagogue for growth hormone.

Stimulation of growth hormone release from rat primary pituitary cells by L-692,429, a novel non-peptidyl GH secretagogue.

L-692,429, a benzolactam derivative, stimulated GH release from rat primary pituitary cells in a dose-dependent manner. The concentration of L-692,429 required for half-maximal stimulation were 59.6 +/- 7.3 nM. Under the same conditions, GHRP-6 and GRF had EC50 values of 10.3 +/- 1.9 nM and 0.47 +/- 0.09 nM, respectively. L-692,428, the enantiomer of L-692,429, was inactive at a concentration as high as 2 microM. Like GHRP-6, L-692,429 had no effect on intracellular cAMP level; however, it synergized with GRF to further increase not only the accumulation of cAMP but also the release of GH. The magnitude of GH release stimulated by maximal concentrations of L-692,429 and GHRP-6 was comparable. Interestingly, when presented together in maximal concentrations, L-692,429 and GHRP-6 did not cause additional GH release when compared with either secretagogue alone. The L-692,429-stimulated GH release was completely inhibited by 20 nM somatostatin. To our knowledge, L-692,429 is the first non-peptidyl GH secretagogue which has a direct effect on the release of growth hormone from rat primary pituitary cells. Its effect is most likely mediated through a mechanism which is similar to that of GHRP-6.

Efficacy and specificity of L-692,429, a novel nonpeptidyl growth hormone secretagogue, in beagles.

L-692,429 is a substituted benzolactam that has recently been shown to stimulate GH secretion from rat pituitary cells in vitro with an ED50 of 60 nM. In the current studies, we evaluated the efficacy and specificity of L-692,429 as a GH secretagogue in beagles. L-692,429 at 0.1, 0.25, or 1.0 mg/kg or saline vehicle was administered iv to four male and four female beagles in a balanced cross-over design. Blood samples were collected up to 75 min posttreatment, and serum was assayed for GH, cortisol, PRL, and LH. Mean peak serum GH levels were significantly increased (P < 0.05) by L-692,429 to 13 +/- 2 (mean +/- SEM) ng/ml (0.1 mg/kg), 39 +/- 6 ng/ml (0.25 mg/kg), or 71 +/- 11 ng/ml (1.0 mg/kg) over the saline control value of 3.6 +/- 0.6 ng/ml. Mean peak GH levels occurred at 15 min and had returned to near-baseline levels by 75 min. There was no difference in response between sexes. Mean peak cortisol levels were significantly increased (P < 0.05) by 2.2-, 2.7-, and 3.1-fold above control levels (3.0 +/- 0.2 micrograms/dl) at 25-35 min and returned to near-baseline levels by 75 min. PRL was slightly decreased after L-692,429 treatment, whereas LH was not affected. In a second study, three groups of three male beagles each were administered 5.0 mg/kg L-692,429, iv; iv saline, or 2.2 U/kg ACTH, im. Blood was collected for 8 h posttreatment and assayed for GH, cortisol, ACTH, aldosterone, PRL, insulin, T3, and T4. L-692,429 administration significantly increased (P < 0.05) GH over the control level (6.0 +/- 3.6 ng/ml) to 133 +/- 14 ng/ml by 15 min, with a return to pretreatment levels by 120 min. Cortisol levels were significantly increased (P < 0.05) by 2.0-fold (L-692,429) or 2.9-fold (ACTH) over the saline control peak concentration of 5.6 +/- 1.6 micrograms/dl and were associated with concurrent increases in ACTH levels of 1.2-fold (L-692,429) or 2.1-fold (ACTH) over the saline control peak concentration of 67 +/- 20 pmol/L. Aldosterone, PRL, T3, and T4 were not significantly affected after L-692,429 administration; however, ACTH treatment significantly increased aldosterone (P < 0.05). These data demonstrate that L-692,429 is a novel nonpeptidyl secretagogue that stimulates a marked, but transient, increase in serum GH levels in the dog.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphinic acid inhibitors of D-alanyl-D-alanine ligase.

We report the synthesis of a series of phosphinic acid dipeptide analogues, NH2CH(R1)PO(OH)CH2CH(R2)CO2H, related to DAla-DAla. The best of these compounds are potent, essentially irreversible inhibitors of DAla-DAla ligase, and their preferred stereochemistry was shown by chiral synthesis of (1(S)-aminoethyl)(2(R)-carboxy-1-n-propyl)phosphinic acid, 12b, and by X-ray crystallography of its derivative benzyl [1(S)-[(benzyloxycarbonyl)-amino]ethyl](2(R)-carbomethoxy-1-propyl) phosphinate, 13, to correspond to the stereochemical configuration of DAla-DAla at both centers. A mechanism for the inhibition of DAla-DAla ligase by these compounds is proposed to involve an ATP-dependent formation of phosphorylated inhibitor within the enzyme's active site. The antibacterial activities of these compounds are modest although their spectra include both Gram-positive and Gram-negative susceptible organisms. The best antibacterial activity was shown by (1(S)-aminoethyl) [2-carboxy-2(R)-(methylthio)-1-ethyl]phosphinic acid, 3e, whose MIC's range from 4-128 micrograms/mL on nine of a panel of 11 bacterial organisms. Combination of one of the more active phosphinic acids 12b with the alanine racemase inhibitor fluoro-D-alanine enhances the antibacterial spectrum of the latter on several strains of bacteria and inhibits fluoro-D-alanine's self-reversal, which normally occurs at concentrations several fold higher than its MIC level. This inhibition of fluoro-D-alanine self-reversal is consistent with an involvement of DAla-DAla ligase inhibition in the antibacterial activity of these compounds.

A novel 3-substituted benzazepinone growth hormone secretagogue (L-692,429).

The 3-substituted benzazepinone, L-692,429 (compound 1), is the prototype compound of a novel class of compounds that stimulate release of growth hormone (GH). The molecule evolved from efforts to identify a non-peptide mimic of the growth hormone-releasing hexapeptide, GHRP-6. Compound 1 is prepared by sequential attachment of dimethyl-beta-alanine and 2'-biphenylyltetrazole side chains to a chiral 3-aminobenzolactam nucleus. Comparison of the biological activity of 1 with the corresponding six- and eight-membered lactam analogs shows the seven-membered benzazepinone skeleton to be preferred. Molecular modeling of the structurally diverse GH secretagogues, L-692,429 and GHRP-6, was performed.

A potent, orally bioavailable benzazepinone growth hormone secretagogue.

The identification of L-739,943 (8b), a potent, orally bioavailable benzolactam growth hormone secretagogue, is obtained from zwitterionic L-692,429 through modification of its amino acid side chain and replacement of the acidic 2'-tetrazole with the neutral and potency enhancing 2'-(N-methylaminocarbonylamino)methyl substituent. L-739,943 is orally active for the release of growth hormone in beagle dogs at doses as low as 0.5 mg/kg. Oral bioavailability in dogs of 8b is 24% at a dose of 2 mg/kg with a mean drug Cmax of 145 +/- 46 ng/mL. L-739,943 represents a significant breakthrough in terms of both potency and oral bioavailability as compared to the prototype benzolactam L-692,429.

N-glucuronidation reactions. II. Relative N-glucuronidation reactivity of methylbiphenyl tetrazole, methylbiphenyl triazole, and methylbiphenyl imidazole in rat, monkey, and human hepatic microsomes.

The relative intrinsic in vitro N-glucuronidation reactivity of three classes of heterocyclic compounds was compared using model compounds incubated with UDP-glucuronic acid-enriched liver microsomes from rats, monkeys, and humans. These compounds, all methylbiphenyl (MB) derivatives, represent three classes of N-containing heterocycles commonly used in the design of new drug entities [i.e MB-tetrazole, MB-triazole, (1,2,3- and 1,2,4-), and MB-imidazole (C2- and C4-substituted)]. The structures of all respective N-glucuronides generated from microsomal incubations were determined by Nuclear Overhauser Effect difference NMR spectroscopy. The chemical and enzymic stabilities of N-glucuronides were also studied. In general, relatively low reactivity was found at nitrogens located next to substituted carbons in heterocycles such as N3 in MB-C4-imidazole, N3 in MB-1,2,3-triazole, N2 (or N4) in MB-1,2,4-triazole, and N1 (or N4) in MB-tetrazole. MB-C2-imidazole, in which both nitrogens are in immediate neighboring positions of the substituted carbon, was unreactive toward N-glucuronidation. When the rate of N-glucuronidation was compared under optimal reaction conditions for each compound, most compounds showed higher reactivity with liver microsomes from monkeys than those from rats, except for N2-glucuronidation of MB-tetrazole and MB-1,2,3-triazole. However, the trend for the relative N-glucuronidation reactivity of these compounds by liver microsomes from humans is quite different from those by monkeys and rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A new class of potent, slowly reversible dehydropeptidase inhibitors.

Dehydrodipeptide analogs whose scissile carboxamide has been replaced with a PO(OH)CH2 group have been found to be potent inhibitors of the zinc protease dehydrodipeptidase 1 (DHP-1, renal dipeptidase, EC 3.4.13.11). The best of these inhibitors, compound 25 (Ki = 0.52 nM), is two hundred times more potent than cilastatin 2 which is used clinically as a component of the broad-spectrum antibiotic combination Primaxin. Compound 25 is a tight binding inhibitor exhibiting slow binding kinetics with a remarkably slow off rate from DHP-1 (half life greater than 8 hours). The kinetics of its binding are consistent with a simple on-off mechanism whereas the less active D-enantiomer 26 appears to bind in an initial loose complex with the enzyme which slowly rearranges to a tighter complex (Ki = 83 nM).

Acyclic structural variants of growth hormone secretagogue L-692,429.

Systematic investigation of acyclic analogs of L-692,429, the prototype benzolactam growth hormone secretagogue, has helped to further define the structural requirements for the release of growth hormone from rat pituitary cells for this class of secretagogues.