**1. Introduction**

Neuroblastoma (NBL) is the most common extracranial solid tumor of childhood. Children with high-risk neuroblastoma currently have long-term survival rates under 50% despite intensive, multimodal treatment regimens that include chemotherapy, surgical tumor resection, autologous stem cell transplantation, radiation therapy, and maintenance immunotherapy combined with 13-*cis*-retinoic acid [1,2]. In addition, the aggressive chemoradiotherapy used for the treatment of these children is associated with severe side effects and multi-organ damage [3]. Therefore, new, less toxic therapeutic combinations directed at relevant targets are needed for these children to reduce relapse rates and improve survival.

**Citation:** Golan, H.; Mechoulam, R.; Smoum, R.; Cohen-Zada, E.; Pri-Chen, S.; Wiener, S.; Grinberg, I.; Bar-Lev, D.D.; Haj, C.G.; Fisher, T.; et al. Anti-Tumorigenic Effect of a Novel Derivative of 2-Hydroxyoleic Acid and the Endocannabinoid Anandamide on Neuroblastoma Cells. *Biomedicines* **2022**, *10*, 1552. https://doi.org/10.3390/ biomedicines10071552

Academic Editor: Wesley M. Raup-Konsavage

Received: 18 May 2022 Accepted: 22 June 2022 Published: 29 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

The cannabinoids are a group of more than 100 chemically related compounds found in the marijuana plant *Cannabis sativa*, that have been found to possess diverse pharmacological activities in cancer, including cytostatic, apoptotic, and antiangiogenic effects [4]. Tetrahydrocannabinol (THC), the main psychoactive constituent in *Cannabis sativa*, acts mainly through the activation of specific cannabinoid receptors CB1 and CB2 and thus mimics the binding of the animal endogenous cannabinoids (named endocannabinoids) [5–7]. Endocannabinoids are known to participate in many biological processes in the immune, respiratory, circulatory, and reproductive systems. On a cellular level, they have been shown to modulate cell proliferation, viability, and differentiation [8]. The cytotoxicity of endocannabinoids on tumoral cells has been frequently reported, hence, their potential use in the treatment of malignant diseases [9]. Several different mechanisms have been implicated in the anti-tumorigenic actions of endocannabinoids and include cytotoxic or cytostatic effects, apoptosis induction, and anti-metastatic effects, such as inhibition of neo-angiogenesis and tumor cell migration [10]. One of the best-known endocannabinoids with anti-tumorigenic effects is anandamide (AEA). AEA has been shown to inhibit cholangiocarcinoma growth [11], to exert cytotoxic and antiproliferative effects on colorectal carcinoma cells [12], and to cause apoptosis of osteosarcoma cells [13] and glioma cells [14].

AEA is the ethanolamide of the fatty acid arachidonic acid. The anti-cancer drug Minerval is a 2-hydroxy derivative of another fatty acid, oleic acid. Minerval (HU-600) is one of the most studied synthetic lipid compounds that was shown to be safe and effective in patients with glioma and other advanced solid tumors [15]. In contrast to most anticancer drugs, HU-600 targets the plasma membrane and mediates its anti-tumor effect by affecting the biophysical properties of membranes [16–18].

Based on the anti-cancer effects of HU-600 and AEA, we assumed that a molecule that is a derivative of both compounds may be a novel anti-cancer drug with a promising anticancer therapeutic profile. Hence, structural modification of HU-600 was done to obtain 2-hydroxy-oleic acid (2-OHOA) ethanolamide (HU-585), a novel compound, which is an HU-600 derivative with the AEA side chain.

To further our research on this subject [19], we explored and compared the antitumorigenic effects of HU-600 and its novel derivate HU-585 on the NBL cell line. The results obtained in our study indicate that of the two compounds tested, HU-585 was indeed more effective on the NBL cell line in comparison to HU-600. Furthermore, HU-585 demonstrated pro-apoptotic and senescence-inducing properties, and combined treatment with senolytic drugs further enhanced its anti-tumorigenic effect. Our findings add contemporary information, attractive strategy, and an effective and less toxic therapeutic option for the treatment of refractory NBL.

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

#### *2.1. Preparation of 2-Hydroxyoleoyl Ethanolamide (HU-585)*

The 2-hydroxy oleic acid (HU-600) was synthesized according to Lazarus et al. [20]. The ethanolamide derivative (HU-585) of HU-600 was prepared according to the following procedure: To a solution of 2-hydroxyoleic acid sodium salt (100 mg, 0.3125 mmol) and N, N-dimethylformamide (23.65 μL, 0.3125 mmol) in dry methylene chloride (4.7 mL) was added dropwise oxalyl chloride (2.0 M solution in methylene chloride, 0.312 mL, 0.62 mmol) under nitrogen atmosphere at 0–5 ◦C. The reaction mixture was stirred for 10 min, and then the solvent was evaporated under nitrogen flow. The crude material in methylene chloride (4.7 mL) was added to an ice-cold solution of ethanolamide (0.214 mL, 3.55 mmol) in methylene chloride (4.7 mL). The reaction mixture was stirred for 10 min, then it was washed with water (3 × 20 mL) and dried (MgSO4), and the solvent was evaporated under reduced pressure. The residue was chromatographed on silica gel. White solid, melting point: 63 ◦C, NMR (CDCl3, ppm): 5.37 [m, 2H]; 4.15 [dd, 1H]; 3.75 [m, 2H]; 3.52–3.45 [m, 2H]; 2.04 [m, 4H]; 1.85–1.66 [m, 2H]; 1.44–1.29 [m, 20H]; 0.92 [t, 3H]. LC-MS (+p) = 342.2.
