Discovery of a potent, orally bioavailable and highly selective human neuronal nitric oxide synthase (nNOS) inhibitor, N-(1-(piperidin-4-yl)indolin-5-yl)thiophene-2-carboximidamide as a pre-clinical development candidate for the treatment of migraine.

We recently reported a series of 1,6-disubstituted indoline-based thiophene amidine compounds (5) as selective neuronal nitric oxide synthase (nNOS) inhibitors to mitigate the cardiovascular liabilities associated with hERG K(+) channel inhibition (IC(50) = 4.7 µM) with previously reported tetrahydroquinoline-based selective nNOS inhibitors (4). The extended structure-activity relationship studies within the indoline core led to the identification of 43 as a selection candidate for further evaluations. The in vivo activity in two different pain (spinal nerve ligation and migraine pain) models, the excellent physicochemical and pharmacokinetic properties, oral bioavailability (F(po) = 91%), and the in vitro safety profile disclosed in this report make 43 an ideal candidate for further evaluation in clinical applications related to migraine pain.

First-in-class, dual-action, 3,5-disubstituted indole derivatives having human nitric oxide synthase (nNOS) and norepinephrine reuptake inhibitory (NERI) activity for the treatment of neuropathic pain.

A family of different 3,5-disubstituted indole derivatives having 6-membered rings were designed, synthesized, and demonstrated inhibition of human nitric oxide synthase (NOS) with norepinephrine reuptake inhibitory activity (NERI). The structure-activity relationship (SAR) within the cyclohexane ring showed the cis-isomers to be more potent for neuronal NOS and selective over endothelial NOS compared to their trans-counterparts. Compounds, such as cis-(+)-37, exhibited dual nNOS and NET inhibition (IC(50) of 0.56 and 1.0 µM, respectively) and excellent selectivity (88-fold and 12-fold) over eNOS and iNOS, respectively. The lead compound (cis-(+)-37) showed lack of any direct vasoconstriction or inhibition of ACh-mediated vasorelaxation in isolated human coronary arteries. Additionally, cis-(+)-37 was effective at reversing both allodynia and thermal hyperalgesia in a standard Chung (spinal nerve ligation) rat neuropathic pain model. Overall, the data suggest that cis-(+)-37 is a promising dual action development candidate having therapeutic potential for the treatment of neuropathic pain.

Novel, druglike 1,7-disubstituted 2,3,4,5-tetrahydro-1H-benzo[b]azepine-based selective inhibitors of human neuronal nitric oxide synthase (nNOS).

A novel class of 1,7-disubstituted 2,3,4,5-tetrahydro-1H-benzo[b]azepine derivatives was designed, synthesized and evaluated as human nitric oxide synthase (NOS) inhibitors. Structure-activity relationship studies based on various basic amine side chains attached at the 1-position of the 2,3,4,5-tetrahydro-1H-benzo[b]azepine ring led to the identification of several potent and highly selective inhibitors (17, 18, 25, (±)-39, and (±)-40) of human neuronal NOS. The potential therapeutic application of one of these new selective nNOS inhibitors (17) was demonstrated in an in vivo spinal nerve ligation model of neuropathic pain, and various in vitro safety pharmacology studies such as the hERG K(+) channel inhibition assay and high throughput broad screen (minimal activity at 79 receptors/transporters/ion channels).

1,2,3,4-tetrahydroquinoline-based selective human neuronal nitric oxide synthase (nNOS) inhibitors: lead optimization studies resulting in the identification of N-(1-(2-(methylamino)ethyl)-1,2,3,4-tetrahydroquinolin-6-yl)thiophene-2-carboximidamide as a preclinical development candidate.

Numerous studies have shown that selective nNOS inhibitors could be therapeutic in many neurological disorders. Previously, we reported a series of 1,2,3,4-tetrahydroquinoline-based potent and selective nNOS inhibitors, highlighted by 1 ( J. Med. Chem. 2011 , 54 , 5562 - 5575 ). Despite showing activity in two rodent pain models, 1 suffered from low oral bioavailability (18%) and moderate hERG channel inhibition (IC(50) = 4.7 µM). To optimize the properties of 1, we synthesized a small focused library containing various alkylamino groups on the 1-position of the 1,2,3,4-tetrahydroquinoline scaffold. The compounds were triaged based on their activity in the NOS and hERG manual patch clamp assays and their calculated physicochemical parameters. From these studies, we identified 47 as a potent and selective nNOS inhibitor with improved oral bioavailability (60%) and no hERG channel inhibition (IC(50) > 30 µM). Furthermore, 47 was efficacious in the Chung model of neuropathic pain and has an excellent safety profile, making it a promising preclinical development candidate.

3,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase (nNOS) inhibitors.

A series of 3,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). Various guanidine isosteric groups were explored at the 5-position of the indole ring, while keeping the basic amine side chain such as N-methylpiperidine ring, fixed at the 3-position of the indole ring. Compounds having 2-thiophene amidine and 2-furanyl amidine groups (7, 8, 10 and 12) showed increased activity for human neuronal NOS and good selectivity over endothelial and inducible NOS isoforms. Compound 8 was shown to reverse (10mg/kg, ip) thermal hyperalgesia in the L(5)/L(6) spinal nerve ligation (neuropathic pain) model and was devoid of any significant drug-drug interaction potential due to cytochrome P450 inhibition or cardiovascular liabilities associated with the inhibition of endothelial NOS.

A medicinal chemistry perspective on structure-based drug design and development.

The application of X-ray crystallography and molecular modeling can provide valuable insight into the optimization of the molecular interactions of a drug-protein complex to achieve potency and selectivity of a drug candidate. For the successful application of SBDD in a drug development program, the impact of these structural modifications required to improve potency and selectivity must be considered in the context of balancing of a multitude of drug properties and other considerations that include solubility, bioavailability, metabolism, distribution, toxicology, chemical stability, and intellectual property space. The utility of structure-based design from the medicinal chemist's perspective is described in this chapter.

Discovery of cis-N-(1-(4-(methylamino)cyclohexyl)indolin-6-yl)thiophene-2-carboximidamide: a 1,6-disubstituted indoline derivative as a highly selective inhibitor of human neuronal nitric oxide synthase (nNOS) without any cardiovascular liabilities.

A series of 1,6-disubstituted indoline derivatives were synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS) designed to mitigate the cardiovascular liabilities associated with previously reported tetrahydroquinoline-based selective neuronal NOS inhibitors due to higher lipophilicity ( J. Med. Chem. 2011 , 54 , 5562 - 5575 ). This new series produced similar potency and selectivity among the NOS isoforms and was devoid of any cardiovascular liabilities associated with QT prolongation due to hERG activity or endothelial NOS mediated vasoconstriction effect. The SAR studies led to the identification of cis-45, which was shown to reverse thermal hyperalgesia in vivo in the spinal nerve ligation model of neuropathic pain with excellent safety profile (off-target activities at 80 CNS related receptors/ion channels/transporters). The results presented in this report make cis-45 as an ideal tool for evaluating the potential role of selective nNOS inhibitors in CNS related disorders where excess NO produced by nNOS is thought to play a crucial role.

Discovery of N-(3-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indol-6-yl) thiophene-2-carboximidamide as a selective inhibitor of human neuronal nitric oxide synthase (nNOS) for the treatment of pain.

3,6-Disubstituted indole derivatives were designed, synthesized, and evaluated as inhibitors of human nitric oxide synthase (NOS). Bulky amine containing substitution on the 3-position of the indole ring such as an azabicyclic system showed better selectivity over 5- and 6-membered cyclic amine substitutions. Compound (-)-19 showed the best selectivity for neuronal NOS over endothelial NOS (90-fold) and inducible NOS (309-fold) among the current series. Compounds 16 and (-)-19 were shown to be either inactive or very weak inhibitors of human cytochrome P450 enzymes, indicating a low potential for drug-drug interactions. Compound 16 was shown to reverse thermal hyperalgesia in vivo in the Chung model of neuropathic pain. Compound 16 was also devoid of any significant vasoconstrictive effect in human coronary arteries, associated with the inhibition of human eNOS. These results suggest that 16 may be a useful tool for evaluating the potential role of selective nNOS inhibitors in the treatment of pain such as migraine and CTTH.

Design, synthesis, and biological evaluation of 3,4-dihydroquinolin-2(1H)-one and 1,2,3,4-tetrahydroquinoline-based selective human neuronal nitric oxide synthase (nNOS) inhibitors.

Neuronal nitric oxide synthase (nNOS) inhibitors are effective in preclinical models of many neurological disorders. In this study, two related series of compounds, 3,4-dihydroquinolin-2(1H)-one and 1,2,3,4-tetrahydroquinoline, containing a 6-substituted thiophene amidine group were synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). A structure-activity relationship (SAR) study led to the identification of a number of potent and selective nNOS inhibitors. Furthermore, a few representative compounds were shown to possess druglike properties, features that are often difficult to achieve when designing nNOS inhibitors. Compound (S)-35, with excellent potency and selectivity for nNOS, was shown to fully reverse thermal hyperalgesia when given to rats at a dose of 30 mg/kg intraperitonieally (ip) in the L5/L6 spinal nerve ligation model of neuropathic pain (Chung model). In addition, this compound reduced tactile hyperesthesia (allodynia) after oral administration (30 mg/kg) in a rat model of dural inflammation relevant to migraine pain.

In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2).

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group between nitrogen atoms (N(1) and N(2)) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhibition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450 isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In that context, 5 might be suitable for further drug development.

Novel 2-aminobenzothiazoles as selective neuronal nitric oxide synthase inhibitors.

A series of substituted 2-aminobenzothiazole compounds have been synthesized and evaluated as nitric oxide synthase (NOS) inhibitors. Compound 14 shows activity in the nM range and is selective for the human neuronal NOS isoform. We have also evaluated the compounds against the rat NOS isoforms. For some of the compounds, there are significant differences in NOS inhibitory activities between the human and rat enzymes. For example, compound 10b has nM activity against the rat nNOS while low microM activity against the human nNOS.

A comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) of anthranilamide derivatives that are multidrug resistance modulators.

In a continuing effort to develop potent and selective modulators of P-glycoprotein (P-gp) activity overcoming the chemoresistance acquired by tumor cells during cancer chemotherapy, we developed 3D quantitative structure-activity relationship (3D QSAR) models using CoMFA and CoMSIA analyses. This study correlates the P-glycoprotein inhibitory activities of 49 structurally related anthranilamide derivatives to several physicochemical parameters representing steric, electrostatic, acceptor, donor, and hydrophobic fields. Both CoMFA and CoMSIA models using three different alignment conformations gave good internal predictions, and their cross-validated r2 values are between 0.503 and 0.644. These most comprehensive CoMFA and CoMSIA models are useful in understanding the structure-activity relationships of anthranilamide derivatives as well as aid in the design of novel derivatives with enhanced modulation of P-gp activity.

In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity at CYP-450.

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency of the P-glycoprotein (P-gp) transporter. The aromatic spacer group between nitrogen atoms (N1 and N2) in the known inhibitor XR9576 was replaced with a flexible alkyl chain of 2 to 6 carbon atoms in length. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline and their open-chain N-methylhomoveratrylamine counterparts were shown to be potent P-gp inhibitors. The maximal inhibition was obtained when using an ethyl or propyl spacer. Several compounds were more potent than verapamil and intrinsically less cytotoxic than XR9576. In addition, in vitro metabolism studies of 23a with a subset of human CYP-450 isoforms revealed that, unlike XR9576, 23a inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of anticancer drugs such as paclitaxel toward metabolism. In this context, 22a might be a suitable candidate for further drug development.

Identification of a novel non-carbohydrate molecule that binds to the ribosomal A-site RNA.

We report the identification of a novel compound that binds to the Escherichia coli 16S ribosomal A-site. Binding by the compound was observed using nuclear magnetic resonance and mass spectrometry techniques. We show that the compound binds in the same position in the A-site RNA as occupied by the aminoglycoside class of antibiotics.