Development of novel selective µ-opioid receptor ligands based on the lead structure PZM21

Abstract

Prescription opioids are used for the treatment of chronic and severe pain. When used appropriately, they are an essential part of pain therapy. But opioids may also pose serious risks. Addiction to opioids and their abuse has led to an epidemic, which demanded about 50.000 people’s lives in 2017 in the USA.44 The µ-opioid receptor (µOR) agonist PZM2118 is functionally selective (biased) towards G protein signaling, which has been associated with an improved side-effect profile in studies with mice.18 Taking this as a starting point, the aim of this thesis was the modification of PZM21 to further elucidate how the structural features of PZM21 determine its unique biological activity and optimize µOR specificity and pharmacokinetic properties, where applicable. The development of novel PZM21 derivatives was guided by a rational design approach taking both structural- and ligand-based information into account. The structure-based design involved molecular docking experiments using the softwares AutoDock Vina 1.1.270 and GOLD71. Ligand-based information, like binding affinity and functional activity, was kindly provided by Dr. Harald Hübner. Bioisosteric replacement of the urea moiety A convergent synthetic route was chosen for the synthesis of new urea derivatives based on three building blocks (Figure 49). The amino acid-derived- and the alkylamine building block were fused by a central urea replacement incorporating thiourea-, N-cyanoguanidine-, 1,1-diaminonitroethylene- and malonodinitrile functionalities. The amino acid-derived building block 1 (Figure 49) was synthesized from commercially available L-tyrosine amide, a semi-synthetic product of the natural amino acid L-tyrosine. The same synthetic route was applied in the synthesis of L-meta-tyrosine, L-tryptophan, L-5-OH-tryptophan and 4-difluor-phenylalanine derivatives. Depending on the commercial availability, the syntheses started from the amino acid, the amino acid ester or the amino acid amide. The urea replacement 2 and the alkylamine building block 3 (R)-β-phenylpropanamine was obtained commercially. In the molecular design the focus was initially set on the modification of the central urea of PZM2118, which formed hydrogen bonds to the amino acid residues Y3267.43, Q1242.60 and D1473.32 in the µOR-PZM21 complex model.18 The urea was replaced by thiourea, N-cyanoguanidine, 1,1-diaminonitroethylene and malonodinitrile groups. The conformational properties of the new structures were derived from NMR spectra and compared to reference compounds in the literature and in the Cambridge Spectral Database (CSD)86. The oxygen atom of the urea group was replaced by a sulfur atom, which features a bigger atomic radius and leads to increased δOR and µOR affinities. Radioligand binding data showed that the thiourea- and the N-cyanoguanidine groups acted as bioisosteres when incorporated into this class of ligands. The introduction of a 1,1-diaminonitroethylene group led to reduced affinities at all receptor subtypes compared to the thiourea analogs, which was attributed to the conformational fixation via an intramolecular hydrogen bond between the nitro- and the amino substituent as observed in NMR spectra (chapter 4.3.1). The malonodinitrile derivative displayed increased binding affinities at all three receptor subtypes, compared to an N-cyanoguanidine derivative. This may point to additional binding energy resulting from interactions of the second nitrile substituent with the receptor or to favorable conformational properties. Replacement of the urea group by amides The urea of PZM21 was replaced by a carbamate, which gave insights into the structure-activity relationships (SAR) of the two hydrogen bond donors of the urea group. The SAR study led to the conclusion, that the hydrogen bond donating property of the thiophenethyl substituted nitrogen (colored red, Figure 47), which forms a hydrogen bond to Q1242.60, is less crucial for µOR binding affinity than the hydrogen bond donor of the tyrosine-derived building block (colored blue, Figure 47). Following up on this rationale, one hydrogen bond donor of the urea was abstracted by the introduction of an amide functionality with different alkyl-, alkenyl-, alkynyl- and aryl-substituents. Molecular docking studies were performed with new amide derivatives, initially a cinnamide (FH163) and an ethynylene amide (FH172), which showed promising results (Figure 50). The amide and the tertiary amine formed a bidentate interaction with D1473.32 and the aromatic appendage addressed the lipophilic binding pocket (see chapter 3.1). The reduced hydrogen bond donor count of the amide group leads to increased logP values (see chapter 9.2) and thereby raises the potential for BBB permeability, which is important when targeting opioid receptors located in the CNS.48 The new amides were synthesized by applying common amide coupling conditions using BOP or PyBOP. Biological investigations conducted by Dr. Harald Hübner revealed high µOR affinity for FH163 (Ki = 13 nM). Therefore, the design was extended towards substituted cinnamide and naphtyl derivatives that could fill empty space in the lipophilic pocket (Figure 50, a)). The naphtyl- and 3-trifluormethyl substituted acryl amides FH210 and FH218 display desired µOR activity together with substantially stronger functional selectivity towards the µOR G protein pathway than PZM21 (chapter 10.1). FH210 is similarly active in the G protein pathway, but remarkably less potent in the β-arrestin-2 recruitment assay in the presence of GRK2. FH218 is a full agonist in the G protein pathway with low β-arrestin-2 recruitment potency compared to PZM21 (Figure 50). The brain permeability of FH210 and FH218 was investigated by the CRO Pharmacelsus GmbH (Saarbrücken, Germany). Although the brain concentration of FH210 was lower than for PZM21, in contrast, the concentration of FH210 increased over the time course of the experiment by about 30 %. Fortunately, the brain concentration of the novel acryl amide FH218 surpassed that of PZM21 up to 3-fold. The design of novel amide analogs was extended towards aryl amides. The 2-carboxamido indole derivative FH178 combined the linear rigidized structure of the cinnamide FH163 with the second hydrogen bond donor of the PZM21 urea group (red, Figure 51). The high µOR affinity of FH178 supports the hypothesis that a hydrogen bond between the indole nitrogen and the receptor is formed, putatively mimicking the interactions observed for the urea of PZM21. All new amide compounds are consistently selective towards the µOR over the κOR, which is in contrast to the subtype specificity of PZM21. Thus, the enhanced µOR selectivity promotes amide derivatives of PZM21 as a pharmacologically very interesting class of compounds. Encouraged by the biological properties of the alkenyl amide derivatives, saturated analogs were designed. The binding affinities of the saturated amides reveal interesting insights into their SARs regarding receptor subtype selectivity. While the binding pocket of the µOR tolerated both, ethyl- and propyl amide derivatives, the κOR active site is better addressed by the longer propyl amide (DD9118, 57, 66, 67) and the δOR by the shorter ethyl amide derivative FH183. Similarly, the short unsubstituted cinnamide FH163 features high selectivity towards the µOR over κOR, but low selectivity over the δOR, confirming the above observations. Conformational restriction of the ethyl linker Conformational restriction is a common concept in medicinal chemistry. The ethyl linker in PZM21 can rotate around three single bonds, which raises the possibility to access many conformations different from the bioactive conformation. The reduction of rotational freedom reduces the entropic penalty for adopting a specific conformation. Thus, it may lead to enhanced affinity and potency by stabilizing a biologically active conformation of the molecule.105 Therefore, enantiomerically pure tetrahydronaphtyl, cyclopropylphenyl and cyclobutylphenyl derivatives were synthesized. In the cyclopropyl structures the methyl-substituted ethyl linker of the thioureas (S,R)/(S,S)-FH120 (chapter 4.1) is formally incorporated into the cyclopropyl ring, leading to four possible stereoisomers, which were synthesized from the cis- and trans-configured cyclopropylamine building blocks followed by separation of the diastereomers via preparative chiral HPLC (Figure 52). FH152transP1 shows high µOR binding affinity and specificity, which validated that the molecule features more specific conformational properties driven by reduced flexibility of the ethyl linker. In essence, the introduction of the cyclopropyl motif into the lead structure PZM21 is an effective way to gain µOR specificity and G protein activity. Replacement of the phenol group by 2,6-dimethyl-L-tyrosine (DMT) derivatives The phenol group of PZM21 was shown to be crucial for strong µOR affinity and potency.18 The 2,6-dimethyl-para-phenol group was associated with even stronger µOR binding affinity for other classes of opioid ligands, taking the peptidic agonist [DMT]-DALDA as a prominent example.109, 113-117, 129 A high affinity DMT derivative of PZM21 was synthesized by a former group member Dr. Viachaslau Bernat (BD131LS57, 66) (Figure 53). Albeit, BD131LS57, 66 is a µOR antagonist in functional assays (unpublished data from Dr. Harald Hübner). Literature research did not reveal any full agonist opioid receptor ligand containing both the DMT scaffold and a tertiary amine forming the salt bridge to Asp1473.32. A structure-based hypothesis for the correlation of the intrinsic activity and the conformational properties of BD131LS57, 66 and PZM21 was drawn: low rotational freedom of the 2,6-dimethyl-4-hydroxy phenyl ring leads to low intrinsic activity. In order to evaluate this idea, N-monomethyl amines were developed as a way of restoring rotational freedom. The synthesis of DMT derivatives urged for the development of a modified synthetic route, which involved the protection of the primary amine by an N-trityl protecting group. Encouragingly, increased intrinsic efficacy of novel N-monomethyl amines compared to N-dimethylamines supported the hypothesis (Figure 53). The µOR binding affinity of (S,S)-FH239 is highly similar to PZM21, showing that the DMT scaffold compensates the loss of affinity resulting from the removal of a methyl group from the tertiary basic amine. Moreover, the meta-trifluormethyl substituted cinnamide scaffold, which was discovered earlier in this project, was incorporated in the DMT series and leads to increased µOR affinity and logP values. Together, these results form a valuable starting point for the design of low efficacy agonists in the future. Introduction of new aromatics replacing the para-phenol group The phenol group of PZM21 was shown to be crucial for strong µOR affinity and potency.18 To further elucidate the influence of the phenol group of PZM21 on its bioactivity, new aromatic moieties such as meta-phenols, meta-phenol ethers, 5- and 7-tetrahydroisoquinolines, indoles-, 5-hydroxyindoles, para-difluoromethyl phenyl- and para-carboxamido pheny derivatives were introduced. Among these, the para-carboxamido pheny derivatives should be highlighted. While phenol groups are subject to glucuronidation in phase II metabolism in vivo, which leads to fast clearance of the drug, glucuronidation of benzamides is not expected.58 In molecular docking, the 4-carboxamido substituent of FH310 addressed the hydrophilic pocket of the µOR, forming a direct interaction with H2976.52 and K2335.39 (Figure 54). This binding mode implicates the displacement of a stable water molecule observed in the µOR-PZM21 complex model. A stable water-mediated hydrogen bond network was also observed in the active state µOR-BU72 crystal structure complex (PDB: 5C1M)29. A new synthetic route was developed starting from the protected amino acid N-Fmoc-L-4-carboxamido-phenylalanine. The acid was first reduced to the alcohol and subsequently oxidized to the aldehyde to perform a reductive amination. The resulting primary amine was coupled to the second building block, the Fmoc-protecting group was cleaved off and the primary amine was converted to the final dimethylamines. Biological data showed strong µOR binding affinity and potency, which can be explained by entropic effects associated with the displacement of a stable water molecule. In comparison to the tertiary amino phenols PZM21, FH210 and FH218, the 4-carboxamido phenyl pendants FH310, FH320 and FH314 feature a higher selectivity towards the µOR over the δOR and the κOR subtype. This is remarkable, since the amino acids H6.52, K5.39 and Y3.33 in the hydrophilic pocket of all three classical opioid receptor subtypes are conserved. A possible reason for the differences in subtype selectivity may be the altered distance between the basic tertiary amine and the hydrogen bond donor of the benzamide compared to the phenol. The phase I metabolic stability of the benzamides FH310 and FH314 was substantially higher than for their phenolic congeners PZM21 and FH218 in a rat liver microsomal assay. Taken together, these pharmacological and pharmacokinetic results promote 4-carboxamido phenyl derivatives of PZM21 as a highly promising class of compounds. N-Alkyl phenyl substituents targeting the lower binding pocket A deep receptor binding pocket below the basic nitrogen of PZM21 was addressed by novel N-alkyl substituted derivatives. Studies on N-phenethyl morphine and N-phenethyl morphinans could show an increased µOR binding affinity and an increased µOR functional potency and efficacy, suggesting that addressing this pocket can improve bioactivity.60, 131 The N-phenethyl and N-phenylpropyl derivatives FH272 and FH278 (Figure 55) were docked to the µOR and showed favorable binding poses. The novel compounds were synthesized from L-tyrosine amide via an SN2 reaction with phenethyl bromide or 1-bromo-3-phenylpropane, respectively, followed by N-methylation. Interestingly, the length of the alkyl linker between the basic nitrogen atom and the aromatic phenyl ring is crucial for the ability of the compound to activate the receptor. The biological results suggest that ethyl linkers are best suited for the design of novel µOR agonists, while methyl- and propyl linkers lead to µOR antagonism (Figure 55). Consequently, it was shown that exactly the right linker length is necessary to address the lower binding pocket of the µOR in order to create the desired activity. Pharmacokinetic properties of novel PZM21 derivatives The pharmacokinetic properties of the novel PZM21 derivatives were assessed. A set of new PZM21 derivatives was investigated in an in vitro model system for phase I metabolism using rat liver microsomes. It turned out, that the para-carboxamido phenyl motif confers substantially enhanced metabolic stability exemplified by the para-phenol FH218 (18 % left after 60 min) and the para-carboxamido phenyl FH314 (61 % left after 60 min). Increased lipophilicity was shown to correlate with a higher propensity of CYP-mediated metabolism in the literature.136 This trend was also observed for the novel acryl amides like FH210 and FH218, which are associated with high logP values and moderate metabolic stability. Notably though, metabolic stability of FH218 was improved by blocking the para-position of the phenyl ring by a fluoro substituent leading to the more stable analog FH315. The logP values were determined as a measure of lipophilicity by three different methods: solid-liquid partitioning in gradient RP-HPLC experiments and isocratic RP-HPLC experiments as well as by computational calculations using an atom-based method developed by Crippen et al.61 The logP value of the majority of novel compounds was higher than for the lead structure PZM21. Most frequent structural elements among these derivatives were the acryl amide, aryl amide, thiourea and the DMT motifs. The 4-carboxamido phenyl and alkyl amide structures are associated with lower logP values. Blood-brain-barrier penetration of PZM21, FH210, FH218 and FH314 was tested by the CRO Pharmacelus GmbH (Saarbrücken, Germany). Encouragingly, the brain concentration of FH218 was found to be 2- to 3-fold higher than for the lead structure PZM21. FH314 displayed brain levels, which were about 2.6-fold (at 30 min) and 2 fold (at 120 min) lower than for PZM21. Albeit, FH314 was cleared more slowly from brain and plasma and the higher functional activity could compensate the lower brain levels. FH210 possesses the highest logP value among the four substances in this experiment (see chapter 10.2). Unexpectedly, the brain concentration of FH210 was lower than for PZM21, but, in contrast to PZM21, increased over the time course of the experiment. Lipinsky’s “rule of 5” describes desired molecular properties for good absorption and permeation of bioactive substances.160 Encouragingly, all of the new PZM21 derivatives fulfilled Lipinsky’s “rule of 5”, promising favorable pharmacokinetic properties. Functional selectivity of novel PZM21 Derivatives calculated by the operational model Functional selectivity, also termed biased agonism, is the phenomenon that GPCR agonists do not uniformly activate all cellular signaling pathways associated with a given receptor.41 The functional bias between G protein signaling and β-arrestin-2 recruitment at the µOR and the δOR was calculated using the operational model of agonism62, 63 derived from the operational model developed by Black and Leff43. The biological data used for the calculations was kindly provided by Dr. Harald Hübner. The novel PZM21 derivatives FH306, FH204, FH210, FH315, FH218, (S,S)-FH100, FH314 and FH320 showed substantial G protein bias at the µOR. In addition, the potent µOR G protein partial agonists (S,S)-FH137, FH163, FH183, FH172, FH200 and FH321 are strongly G protein biased, which can be attributed without the use of the operational model41, but due to their low or absent β-arrestin-2 recruitment even when co-expressing GRK2. At the δOR, the novel amide derivatives FH314, FH218, FH320, FH163 and FH178 as well as the reference substances mitragynine pseudoindoxyl and buprenorphine display substantial functional selectivity towards G protein activation over β-arrestin-2 recruitment. Among all µOR and/or δOR G protein biased compounds, the acryl amide structural motif was frequently represented. Of the ten acryl amides synthesized in the course of this project, seven were fully functionally characterized by Dr. Harald Hübner and five turned out to be substantially G protein biased µOR- or µOR/δOR agonists. Taken together, this thesis identified novel PZM21 derivatives with distinct pharmacology and pharmacokinetics, some of which are highlighted in Figure 56. The SARs derived from these compounds contribute to the understanding of how the structural features of PZM21 determine its unique biological activity and aid in the development of novel opioids with potentially diminished side effects in the future. Show more