Highly functionalized β-lactams and 2-ketopiperazines as TRPM8 antagonists with antiallodynic activity

Abstract

The cool sensor transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia, playing a key role in cold hypersensitivity associated to different peripheral neuropathies. Moreover, these channels are aberrantly expressed in different cancers, and seem to participate in tumor progression, survival and invasion. Accordingly, the search for potent and selective TRPM8 modulators attracted great interest in recent years. We describe new heterocyclic TRPM8 antagonist chemotypes derived from N-cloroalkyl phenylalaninol-Phe conjugates. The cyclization of these conjugates afforded highly substituted β-lactams and/or 2-ketopiperazine (KP) derivatives, with regioselectivity depending on the N-chloroalkyl group and the configuration. These derivatives behave as TRPM8 antagonists in the Ca 2+ microfluorometry assay, and confirmed electrophysiologically for the best enantiopure β-lactams 24a and 29a (IC 50 , 1.4 and 0.8 µM). Two putative binding sites by the pore zone, different from those found for typical agonists and antagonists, were identified by in silico studies for both β-lactams and KPs. β-Lactams 24a and 29a display antitumor activity in different human tumor cell lines (micromolar potencies, A549, HT29, PSN1), but correlation with TRPM8 expression could not be established. Additionally, compound 24a significantly reduced cold allodynia in a mice model of oxaliplatin-induced peripheral neuropathy.

Phenylalanine-Derived β-Lactam TRPM8 Modulators. Configuration Effect on the Antagonist Activity

“…N -Dibenzylphenylalaninol 2-azetidinones 13 – 16 were prepared from commercial alcohols 3 and 4 in three steps ( Scheme 1 ), following the previously described synthetic procedure [ 41 ].…”

“…As expected, enantiomeric pairs ( 13 / 16 and 14 / 15 ) show similar values of optical rotation, but of opposite sign. As KPs are not possible in this case, the yield of β-lactams is in general higher than those obtained for the corresponding 2′-benzyloxycarbonyl derivatives [ 41 ], but some hydrolysis of the benzyl ester was observed during the cyclization step workup (higher for 2′ R -diastereoisomers compared to 2′ S -counterparts).…”

“…The prototype β-lactams bear four hydrophobic substituents very important for activity, three benzyl groups and a tert -butoxycarbonyl (Boc) moiety, and a short N -alkyl chain (1–2 carbons). Very recently, we reported on the cyclization of a series of shorter phenylalaninol-Phe conjugated, affording 2-azetidinones and/or 2-ketopiperazine (KP) derivatives, depending on the N -chloroalkyl group and on the configuration of linear intermediates [ 41 ]. Cyclization of all S -configured linear choroppropanoyl derivatives afforded the expected β-lactam 1 along with a small quantity of its regiomeric 2-ketopiperazine (KP), due to the cyclization through the 2′-NH group (indicated by a red arrow, Figure 1 ).…”

“…In addition, for 2″- R -chloropropanoyl derivatives, the corresponding β-lactams were obtained as inseparable mixtures of diastereoisomers, thus precluding the study on the configuration influence. The prototype β–lactam ( 1 , configuration 3 S ,4 S ,2′ S ) blocks the channel activation by menthol (Ca 2+ entry assay) with micromolar values, significantly reduces the oxaliplatin-induced allodynia from 15 to up to 60 min in a mice model in vivo, and exhibits non-selective antitumor activity (micromolar potency in four tumor cell lines) [ 41 ]. More importantly, docking studies indicated that compound 1 localizes preferentially at two possible locations by the pore zone of the TRPM8 channel, suggesting either an allosteric or a pore blocking mechanism, different to that of other described antagonists.…”

“…The dibenzylamino moiety is present in other potent tryptamine- and tryptophan-derived TRPM8 antagonists [ 32 , 33 ], and some conformationally restricted tetrahydro-β-carboline derivatives [ 42 ]. The 3 S ,4 S ,2′ R -diastereoisomer of β–lactam 1 showed slightly better TRPM8 antagonist activity than the N -2’S-isomer ( 1 ), while the corresponding 3 R ,4 R ,2′ S - and 3 R ,4 R ,2′ R -isomers could not be obtained in diasteroisomeric pure form [ 41 ]. Given the intrinsic chirality of biological systems, the absolute configuration of drugs is normally an important issue.…”