**1. Introduction**

In the United States, 150,000 new cases of colorectal cancer will be diagnosed in 2022 with an estimated 55,000 deaths caused by the disease [1]. Cisplatin, a commonly used chemotherapeutic agent, is used to treat colorectal cancer, albeit with negative side effects, including chemotherapeutic-induced peripheral neuropathy (CIPN), for which there is a lack of treatment options [2–4]. The prevalence of CIPN is agent-dependent, with reported rates varying from 19% to more than 85% [5] and is the highest in the case of platinumbased drugs (70–100%), including cisplatin [6]. This outcome results in an impaired quality of life for affected patients. Therefore, there is a need to identify alternative treatments for colorectal cancer, as well as identify anti-nociceptive agents capable of treating CIPN in patients undergoing cisplatin-based chemotherapy for colorectal cancer.

A number of cannabinoids have been found to reduce cancer cell growth with Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD)—the two most often studied cannabinoids for anti-cancer activity [7–12]. In addition to anti-cancer effects, evidence suggests that cannabinoids (e.g., cannabigerol (CBG), THC, and CBD) have anti-nociceptive properties, resulting in ongoing clinical investigations related to cannabinoid effects on pain [13–18]. The mechanisms by which cannabinoids reduce cancer cell growth as well as mitigate pain are currently under investigation. However, it is known that cannabinoids

**Citation:** Raup-Konsavage, W.M.; Sepulveda, D.E.; Morris, D.P.; Amin, S.; Vrana, K.E.; Graziane, N.M.; Desai, D. Efficient Synthesis for Altering Side Chain Length on Cannabinoid Molecules and Their Effects in Chemotherapy and Chemotherapeutic Induced Neuropathic Pain. *Biomolecules* **2022**, *12*, 1869. https://doi.org/10.3390/ biom12121869

Academic Editors: Marialuigia Fantacuzzi, Barbara De Filippis and Alessandra Ammazzalorso

Received: 3 October 2022 Accepted: 9 December 2022 Published: 13 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are promiscuous molecules with the ability to bind to a number of receptors involved in pain processing and inflammation (e.g., CB1, CB2, 5-HT1A, α2-adrenergic receptor) as well as induce immunogenic cell death [15,18–20].

Cannabis produces a number of bioactive compounds that are of pharmaceutical interest. Broadly, these molecules fall into one of three classes: terpenes, flavonoids, and cannabinoids (which are unique to the genus *Cannabis*). Over 100 different types of cannabinoids are produced by cannabis, with CBD and THC being the two most abundant and most well studied. The predominant cannabinoids in *Cannabis* cultivars have a 5-carbon side chain off of the aromatic ring; however, shortly after the discovery of the major 5-carbon phytocannabinoids, CBD and THC, two additional variations of these molecules were described with a shorter, 3-carbon, side chain, cannabidivarin (CBDV) and tetrahydrocannabivarin (THCV) [21]. More recently, CBD and THC variants with carbon side chains of 4, 6, and 7 carbons have been isolated from cannabis, albeit in much lower concentrations [22–24].

The biosynthesis of all cannabinoids begins with geranyl pyrophosphate and a benzoic acid, olivetolic acid in the case of the 5-carbon cannabinoids, by the enzyme geranylpyrophosphate-olivetolic acid geranyltransferase (GOT) [25]. This reaction produces CBG, and the respective variations, which serve as the precursor molecules to all other cannabinoids produced in the plant. However, in most cultivars, CBG is found at low levels because this precursor is efficiently converted to downstream products, which means that the variant molecules will therefore be present in trace amounts. Novel and trace side chain length variants of CBD and THC have previously been isolated from plant material [22]. Here, we set out to develop a novel synthetic mechanism to produce side-chain variants of CBG. Additionally, we tested CBG and CBG variants for potential anti-cancer activity in colorectal cancer cell lines, as well as anti-nociceptive properties in a model of chemotherapy-induced neuropathic pain (CIPN).

#### **2. Materials and Methods**

Thin-layer chromatography (TLC) was performed on aluminum-supported, precoated silica gel plates (EM Industries, Gibbstown, NJ, USA). Flash column chromatography was performed using silica gel SI 60. 1H NMR spectra were recorded on a 500 MHz Bruker mass spectrometer (Billerica, MA, USA). Proton chemical shifts are reported in parts per million (δ). The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, dd = double doublet, t = triplet, dt = doublet of triplet, m = multiplet. Mass spectrometry analysis was performed on a 4000 Q-trap hybrid triple quadrupole/linear ion trap instrument (Applied Biosystems/MDS Sciex, Waltham, MA, USA) at the proteomic facility of the Penn State College of Medicine, Hershey, PA. High Resolution Mass Spectrometry (HRMS) was performed on AB Sciex TripleTOF 5600 mass spectrometer (Farmington, MA, USA) with electrospray ionization (ESI) in positive-ion mode at the metabolomics core at Penn State University, University Park, PA. The sample was analyzed by flow infusion with a Prominence UFLC system (Shimadzu, Kyoto, Japan) at flow 300 μL/min rates of 0.1% formic acid in a mixture of methanol and water 60:40. MS1 and MS2 data were acquired using a declustering potential (DP) of 80 V. MS2 data were collected in IDA mode with collision energy (CE) of 40 V and collision energy spread (CES) 20 V. During the analysis, an ion spray voltage (IS) of 5500 V, curtain gas (CUR) of 35 psi, nebulizer gas (GS1) of 50 psi, heater gas 2 (GS2) of 55, and heater temperature of 550 C were applied. CBG was purchased from a commercial source (Cayman Chemical, Ann Arbor, MI, USA). 3,5-dimethoxybenzyltriphenylphosphonium bromide (**1**) was prepared as reported in the literature [26]. Briefly, 3,5-dimethoxybenzylbromide (10 g, 43.2 mmol) was heated under reflux with triphenyl phosphine (12.6 g, 47.6 mmol) in toluene (60 mL) for 6 h to give a quantitative yield of compound **1**. All starting materials were obtained from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Synthesis of 5-alkyl substituted-1,3-dihydroxybenzene precursors was conducted as described below (and illustrated in Scheme 1).

**Scheme 1.** Synthesis of 5-alkyl substitute-1,3-dihydroxybenezene.
