General Approach to Enantiopure 1-Aminopyrrolizidines: Application to the Asymmetric Synthesis of the Loline Alkaloids.

The synthesis of a range of loline alkaloids is reported. The C(7) and C(7a) stereogenic centers for the targets were formed by the established conjugate addition of lithium (S)-N-benzyl-N-(α-methylbenzyl)amide to tert-butyl 5-benzyloxypent-2-enoate, ensuing enolate oxidation to give an α-hydroxy-ß-amino ester, and then formal exchange of the resultant amino and hydroxyl functionalities (via the intermediacy of the corresponding aziridinium ion) to give an α-amino-ß-hydroxy ester. Subsequent transformation gave a 3-hydroxyprolinal derivative which was converted to the corresponding N-tert-butylsulfinylimine. Mannich-type reaction with the enolate derived from O-Boc protected methyl glycolate then formed the remaining C(1) and C(2) stereogenic centers for the targets. The 2,7-ether bridge was formed by a displacement reaction, completing construction of the loline alkaloid core. Facile manipulations then gave a range of loline alkaloids, including loline itself.

ß-Peptides incorporating polyhydroxylated cyclohexane ß-amino acid: synthesis and conformational study.

We describe the synthesis of trihydroxylated cyclohexane ß-amino acids from (-)-shikimic acid, in their cis and trans configuration, and the incorporation of the trans isomer into a trans-2-aminocyclohexanecarboxylic acid peptide chain. Subsequently, the hydroxyl groups were partially or totally deprotected. The structural study of the new peptides by FTIR, CD, solution NMR and DFT calculations revealed that they all fold into a 14-helix secondary structure, similarly to the homooligomer of trans-2-aminocyclohexanecarboxylic acid. This means that the high degree of substitution of the cyclohexane ring of the new residue is compatible with the adoption of a stable helical secondary structure and opens opportunities for the design of more elaborate peptidic foldamers with oriented polar substituents at selected positions of the cycloalkane residues.

Structure-activity relationships of 2-pyrimidinecarbohydrazides as utrophin modulators for the potential treatment of Duchenne muscular dystrophy.

A therapeutic approach that holds the potential to treat all Duchenne muscular dystrophy (DMD) patient populations is utrophin modulation. Ezutromid, a first generation utrophin modulator which was later found to act via antagonism of the arylhydrocarbon receptor, progressed to Phase 2 clinical trials. Although interim data showed target engagement and functional improvements, ezutromid ultimately failed to meet its clinical endpoints. We recently described the identification of a new class of hydrazide utrophin modulators which has a different mechanism of action to ezutromid. In this study we report our early optimisation studies on this hydrazide series. The new analogues had significantly improved potency in cell-based assays, increased sp3 character and reduced lipophilicity, which also improved their physicochemical properties. A representative new analogue combining these attributes increased utrophin protein in dystrophic mouse cells showing it can be used as a chemical tool to reveal new insights regarding utrophin upregulation as a strategy for DMD therapeutic intervention.

Microgrewiapine C: Asymmetric Synthesis, Spectroscopic Data, and Configuration Assignment.

The first asymmetric synthesis of microgrewiapine C, a piperidine alkaloid isolated from Microcos paniculata, is reported. This synthesis prompted correction of the 1H and 13C NMR data for the natural sample of the alkaloid, which was achieved by reanalysis of the original spectra. The corrected data for the natural product were found to be identical to those of the synthetic sample prepared herein, thus confirming the structural and relative configurational assignment of microgrewiapine C. Although comparison of specific rotation values indicates that the (1R,2S,3S,6S) absolute configuration should be assigned to the alkaloid, consideration of potential common biosynthetic origins of microgrewiapine C and congeners suggests that further phytochemical investigations are warranted.

Synthesis and Configuration of O-Acetyl Microgrewiapine A: Phantomization of O-Acetyl 6-epi-Microgrewiapine A.

The formation of O-acetyl microgrewiapine A is investigated. NMR data for the authentic sample derived from the natural product are corrected. Wholly synthetic samples, produced from reductive N-methylation of synthetic microcosamine A (to give synthetic microgrewiapine A) followed by O-acetylation, exhibit NMR data that are identical to those of the authentic sample. The previous report that this two-step transformation proceeds with epimerization at C-6 is thus shown to be in error: the purported sample of O-acetyl 6-epi-microgrewiapine A is structurally misassigned and is, in fact, O-acetyl microgrewiapine A. A plausible rationale for the structural misassignment is advanced.

Mutual kinetic resolution: probing enantiorecognition phenomena and screening for kinetic resolution with racemic reagents.

Enantiorecognition between a racemic reagent and a racemic substrate can be a valuable process in organic synthesis. This review highlights representative examples of this phenomenon and the use of mutual kinetic resolution as a method for screening of kinetic and/or parallel kinetic resolutions.

Identification and Preliminary Structure-Activity Relationship Studies of 1,5-Dihydrobenzo[e][1,4]oxazepin-2(3H)-ones That Induce Differentiation of Acute Myeloid Leukemia Cells In Vitro.

Acute myeloid leukemia (AML) is the most aggressive type of blood cancer, and there is a continued need for new treatments that are well tolerated and improve long-term survival rates in patients. Induction of differentiation has emerged as a promising alternative to conventional cytotoxic chemotherapy, but known agents lack efficacy in genetically distinct patient populations. Previously, we established a phenotypic screen to identify small molecules that could stimulate differentiation in a range of AML cell lines. Utilising this strategy, a 1,5-dihydrobenzo[e][1,4]oxazepin-2(3H)-one hit compound was identified. Herein, we report the hit validation in vitro, structure-activity relationship (SAR) studies and the pharmacokinetic profiles for selected compounds.

Aminothiazolones as potent, selective and cell active inhibitors of the PIM kinase family.

We have previously reported the discovery of a series of rhodanine-based inhibitors of the PIM family of serine/threonine kinases. Here we described the optimisation of those compounds to improve their physicochemical and ADME properties as well as reducing their off-targets activities against other kinases. Through molecular modeling and systematic structure activity relationship (SAR) studies, advanced molecules with high inhibitory potency, reduced off-target activity and minimal efflux were identified as new pan-PIM inhibitors. One example of an early lead, OX01401, was found to inhibit PIMs with nanomolar potency (15 nM for PIM1), inhibit proliferation of two PIM-expressing leukaemic cancer cell lines, MV4-11 and K562, and to reduce intracellular phosphorylation of a PIM substrate in a concentration dependent manner.

Synthesis of SMT022357 enantiomers and in vivo evaluation in a Duchenne muscular dystrophy mouse model.

Following on from ezutromid, the first-in-class benzoxazole utrophin modulator that progressed to Phase 2 clinical trials for the treatment of Duchenne muscular dystrophy, a new chemotype was designed to optimise its physicochemical and ADME profile. Herein we report the synthesis of SMT022357, a second generation utrophin modulator preclinical candidate, and an asymmetric synthesis of its constituent enantiomers. The pharmacological properties of both enantiomers were evaluated in vitro and in vivo. No significant difference in the activity or efficacy was observed between the two enantiomers; activity was found to be comparable to the racemic mixture.

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

SuperQuat chiral auxiliaries: design, synthesis, and utility.

The SuperQuat (4-substituted 5,5-dimethyloxazolidine-2-one) family of chiral auxiliaries was first developed by us in the 1990s to address the shortcomings of the Evans (4-substituted oxazolidin-2-one) family of chiral auxiliaries. The incorporation of geminal dimethyl substitution at C(5) has two effects: (i) it induces a conformational bias on an adjacent, otherwise conformationally labile C(4)-substituent so that it projects towards the N-acyl fragment, thus offering superior diastereofacial selectivity in a range of transformations; and (ii) it hinders nucleophilic attack at the endocyclic carbonyl group, facilitating recovery and recyclability of the auxiliary, with enhanced cleavage properties. This review summarises the development and some of the most common uses of the SuperQuat family of chiral auxiliaries, particularly in the synthesis of natural products or other targets having bioloigcal interest. Where possible, comparisons with the performances of the corresponding Evans auxiliaries are presented.

N-Acetylcolchinol Methyl Ether (a Natural Product); Suhailamine (a Phantom Natural Product).

The reported characterization data for the allocolchicinoid alkaloid suhailamine, isolated from Colchicum decaisnei and known to have an erroneous structure, have been reanalyzed. This analysis has led to the current proposal that suhailamine has the same structure as N-acetylcolchinol methyl ether (NCME), an assertion that is supported by comparison with previously reported data for NCME. Suhailamine is therefore a phantom natural product, while NCME represents a naturally occurring allocolchicinoid rather than a purely synthetic entity.

Diastereoselective Ammonium-Directed Epoxidation in the Asymmetric Syntheses of Dihydroconduramines (+)-C-2, (-)-C-2, (+)-D-2, (+)-E-2, (+)-F-2, and (-)-F-2.

Epoxidations (40% aq HBF4 then m-CPBA) of racemic cis-2-( N-benzylamino)cyclohex-3-en-1-ol and racemic cis-2-( N, N-dibenzylamino)cyclohex-3-en-1-ol proceed with very high levels of diastereoselectivity (>95:5 dr). The latter is in direct contrast to the epoxidation of the corresponding trans-diastereoisomer (which proceeds with essentially no selectivity), showing that the relative configuration of the substrate dramatically influences the diastereoselectivity in these instances. Meanwhile, epoxidations of enantiopure (1 R,2 S,α R)-2-[( N-α-methylbenzyl)amino]cyclohex-3-en-1-ol and (1 S,2 R,α R)-2-[( N-α-methylbenzyl)amino]cyclohex-3-en-1-ol [surrogates for the enantiomers of cis-2-( N-benzylamino)cyclohex-3-en-1-ol] proceed with complete diastereoselectivity (>95:5 dr) under the same conditions, showing that neither the presence of the α-methyl group nor the relative configuration of the α-methylbenzyl stereocenter have an effect upon the established level of diastereoslectivity in these cases. In contrast, epoxidations of enantiopure (1 R,2 S,α R)-2-[ N-benzyl- N-(α-methylbenzyl)amino]cyclohex-3-en-1-ol and (1 S,2 R,α R)-2-[ N-benzyl- N-(α-methylbenzyl)amino]cyclohex-3-en-1-ol [surrogates for the enantiomers of cis-2-( N, N-dibenzylamino)cyclohex-3-en-1-ol] proceed with lower diastereoselectivity (∼70:30 dr). Thus, the presence of the α-methyl group has a detrimental effect on the established level of diastereoselectivity in these cases (although again the relative configuration of the α-methylbenzyl stereocenter is unimportant). The diastereoselective epoxidation pathway is used to enable the asymmetric syntheses of six hitherto unknown, enantiopure dihydroconduramines (+)-C-2, (-)-C-2, (+)-D-2, (+)-E-2, (+)-F-2, and (-)-F-2 (>99% ee in each case).

The Hancock Alkaloids (-)-Cuspareine, (-)-Galipinine, (-)-Galipeine, and (-)-Angustureine: Asymmetric Syntheses and Corrected 1H and 13C NMR Data.

The asymmetric syntheses of all members of the Hancock alkaloid family based upon a 2-substituted N-methyl-1,2,3,4-tetrahydroquinoline core are delineated. The conjugate addition of enantiopure lithium N-benzyl- N-(α-methyl- p-methoxybenzyl)amide to 5-( o-bromophenyl)- N-methoxy- N-methylpent-2-enamide is used to generate the requisite C-2 stereogenic center of the targets, while an intramolecular Buchwald-Hartwig coupling is used to form the 1,2,3,4-tetrahydroquinoline ring. Late-stage diversification completes construction of the C-2 side chains. Thus, (-)-cuspareine, (-)-galipinine, (-)-galipeine, and (-)-angustureine were prepared in overall yields of 30%, 28%, 15%, and 39%, respectively, in nine steps from commercially available 3-( o-bromophenyl)propanoic acid in all cases. Unambiguously corrected 1H and 13C NMR data for the originally isolated samples of (-)-cuspareine, (-)-galipinine, and (-)-angustureine are also reported, representing a valuable reference resource for these popular synthetic targets.

Second-generation compound for the modulation of utrophin in the therapy of DMD.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches are progressing through human clinical trials. By pharmacologically modulating the expression of the dystrophin-related protein utrophin, we have previously demonstrated in dystrophin-deficient mdx studies, daily SMT C1100 treatment significantly reduced muscle degeneration leading to improved muscle function. This manuscript describes the significant disease modifying benefits associated with daily dosing of SMT022357, a second-generation compound in this drug series with improved physicochemical properties and a more robust metabolism profile. These studies in the mdx mouse demonstrate that oral administration of SMT022357 leads to increased utrophin expression in skeletal, respiratory and cardiac muscles. Significantly, utrophin expression is localized along the length of the muscle fibre, not just at the synapse, and is fibre-type independent, suggesting that drug treatment is modulating utrophin transcription in extra-synaptic myonuclei. This results in improved sarcolemmal stability and prevents dystrophic pathology through a significant reduction of regeneration, necrosis and fibrosis. All these improvements combine to protect the mdx muscle from contraction induced damage and enhance physiological function. This detailed evaluation of the SMT C1100 drug series strongly endorses the therapeutic potential of utrophin modulation as a disease modifying therapeutic strategy for all DMD patients irrespective of their dystrophin mutation.

Structural Revision of the Hancock Alkaloid (-)-Galipeine.

The 1H and 13C NMR data of synthetic samples of (S)-N(1)-methyl-2-[2'-(3″-hydroxy-4″-methoxyphenyl)ethyl]-1,2,3,4-tetrahydroquinoline, the originally proposed structure of the Hancock alkaloid (-)-galipeine, do not match those of the natural product. Herein, the preparation of the regioisomer (S)-N(1)-methyl-2-[2'-(3″-methoxy-4″-hydroxyphenyl)ethyl]-1,2,3,4-tetrahydroquinoline is reported, the 1H and 13C NMR data of which are in excellent agreement with those of (-)-galipeine. Comparison of specific rotation data enables assignment of the absolute (S)-configuration of the alkaloid, and together, these data engender the structural revision of (-)-galipeine to (S)-N(1)-methyl-2-[2'-(3″-methoxy-4″-hydroxyphenyl)ethyl]-1,2,3,4-tetrahydroquinoline.

Asymmetric Synthesis of the Tetraponerine Alkaloids.

The asymmetric syntheses of all eight tetraponerine alkaloids (T1-T8) were achieved using the diastereoselective conjugate additions of lithium amide reagents in the key stereodefining steps. Conjugate addition of either lithium (R)-N-allyl-N-(α-methylbenzyl)amide or lithium (R)-N-(but-3-en-1-yl)-N-(α-methylbenzyl)amide to tert-butyl sorbate was followed by ring-closing metathesis of the resultant N-alkenyl ß-amino esters, reduction to the corresponding aldehydes, and reaction with tert-butyl (triphenylphosphoranylidene)acetate. Subsequent conjugate addition of the requisite antipode of lithium N-benzyl-N-(α-methylbenzyl)amide to the resultant α,ß-unsaturated esters gave a range of diamines for elaboration to T1-T8 via a sequence involving reduction of the ester moiety to give the corresponding aldehyde, olefination, tandem hydrogenation/hydrogenolysis, and cyclization upon reaction with 4-bromobutanal to give the tricyclic skeleton.

Asymmetric Syntheses of 3-Deoxy-3-aminosphingoid Bases: Approaches Based on Parallel Kinetic Resolution and Double Asymmetric Induction.

The asymmetric syntheses of a range of N- and O-protected 3-deoxy-3-aminosphingoid bases have been achieved using two complementary approaches. dl-Serine was converted to a racemic N,N-dibenzyl-protected γ-amino-α,ß-unsaturated ester which was resolved using a parallel kinetic resolution (PKR) strategy upon reaction with a pseudoenantiomeric mixture of lithium (R)-N-benzyl-N-(α-methylbenzyl)amide and lithium (S)-N-3,4-dimethoxybenzyl-N-(α-methylbenzyl)amide, giving the corresponding enantio- and diastereoisomerically pure ß,γ-diamino esters. Alternatively, elaboration of l-serine gave the corresponding enantiopure N,N-dibenzyl-protected γ-amino-α,ß-unsaturated ester, and doubly diastereoselective conjugate addition of the antipodes of lithium N-benzyl-N-(α-methylbenzyl)amide was found to proceed under the dominant stereocontrol of the lithium amide reagent in both cases, thus augmenting the accessible range of ß,γ-diamino esters. Both of these protocols were expanded to include in situ oxidation of the enolate formed upon conjugate addition, giving access to the corresponding α-hydroxy-ß,γ-diamino esters. Elaboration of these ß,γ-diamino and α-hydroxy-ß,γ-diamino esters gave the protected forms of the 3-deoxy-3-aminosphingoid base targets.

Probing Competitive and Co-operative Hydroxyl and Ammonium Hydrogen-Bonding Directed Epoxidations.

The diastereoselectivities and rates of epoxidation (upon treatment with Cl3CCO2H then m-CPBA) of a range of cis- and trans-4-aminocycloalk-2-en-1-ol derivatives (containing five-, six-, and seven-membered rings) have been investigated. In all cases where the two potential directing groups can promote epoxidation on opposite faces of the ring scaffold, evidence of competitive epoxidation pathways, promoted by hydrogen-bonding to either the in situ formed ammonium moiety or the hydroxyl group, was observed. In contrast to the relative directing group abilities already established for the six-membered ring system (NHBn ≫ OH > NBn2), an N,N-dibenzylammonium moiety appeared more proficient than a hydroxyl group at directing the stereochemical course of the epoxidation reaction in a five- or seven-membered system. In the former case, this was rationalized by the drive to minimize torsional strain in the transition state being coupled with assistance from hydrogen-bonding to the ammonium moiety. In the latter case, this was ascribed to the steric bulk of the ammonium moiety disfavoring conformations in which hydrogen-bonding to the hydroxyl group results in direction of the epoxidation to the syn face. In cases where the two potential directing groups can promote epoxidation on the same face of the ring scaffold, an enhancement of epoxidation diastereoselectivity was not observed, while introduction of a second, allylic heteroatom to the substrate results in diminishment of the rate of epoxidation in all cases. Presumably, reduction of the nucleophilicity of the olefin by the second, inductively electron-withdrawing heteroatom is the dominant factor, and any assistance to the epoxidation reaction by the potential to form hydrogen-bonds to two directing groups rather than one is clearly unable to overwhelm it.

Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family.

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75µM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.