Synthesis and Characterization of the Diastereomers of HHC and H4CBD

The characterization of any compound is important in the field of chemistry. As the field of cannabinoid chemistry grows so does the need for the characterization of new cannabinoids or rare cannabinoids that gain popularity within the consumer and research fields. Hexahydrocannabinol (HHC) a hydrogenated analogue of Δ9-tetrahydrocannabinol (THC), also found in trace amounts naturally within the Cannabis sativa plant, has been gaining attention and popularity within the cannabis industry. Hexahydrocannabidiol (H4CBD) is a synthetic hydrogenated analogue to cannabidiol (CBD). Identifying the diastereomers of the cannabinoids with instrumentation plays a huge role within the chemistry field adding valuable information of the structure and the parameters for others to identify such cannabinoids. Elucidation and characterization of HHC and H4CBD were performed using current analytical techniques such as 1D and 2D nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS), effectively characterizing both the diastereomers of HHC and H4CBD.


Introduction
Currently, there are many studies that are concentrated on the potential of cannabinoids to treat various inflammatory and neurodegenerative diseases and cancer. 1,2 Cannabinol receptors have been identified in pancreatic cancer with several studies showing in vitro antiproliferative and proapoptotic effects. The main active substances found in cannabis plants are cannabidiol (CBD) and tetrahydrocannabinol (THC). [3][4][5][6] Even though, they have shown synergistic treatment effects in studies with the combination of CBD/synthetic cannabinoid receptor ligands and chemotherapy in xenograft and genetically modified spontaneous pancreatic cancer models, 7 no clinical studies to date showing treatment benefits of CBD or THC in patients with pancreatic cancer because of their weaker efficacy against the pancreatic cancer cells. [7][8][9][10] Recently, we have shown that the hydrogenated cannabinoids based on hexahydrocannabinol (HHC) and hexahydrocannabidiol (H4CBD) structures demonstrated a better potency than marketed poly(ADP-ribose) polymerase (PARP) inhibitors (Olaparib and Veliparib) and the parental molecules CBD and THC in human pancreatic ductal adenocarcinoma (PDAC) cell lines especially PANC1 and MiaPaCa2 thus, we believe that these new molecules, could serve as promising agents for better treatment outcome of patients diagnosed with pancreatic cancer. 11,12 We found that a mixture of the diastereomers of H4CBD have IC 50 values of 6.1 and 2.15 μM in MiaPaCa2 and PANC1 cell lines when compared with the IC 50 values of 9.11 μM and 25.35 μM for Olaparib and veliparib in MiaPaCa2 cell line respectively. HHC is a naturally occurring cannabinoid found in trace amounts within the Cannabis sativa plant. [13][14][15] It is structurally similar to the Δ9-THC compound, except HHC is lacking the double bond within the cyclohexyl ring, making it a hydrogenated analogue to Δ9-THC. Since the HHC is found in trace amounts, it is synthesized in multi-gram scale for research purposes. H4CBD is a synthetic hydrogenated analogue of CBD. 16,17 The cannabinoid has several studies providing evidence on the safety of the cannabinoid. 18,19 As the field of cannabinoid chemistry grows, the need for characterization of cannabinoids and constituents of the extracts are pertinent to the fields of medicinal chemistry. As the field of cannabinoid chemistry grows so does the need for the characterization of new cannabinoids and/or rare cannabinoids that gain popularity among consumers and researchers. Providing information on the parameters used as well as the structure allows for others to identify such cannabinoids within extract mixtures, or for purification measures, including identification of product during purification methods and for pharmaceutical research.

Results and Discussions
HHC and H4CBD have been the subject to a limited number of studies involving the pharmacology of these rare cannabinoids. 20,21 Herein, we describe the synthesis and characterization of HHC and H4CBD, synthetic cannabinoids that differ structurally from THC and CBD by the saturation of the carbon-carbon double bonds on cyclohexene ring. Despite the similarity of H4CBD and HHC to CBD and THC, they are not present in cannabis extracts and therefore not a controlled substance. Also, there is no reported synthetic route for the conversion of H4CBD to THC, like CBD. While there is evidence to show the sedative effects of CBD due to in vivo conversion to psychoactive THC in the acidic gastric environment, unlike CBD, H4CBD lacks the exocyclic double bond and due to that, it cannot undergo cyclization reaction.

Materials and Methods
CBD was used as a starting material for the synthesis of HHC and H4CBD. CBD was purchased in bulk from GVB Biopharma and converted to delta-8 THC following the literature procedures. 16,17 Purification of the completed reaction crude afforded the desired product. HHC and H4CBD were dissolved in acetonitrile-d3 and 1H, 13C, COSY, HSQC, HMBC, and NOESY data were acquired on a 500 MHz Bruker AVANCE II system at 25°C. 1H, 13C, COSY, HSQC, and HMBC data sets were analyzed to yield complete 1 H and 13 C peak assignments. Once the peaks were assigned, NOESY data were analyzed to yield stereochemistry information about the orientation of the hexanyl methyl group. All GC-MS/MS analysis was performed using a Shimadzu Nexis GC-2030 coupled with a GC-MS-TQ8050NX detector. The analytical column is a Restek Rxi-35Sil MS (30 m × 0.25 mm × 0.25 µm) (35%silphenylene). Injection volume is 0.5 µL on a 33:1 split and injection port temperature of 200°C. Carrier gas is ultra-high purity helium at 1.2 ml/min (Constant Linear Velocity mode). Oven program is as follows: 240°C with a 9.0 min hold, first ramp 10°C/min to 280°C, no hold, second ramp 25°C/min to 300°C, hold 3.0 min (run time: 17.30 min). Transfer line temperature is 280°C. Solvent delay is 2.75 min. MS acquisition was performed in both Q3 full scan mode (mass range: 40-400 amu) and MRM mode (Supplemental material).

Experimental Section
General procedure: The synthesis of (R) and (S)-HHC and H4CBD was followed by general procedure. A 20 L flask equipped with a reflux condenser and an addition funnel was purged with argon for 10 min at 1 bar. Pd/C (0.1 molar %) was added to the reaction slowly using a powder funnel under argon. The flask was then purged with argon for 10 min at 1 bar. Ethanol (1L) was added slowly to avoid sparking the solvent. THC (100 g) was dissolved in ethanol (300 mL). The solution was added to the flask under argon and purged for 10 min at 1 bar. Afterward, the atmosphere of argon was stopped, and an atmosphere of hydrogen (1 bar) was introduced. The reaction was then stirred at 25°C for 3 h or until complete by high performance liquid chromatography (HPLC) with a diode array detector. Upon completion, the reaction was purged with argon for 10 min at 1 bar. The reaction mixture was poured over 1-3-micron filter paper on a Buchner funnel and then concentrated in vacuo. The crude oil was then dissolved in hexane and purified over silica (0%-5% ethyl acetate). The fractions of interest were concentrated in vacuo and then distilled to afford a mixture of R and S diastereomers of HHC as a yellow oil. 1  Chiral separation of (R) and (S) HHC: The diastereomers of HHC were separated by supercritical fluid chromatography (SFC). SFC is used in the industry primarily for the separation of chiral molecules, and uses the same columns as standard HPLC systems and CO2 as the mobile phase. HHC (5 g) was submitted for diastereomeric resolution. The analytical and preparative SFC methods described below were used for the purification.
Analytical SFC method: Column 4.6 × 100 mm Chiralpak AD-H from Chiral Technologies (West Chester, PA); CO2 Co-solvent (Solvent B) Isopropanol; Isocratic Method 25% Co-solvent at 4 mL/min System Pressure 125 bar; Column Temperature 40°C; and Sample Diluent Isopropanol Preparative SFC method: Column 2.1 × 25.0 cm Chiralpak AD-H was procured from Chiral Technologies (West Chester, PA); CO2 Co-solvent (Solvent B) isopropanol; isocratic method 30% Co-solvent at 80 g/min; system pressure 100 bar; column temperature 25°C; and sample diluent isopropanol  The chromatogram of the crude is shown in Figure 1. The HHC was purified using the preparative method described above. The collected fractions were dried in a rotary evaporator at 40°C, and transferred to the final container using isopropanol. The isolates were analyzed and recovery details for each isolate is shown in Table 1. The percentage of each isomer was calculated by SFC (UV220 nM) and was found to be 4.6%:71.8%:23.5%.
Following the same procedure, the mixture of diastereomers of H4CBD was synthesized from CBD.
Chiral separation of (R) and (S) H4CBD: Following the same analytical and preparative SFC methods of HHC, the diastereomers of H4CBD were separated by SFC.
The chromatogram of the crude H4CBD is shown in Figure 2. The pure fractions were dried in a rotary evaporator at 40°C, and transferred to the final container using isopropanol. The isolates were analyzed and recovery details for each isolate are shown in Table 2.

Results
For the identification of the diastereomers of HHC and H4CBD, approximately 20 mg of each diastereomer was sent for NMR analysis. Samples were dissolved in acetonitrile-d3 and 1 H, 13 C, COSY, HSQC, HMBC, and NOESY data were acquired on a 500 MHz Bruker AVANCE II system at 25°C. In order to avoid a replacement of hydroxyl protons with deuterium, and an easy recovery of the product from CD3CN from NMR sample, deuterated acetonitrile was preferred over other NMR solvents. 1 H, 13 C, COSY, HSQC, and HMBC data sets were analyzed to yield complete 1 H and 13 C peak assignment. Once the peaks were assigned, NOESY data were analyzed to yield stereochemistry information about the orientation of the hexanyl methyl group.
The data are provided in Supplemental material, and each carbon atom of the molecules is numbered and peak assignment results from 1H and 13C data of each isomer are listed in Supplementary Tables 1 to 4. In the NOESY data of HHC-Peak3-9S-1 (Fraction 1), proton 10a shows NOE to protons at 12, 13, 7, or 8 (one of the CH2 protons), and 9 and 10 (one of the 2), but it doesn't have NOE to methyl protons located at 11. This indicates that they are pointing in the opposite directions. Therefore the structure proposed for Fraction 1 is the (S)-HHC. In the NOESY data for HHC-peak 2-9R (Fraction 2), proton 10a has NOE to   protons at 12, 13, and 10, and it does have NOE to methyl protons at 11. Therefore, the structure proposed for this fraction is (R)-HHC. The designations of 9R and 9S are further confirmed, since proton 10a is pointing away, proton 11 in Fraction 2 (R) is also pointing away, while in Fraction 1 (S) the methyl 11 is pointing up. The pertinent NMR spectra and all other in-depth spectra are in the supporting information. The conformation analysis was configured through the various forms of NMR techniques that allowed for specific conformation analysis to be pieced together. The elucidation of the isomers R and S of HHC and H4CBD were presented through the analysis of GC-MS, NMR, and mass spectrometry, to identify the fragments and to identify the parameters of the spectra of the given compounds. The R and S isomers of HHC and H4CBD can be easily identified with the help of NOESY and COSY NMR spectra techniques, which might not be easily accessible to some labs, the utility of proton NMR the shifts of the given isomers change slightly and can be deduced from the given changes of which isomer the given compounds are. Through mass spectrometry, the identification of the isomers is not clearly shown since similar fragmentation patterns occur for diastereomers. HPLC using RP-C18 column with an isocratic mobile phase, the separation of the isomer is possible showing distinct R/S peaks of HHC and H4CBD but not better than SFC.
HPLC, liquid chromatography-mass spectrometry (LC-MS), and GC-MS data of the samples characterized by a pseudomolecular ion at m/z 317 based on positive ionization electrospray LC-MS analysis indicating an apparent molecular weight of 316, consistent with that of the HHC isomers. Analysis of sample by GC/MS indicated the presence of molecular mass of 316 and characterized by fragment ions at m/z 193, m/z 260, and m/ z 273. These fragment ions are consistent with reported fragment ions of HHC. In the case of H4CBD isomers, a pseudomolecular ion at m/z 318 based on positive ionization electrospray LC-MS analysis indicates an apparent molecular weight of 318, and is consistent with that of the H4CBD isomers. Analysis of sample by GC/MS indicated the presence of molecular mass of 318 and is characterized by fragment ions at m/z 193, m/z 233, and m/z 262.

Conclusion
The isolation and elucidation of the R and S isomers of H4CBD and HHC can be deduced definitively through proton and carbon NMR and various 2D NMR techniques, when the 2D NMR techniques are lacking, the use of 13 C and 1 H NMR techniques can help provide insight into the deduction of the isomers. The other instrumental techniques used help provide an in-depth understanding of how the isomers look under various conditions. Although the IC 50 values are lower compared to other active antineoplastic compounds on the market the treatment of Pancreatic cancer is still evolving and the need to produce antineoplastics is pertinent. Continued SAR studies of the synthetic analogs of HHC and H4CBD are currently being conducted to increase bioavailability as well as potency in xenograft models.