Alan Duncan Robertson

PI 3-kinase p110β : a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin αIIbβ3 (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110β isoform in regulating the formation and stability of integrin αIIbβ3 adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110β inhibitors have been developed which prevent formation of stable integrin αIIbβ3 adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110β as an important new target for antithrombotic therapy.

The classification of prostaglandin DP‐receptors in platelets and vasculature using BW A868C, a novel, selective and potent competitive antagonist

1 BW A868C, a novel compound, behaved as a simple competitive antagonist in a human washed platelet aggregation assay. Anti‐aggregatory concentration‐effect curves to BW 245C were displaced in a parallel manner. The shifts accorded with a Schild plot slope of unity and a pKB of 9.26. 2 Inhibition of platelet aggregation by prostaglandin D2 (PGD2) was antagonized with a similar potency, as were the relaxation effects of BW 245C and PGD2 in the rabbit jugular vein. BW A868C can, therefore, be classified as a DP‐receptor antagonist. 3 Actions of BW A868C at other prostaglandin receptors (IP, EP1, EP2, TP and FP) were excluded at concentrations up to 1,000 times higher than the DP‐receptor affinity. 4 Analyses of BW 245C‐ and PGD2‐mediated effects were complicated by additional agonist receptor interactions which were revealed by BW A868C. In rabbit jugular vein a resistant phase of agonism was detectable, indicating that both agonists exerted effects through another receptor (possibly EP2). Also, PGD2, in addition to its anti‐aggregatory effect on platelets, demonstrated a pro‐aggregatory action in the presence of BW A868C. 5 The contractile effects of PGD2 in guinea‐pig tracheal strips were resistant to 10 μm BW A868C indicating that they were not mediated through DP‐receptors. 6 To our knowledge this is the first account of a well‐classified competitive antagonist at the DP‐receptor. Its potency and selectivity make it an important new tool in prostanoid receptor classification and identification.

Receptor specificity and trigemino-vascular inhibitory actions of a novel 5-HT1B/1D receptor partial agonist, 311C90 (zolmitriptan)

311C90 (zolmitriptan zomig: (S)‐4[[3‐[2‐(dimethylamino)ethyl]‐1H‐indol‐5‐yl]methyl]‐2‐oxazolidinone) is a novel 5‐HT1B/1D receptor agonist with proven efficacy in the acute treatment of migraine. Here, we describe the receptor specificity of the drug and its actions on trigeminal‐evoked plasma protein extravasation into the dura mater of the anaesthetized guinea‐pig. At the ‘5‐HT1B‐like’ receptor mediating vascular contraction (rabbit saphenous vein), the compound was a potent (p[A50]=6.79±0.06) partial agonist achieving 77±4% of the maximum effect to 5‐hydroxytryptamine (5‐HT). In the same experiments, sumatriptan (p[A50]=6.48±0.04) was half as potent as 311C90 and produced 97±2% of the 5‐HT maximum effect. Studies in which receptor inactivation methods were used to estimate the affinity (pKA) and efficacy relative to 5‐HT (τrel.) for each agonist confirmed that 311C90 exhibits higher affinity than sumatriptan (pKA=6.63±0.04 and 6.16±0.03, respectively) and that both drugs are partial agonists relative to 5‐HT (τrel=0.61±0.03 and 0.63±0.10, respectively, compared to 5‐HT=1.0). Consistent with its effects in rabbit saphenous vein, 311C90 also produced concentration‐dependent contractions of primate basilar artery and human epicardial coronary artery rings. In basilar artery, agonist potency (p[A50]=6.92±0.07) was similar to that demonstrated in rabbit saphenous vein, again being 2–3 fold higher than for sumatriptan (p[A50]=6.46±0.03). Both agonists produced about 50% of the maximum response obtained with 5‐HT in the same preparations. In rings of human coronary artery, the absolute potency of 311C90 and sumatriptan was higher than in primate basilar artery (p[A50]=7.3±0.1 and 6.7±0.1, respectively), but maximum effects relative to 5‐HT were lower (37±8% and 35±7%, respectively). In both types of vessel, the inability of 5‐HT1B/1D agonists to achieve the same maximum as the endogenous agonist 5‐HT is explained by the additional presence of 5‐HT2A receptors. 311C90 displayed high affinity at human recombinant 5‐HT1D (formerly 5‐HT1Dα) and 5‐HT1B (formerly 5‐HT1Dβ) receptors in transfected CHO‐K1 cell membranes (pIC50 values=9.16±0.12 and 8.32±0.09, respectively). In intact cells, the drug produced concentration‐dependent inhibition of forskolin‐stimulated adenylyl cyclase (p[A50]=9.9 and 9.5, respectively) achieving the same maximum effect as 5‐HT. Excepting human recombinant 5‐HT1A and 5‐ht1F receptors at which the drug behaved as an agonist with modest affinity (pIC50=6.45±0.11 and 7.22±0.12, respectively), 311C90 exhibited low, or no detectable affinity (pKi or pKB 5.5) at numerous other monoamine receptors, including other 5‐HT receptor subtypes. When administered to anaesthetized guinea‐pigs ten minutes before unilateral electrical stimulation of the trigeminal ganglion (1.2 mA, 5 Hz, 5 ms, 5 min), 311C90 (3–30 μg kg−1, i.v.) caused a dose‐dependent inhibition of [125I]‐albumin extravasation within the ipsilateral dura mater. At the same doses, the drug also produced dose‐dependent falls in cranial vascular conductance (32.3±7.5% at 30 μg kg−1), as measured in the ear by laser doppler flowmetry. These results show that 311C90, a novel member of the 5‐HT1B/1D agonist drug class, exhibits a high degree of pharmacological specificity. Its potent partial agonist action at ‘5‐HT1B‐like’ receptors in intracranial arteries, coupled with potent agonism at 5‐HT1D and 5‐HT1B receptors and an ability to inhibit neurogenic plasma protein extravasation in the dura, are consistent with its utility as an effective acute treatment for migraine.

Cardiovascular and autonomic effects of ω-conotoxins MVIIA and CVID in conscious rabbits and isolated tissue assays

The effects of a novel N‐type voltage‐operated calcium channel antagonist, ω‐conotoxin CVID, were compared with ω‐conotoxin MVIIA on sympathetic‐evoked activation of right atria (RA), small mesenteric arteries (MA) and vasa deferentia (VD) isolated from the rat. Their effects were also compared on blood pressure and cardiovascular reflexes in conscious rabbits. The pIC50 values for MVIIA and CVID, respectively, for inhibiting sympathetic‐evoked responses were equivalent in RA (8.7 and 8.7) and VD (9.0 and 8.7); however, in MA the values were 8.4 and 7.7. The cardiac to vascular (RA/MA) potency ratios, antilog (plog RA–plog MA), for MVIIA and CVID were 2 and 10. The offset rates for CVID and MVIIA were rapid, and peptide reapplication caused rapid onset of blockade, suggesting limited desensitization. In the conscious rabbit, CVID and MVIIA (100 μg kg−1 i.v.) caused a similar fall in blood pressure and a tachycardia that rapidly reached maximum. Both peptides decreased the vagal‐ and sympathetic‐mediated components of the baroreflex, but had no effect on the vagal nasopharyngeal reflex. The orthostatic reflex to 90° tilt was blocked by MVIIA with sustained postural hypotension for 90 min after administration. In contrast, CVID caused postural hypotension at 30 min which recovered rapidly. Neither CVID nor MVIIA (3 μg kg−1 i.t.) significantly altered cardiovascular variables or autonomic reflexes. In conclusion, CVID appears to be relatively weak at inhibiting the reflex response to tilt consistent with its weaker inhibition of rat mesenteric artery constriction to perivascular nerve stimulation. This may point to subtype N‐type calcium channel selectivity.

Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas.

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.

Targeting conserved water molecules: design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization.

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.

Antiviral agents 2. Synthesis of trimeric naphthoquinone analogues of conocurvone and their antiviral evaluation against HIV

The synthesis of a new series of conocurvone analogues is presented that explores the importance of the pyran rings of conocurvone, their degree of unsaturation as well as the role of alkoxy functionalities as pyran ring replacements, for the inhibition of the HIV-1 integrase (IN) enzyme. Difficulties in synthesising a trimeric naphthoquinone where the central quinone bears a peri-dihydropyran ring was attributed to distortion of the electrophilic dihaloquinone successfully utilised in the past. Increased electron density could also be a factor in reducing reactivity. The desired central dihydropyran bearing trimeric naphthoquinone was successfully synthesised by using a more reactive bromo-tosyloxyquinone intermediate. A maleimide derivative, where the central quinone between the pendant hydroxyquinones was replaced, was successfully synthesised and although it exhibited comparable enzyme inhibitory activity it had negligible HIV inhibitory cellular activity. Compounds were assessed for activity in both in vitro assays using purified recombinant HIV-1 IN and demonstrated superior or comparable activity to conocurvone derivatives previously reported.

Synthesis of hexahydrocyclopentimidazol-2-(1H)-one derivatives displaying selective DP-receptor agonist properties

The rationale for investigating conformationally restricted analogues of BW245C as DP-receptor ligands and the syntheses of three such racemic bicyclic imidazolidinone analogues are described. Compounds 7 (BW587C), 8 (BW480C85), and 9 (BW572C85) were found to be potent inhibitors of human platelet aggregation and selective DP-receptor agonists in washed platelet and jugular vein isolated tissue assays.

Antiviral Agents. I. Synthesis and Antiviral Evaluation of Trimeric Naphthoquinone Analogues of Conocurvone

Conocurvone, a novel natural product isolated from the endemic Australian shrub Conosperum sp. (Proteaceae), exhibits anti-HIV activity but is a highly lipophilic compound, which suggests that there may be problems with its aqueous solubility and bioavailability. A general and convenient synthesis of trimeric naphthoquinones using the condensation of 2-hydroxynaphthoquinones and 2,3-dihaloquinones is described. The application of this method to the synthesis of a series of simpler and less lipophilic trimeric naphthoquinone simple analogues of conocurvone is also reported together with their anti-HIV activity.

The Synthesis of New Dibenzothiophen Amino Acid and Cyclophane Derivatives

Some novel dibenzothiophen amino acid and cyclophane derivatives have been produced as a result of synthetic studies on the development of dibenzothiophen-based antibacterial compounds. Allylation through a Stille reaction has produced 2-allyl-8-bromodibenzothiophen (3a) and 2,8-diallyldibenzothiophen (3b). Under different conditions, the Stille reaction also produced 2-bromo-8-(prop-1-enyl)dibenzothiophen (4a) and 2,8-di(prop-1- enyl)dibenzothiophen (4b) via double bond isomerization. Olefin metathesis with (3b) and methyl N-acetylallylglycinate (5) produced two homodimers, the novel [4](5,11)[4](18,24)dibenzothiophenophane-2,15-diene (6), as a mixture of geometrical isomers, and dimethyl (E)- and (Z)-(R,S)-2,7-diacetamidooct-4-enedioate 8), and the two cross-metathesis products methyl (E)- and (Z)-(R,S)-2-acetamido-6-(8-allyldibenzothien-2-yl)hex-4-enoate (7a) and dimethyl (E)- and (Z)-(R,S)-6,6¢-(dibenzothiophen-2,8-diyl)bis(2-acetamidohex-4-enoate) (7b). Palladium-catalysed hydrogenation of the homodimer (6) and the cross-metathesis products (7a,b) yielded the corresponding reduced compounds (9) and (10a,b), respectively.