Search

The development of a commercial manufacturing route to verubecestat (MK-8931) is described, highlights of which include the application of a continuous processing step to outcompete fast proton transfer in a Mannich-type ketimine addition, a copper-catalyzed amidation reaction, and an optimized guanidinylation procedure to form the key iminothiadiazine dioxide core.

Verubecestat is an inhibitor of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimer's disease. Synthetic route development involves diastereoselective transformations with a need for enantiomeric excess (ee) determination of each intermediate and final active pharmaceutical ingredient (API). The analytical technical package of validated methods relies on enantioselective SFC and RPLC separations using multiple 3 and 5 μm coated polysaccharide-based chiral stationary phases (CSPs) and mobile phases combinations. Evaluation of recently developed chiral columns revealed a single chiral selector (Teicoplanin) bonded to 2.7 μm core-shell particles using H 3 PO 4 in H 2 O/ACN and triethylammonium acetate: methanol based eluents at different isocratic compositions allowed good enatioseparation of all verubecestat intermediates. EE determination of verubecestat is easily performed on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 μm superficially porous particles. This approach enables fast and reliable enantiopurity analysis of the entire verubecestat synthetic route using only two chiral columns and mobile phases on a conventional HPLC system, simplifying technical package preparation, method validation and transfer to manufacturing facilities., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aβ levels in rats and nonhuman primates and CSF Aβ levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Verubecestat is an inhibitor of β-secretase being evaluated for the treatment of Alzheimer's disease. The first-generation route relies on an amide coupling with a functionalized aniline, the preparation of which introduces synthetic inefficiencies. The second-generation route replaces this with a copper-catalyzed C-N coupling, allowing for more direct access to the target. Other features of the new route include a diastereoselective Mannich-type addition into an Ellman sulfinyl ketimine and a late-stage guanidinylation.

The stereoselective synthesis of 1,2,11,11a-tetrahydrobenzo[e]pyrazino[1,2-b][1,2,4]thiadiazin-3(4H)-one 6,6-dioxides on a solid support via tandem N-sulfonyl iminium ion cyclization, followed by nucleophilic addition is reported. The synthesis proceeded with full control of stereoselectivity at the newly formed asymmetric carbon, under mild conditions, and using commercially available building blocks. The synthetic route provided high-purity crude products.

Diversely substituted 3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides are known to be potent KATP channel openers, with several drugs being selective for the SUR1/Kir6.2 channel subtype. This work examined the biological activity, tissue selectivity, and in vitro metabolic stability of hydroxylated analogues of 3-isopropylaminobenzothiadiazine dioxides. Because of the presence of a chiral center, the R and S isomers were prepared separately and characterized. R isomers were systematically found to be more potent and more selective than S isomers on pancreatic tissue (compared to vascular smooth muscle tissue), leading to compounds with an improved sulfonylurea receptor 1 (SUR1) selectivity. An in vitro metabolic study revealed that 7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (1a) was rapidly biotransformed and led in part to a mixture of the corresponding (R)- and (S)-3-(1-hydroxy-2-propyl)amino-substituted derivatives. Radioisotopic experiments characterized one of the most potent and SUR1-selective enantiomers, (R)-7-chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide 13a, as being a KATP channel opener. Moreover, 13a exhibited an enhanced metabolic stability. Such a compound can be considered as a new lead candidate displaying improved physicochemical (hydrosolubility) and pharmacological (tissue selectivity) properties as well as improved metabolic stability compared to its nonhydroxylated counterpart, 1a.

The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.

A series of novel 2,4-disubstituted 7-methyl-1,1,3-trioxo-2,4-dihydro-pyrazolo[4,5-e] [1,2,4]thiadiazines (PTDs) was synthesized, structurally confirmed by spectral analysis, and evaluated for their anti-HIV activities by inhibition of HIV-induced cytopathogenicity in MT-4 cell culture. The results showed that some compounds exhibited inhibitory activity specifically against HIV-2 replication. The most active HIV-2 inhibitor was compound 7i (R1 = benzyl, R2 = 4-t-butyl-benzyl) with an EC50 value of 18.7 microM and SI=15, which may provide a useful lead for further molecular optimization.

A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides have been synthesized and evaluated as potentiators of AMPA receptors. Attention was paid to the impact of the substituent introduced at the 7-position of the heterocycle. The biological evaluation was achieved by measuring the AMPA current in rat cortex mRNA-injected Xenopus oocytes. The most potent compound, 4-ethyl-7-fluoro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (12a) was found to be active in an object recognition test in rats demonstrating cognition enhancing effects in vivo after oral administration.

Compound 1a (NN414) is a potent opener of Kir6.2/SUR1 K(ATP) channels. Compound 1a inhibits insulin release in vitro and in vivo and preserves beta cell function in preclinical animal models suggesting that such a compound could find use in treatment or prevention of type 1 and type 2 diabetes. The crystal structure and a convergent synthesis of 1a are presented together with a range of new analogues of 1a. Several compounds, e.g., 6-chloro-3-(1-methyl-1-phenylethyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (1h), were found to be potent openers of Kir6.2/SUR1 K(ATP) channels and were able to suppress glucose-stimulated insulin release from rat islets in vitro (EC(50) = 0.04 +/- 0.01 muM) and in vivo after intravenous or peroral administration to hyperinsulinemic obese Zucker rats (ED(50) = 4.0 mg/kg). Structural modifications of this series of K(ATP) channel openers have provided compounds with promising pharmacokinetic properties indicating that brief periods of beta cell rest can be achieved.

A series of 3-alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides structurally related to diazoxide and pinacidil were synthesized and tested as possible K(ATP) channel openers on isolated pancreatic endocrine tissue as well as on isolated vascular, intestinal, and uterine smooth muscle. In contrast to previously described 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1, 1-dioxides, most of the new compounds were found to be poorly active on B-cells but exhibited clear vasorelaxant properties. 3-(3, 3-Dimethyl-2-butylamino)-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxide (4d) and 7-chloro-3-(3, 3-dimethyl-2-butylamino)-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxide (5d), two compounds bearing the alkyl side chain of pinacidil, were found to be the most active representatives of their respective series on rat aorta rings. 3-Cycloalkylalkylamino- and 3-aralkylamino-7-chloro-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides also expressed myorelaxant activity on electrically stimulated guinea pig ileum and on oxytocin-induced contractions of the rat uterus. Further biological investigations ((86)Rb efflux measurements, vasodilator potency on 30 and 80 mM KCl-induced contractions in the absence and presence of glibenclamide) revealed that compounds 4d and 5d, but not compound 5f, expressed the pharmacological profile of classical K(ATP) channel openers. In conclusion, by changing the position of the nitrogen atom in the pyridine ring, we now have obtained a family of drugs expressing an opposite tissue selectivity. Taken as a whole, the present findings also suggest that 3-alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides such as 4c, 4d, 5c, and 5d may be considered as new examples of K(ATP) channel openers expressing a pharmacological profile similar to that of pinacidil and diazoxide.

The preparation and the pharmacological evaluation of the R- and S-isomers of 3-(2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (BPDZ 42) and 3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (BPDZ 44), two potassium channel openers, is described. Their optical purity was estimated by means of capillary electrophoresis (R- and S-BPDZ 42) and chiral HPLC (R- and S-BPDZ 44). The absolute configuration of each isomer of BPDZ 44 was deduced from crystallographic data. Pharmacological assays performed with the R- and S-isomers of BPDZ 44 revealed only slight differences in their activity on pancreatic B-cells but significant differences in their activity on vascular smooth muscle cells: the R-isomer being sixfold more potent than its corresponding S-isomer. The R-isomer of BPDZ 42 was shown to be more potent than its corresponding S-isomer on the endocrine pancreas. S-BPDZ 44 as well as R- and S-BPDZ 42 were found to exhibit tissue selectivity for the pancreatic versus the vascular smooth muscle tissue.

A series of N-1-substituted derivatives of pyrazino[2,3-c][1,2, 6]thiadiazine 2,2-dioxides bearing aryl groups at the pyrazino moiety have been prepared. The synthesis involves ring formation between the diaminothiadiazine and suitable dicarbonyl compounds and subsequent introduction of the substituent at N-1. The compounds have been tested in vitro, as inhibitors of rabbit and human platelet aggregation, and ex vivo against rat platelet aggregation induced by arachidonic acid, ADP, collagen, U46619, and I-BOP. The results obtained indicate that some pyrazino[2,3-c][1,2, 6]thiadiazine derivatives show significant platelet aggregation inhibition similar to other antithrombotic agents and that the antiplatelet properties may be mediated by interference with the arachidonic acid pathway.

A series of 4H-1,2,4-pyridothiadiazine 1,1-dioxides and 2, 3-dihydro-4H-1,2,4-pyridothiadiazine 1,1-dioxides bearing various alkyl and aryl substituents on the 2-, 3-, and 4-positions was synthesized and tested as possible positive allosteric modulators of the (R/S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. Many compounds were found to be more potent than the reference compounds diazoxide and aniracetam as potentiators of the AMPA current in rat cortex mRNA-injected Xenopus oocytes. The most active compound, 4-ethyl-2,3-dihydro-4H-pyrido[3,2-e]-1,2, 4-thiadiazine 1,1-dioxide (31b), revealed an in vitro activity on Xenopus oocytes not far from that of cyclothiazide, the most potent allosteric modulator of AMPA receptors reported to date. Moreover, 31b, but not cyclothiazide, was found to potentiate the duration and the amplitude of the excitatory postsynaptic field potentials induced by electric stimulation in rat hippocampal slices. Such an effect could indicate, for 31b, but not for cyclothiazide, a possible interaction with postsynaptic AMPA receptor binding sites located on hippocampal CA1 neurons. Structure-activity relationships indicated that the structural requirements responsible for a biological activity on AMPA receptors are different from those responsible for an inhibitory activity on the insulin releasing process (putative ATP-sensitive K+-channel openers). For instance, 31b and other related dihydropyridothiadiazines were found to be ineffective as inhibitors of insulin release from rat pancreatic B-cells, in contrast to diazoxide and known pyridothiadiazines reported as ATP-sensitive K+-channel openers. Conversely, the pyridothiadiazines active on B-cells were found to be ineffective as potentiators of the AMPA currents in Xenopus oocytes. Thus, 31b appeared to be more specific than diazoxide as an AMPA receptor modulator. This compound may be considered as a new pharmacological tool, different from diazoxide and cyclothiazide, for studying AMPA receptors. Moreover, 31b can also constitute a new therapeutic agent for the treatment of cognitive disorders.

Several novel substituted tetrahydro- and hexahydro-1,2-benzisothiazol-3-one 1,1-dioxides and thiadiazinones were prepared and examined in a series of in vitro and in vivo tests to determine their pharmacological profile. Most compounds were orally active in blocking the conditioned avoidance response (CAR) but did not antagonize apomorphine-induced stereotyped behavior. Several compounds demonstrated moderate to high affinity for the 5-HT1A receptor binding site, with compounds 37 and 38 containing 2-pyrimidinylpiperazinyl and [3-(trifluoromethyl)phenyl]piperazinyl moieties and compound 47 containing the 2-pyrazinylpiperazinyl moiety displaying the highest affinity (Ki values of 10, 4, and 9 nM, respectively). Compound 37, 3-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]hexahydro-4, 7-etheno-1H-cyclobut [f]-1,2-benzisothiazol-3(2H)-one 1,1-dioxide, buspirone, and ipsapirone showed similarities in their neurochemical and behavioral profiles. They were similar in potency in blocking CAR with AB50 values of 39, 32, and 42 mg/kg, respectively. They also demonstrated high affinity and selectivity for the 5-HT1A receptor site (Ki = 10 nM) and exhibited partial agonist/antagonist activity in the serotonin syndrome test. In addition, compound 37 inhibited apomorphine-induced climbing behavior much more potently (ED50 of 3.4 mg/kg) than stereotyped behavior (ED50 of 32.2 mg/kg) and will be evaluated further. Structure-activity relationships within this series of compounds are discussed.