Harmaline analog; semisynthetic alkaloid

In this study, researchers report the design, synthesis, and evaluation of a series of harmaline analogs as selective inhibitors of 2-arachidonylglycerol (2-AG) oxygenation over arachidonic acid (AA) oxygenation by purified cyclooxygenase-2 (COX-2). A fused tricyclic harmaline analog containing a CH3O substituent at C-6 and a CH3 group at the C-1 position of 4,9-dihydro-3H-pyrido[3,4-b]indole (compound 3) was the best substrate-selective COX-2 inhibitor of those evaluated, exhibiting a 2AG-selective COX-2 inhibitory IC50 of 0.022 μM as compared to >1 μM for AA. The 2.66 Å resolution crystal complex of COX-2 with compound 3 revealed that this series of tricyclic indoles binds in the cyclooxygenase channel by flipping the side chain of L531 toward the dimer interface. This novel tricyclic indole series provides the foundation for the development of promising substrate-selective molecules capable of increasing endocannabinoid (EC) levels in the brain to offer new treatments for a variety of diseases, from pain and inflammation to stress and anxiety disorders.[1]

The inhibitory potencies of the synthesized compounds (e.g. Harmaline analog/compound 3) against purified human COX-2 or ovine COX-1 were determined by a thin layer chromatography (TLC)-based assay that measures the conversion of [1-14C]-AA to radiolabeled PGs. Briefly, reaction mixtures of 200 μL contained hematin-reconstituted protein in 100 mM Tris-HCl, pH 8.0, 500 μM phenol, and [1-14C]AA (5 μM, ∼55–57 mCi/mmol, PerkinElmer). Reactions were terminated by solvent extraction in diethyl ether/methanol/1 M citrate buffer, pH 4.0 (30:4:1). The phases were separated by centrifugation at 2000 rpm for 2 min, and the organic phase was spotted on a TLC plate (EMD Kieselgel 60, VWR). Following development in ethyl acetate/methylene chloride/glacial acetic acid (75:25:1) at 4 °C, radiolabeled products were quantified with a radioactivity scanner.[1]

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Compounds were next evaluated for substrate-selective COX-2 inhibitory activity using a mass spectrometry (MS)-based assay. The final assay reaction mixture contained 50 nM purified murine COX-2, 100 nM heme, 5 μM AA or 2-AG, 1 μM 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), and inhibitor or vehicle (DMSO at a final concentration of 5%) in a buffer of 50 mM Tris-HCl, pH 8.0, 0.5 mM phenol. Following addition of inhibitor, the mixture was incubated for 15 min, and then the reaction was initiated by the addition of AA or 2-AG. After 30 s, the reaction was quenched by addition of ethyl acetate or acetonitrile containing internal standards (PGE2-d4 and PGE2G-d5) at 0.3 μM. The samples were injected onto a C18 5 cm × 0.2 cm, 3 μm particle size column connected to a Shimadzu LC system coupled with an ABSCIEX MS. Elution solvents were solvent A (5 mM ammonium acetate, pH 3.6) and solvent B (94% acetonitrile with 6% solvent A) applied in a gradient from 30% to 100% B over 1.5 min followed by 100% B for 1 min. Analytes of interest were detected by selected reaction monitoring MS/MS using the following transitions: PGE2/D2m/z 370 → 317; PGE2-d4m/z 374 → 321; PGE2/D2-G m/z 444 → 391; PGE2-G-d5m/z 449 → 396. Analyte peak areas were normalized to those of their deuterated internal standards for the quantification of product formation and inhibition. Note that PPHP was included in this assay to eliminate possible effects of inhibitors on peroxide-mediated activation of COX-2, a mechanism that has been implicated in substrate-selective inhibition of 2-AG oxygenation by some inhibitors. The presence of PPHP tends to increase the IC50 for inhibition of both AA and 2-AG oxygenation; however, its effect is greater in the case of 2-AG because higher peroxide tone is required for activation in the case of that substrate.[1]

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Compounds 1–6 possessed a tricyclic indole core (4,9-dihydro-3H-pyrido[3,4-b]indole) containing a methoxy substitution at the C-6 or C-7 position. The p-chlorobenzyl substitution was introduced at C-9 position of compounds 3–4 and 6 in order to achieve improved in vitro and in vivo metabolic stability. In the purified COX inhibition assay, compounds 1 and 2 (harmaline) showed no COX inhibitory activity. However, 9-(4-chlorobenzyl)-6-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole (Harmaline analog/compound 3) was a selective COX-2 inhibitor (IC50 = 0.2 μM), whereas its regioisomer 9-(4-chlorobenzyl)-7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole (compound 4) exhibited COX-2 selectivity with a poor potency (IC50 = 2.1 μM). Although the carboxylic acid-containing compound 5 showed no COX inhibitory activity, 9-(4-chlorobenzyl)-6-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indole-1-carboxylic acid (compound 6, Table 1) exhibited mild inhibitory activity against COX-1 with an IC50 value of 2.9 μM.[1]

[1].Harmaline Analogs as Substrate-Selective Cyclooxygenase-2 Inhibitors. ACS Med Chem Lett. 2020 Feb 14;11(10):1881-1885.

In this study, researchers evaluated all the compounds for their ability to inhibit 2-AG oxygenation selectively over AA oxygenation by COX-2. The evaluation was performed using an MS-based assay, as described above with added inhibitor concentrations up to 1 μM. Under these conditions, no compound reached its IC50 for COX-2-dependent AA oxygenation. Only compounds 3 and 6 exhibited substrate-selective inhibitory activity against 2-AG oxygenation, with IC50 values of 0.022 μM and 0.8 μM, respectively (Table 2). In addition, we evaluated Harmaline analog/compound 3 in the presence of both AA and 2-AG (see Supporting Information for details), where it showed substrate-selective COX-2 inhibitory activity against 2-AG oxygenation with an IC50 value of 0.145 μM.[1]

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To explore the structural basis for substrate-selective inhibition of COX-2 by Harmaline analog/compound 3, we obtained a 2.66 Å resolution X-ray crystal structure of the inhibitor in complex with the protein (PDB code 6V3R). Statistics of X-ray data collection and structure refinement is described in the Supporting Information (Table 1s). As seen in Figure​Figure11, compound 3 binds in the cyclooxygenase active site of COX-2, resting above a “constriction” designated by Arg-120, Tyr-355, and Glu-524, as is the case for the vast majority of COX inhibitors. Notably, however, steric hindrance between the tricyclic indole core and Leu-531 induces a movement of the side chain of this amino acid relative to the position it occupies in most COX-inhibitor crystal structures. A similar movement of Leu-531 has been observed in other COX-2:ligand complexes, including COX-2:AA (PDB files 1CVU and 3HS5, unproductive binding mode), COX-2:eicosapentaenoic acid (PDB file 3HS6), COX-2:1-AG (PDB file 3MDL), COX-2:3(S)- methylarachidonic acid (PDB file 4RUT), and complexes with inhibitors of the oxicam class (PDB files 4M10, 4M11, and 4O1Z). In each case, displacement of the Leu-531 side chain helps to accommodate a bulky ligand or otherwise unfavorable binding pose. This finding suggests that movement of Leu-531 may play a role in substrate-selective inhibition, but such a displacement was not observed in the crystal structures of other such inhibitors [e.g., ibuprofen (PDB file 4RSO)40 and mefenamic acid (PDB file 5IKR). Furthermore, the oxicams induce a similar displacement of Leu-531, but they are not substrate-selective inhibitors. It is also notable that in the crystal structure, compound 3 is bound to both subunits of COX-2. Thus, the structure does not provide support for the hypothesis that substrate-selective inhibition results from binding of inhibitor to the allosteric site only. This may be the result of constraints present during crystallization, however, and may not reflect the behavior of the enzyme in the cellular environment.[1]

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In conclusion, we have described the design and synthesis of a novel series of harmaline analogs that are derived from the 4,9-dihydro-3H-pyrido[3,4-b]indole core. These compounds have been evaluated for their COX inhibitory activity with respect to both isoform- and substrate-selectivity. The SAR identified a fused tricyclic rigid indole derivative, Harmaline analog/compound 3, as a promising substrate-selective COX-2 inhibitor. The crystal complex of COX-2 with Harmaline analog/compound 3 revealed a movement of Leu-531 in the COX active site to accommodate inhibitor binding, suggesting that subtle structural changes in this region may contribute to its substrate-selectivity. This molecule will serve as a useful tool compound for further exploration of the biological relevance of COX-2-dependent endocannabinoid oxygenation.[1]

C20H19CLN2O

338.830663919449

338.11859

2411677-02-4

145927371

Typically exists as solid at room temperature

4.2

0

2

3

24

474

0

ClC1C=CC(=CC=1)CN1C2C=CC(=CC=2C2CCN=C(C)C1=2)OC

SRMUEFRMPPZYOH-UHFFFAOYSA-N

InChI=1S/C20H19ClN2O/c1-13-20-17(9-10-22-13)18-11-16(24-2)7-8-19(18)23(20)12-14-3-5-15(21)6-4-14/h3-8,11H,9-10,12H2,1-2H3

9-[(4-chlorophenyl)methyl]-6-methoxy-1-methyl-3,4-dihydropyrido[3,4-b]indole

2411677-02-4; Harmaline analog; CHEMBL4758939; compound 3 [Uddin et al., 2020]; 9-[(4-chlorophenyl)methyl]-6-methoxy-1-methyl-4,9-dihydro-3H-beta-carboline; 9-(4-Chlorobenzyl)-6-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole; 3H-Pyrido[3,4-b]indole,9-[(4-chlorophenyl)methyl]-4,9dihydro-6-methoxy-1-methy;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)