Science, art and drug discovery: a personal perspective.

The research programme that started in 1985 led to the approval of Sildenafil (Viagra), in 1998, as the first oral treatment for male erectile dysfunction. The initial project objective was the design and synthesis of novel inhibitors of phosphodiesterase that would increase tissue levels of cGMP, and that could be beneficial for the treatment of cardiovascular conditions. Starting from zaprinast, a weak phosphodiesterase inhibitor, computer modelling guided rational medicinal chemistry to achieve significant increases in potency and selectivity for the 5-isoenzyme within a novel series of pyrazolopyrimidinones. Optimization of structure-activity relationships and pharmacokinetic properties led to sildenafil, which proved essentially devoid of cardiovascular activity in clinical trials. However, the emerging role of nitric oxide and cGMP in controlling blood flow in the penis suggested that sildenafil would have a beneficial effect on erectile function. This hypothesis was confirmed by extensive clinical trials in nearly 5000 patients and the Food and Drug Administration approved sildenafil in March 1998 for male erectile dysfunction. Sildenafil is now available in over 100 countries and more than 150 million tablets have been dispensed worldwide. The sildenafil research programme reflects a traditional approach to drug discovery, but pressures to improve productivity have prompted major investments in genome sciences and new technologies. The impact of these initiatives on the drug discovery paradigm will be discussed, particularly with respect to shortening time scales between identifying gene sequences and submitting innovative products for regulatory approval.

Transoral resection of retro-odontoid disc sequestration: case report and review of the literature.

A rare case of retro-odontoid disc sequestration causing significant cord compression and progressive neurological deterioration is presented. The clinical history, radiology, treatment and pathogenesis of the case are described, along with a review of the relevant literature.

Head trauma and brain tumours revisited.

The authors report a case of an anaplastic astrocytoma which on magnetic resonance imaging and direct visualisation was continuous with an area of gliosis in the left frontal lobe. This gliosis was secondary to a head injury received 19 years earlier that required evacuation of an intracerebral haematoma. This case largely meets the accepted criteria for brain tumour associated with head trauma.

Prevalence of cutaneous findings in hospitalized medical patients.

BACKGROUND: Cutaneous signs may represent a systemic process or a primary cutaneous disorder. Prompt observation and identification of cutaneous abnormalities should improve care of hospitalized medical patients. OBJECTIVE: Our purpose was to determine the prevalence of cutaneous abnormalities in newly hospitalized medical patients and the frequency with which these findings were noted by the admitting team. METHODS: All new medical patients were offered a complete skin examination within 48 hours after admission to the hospital, and 231 participated. Cutaneous diagnoses were based on characteristic clinical features or skin biopsy in patients in whom a diagnosis could not be made clinically. RESULTS: Ninety-three cutaneous findings were present in 83 (35.9%) of 231 patients. In 31 (13.4%) we found cutaneous signs related to the reason for hospitalization or associated with a systemic disorder. These were not noted by the admitting medical service in 14 patients. In two patients, one with metastatic adenocarcinoma and one with sclerosis, the cutaneous findings were manifestations of the new diagnosis. In 52 patients (22.5%) we found 62 primary cutaneous disorders. Fifty-eight disorders (93.5%), including 10 nonmelanoma skin cancers, were unrecognized at the time of admission. CONCLUSION: Cutaneous findings representative of systemic disease or primary cutaneous disorders are commonly present and frequently overlooked in medical patients newly admitted to the hospital. These data suggest that a complete skin examination is necessary in all newly hospitalized medical patients.

Severe amoebic colitis requiring colonic resection.

A 54 year old Australian man presented with fulminant amoebic colitis. Although the amoebae were eradicated, his colonic function did not recover. Colectomy was required. Situations where the surgeon may encounter amoebiasis are discussed.

Substrate recognition by proteinases.

The molecular recognition of limited proteolytic site substrates by serine proteinases has been compared and contrasted to the recognition of serine proteinase inhibitors, utilising the coordinate sets contained in the Brookhaven Protein Databank. Most families of these inhibitors are known to possess a structurally conserved recognition motif at their reactive site-binding loops. Structural comparisons with trypsin limited proteolytic sites revealed that the in situ conformation of these substrates bears little resemblance to the inhibitor-binding loops. Assuming that both inhibitors and substrates bind to the proteinase in the same manner, segmental mobility would be required to permit substrates to adopt an 'inhibitor-like' binding conformation, which is presumed to be necessary for proteolysis. Modelling experiments have been conducted to attempt to introduce such a conformation into tryptic limited proteolytic segments of the native proteins, to test the ability of the limited proteolytic sites to alter their geometry. Further to this, the conformational parameters of accessibility, protrusion, mobility and secondary structure have been analysed and incorporated into a predictive algorithm to assign likely limited proteolytic sites within native protein structures.

Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors.

The conformations of known tryptic limited proteolytic sites have been analysed and compared to the structures of the binding regions of serine proteinase inhibitors, as they are found when complexed to a serine proteinase. Conformational parameters studied include main-chain torsion angles, root-mean-square fits, accessibility, mobility and protrusion indices. As observed before, the inhibitors share a common main-chain conformation at the binding loop from P3-P'3 (Schechter & Berger notation), which is maintained throughout all the serine proteinase inhibitor families for which X-ray data is available, despite lack of similarity in the rest of the protein. This canonical structure is not found amongst the limited proteolytic sites (or nicksites), which differ markedly from the inhibitor binding loop conformation, and also amongst themselves. The experimentally determined nicksites are in general both accessible and protruding; as are the inhibitor binding loops, as well as being typically flexible regions of structure, as denoted by elevated temperature factors from crystallographic determinations. For cleavage by serine proteinases these loops must radically alter their local conformations and a large motion of the loop relative to the structure, in some cases, would be required to orientate these sites for cleavage.

Long-acting dihydropyridine calcium antagonists. 6. Structure-activity relationships around 4-(2,3-dichlorophenyl)-3-(ethoxycarbonyl)-2-[(2-hydroxyethoxy)methyl]-5 -(methoxycarbonyl)-6-methyl-1,4-dihydropyridine.

The preparation of 4-(2,3-dichlorophenyl)-3-(ethoxycarbonyl)-2-[(2-hydroxyethoxy)methyl]-5- (methoxycarbonyl)-6-methyl-1,4-dihydropyridine (2) is described, and its potent calcium antagonist activity on rat aorta (IC50 = 4 x 10(-9) M) and marked tissue selectivity in vitro for vascular smooth muscle over cardiac smooth muscle are established. In order to exploit the excellent in vitro profile of compound 2, a range of analogues were prepared but none were found to have superior calcium antagonist potency and tissue selectivity. Compound 2 has excellent in vivo activity in the anesthetized dog (ED50 = 12 micrograms/kg for reduction of CVR) and a plasma half-life in the conscious dog of 7.2 h. The pharmacokinetic parameters of 2 are compared to those determined for the structurally related compounds amlodipine and felodipine. The plasma clearance for 2 (9.6 mL/min/kg) is similar to that of amlodipine and is consistent with the extended 2-substituent hindering approach to the cytochrome P-450 enzyme responsible for oxidation of the DHP ring to the corresponding pyridine.

Long-acting dihydropyridine calcium antagonists. 5. Synthesis and structure-activity relationships for a series of 2-[[(N-substituted-heterocyclyl)ethoxy]methyl]-1,4-dihydropyridine calcium antagonists.

The synthesis of a series of 1,4-dihydropyridines which have N-linked heterocycles at the terminus of an ethoxymethyl chain at the 2-position is described. The calcium antagonist activity on rat aorta of this class of DHPs is compared with their negative inotropic activity as determined by using a Langendorff-perfused guinea pig heart model. The compounds examined show a wide range of selectivity for vascular over cardiac tissue, with those analogues which possess an amide group at the terminus of the 2-substituent proving the most selective. From the in vitro data obtained for a series of 1,2,3-triazoles, it is possible to conclude that the SARs for binding to the calcium channels in vascular and cardiac tissue are different. One of the compounds, 2-amino-1-[2-[[4-(2,3-dichlorophenyl)-3-(ethoxycarbonyl)-5- (methoxycarbonyl)-6-methyl-1,4-dihydropyrid-2-yl]methoxy]ethyl]-4( 3H)- imidazolone (20b, UK-55,444), was identified as a potent (IC50 = 8 x 10(-9) M) calcium antagonist which is 40-fold selective for vascular over cardiac tissue and which has a significantly longer duration of action (greater than 3 h) than nifedipine in the anesthetized dog on intravenous administration.

Long-acting dihydropyridine calcium antagonists. 4. Synthesis and structure-activity relationships for a series of basic and nonbasic derivatives of 2-[(2-aminoethoxy)methyl]-1,4-dihydropyridine calcium antagonists.

The preparation of a series of 1,4-dihydropyridines (DHPs) which have polar, acyclic, nonbasic substituents on an ethoxymethyl chain at the 2-position is described. In addition, in order to assess the effects of incorporating a basic center into DHPs of this type, a series of glycinamides were also prepared. The calcium antagonist activity on rat aorta of both these classes of DHP is compared with their negative inotropic activity as determined by using a Langendorff perfused guinea pig heart model. A number of the compounds evaluated have activity of the same order as nifedipine although those with more extended substituents have lower potency, particularly when a basic substituent is present. The compounds examined displayed a wide variation in selectivity for vascular over cardiac tissue. A number of structure-activity relationship trends were identified and possible explanations to account for the differences in selectivity observed are advanced. One of the compounds, 2-[[2-[[4-(2-chlorophenyl)-3-(ethoxycarbonyl)-5-(methoxycarbonyl)-6- methyl-1,4-dihydropyrid-2-yl]methoxy]ethyl]amino]acetamide (26, UK-51,656), was identified as a potent (IC50 = 4 x 10(-9) M) calcium antagonist which is 20-fold selective for vascular over cardiac tissue and which has a markedly longer duration of action (greater than 5 h) than nifedipine in the anesthetized dog on intravenous administration. The pharmacokinetic half-life of 26 was established as 4.7 h and possible explanations are advanced to account for 26 having a shorter plasma half-life than amlodipine and a longer plasma half-life than felodipine.

UK-69,578, a novel inhibitor of EC 3.4.24.11 which increases endogenous ANF levels and is natriuretic and diuretic.

A search for potent inhibitors of EC 3.4.24.11, an enzyme which is found most abundantly in the kidney and which degrades atrial natriuretic factor, has led to the identification of UK-69,578. Structure-activity studies starting from substituted N-carboxymethyl dipeptide inhibitors resulted in the introduction of a cyclo-alkane P1' residue and in the replacement of the aza-link between P1 and P1' residues by a methylene group, with a net ten-fold potency gain. UK-69,578 increases endogenous ANF levels and produces natriuretic and diuretic responses intravenously in mice.

Comparison of location and binding for the positively charged 1,4-dihydropyridine calcium channel antagonist amlodipine with uncharged drugs of this class in cardiac membranes.

The distinctive pharmacokinetic and pharmacodynamic activity of amlodipine, including long onset and duration of activity as a calcium channel antagonist, may be related to its interactions with membranes. We have used X-ray crystallography and small-angle X-ray scattering to examine and compare the crystal structure of amlodipine and its location in cardiac sarcolemmal lipid bilayers with that of uncharged dihydropyridines (DHPs) such as nimodipine. Crystallographic analysis demonstrated that the DHP ring of amlodipine is considerably more planar than that of nimodipine, that amlodipine has a greater torsion angle between the DHP and aryl rings, and that the protonated amino group extends away from the DHP ring structure. Despite the positive charge of amlodipine at physiological pH, membrane electron density profile structures showed amlodipine to have a time-averaged location near the hydrocarbon core/water interface similar to that observed for several uncharged DHPs. However, unlike uncharged DHPs, this location is consistent with an ionic interaction between the protonated amino function of amlodipine and the negatively charged phospholipid headgroup region, in addition to a hydrophobic interaction with the fatty acyl chain region near the glycerol backbone similar to other DHPs. This location may also provide an appropriate conformation and orientation for amlodipine binding to its receptor site at this depth in the membrane. Finally, we have measured the nonspecific partitioning of amlodipine into native sarcoplasmic reticulum membranes from rabbit skeletal muscle and compared these data with those for the uncharged DHPs. The partition coefficient into light sarcoplasmic reticulum for amlodipine was higher than that observed for most uncharged DHPs and rates of incorporation of amlodipine into membranes were very high, as with other DHPs, whereas the "washout time" of amlodipine from these membranes was longer by over 1 order of magnitude. These data suggest differences in membrane interactions for amlodipine, compared with uncharged DHPs, that may be correlated with its novel pharmacodynamic and pharmacokinetic profile.

Long-acting dihydropyridine calcium antagonists. 3. Synthesis and structure-activity relationships for a series of 2-[(heterocyclylmethoxy)methyl] derivatives.

The preparation of 1,4-dihydropyridines containing (heterocyclylmethoxy)methyl groups in the 2-position is described and the structural identification of certain of the compounds using 1H NMR spectroscopic methods is reported. The calcium antagonist activity of the compounds on rat aorta is listed and is compared with the negative inotropic potency as determined by using a Langendorff-perfused guinea pig heart model. Several compounds are more potent than nifedipine and show greater selectivity for the vasculature over the heart. One compound, 2-[(2-amino-4-hydroxypyrimidin-6-yl)methoxy]-4- (2,3-dichlorophenyl)-3-(ethoxycarbonyl)-5-(methoxycarbonyl)-6-methyl- 1,4-dihydropyridine (27, UK-56,593), was identified as a potent (IC50 = 1.6 x 10(-9) M), tissue-selective calcium antagonist which proved to have a markedly longer duration of action (greater than 4.5 h) than nifedipine in the anesthetized dog on intravenous administration.

7-Heteroaryl-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin -2(1H)-one derivatives with cardiac stimulant activity.

A series of 7-heteroaryl-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin++ +-2 (1H)-ones was synthesized and evaluated in dogs for cardiac stimulant activity. Compounds were obtained by a palladium-catalyzed cross-coupling reaction between a heteroarylzinc chloride and a 7-iodo-1,2,3,5-tetrahydroimidazo [2,1-b]quinazolin-2(1H)-one or by cyclization of an N-[(2-aminophenyl)methyl]glycinate with cyanogen bromide. Compared to the parent ring system (3), introduction of a 2,6-dimethylpyridin-3-yl (6), 2,4-dimethylimidazol-1-yl (7), or 1,2,4-triazol-1-yl (8) moiety at the 7-position led to a 13-17-fold increase in positive inotropic activity (percentage increase in dP/dtmax) in anesthetized dogs. Potency could be further enhanced with a 9-methyl substituent (10-12). The most potent member of the series, 7-(2,4-dimethylimidazol-1-yl)-9-methyl-1,2,3,5-tetrahydroimidaz o [2,1-b]quinazolin-2(1H)-one (11) (23% increase in dP/dtmax, 2 micrograms/kg), was 80 times more active than 3 and displayed a 5-fold advantage over milrinone. In conscious dogs, 6 elicited marked and sustained positive inotropic activity (decrease in QA interval) after oral administration (1 mg/kg), whereas 10-12 were 10 times more potent. 11 produced an obvious increase in cardiac contractility (20% increase in dP/dtmax) at low dose levels (25 micrograms/kg) while, after 100 micrograms/kg, the marked response (50% increase in dP/dtmax) was maintained for the whole 7-h test period. In these experiments, 11 had no effect on heart rate, and the compound also displayed exceptional selectivity for increasing the force rather than the rate of cardiac contraction (greater than 150% increase in dP/dtmax) in the Starling heart-lung preparation. These studies demonstrate that the tetrahydroimidazoquinazolinone nucleus is an effective bioisostere for the 2(1H)-quinolinone system and that 11 displays improved cardiac stimulant activity and duration of action when compared to milrinone.

2(1H)-quinolinones with cardiac stimulant activity. 3. Synthesis and biological properties of 6-imidazol-1-yl derivatives.

A series of 6-imidazol-1-yl-8-methyl-2(1H)-quinolinones was synthesized and evaluated for cardiac stimulant activity in dogs. The majority of compounds were prepared from an appropriate 6-imidazol-1-yl-2(1H)-quinolinone precursor or by sulfuric acid catalyzed cyclization of an N-(4-heteroarylphenyl)-3-ethoxypropenamide. Introduction of a range of 5-substituents into 6-(2,4-dimethylimidazol-1-yl)-8-methyl-2(1H)-quinolinone (1) reduced inotropic activity in anesthetized dogs (percentage increase in dP/dtmax) although replacement of the 2-methyl group by iodo (10) or cyano (11) substituents was well tolerated. The 2-methyl-4-chloro (15) and 2-methyl-4-(methylthio) (22) derivatives displayed similar potency to 1 (40-50% increase in dP/dtmax, 10-12.5 micrograms/kg) and these compounds were 3-5 times more potent than milrinone. Introduction of iodo (14), cyano (16), or acetyl (17) substituents into the 4-position approximately halved inotropic activity. In conscious dogs, 11 (0.25 mg/kg) and 16 and 17 (0.125 mg/kg) produced similar increases in cardiac contractility (decrease in the QA interval) to 1 (0.125 mg/kg) and maximum responses were maintained for at least 3 h. Dose-related (25, 125, 250 micrograms/kg) cardiac stimulant activity was demonstrated by 17 and after the higher doses a marked response (approximately 30% increase in dP/dtmax) was still observed after 7 h, in contrast to milrinone. The substantial increases in cardiac contractility observed with 16 and 17 in the conscious dog were not accompanied by any tachycardia. These compounds also displayed an overwhelming selectivity for increasing the force of cardiac contraction (greater than 120% increase in dP/dtmax) rather than heart rate (5-10 beats/min decrease) in the Starling heart-lung preparation. As a result of this beneficial pharmacological profile, 6-(4-acetyl-2-methylimidazol-1-yl)-8-methyl-2(1H)-quinolinone (17, UK-66,838) was selected for preclinical development studies.

2(1H)-quinolinones with cardiac stimulant activity. 2. Synthesis and biological activities of 6-(N-linked, five-membered heteroaryl) derivatives.

A series of 6-(N-linked, five-membered heteroaryl)-2(1H)-quinolinone derivatives was synthesized and evaluated for cardiotonic activity. Most compounds were prepared by sulfuric acid catalyzed cyclization of an N-(4-heteroarylphenyl)-3-ethoxypropenamide or by condensation of a 2-amino-5-heteroarylbenzaldehyde or -acetophenone derivative with the ylide derived from triethyl phosphonoacetate. In anesthetized dogs, 6-imidazol-1-yl-8-methyl-2(1H)-quinolinone (3; 25 micrograms/kg) produced a greater increase in cardiac contractility (percentage increase in dP/dt max) than alternative 6-(five-membered heteroaryl)-substituted analogues (4-8). Introduction of 4-methyl (10) or 2,4-dimethyl (13) substituents into the imidazole ring of 3 produced a marked increase in inotropic activity, and these compounds were some 10 and 5 times more potent than milrinone. Most of these quinolinones also displayed positive inotropic effects (decrease in QA interval) in conscious dogs after oral administration (0.0625-1 mg/kg) and in many cases (3, 5-7, 9, 11, 13, 16) there was little difference in activities at both the 1- and 3-h time points. Compound 13 (62.5, 125, 250 micrograms/kg po) demonstrated dose-related cardiac stimulant activity which, in contrast to milrinone, was maintained over the whole 7-h test period. No changes in heart rate were detected at any dose level and compounds 3, 9, 10, and 13 also displayed high selectivity for the stimulation of cardiac contractile force rather than heart rate in the Starling dog heart-lung preparation. Increases in dP/dt max of approximately 50% were accompanied by heart rate changes of less than 10 beats/minute. Physicochemical measurements gave a log P of 1.64 for 13 with pKa values of 7.13 +/- 0.04 and 11.5 +/- 0.2 for the imidazole and quinolinone moieties, respectively. X-ray structural analysis of 13 showed the imidazole and quinolinone rings at 52 degrees to one another in close agreement with the minimum-energy conformation (30 degrees) suggested by PCILO calculations. 6-(2,4-Dimethylimidazol-1-yl)-8-methyl-2-(1H)-quinolinone (13, UK-61,260) is currently undergoing phase II clinical evaluation in congestive heart failure patients.

Long acting dihydropyridine calcium antagonists. 2. 2-[2-Aminoheterocycloethoxy]methyl derivatives.

A series of [(2-aminoheterocycloethoxy)methyl]dihydropyridines were prepared as selective coronary vasodilators. Results showed that a wide variety of five- and six-membered heterocycles were acceptable at the 2-position of the dihydropyridine ring and in vitro potency and tissue selectivity was independent of the basicity of these heterocycles. The SAR indicated that activity was optimum when the largest ester group was placed at the 3 rather than 5 position. 2-[[2-[(3-Amino-1H-1,2,4-triazol-5-yl)amino]ethoxy]methyl]-4- (2,3-dichlorophenyl)-3-(ethoxycarbonyl)-5-(methoxycarbonyl)-6-methyl- 1,4-dihydropyridine (3b) (UK-52,831) emerged as a potent (IC50 = 6.3 X 10(-9) M) and tissue-selective calcium channel blocker with a duration of action greater than 7 h in the anaesthetized dog.

2(1H)-quinolinones with cardiac stimulant activity. 1. Synthesis and biological activities of (six-membered heteroaryl)-substituted derivatives.

A series of (six-membered heteroaryl)-substituted 2(1H)-quinolinones (1) was synthesized, and structure-activity relationships for cardiac stimulant activity were determined. Most compounds were prepared by acidic hydrolysis of a heteroaryl-2-methoxyquinoline obtained by palladium-catalyzed cross-coupling methodology. Direct reaction of a pyridinylzinc reagent with a 6-haloquinolinone also proved successful. In anesthetized dogs, 6-pyridin-3-yl-2(1H)-quinolinone (3; 50 micrograms/kg) displayed greater inotropic activity (percentage increase in dP/dt max) than positional isomers (2, 4-6), and potency was maintained with either mono- (13, 15) or di- (16) alkylpyridinyl substituents. Introduction of a 4- (24) or 7- (25) methyl group into 3 reduced inotropic activity, whereas the 8-isomer (26) proved to be the most potent member of the series. Compound 26 and the 2,6-dimethylpyridinyl analogue (27) were approximately 6 and 3 times more potent than milrinone. Several quinolinones displayed positive inotropic activity (decrease in QA interval) in conscious dogs after oral administration (1 mg/kg), and 26, 27 were again the most potent members of the series. Compound 27 (0.25, 0.5, 1.0 mg/kg po) demonstrated dose-related cardiac stimulant activity, which was maintained for at least 4 h. No changes in heart rate were observed. Compounds 3, 4, 26, and 27 also selectively stimulated the force of contraction, rather than heart rate, in the dog heart-lung preparation. For a 50% increase in dP/dt max with 27, heart rate changed by less than 10 beats/min. In norepinephrine contracted rabbit femoral artery and saphenous vein, 27 produced dose related (5 X 10(-7) to 5 X 10(-4) M) vasorelaxant activity. The combined cardiac stimulant and vasodilator properties displayed by 27, coupled with a lack of effect on heart rate, should be beneficial for the treatment of congestive heart failure.