N‐(Anilinoethyl)amides: Design and Synthesis of Metabolically Stable,Selective Melatonin Receptor Ligands

The class of N‐(anilinoethyl)amides includes melatonin receptor ligands with varied subtype selectivity and intrinsic activity. One of these ligands, the MT₂‐selective partial agonist UCM765 (N‐{2‐[(3‐methoxyphenyl)phenylamino]ethyl}acetamide), had evidenced hypnotic effects in rodents at doses ≥40 mg kg^?1 (s.c.), in spite of its sub‐nanomolar affinity for human melatonin receptors. Supposing that its low in vivo potency could be due, at least in part, to metabolic liability in rat liver, UCM765 was incubated with rat liver S9 fraction and rat, mouse, or human microsomes, and the major metabolites were identified by LC–MS, synthesized, and in vitro tested for their affinity toward MT₁ and MT₂ receptors. The obtained information was exploited to design novel analogues of UCM765 that are more resistant to in vitro oxidative degradation, while maintaining a similar binding profile. The analogue UCM924 (N‐{2‐[(3‐bromophenyl)‐(4‐fluorophenyl)amino]ethyl}acetamide) displayed a binding profile similar to that of UCM765 on cloned human receptors (MT₂‐selective partial agonist) and a significantly longer half‐life in the presence of rat liver S9 fraction.     

MT1‐Selective Melatonin Receptor Ligands: Synthesis,Pharmacological Evaluation,and Molecular Dynamics Investigation of N‐{[(3‐O‐Substituted)anilino]alkyl}amides

The design of compounds selective for the MT₁ melatonin receptor is still a challenging task owing to the limited knowledge of the structural features conferring selectivity for the MT₁ subtype, and only few selective compounds have been reported so far. N‐(Anilinoalkyl)amides are a versatile class of melatonin receptor ligands that include nonselective MT₁/MT₂ agonists and MT₂‐selective antagonists. We synthesized a new series of N‐(anilinoalkyl)amides bearing 3‐arylalkyloxy or 3‐alkyloxy substituents at the aniline ring, looking for new potent and MT₁‐selective ligands. To evaluate the effect of substituent size and shape on binding affinity and intrinsic activity, both flexible and conformationally constrained derivatives were prepared. The phenylbutyloxy substituent gave the best result, providing the partial agonist 4 a , which was endowed with high MT₁ binding affinity (pK_i=8.93) and 78‐fold selectivity for the MT₁ receptor. To investigate the molecular basis for agonist recognition, and to explain the role of the 3‐arylalkyloxy substituent, we built a homology model of the MT₁ receptor based on the β₂ adrenergic receptor crystal structure in its activated state. A binding mode for MT₁ agonists is proposed, as well as a hypothesis regarding the receptor structural features responsible for MT₁ selectivity of compounds with lipophilic arylalkyloxy substituents.     

Synthesis and Pharmacological Evaluation of a series of the Agomelatine Analogues as Melatonin MT1/MT2 Agonist and 5‐HT2C Antagonist

Agomelatine is a naphthalenic analogue of melatonin that is in clinical use for the treatment of major depressive disorders. Interestingly, while agomelatine exhibits potent affinity for melatonin receptors, it binds with only moderate affinity to the serotonin 5‐HT_2C receptor. Optimization of agomelatine toward this target could further potentiate its clinical efficacy. To explore this hypothesis and to access derivatives in which a key point of agomelatine metabolism is blocked, a series of naphthalenic derivatives was designed and synthesized as novel analogues of agomelatine. Most of the prepared compounds exhibited good binding affinity at the melatonin MT₁ and MT₂ receptor subtypes. Two compounds, an acetamide and an acrylamide derivative, exhibited good binding affinities at both the human melatonin (MT) receptors and the serotonin 5‐HT_2C receptor subtype, with pK_i values of 7.96 and 7.95 against MT₁, 7.86 and 8.68 against MT2, and 6.64 and 6.44 against 5‐HT_2C, respectively.     

Synthesis and SAR Studies of Isoquinoline and Tetrahydroisoquinoline Derivatives as Melatonin Receptor Ligands

In our constant search for new successors of agomelatine, we report herein a new series of compounds resulting from bioisosteric modulation of the naphthalene ring. The isoquinoline and tetrahydroisoquinoline derivatives were synthesized and pharmacologically evaluated. This isosteric replacement of the naphthalene group of agomelatine has led to potent agonist and partial agonist compounds with nanomolar melatonergic binding affinities. Overall, the presence of a nitrogen atom was accompanied with a decrease in the binding affinity toward both MT₁ and MT₂ and the loss of 5HT_2C response, especially for tetrahydroisoquinoline in comparison with the parent compound. Interestingly, due to the presence of this nitrogen atom, a notable improvement in the pharmacokinetic properties was observed for all compounds.     

Synthesis and Characterization of New Bivalent Agents as Melatonin- and Histamine H3-Ligands

Melatonin is an endogenous molecule involved in many pathophysiological processes. In addition to the control of circadian rhythms, its antioxidant and neuroprotective properties have been widely described. Thus far, different bivalent compounds composed by a melatonin molecule linked to another neuroprotective agent were synthesized and tested for their ability to block neurodegenerative processes in vitro and in vivo. To identify a novel class of potential neuroprotective compounds, we prepared a series of bivalent ligands, in which a prototypic melatonergic ligand is connected to an imidazole-based H₃ receptor antagonist through a flexible linker. Four imidazolyl-alkyloxy-anilinoethylamide derivatives, characterized by linkers of different length, were synthesized and their binding affinity for human MT₁, MT₂ and H₃ receptor subtypes was evaluated. Among the tested compounds, 14c and 14d, bearing a pentyl and a hexyl linker, respectively, were able to bind to all receptor subtypes at micromolar concentrations and represent the first bivalent melatonergic/histaminergic ligands reported so far. These preliminary results, based on binding affinity evaluation, pave the way for the future development of new dual-acting compounds targeting both melatonin and histamine receptors, which could represent promising therapeutic agents for the treatment of neurodegenerative pathologies.     

Benzocyclobutane,Benzocycloheptane and Heptene Derivatives as Melatonin Agonists and Antagonists

Two series of analogues were designed, synthesised and evaluated as potential human melatonin type 1 and 2 receptor (hMT₁ and hMT₂) ligands. Their biological effects were assessed by a well‐established, specific model of melatonin action, the pigment response of Xenopus laevis melanophores. Compounds containing a benzocyclobutane scaffold and a methoxy group in the “melatonin” orientation were found to be potent agonists, with one of the analogues exhibiting activity comparable to melatonin. In contrast, analogues with a methoxy group in non‐melatonin positions or with multiple methoxy groups showed either weaker agonist activity or were antagonists. Benzocycloheptene derivatives with one methoxy group are found to be weak agonists, whereas those with two methoxy groups were found to be antagonists, as were all of the benzocycloheptane derivatives evaluated. The most active compounds were assessed in a human receptor radio ligand binding assay but showed little discrimination between MT₁ and MT₂. These results again show that the indole nitrogen of melatonin is not a necessary component for analogue activity and also illustrate that replacement of the indole ring with a 4‐membered carbocycle can provide highly active compounds when the methoxy group is in the melatonin position.     

Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor

A novel series of 30 symmetric bispyridinium and related N‐heteroaromatic bisquaternary salts with a propane‐1,3‐diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4‐tert‐butyl‐substituted bispyridinium salt MB327 was previously identified as a nAChR re‐sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N‐alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)‐based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pK_i=4.73±0.03). Notably, the newly prepared lipophilic 4‐tert‐butyl‐3‐phenyl‐substituted bispyridinium salt PTM0022 ( 3 h ) was found to have significantly higher binding affinity, with a pK_i value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re‐sensitizers.     

Synthesis and Pharmacological Evaluation of SNC80 Analogues with a Bridged Piperazine Ring

To discover novel δ‐opioid receptor ligands derived from SNC80 ( 1 ), a series of 6,8‐diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, and the affinity toward δ, μ, and κ receptors, as well as σ receptors, was investigated. After removal of the 4‐methoxybenzyl and 2,4‐dimethoxybenzyl protecting groups, the pharmacophoric N,N‐diethylcarbamoylbenzyl residue was attached to the 6,8‐diazabicyclo[3.2.2]nonane framework to yield the designed δ receptor ligands. In a first series of compounds the benzhydryl moiety of SNC80 was dissected, and one phenyl ring was attached to the bicyclic framework. In a second series of δ ligands the complete benzhydryl moiety was introduced into the bicyclic scaffold. The determined δ receptor affinities show that compounds based on an (R)‐glutamate‐derived bicyclic scaffold possess higher δ receptor affinity than their (S)‐glutamate‐derived counterparts. Furthermore, an intact benzhydryl moiety leads to δ receptor ligands that are more potent than compounds with two separated aromatic moieties. Compound 24 , with the same spatial arrangement of substituents around the benzhydryl stereocenter as SNC80, shows the highest δ receptor affinity of this series: K_i=24 nM . Whereas the highly potent δ ligands reveal good selectivity against μ and κ receptors, the σ₁ and/or σ₂ affinities of some compounds are almost in the same range as their δ receptor affinities, such as compound 25 (σ₂: K_i=83 nM ; δ: K_i=75 nM ). In [³⁵S]GTPγS assays the most potent δ ligands 24 and 25 showed almost the same intrinsic activity as the full agonist SNC80, proving the agonistic activity of 24 and 25 . The enantiomeric 4‐benzylidene derivatives 15 and ent‐ 15 showed selective cytotoxicity toward the 5637 (bladder) and A‐427 (small‐cell lung) human tumor cell lines.     

Application of the guanidine-acylguanidine bioisosteric approach to argininamide-type NPY Y₂ receptor antagonists

Strongly basic groups such as guanidine moieties are crucial structural elements, but they compromise the drug‐likeness of numerous biologically active compounds, including ligands of G‐protein‐coupled receptors (GPCRs). As part of a project focused on the search for guanidine bioisosteres, argininamide‐type neuropeptide Y (NPY) Y₂ receptor (Y₂R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, N^G‐acylated argininamides were obtained by guanidinylation with tailor‐made mono‐Boc‐protected N‐acyl‐S‐methylisothioureas. The compounds were investigated for Y₂R antagonism (calcium assays), Y₂R affinity, and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the N^G‐substituted (S)‐argininamides showed Y₂R antagonistic activities and binding affinities similar to those of the parent compound, whereas N^G‐acylated or ‐carbamoylated analogues with a terminal amine were superior (Y₂R: K_i and K_B values in the low nanomolar range). This demonstrates that the basicity of the compounds, although 4–5 orders of magnitude lower than that of guanidines, is sufficient to form key interactions with acidic amino acids of the Y₂R. The acylguanidines bind with high affinity and selectivity to Y₂R over the Y₁, Y₄, and Y₅ receptors. As derivatization of the amino group is tolerated, these compounds can be considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y₂R. The results support the concept of bioisosteric guanidine–acylguanidine exchange as a broadly applicable approach to retain pharmacological activity despite decreased basicity.     

Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

Herein we report the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective agonists of the dopamine‐3 (D₃) receptor. A number of these new compounds bind to the D₃ receptor with sub‐nanomolar affinity and show excellent selectivity (>10 000) for the D₃ receptor over the D₁ and D₂ receptors. For example, compound 23 (N‐(cis‐3‐(2‐(((S)‐2‐amino‐4,5,6,7‐tetrahydrobenzo[d]thiazol‐6‐yl)(propyl)amino)ethyl)‐3‐hydroxycyclobutyl)‐3‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)benzamide) binds to the D₃ receptor with a K_i value of 0.53 nM and shows a selectivity of >20 000 over the D₂ and D₁ receptors in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes. Moreover, in vitro functional assays showed it to be a full agonist for the human D₃ receptor.     

A Molecular and Chemical Perspective in Defining Melatonin Receptor Subtype Selectivity

Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT₁ and MT₂ transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT₁ and MT₂ receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT₁ and MT₂ that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT₁ and MT₂ receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands.     

Structure–Affinity Relationships of Fluorinated Spirocyclic σ2 Receptor Ligands with an Exocyclic Benzylamino Moiety

To identify a potent and selective σ₂ receptor ligand appropriate for development as a positron emission tomography (PET) tracer, several fluorinated analogues of the spirocyclic lead compounds trans- and cis- 6 (N-(2,4-dimethylbenzyl)-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1′-cyclohexan]-4′-amine) were designed. In multistep syntheses, a fluorine atom was introduced directly or as a 2-fluoroethoxy moiety on the 2-benzopyran scaffold, on the dimethylbenzylamino moiety, or on the central amino moiety. The σ₁ and σ₂ receptor affinity was determined in receptor binding studies with radioligands. With respect to σ₂ affinity and σ₂/σ₁ selectivity, cis-N-(2,4-dimethylbenzyl)-5-fluoro-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1′-cyclohexan]-4′-amine (cis- 15 c , K_i(σ₂)=51 nm ) and cis-N-[4-(fluoromethyl)-2-methylbenzyl]-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1′-cyclohexan]-4′-amine (cis- 28 e , K_i(σ₂)=57 nm ) are the most promising ligands. The combination of both structural elements in one molecule, cis-N-[4-(fluoromethyl)-2-methylbenzyl]-5-fluoro-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1′-cyclohexan]-4′-amine (cis- 28 c : K_i(σ₂)=874 nm ), resulted in decreased σ₂ and σ₁ affinity. Methylation of secondary amines led to three tertiary methylamines with moderate affinity for both σ receptor subtypes.     

Light-Switchable Antagonists for the Histamine H1 Receptor at the Isolated Guinea Pig Ileum

The histamine H₁ G protein-coupled receptor (GPCR) plays an important role in allergy and inflammation. Existing drugs that address the H₁ receptor differ in their chemical structure, pharmacology, and side effects. Light-controllable spatial and temporal activity regulation of photochromic H₁ ligands may contribute to a better mechanistic understanding and the development of improved correlations between ligand structure and pharmacologic effects. We report photochromic H₁ receptor ligands, which were investigated in an organ-pharmacological assay. Initially, five photochromic azobenzene derivatives of reported dual H₁–H₄ receptor antagonists were designed, synthesized, photochemically characterized, and organ-pharmacologically tested on the isolated guinea pig ileum. Among them, one compound [trans- 19 : (Z)-1-(4-chlorophenyl)-1-(4-methylpiperazin-1-yl)-N-(4-((E)-phenyldiazenyl)phenyl)methanimine] retained the antagonistic activity of its non-photochromic lead, and trans–cis isomerization by irradiation induced a fourfold difference in the pharmacological response. Further structural optimization resulted in two bathochromically shifted derivatives of 19 [NO₂-substituted 35 {(Z)-1-(4-chlorophenyl)-1-(4-methylpiperazin-1-yl)-N-(4-((E)-(4-nitrophenyl)diazenyl)phenyl)methanimine} and SO₃⁻-substituted 41 {4-((E)-(4-(((Z)-(4-chlorophenyl)(4-methylpiperazin-1-yl)methylene)amino)phenyl)diazenyl)benzenesulfonate}], which do not require the use of UV light for photoisomerization and which also have improved solubility and show reduced tissue impairment. The trans isomers of both compounds showed a remarkable increase in antagonistic activity relative to their lead trans- 19 ; furthermore, a 46-fold difference in activity on the isolated guinea pig ileum was observed between trans- and cis- 35 .     

Substituted Aminoacetamides as Novel Leads for Malaria Treatment

Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4-methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28 ) with low-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter-screen up to 25 μm . Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.     

Development of a Metabolically Stable Neurotensin Receptor 2 (NTS2) Ligand

Subtype‐selective neurotensin receptor 2 (NTS2) ligands can be used as molecular probes to investigate the physiological role of neurotensinergic systems and serve as lead compounds to initiate the development of drugs for the treatment of tonic pain. Starting from our recently described NTS2 ligand 1 , structural variants of type 2 were synthesized to further improve binding affinity and selectivity to gain metabolic stability. The peptide–peptoid hybrid 2 b showed excellent NTS2 binding affinity (K_i=2.8 nM ) and 22 000‐fold selectivity over NTS1, as well as metabolic stability over 32 h in a serum degradation assay. Employing a MAPK‐driven luciferase reporter gene assay and an IP accumulation assay, the neurotensin mimetic 2 b displayed respective inhibitions of constitutive activity exceeding 4.3‐ and 3.9‐fold that of the inverse agonist activity of the endogenous ligand neurotensin.     

Solubilization and release of fragrance agents in the aqueous solution of CO2 switchable surfactants

This study describes the enhancement in the solubility and controlled-release of fragrance agents (menthol, four n-alkanols, and three aromatic esters) using three CO₂ switchable surfactants (N-alkylimidazolium bicarbonates). The surfactants significantly improved the solubility of fragrance agents in water. N-Dodecylimidazolium bicarbonate was the most effective surfactant to solubilize menthol. A stability test indicated that the surfactant could stably disperse menthol in water. Moreover, the surfactants improved the solubility of n-alkanols to different levels, however, the solubility of the aromatic esters equally. The release of menthol from the surfactant solution under N₂ at different durations of bubbling time, temperatures, and flow velocities were studied.     

Insight into the Interactions between Novel Coumarin Derivatives and Human A3 Adenosine Receptors

A study focused on the discovery of new chemical entities based on the 3‐arylcoumarin scaffold was performed with the aim of finding new adenosine receptor (AR) ligands. Thirteen synthesized compounds were evaluated by radioligand binding (A₁, A_2A, and A₃) and adenylyl cyclase activity (A_2B) assays in order to study their affinity for the four human AR (hAR) subtypes. Seven of the studied compounds proved to be selective A₃AR ligands, with 3‐(4′‐methylphenyl)‐8‐(2‐oxopropoxy)coumarin ( 12 ) being the most potent (K_i=634 nM ). None of the compounds showed affinity for the A_2B receptor, while four compounds were found to be nonselective AR ligands for the other three subtypes. Docking simulations were carried out to identify the hypothetical binding mode and to rationalize the interaction of these types of coumarin derivatives with the binding site of the three ARs to which binding was observed. The results allowed us to conclude that the 3‐arylcoumarin scaffold composes a novel and promising class of A₃AR ligands. ADME properties were also calculated, with the results suggesting that these compounds are promising leads for the identification of new drug candidates.     

Synthesis and investigations on the oxidative degradation of C3/C5-alkyl-1,2,4-triarylpyrroles as ligands for the estrogen receptor

In this study, we synthesized 1,2,4‐triarylpyrroles as ligands for the estrogen receptor (ER). Two pyrrole series were prepared with either C3‐alkyl or C3/C5‐dialkyl residues. Compounds from both series were susceptible to oxidative degradation—dialkylated compounds (t_1/2=33–66 h) to a higher extent than their monoalkylated congeners (t_1/2=140–211 h). Nevertheless, stability was sufficient for determination of in vitro ER binding affinity. The most active agonist in hormone‐dependent, ERα‐positive MCF‐7/2a and U2‐OS/α cells was 1,2,4‐tris(4‐hydroxyphenyl)‐3‐propyl‐1H‐pyrrole ( 6 d ) (MCF‐7/2a: EC₅₀=70 nM ; U2‐OS/α: EC₅₀=1.6 nM ). A corresponding inactivity in U2‐OS/β cells demonstrated the high ERα selectivity. This trend was confirmed in a competition experiment using estradiol (E2) and purified hERα and hERβ proteins (relative binding affinity (RBA) calculated for 6 d : RBA(ERα)=1.85 %; RBA(ERβ) <0.01 %). Generally, C3/C5‐dialkyl substitution led to reduction of activity, possibly due to lower stability.     

Structure‐Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics

Structure‐based virtual screening using a D₂ receptor homology model was performed to identify dopamine D₂ receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D₂ ligands were identified (47.6 % success rate, among them D₂ receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5‐HT_2A receptors. The affinity (K_i values) of the compounds ranged from 58 nm to about 24 μm . Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D₂ receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D₂ pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20‐fold binding selectivity for the D₂ receptor over the D₃ receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction.     

Derivatives of Di-O-octanoylglycerol and mono-O-octylglycerol as modulators of protein kinase C and diacylglycerol kinase activities

Twelve analogs of 1,2-di-O-octanoylglycerol modified at C-3 and three quaternaryN-alkyl-ammonium derivatives of glycerol were synthesized. The compounds were testedin vitro as potential modulators of the calcium activated, phospholipid dependent protein kinase C (PKC) and diacylglycerol (DAG) kinase activities in order to understand the molecular interactions of these enzymes with their natural activators, inhibitors, or substrates. PKC activity was assayed by measuring histone H₁ phosphorylation, and the compounds synthesized were tested either in the presence (inhibitors) or in the absence (activators) of 1,2-di-O-octanoyglycerol analogs with the phosphatidylserine/Ca²⁺ mixture. DAG kinase activity was measured by the incorporation of phosphate into 1,2-di-O-oleoyl-sn-glycerol in the presence of the various analogs synthesized. In regard to PKC activity, the assays revealed that 1,2-di-O-octanoylglycerol analogs are inactive when modified at C-3 with groups which do not permit hydrogen bonding. Under our conditions, di-O-octanoylthioglycerol, which has been reported as inactive, was able to activate PKC in the presence of phosphatidylserine. It has been shown to give a synergistic activation with diacylglycerol and had no affinity for the phorbol ester receptor binding site, suggesting thatO-octanoylthioglycerol interacts with the enzyme at a different site from the phorbol ester receptor binding site. PKC and DAG kinase activities are inhibited byN-alkyl-ammonium compounds (IC₅₀ 24 μM) only when either two 8-carbon alkyl or acyl chains are present at the 1- and 2-positions of the glycerol backbone. The fact that these compounds have a strong effect on the binding of [³H]phorbol 12,13-dibutyrate to protein kinase C, and also inhibit DAG kinase, may suggest binding to the DAG site of the regulatory domain of PKC.