Opioids are the most effective drugs for the treatment of acute and chronic pain, and they occupy the largest market share of analgesic medications. Medical uses of opioids for pain relief include opioid alkaloids such as morphine and synthetic opioids like fentanyl. Although opioids are potent painkillers, they also result in severe adverse effects such as addiction, respiratory suppression, and constipation, thus limiting their clinical use.

The pharmacological actions of such opioids are mediated by agonism of the μ-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) that is highly expressed in the central nervous system (CNS). GPCRs are seven-transmembrane (7TM) receptors. The receptor protein is embedded within the membrane, with the binding site for the chemical messenger exposed on the outer surface. On the inner surface, there is another binding site which is normally closed. When a chemical messenger binds to its binding site, the receptor protein undergoes a conformational change. This change of shape opens up the binding site on the inner surface. This new binding site is then recognized by a specific G-protein. G-proteins are attached to the inner surface of the cell membrane and are made up of three protein subunits. Upon binding to the receptor, the G-protein changes shape, allowing the release of the GDP located at the alpha subunit. The empty binding pocket has now the right shape to bind GTP. Binding of GTP results in another conformational change, and the subunits dissociate to form a monomer and a dimer. These subunits depart the receptor and initiate a signaling cascade. There is substantial amplification of the signal in this process, because one activated receptor activates several G-proteins before the ligand departs, switching off the receptor.

There are several different G-proteins, which are recognized by different types of receptors. Some of the activated subunits from these G-proteins have an inhibitory effect, while others have a stimulatory effect. For example, the alpha s subunit of the Gs-protein binds to a membrane-bound enzyme called adenylate cyclase. Upon binding, the enzyme catalyzes the synthesis of cyclic AMP also known as cAMP. cAMP is a secondary messenger that moves into the cell’s cytoplasm and carries the signal from the cell membrane into the cell itself.

Signaling of the μ-opioid receptor is primarily transduced through Gi proteins to inhibit cAMP production. On the other hand, activation of the opioid receptor also leads to receptor phosphorylation by protein kinases such as GRK2. This triggers binding of beta-arrestin 2 to the receptor, which sterically inhibits further G protein activation and results in the activation of other downstream signaling pathways.

It has been proposed that the opioid-induced analgesia is attributed to Gi signaling, whereas the undesired side effects such as respiratory suppression and constipation might be caused by β-arrestin signaling. The proposed role of β-arrestins in the unwanted effects of opioid receptor agonists has led to the development of ligands that do not recruit β-arrestin 2 to the receptor, under the assumption that this would avoid on-target side effects mediated by the proposed β-arrestin–dependent mechanism. Oliceridine acts as a biased agonist at the μ-opioid receptor by preferentially activating the G-protein pathway with minimal receptor phosphorylation and recruitment of β-arrestin. [1]

It is proposed that oliceridine binding induces a different intracellular conformation of the μ-opioid receptor, due to a lack of coupling with transmembrane helix six and seven, which confers the specificity for G-protein over β-arrestin interaction. Therefore, ligands designed to reduce TM6/7 interactions display preferential G protein signaling. [2]

Some studies compared the efficacy of opioid agonists such as fentanyl, morphine, and oliceridine, using an analog of the endogenous opioid peptide, DAMGO, which was used as a reference, and arrived at the conclusion that oliceridine displayed lower intrinsic efficacy, acting as a partial agonist. No evidence of statistically significant bias was observed toward G protein signaling compared to β-arrestin 2 recruitment and concluded that low-efficacy agonists, such as oliceridine will produce strong responses in highly amplified G protein assays while having little activity in no-amplification, protein-protein interaction assays such as β-arrestin recruitment. [3]

Some recent studies also demonstrated that the μ-opioid agonists’ morphine and fentanyl induced respiratory depression and constipation in β-arrestin 2 knockout mice to the same extent as that observed in wild-type mice. These studies do not support the original suggestion that β-arrestin 2 signaling plays a key role in opioid-induced respiratory depression and call into question the concept of developing G protein-biased μ-opioid receptor agonists as a strategy for the development of safer opioid analgesic drugs. On the other hand, there are in vitro, in vivo, and clinical studies supporting the view that this biased agonism results in comparable analgesia compared with traditional opioids at a comparable or decreased risk of adverse effects. [4]

In any case, regardless of the mechanism of action, oliceridine is a potent compound that does exhibit to a lesser extent undesired side effects. Therefore, there is still room for improvement. And next-generation opioid drugs devoid of these undesired effects remain to be seen. [5]

Oliceridine is a T-shaped chiral compound that presents a pyridine and a thiophene ring and a spirocycle. The synthesis of the molecule was first described by Trevena, and it involves a reductive amination. A 1,4-addition of the pyridyl ring to this intermediate followed by decarboxylation and reduction of the nitrile to the primary amine. A Knoevenagel condensation reaction. And a Ley-Griffith oxidation reaction to the alcohol obtained from a Prins cyclization.

The forward synthesis begins with a Prins reaction between pentanone and butenol in 75% sulfuric acid. The Prins reaction is the acid-catalyzed condensation of alkenes with aldehydes or ketones. The reaction involves the electrophilic addition of a ketone to an alkene followed by the capture of water. The resulting alcohol was further oxidized to the corresponding ketone via Ley-Griffith oxidation. The Ley-Griffith reaction is the oxidation of primary or secondary alcohols to aldehydes or ketones. The central catalyst is tetra propylammonium perruthenate which is used in combination with the co-oxidant N-methylmorpholine N-oxide. Knoevenagel condensation between the cyclic ketone and methyl cyanoacetate provided a mixture of regioisomers, which were not separated. The aryl Grignard reagent was then added to the unsaturated cyanopropenoate in the presence of catalytic amount of copper iodide to give the conjugate addition product. Decarboxylation was achieved under strong basic conditions with heating to afford the nitrile, which was subsequently separated by SFC chiral separation. Reduction of the nitrile to its primary amine was accomplished with lithium aluminum hydride in ether. Reductive amination with the commercial aldehyde and fumarate salt formation provided oliceridine fumarate in 41% yield over two steps.

1. J. Med. Chem. 2013, 56, 8019.

2. Cell. 2022, 185, 4361. Open access.

3. Sci. Signal. 13, eaaz3140 (2020)

4. Br J Pharmacol. 2020, 177, 2923. Open access.

5. Mol Pharmacol. 2019, 96, 542. 

6. J. Med. Chem. 2022, 65, 9607.

Oliceridine-bound mu-opioid receptor-Gi complex: PDB ID: 8EFB.

Fibroblast growth factor receptors (FGFR) are a family of receptors consisting of an extracellular region composed of a ligand-binding domain, a transmembrane segment, and a cytoplasmic tyrosine kinase domain. FGF receptor activation is triggered by the binding of one of the fibroblast growth factors to the extracellular receptor domain. Upon binding, the tyrosine kinase domains get phosphorylated by adenosine triphosphate, ATP, initiating a downstream cascade of intracellular signals. On a molecular level, these signals mediate cell division, growth, and differentiation. Alterations of FGF receptors have been identified to be important for progression and development of several types of cancer, including cholangiocarcinoma. In this setting, inhibition of FGF receptor signaling is a promising therapeutic strategy for cancer treatment. One modality of FGF receptor inhibitor is the use of small molecule compounds that bind to the ATP-binding site of the receptor at the tyrosine kinase domain and successfully block cross-phosphorylation and subsequent cell signaling.

Binding at the ATP-binding site, it is no surprise that the molecule presents an ATP-mimicking pyrazolopyrimidine core structure in the middle, a pyrrolidine moiety overlapping with the sugar part of ATP, an acrylamide functional group at the edge, and a straight-chain alkyne to add the necessary chemical space for a dimethoxyphenyl ring to bind into a hydrophobic pocket of the receptor. The dimethoxyphenyl ring is stabilized by extensive van der Waals contacts and a single hydrogen bond (to Asp 641). The core pyrazolopyrimidine presents two hydrogen bonds (to Glu562 O and Ala564 N). And the acrylamide makes a covalent bond with the Cys488 S. (Futibatinib-FGFR 1 Covalent Complex: PDB ID: 6MZW.)

Futibatinib is a targeted covalent inhibitor (TCI). TCIs are typically generated by screening potential covalent binding compounds or by structure-based design from optimized reversible ligands, which are modified by attachment of an electrophilic covalent reactive group also known as warhead. Warheads are functional groups within a ligand that form covalent bonds with the targeted amino acid side chains in proteins. Futibatinib is a drug that was designed to undergo an irreversible hetero-Michael addition reaction with a unique cysteine residue of the specific protein.

TCI binding involves a two-step process in which an initial reversible binding event takes place, followed by the covalent bond-forming reaction. Irreversible inhibitors have the potential to be highly selective because they target a unique residue of a specific kinase by design, but warheads require a balanced reactivity profile. They should be reactive enough to form a covalent bond with the target residue, but only after the proper alignment of both reaction partners by the reversible binding event. Reactivity should be reduced to a necessary minimum in order to prevent nonspecific off-target labeling. In addition, warheads intended for in vivo use, either in drugs or in chemical probes, should also be chemically and metabolically stable and nontoxic.

Unfunctionalized acrylamides are weakly electrophilic and relatively unreactive toward thiols; this has led to acrylamides being the most successful electrophile used in targeted covalent inhibitors. On the other hand, the neutral cysteine thiol group is only moderately nucleophilic. However, this nucleophilicity is increased by several orders of magnitude for the thiolate form of the cysteine side chain, making it the strongest nucleophile among the 20 canonical amino acids. The thiol group can easily be deprotonated (pKa = 8–9 for cysteine), and the pKa can shift several orders of magnitude within proteins. Being highly polarizable, thiols and thiolates are soft bases. And the increased nucleophilicity and polarizability of thiolates compared to alkoxides are owed to the size and the high energy of the 3sp3 lone pairs.

The discovery of futibatinib started off with the screening of an in-house compound collection where this pyrrolopyrimidine analog was identified as a unique hit with inhibitory activity of FGF receptor 2 with a half maximal inhibitory concentration (IC50) of 57 nM. The enantiomers of this compound and those of the pyrazolopyrimidine were prepared to investigate the effect of the stereochemistry of the pyrrolidine moiety and the additional nitrogen atom. The S-pyrrolidine analogs showed better inhibition. Although the pyrrolopyrimidine showed 15-fold more potent inhibitory activity than the pyrazolopyrimidine, the latter was selected as an initial lead molecule for rapid structure−activity relationship (SAR) generation, due to the synthetic accessibility of the pyrazolopyrimidine core. Considering that All FDA-approved FGFR-selective inhibitors possess a 3,5-dimethoxybenzene ring in their compounds, the Kinase inhibitory activity of 3,5-dimethoxybenzene analogs was tested. The straight-chain alkyne could correctly place the 3,5-dimethoxybenzene ring in the well-known unique hydrophobic pocket of the FGF receptor. Other linkers and warheads were also tested during this drug discovery campaign, which gave valuable information, but led to less potent compounds. For example, the propionamide analog, without covalent binding ability, was prepared to determine the effect of the acrylamide warhead moiety. This compound reduced the inhibitory activity by 240-fold, indirectly confirming the covalent inhibitory mechanism of futibatinib.

A short synthesis of the molecule was disclosed by researchers at Taiho pharmaceuticals. The key steps involved an acylation reaction to install the warhead, a carbon-carbon cross-coupling reaction, and an alkylation reaction. The first step of the synthesis was the nucleophilic substitution of the activated alcohol, which was generated in situ by the reaction of the corresponding alcohol with mesyl chloride. The next step was a Sonogashira reaction to install the internal alkyne. The reaction was accomplished in 96% yield under these conditions. Finally, boc cleavage and acylation reaction with acryloyl chloride delivered futibatinib in 84% yield over two steps.

Futibatinib:
1. ACS Med. Chem. Lett. 2023, 14, 4, 396–404. 
2. ChemMedChem 2019, 14, 494. 

FGFR:
1. Nat Rev Cancer 10, 116–129 (2010). 

Covalent Inhibitors and Warheads:
1. J. Med. Chem. 2022, 65, 1, 58–83. 
2. Current Opinion in Chemical Biology 2017, 39:54–63. 
3. J. Med. Chem. 2019, 62, 12, 5673–5724. 

Futibatinib-FGFR 1 Covalent Complex: PDB ID: 6MZW.

Cholesterol is an essential structural component of cell membranes in the human body and serves as a precursor for the biosynthesis of numerous compounds such as steroid hormones, cell membranes, and vitamin D. Cholesterol can be obtained through diet or endogenously synthesized within hepatocytes. Disruption in cholesterol homeostasis due to hereditary factors or diet can lead to elevated plasma cholesterol levels that can produce fatty deposits in blood vessels, which, over time, can impede proper blood flow.

Bempedoic acid is a medicine that lowers low-density lipoprotein cholesterol (LDL-c; also known as ‘bad’ cholesterol). It does so by blocking adenosine triphosphate citrate lyase, which is an enzyme in the liver that is responsible for making cholesterol. As a result, bempedoic acid lowers blood levels of cholesterol, including LDL-cholesterol and other fatty substances made by the liver. Bempedoic acid is a prodrug. A prodrug is a pharmacologically inactive medication that, after intake, is metabolized into a pharmacologically active drug. It is activated to the thioester with coenzyme A in the liver. According to the mechanism of action, the active form of bempedoic acid reduces the production of cytosolic acetyl-coenzyme A (acetyl-CoA), the final substrate for both fatty acid and sterol synthesis. [2]

The chemical structure of the molecule resembles that of oleic acid. It has been classified in the literature as a fraudulent fatty acid, because it belongs to a family of molecules that activate regulatory pathways in the liver, resulting in enhanced fatty acid catabolism. [3] These molecules are characterized by elevated hydrophobicity and the frequent presence of carboxylic acid functional groups. In fact, bempedoic acid presents two carboxylic acids, two spacer groups or symmetrical alkyl chains, and a central group. 

A convenient route for the synthesis of this molecule would involve breaking the two C–C bonds adjacent to the hydroxyl group, because that would deliver two identical fragments. However, both the carbonyl group and the alkyl halide are electrophiles. Therefore, umpolung of one of the functional groups is required. Researchers at Esperion Therapeutics made use of a ketone surrogate that was employed as the nucleophilic counterpart. 

Kidneys produce a hormone called erythropoietin, which signals the body to make red blood cells. In a person with chronic kidney disease, the kidneys cannot produce enough erythropoietin, leading to reduced numbers of red blood cells.

Anemia is a condition in which you lack enough healthy red blood cells to carry adequate oxygen to your body’s tissues. The primary cause of renal anemia is the depression of erythropoietin production in the kidney associated with the kidney disorder. A promising strategy for the treatment of renal anemia involves the inhibition of prolyl hydroxylase, because inhibition of this enzyme may increase internal erythropoietin levels as a result of hypoxia inducible factor stabilization (HIF-PH), which is a key transcription factor that plays a central role in regulating erythropoietin production.

Enarodustat functions as a inhibitor of (HIF-PH).

Enarodustat is a non-chiral compound that contains a phenyl ring attached to a triazolopyridine through a two-carbon alkyl chain. The molecule contains a carboxylic acid, an amide bond, and a hydroxyl group.

A large-scale synthesis of the drug was reported by Japan Tobacco. The key steps for the synthesis of this molecule involve an amide coupling with glycine and a C–C bond formation. Formation of the triazole ring and regioselective halogenation was accomplished through an intermediate that could be easily prepared from commercial 2,4-dichloropyridine.

Lumateperone is a chiral compound that contains 4 fused rings in the molecule, 3 of which are heterocycles. The key steps for the synthesis of Lumateperone involve a simple alkylation reaction and a ring formation reaction giving rise to the core of the molecule and exposing an indole fragment that could be forged via a Fischer indole synthesis.