J. Med. Chem. 2003, 46, 913-916
1,2-Dihydro-4-quinazolinamines: Potent, Highly Selective Inhibitors of Inducible Nitric Oxide Synthase Which Show Antiinflammatory Activity in Vivo Alan C. Tinker,*,† Haydn G. Beaton,† Nigel Boughton-Smith,‡ Tony R. Cook,† Sally L. Cooper,† Lynne Fraser-Rae,‡ Kay Hallam,‡ Peter Hamley,† Tom McInally,† David J. Nicholls,‡ Austen D. Pimm,† and Alan V. Wallace‡
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potency in vitro. However, their use in in vivo models has generally been limited by poor bioavailability or toxicity. We have previously reported the discovery of two new inhibitor classes, the dihydroisoquinolines (e.g., 5)12 and thienopyridines (e.g., 6),13 as part of a program aimed at the development of potent, truly selective i-NOS inhibitors for use in the treatment of inflammatory arthritis, preferably by oral administration. Therefore, the reported14 dihydroquinazoline (7), directly analogous to (5), was of obvious interest to us.
Departments of Medicinal Chemistry and BioScience, AstraZeneca R & D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, UK Received October 9, 2002 Abstract: The discovery of a novel class of nitric oxide synthase (NOS) inhibitors, 2-substituted 1,2-dihydro-4-quinazolinamines, and the related 4′-aminospiro[piperidine-4,2′(1′H)quinazolin]-4′-amines is described. Members of both series exhibit nanomolar potency and high selectivity for the inducible isoform of the enzyme (i-NOS) relative to the constitutive isoforms in vitro. Efficacy in acute and chronic animal models of inflammatory disease following oral administration has also been demonstrated using these compounds.
Introduction. Nitric oxide synthases (NOS) are a family of closely related heme-based oxygenases, which synthesize nitric oxide from the natural amino acid L-arginine.1 Three isoforms of the enzyme have been characterized. Two of these, endothelial NOS (e-NOS) and neuronal NOS (n-NOS), are constitutive and calcium dependent. The third isoform, inducible NOS (iNOS), is formed in response to pathological challenges. It is not dependent on calcium and produces much higher concentrations of nitric oxide than the others. Overexpression of i-NOS has been implicated in a number of inflammatory diseases,2 for example, septic shock and rheumatoid arthritis. Inhibition of i-NOS should be a useful approach to treatment of these conditions. However, reported studies using i-NOS inhibitors in disease models have not demonstrated efficacy unequivocally.3 This can largely be accounted for by the fact that the inhibitors studied lack sufficient selectivity over the other isoforms, particularly e-NOS, which is essential in maintaining vascular homeostasis, to produce uncomplicated pharmacology. The best known inhibitors of NOS are amino acids related to the substrate arginine, for example L-NMMA (1),4 L-NA (2)5 (or its methyl ester L-NAME, 2′), and L-NIL (3).6 However, none of them is particularly potent or selective, and cardiovascular effects due to e-NOS inhibition7 limit their usefulness in vivo. Aminoguanidine (4) selectively inhibits i-NOS relative to e-NOS but has many additional biological effects that are not related to NOS inhibition.8 More recently, other inhibitors based on guanidines,9 isothioureas,10 or amidines11 have been reported with various levels of selectivity and * For correspondence: Tel: (44)1509-644882, Fax: (44)1509-645576. E-mail:
[email protected]. † Department of Medicinal Chemistry. ‡ Department of BioScience.
While (7) had only modest i-NOS inhibitory activity (IC50 ) 2.5 µM against human i-NOS enzyme), this compared sufficiently favorably with the measured potency of (5) (IC50 ) 8.5 µM)12 to encourage further investigation. The elaboration of this lead to afford selective, potent inhibitors of i-NOS that display antiinflammatory activity in vivo is described here. Chemistry. Very few examples of the 1,2-dihydro-4quinazolinamine ring-system have been reported in the literature, and only one practical synthetic approach is described, the reaction of 2-aminobenzamidine with benzaldehyde14 or acetone15 (Scheme 1). Fortunately, this method has proved to be very versatile in our hands: a variety of amidines (9) react with a wide range of aldehydes and ketones in refluxing ethanol to produce the required products (8) in generally excellent yield and purity. The reaction is quite insensitive to solvent, and the method is easily adapted to parallel synthesis in 96well 2 mL microtiter plates by using DMSO solutions heated to 65 °C. Amidines (9) themselves can be prepared from 2-aminobenzonitriles (10) by reaction with hydroxylamine followed by hydrogenation of the resulting amidoxime over a Raney nickel catalyst.15 Biology. Inhibition of NOS enzymes was determined by measuring the formation of L-[3H]citrulline from L-[3H]arginine using an adaptation of the method of Fo¨rstermann et al.16 Compound libraries in solution were first tested at a single concentration against an
10.1021/jm0255926 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/07/2003
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Journal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Scheme 1a
Letters
Scheme 2a
a Reagents (i) (a) NH OH, NaOMe, MeOH. (b) H Raney Ni, 60 2 2 °C (ii) R1R2CdO, ethanol, reflux.
Table 1. i-NOS Inhibitor Activity of Representative Quinazolinamines
a Reagents. (i). RCOCl, base. (ii) RSO Cl, base. (iii) RNCO (for 2 R′ ) H) or RR′NCOCl, base.
compd no.
R1
R2
i-NOS IC50 (µM)a
7 8a 8b 8c 8d 8e 8f 8g 8h 8i 8j
H H H H H H H H H CH3 CH3
Ph H CH3 C2H5 c-propyl c-butyl c-pentyl 2-furanyl 2-thienyl CH3 C2H5
2.5 40 9.8 0.9 1.1 1.1 4.5 0.2 0.4 52 32
a Inhibition of isolated human enzyme (Mean of at least n ) 2). Data for single, purified compounds.
isolated i-NOS enzyme preparation. Compounds from wells showing inhibition similar to or greater than the product from an included control reactant (benzaldehyde or ethyl chloroformate) were prepared individually for accurate IC50 and selectivity measurements against human recombinant enzymes. Test compounds which showed IC50 < 0.5 µM vs i-NOS were also assayed for ability to inhibit NO production in human DLD-1 cells preincubated with a cytokine cocktail.17 Those with IC50 100e >100e n.s. @ 100e >100e >100e >100e 100 0.33 2.7 2.6 100
1 0.06 3.5 40 >100e >100e 70 12 3.3 1 50 0.09 0.15 1.5 5.9
1.2 0.2 4.0 9.6 9.2 11 12 4.5 1.6 0.9 >500 21 >300 2.8 >1000
>30 16 >30 n.d. n.d. n.d. n.d. 14 3 3 n.d. 700 >300 9 n.d.
a All compounds gave satisfactory spectral and analytical data. b Inhibition of recombinant human enzyme at 37 °C. c Inhibition of NO production by intact human DLD-1 cells. d Inhibition of LPS induced nitrite production in rat following oral administration. e >100 indicates between 25% and 50% inhibition observed by 100 µM compound. n.s. )
