Synthesis , Characterization , and In Vitro and In Vivo Evaluations of 4-( N )-Docosahexaenoyl 2 ′ , 2 ′-Difluorodeoxycytidine with Potent and Broad-Spectrum Antitumor Activity 1 , 2

In this study, a new compound, 4-(N)-docosahexaenoyl 2′, 2′-difluorodeoxycytidine (DHA-dFdC), was synthesized and characterized. Its antitumor activity was evaluated in cell culture and in mouse models of pancreatic cancer. DHA-dFdC is a poorly soluble, pale yellow waxy solid, with a molecular mass of 573.3 Da and a melting point of about 96°C. The activation energy for the degradation of DHA-dFdC in an aqueous Tween 80–based solution is 12.86 kcal/mol, whereas its stability is significantly higher in the presence of vitamin E. NCI-60 DTP Human Tumor Cell Line Screening revealed that DHA-dFdC has potent and broad-spectrum antitumor activity, especially in leukemia, renal, and central nervous system cancer cell lines. In human and murine pancreatic cancer cell lines, the IC50 value of DHA-dFdC was up to 10 -fold lower than that of dFdC. The elimination of DHA-dFdC in mouse plasma appeared to follow a biexponential model, with a terminal phase t1/2 of about 58 minutes. DHA-dFdC significantly extended the survival of genetically engineered mice that spontaneously develop pancreatic ductal adenocarcinoma. In nude mice with subcutaneously implanted human Panc-1 pancreatic tumors, the antitumor activity of DHA-dFdC was significantly stronger than the molar equivalent of dFdC alone, DHA alone, or the physical mixture of them (1:1, molar ratio). DHA-dFdC also significantly inhibited the growth of Panc-1 tumors orthotopically implanted in the pancreas of nude mice, whereas the molar equivalent dose of dFdC alone did not show any significant activity. DHA-dFdC is a promising compound for the potential treatment of cancers in organs such as the pancreas. Neoplasia (2016) 18, 33–48 Address all correspondence to: Zhengrong Cui, Pharmaceutics Division, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712. E-mail: zhengrong.cui@austin.utexas.edu This work was supported in part by grants from the U.S. National Institutes of Health (CA179362 and CA135274) to Z. C. Y. W. N. was supported by a doctoral scholarship from the Egyptian Ministry of Higher Education. Conflict of interest: None. Received 11 August 2015; Revised 11 November 2015; Accepted 11 November 2015 © 2015 The Authors. Published by Elsevier Inc. on behalf of Neoplasia Press, Inc. This is an open access article under theCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1476-5586/15 http://dx.doi.org/10.1016/j.neo.2015.11.012 Introduction Docosahexaenoic acid (DHA, 22:6, n-3) is a polyunsaturated fatty acid (PUFA) that has been extensively investigated for its potential antitumor activity, either as a single agent or in combination with other cancer chemotherapeutic agents [1–5]. Omega-3 PUFAs are known to induce apoptosis in various cancer cells [6,7], inhibit cancer cell invasiveness [8], and inhibit metastasis and angiogenesis in tumor tissues [9–11]. The exact mechanism underlying the antitumor activity of omega-3 PUFAs remains unknown, but it is thought to be in part related to its potent antioxidant activity [6]. To better take 34 In Vitro and In Vivo Evaluations of DHA-dFdC Naguib et al. Neoplasia Vol. 18, No. 1, 2016 advantage of the antitumor activity of DHA, it was previously conjugated with some commonly used cancer chemotherapeutic agents, such as paclitaxel, doxorubicin, and camptothecin [12–14]. For example, Bradley et al. showed that conjugation of DHA to paclitaxel significantly modified the pharmacokinetics and biodistribution of paclitaxel, prompting the testing of the DHA-paclitaxel conjugate (i.e., Taxoprexin) in clinical trials [14–16], although the DHA-paclitaxel conjugate was not more cytotoxic than paclitaxel alone against many tumor cells in culture [14,17,18]. Gemcitabine HCl (2′, 2′-difluorodeoxycytidine HCl, dFdC) is a fluorinated deoxycytidine analogue. It is one of the standard treatments of locally advanced and metastatic pancreatic cancer [19,20] and is also used in combination therapy of other solid tumors including breast, bladder, lung, and ovarian cancers [19,21,22]. Extensive deamination at the 4-amino site, which takes place both intracellularly and extracellularly by the action of cytidine deaminase, is responsible for the loss of about 90% of dFdC after intravenous administration, and the deaminated metabolite difluorodeoxyuridine is almost inactive [19,23–26]. More than 99%of administered dFdC is excreted in the urine, with unchanged dFdC comprising only 5% [19,27]. Over the years, there have been reports showing that chemical modifications of this fluorinated deoxycytidine analogue may potentially improve its efficacy and/or safety profiles. For example, it was shown that conjugation of a fatty acid, such as stearic acid, to dFdC at the 4-NH2 group decreases the sensitivity of the latter to deaminase; modifies its pharmacokinetics; and, in some cases, improves its in vivo antitumor activity [24,28–41]. In the present study, we report the synthesis, characterization, and in vitro and in vivo evaluations of 4-(N)-docosahexaenoyl 2′, 2′-difluorodeoxycytidine (DHA-dFdC) conjugate. DHA-dFdC showed potent and broad spectrum antitumor activity in various human cancer cell lines in culture. DHA-dFdC also showed an unexpectedly longer residence time in mouse pancreas compared with dFdC. Because pancreatic cancer is one of the most aggressive, and in most cases fatal, types of cancer, with a mortality rate almost equal to incidence rate [42,43], the antitumor activity of the DHA-dFdC was primarily evaluated in mouse models of pancreatic cancer. Materials and Methods Materials Gemcitabine HCl (dFdC) was from Biotang, Inc. (Lexington, MA). Cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCI and trifluoroacetic acid (TFA) were from Acros Organics (Morris Plains, NJ). Di-tert-butyl-dicarbonate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), vitamin E, HPLC-grade methanol, sodium dodecyl sulfate (SDS), Triton X-100, and Tween 80 were from Sigma-Aldrich (St. Louis, MO). Hydroxy-7-azabenzotriazole was from CreoSalus, Inc. (Louisville, KY). Isopropylmyristate (IPM)was fromTCIAmerica (Montgomeryville, PA). Anhydrous sodium sulfate, ammonium chloride, monoand di-basic sodium phosphates, ethyl acetate, HPLC-grade acetonitrile, dichloromethane (DCM), acetone, hexane, and octanol were from Thermo Fisher (Waltham,MA). BDMatrigel BasementMembraneMatrix was fromBD Biosciences (San Jose, CA). D-Luciferin K salt bioluminescent substrate was from Perkin Elmer (Waltham, MA). Guava Nexin reagent for flow cytometry was from EMD Millipore (Billerica, MA). Lactate dehydrogenase (LDH) cytotoxicity detection kit was from Takara-Clontech Laboratories, Inc. (Mountain View, CA). Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI 1640) medium, fetal bovine serum (FBS), penicillin, streptomycin, horse serum, and Dulbecco’s phosphate buffer saline were all from Invitrogen-Life Technologies (Carlsbad, CA). All other chemicals, reagents, and solvents were of analytical grade and used as received without further purification. Cell Lines Panc-02 (mouse pancreatic cancer cell line), BxPC-3 (humanpancreatic cancer cell line), MIA PaCa-2 (human pancreatic cancer cell line), and TC-1 (mouse lung cancer cell line) were from the American Type Culture Collection (ATCC, Manassas, VA). Panc-1-luc human pancreatic cancer cell line was generously provided byDr. Dawn E. Quelle at the University of Iowa [44]. TC-1 andPanc-02 cells were grown inRPMI1640medium. BxPC-3, MIA PaCa-2, and Panc-1-luc cells were grown in DMEM. All media were supplemented with 10% FBS, 100 U/ml of penicillin, and 100 g/ml of streptomycin, and the DMEM for MIA PaCa-2 cells was supplemented additionally with 2.5% horse serum. Synthesis of DHA-dFdC DHA-dFdC was synthesized following a previously reported conjugation scheme with slight modifications [24,25,29] (Scheme 1). Briefly, dFdC (1) (200 mg, 0.67 mmol) in 13.3 ml of 1 N potassium hydroxide was cooled to 4°C. To this solution, di-tert-butyl dicarbonate (Boc2O, 1.483 g, 6.8 mmol) in 13.3 ml of anhydrous dioxane was added over 10 minutes under argon atmosphere as previously reported [45]. The reaction mixture was stirred at room temperature (~22°C) for 1 hour and extracted with ethyl acetate (EtOAc). The organic layer was washed with brine, dried over anhydrous sodium sulfate (Na2SO4), and filtered. Solvent was removed under reduced pressure. The residue was added to Boc2O (1.483 g, 6.8 mmol) in 13.3 ml of anhydrous dioxane and 13.3 ml of 1 M KOH at room temperature. The reaction was monitored by thin-layer chromatography. After 1 hour, the reaction mixture was extracted to EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, and filtered. Solvent was then removed, and the crude product was purified by column chromatography (DCM to acetone, 1:1, v/v). The desired product fractions were pooled and dried to yield 219 mg (71%) of 3′,5′-O-bis(tert-butoxycarbonyl) dFdC (2). H nuclear magnetic resonance (NMR) (500 MHz, acetone-d) δ 7.60 (1 H, d, J = 7.6 Hz, 6-CH), 6.34 (1 H, brs, 1′-CH), 5.97 (1 H, d, J = 7.6 Hz, 5-CH), 5.29 (1 H, brs, 3′-CH), 4.53-4.39 (3 H, m, 4′-CH, 5′A-CH, 5′B-CH), 2.82 (2 H, s, NH2) 1.50, 1.47 (18 H, two s, (CH3)3C). A solution that contains 3′,5′-O-bis(tert-butoxycarbonyl) dFdC (150 mg, 324 μmol), DHA (123 mg, 373.9 μmol), and hydroxy-7-azabenzotriazole (75 mg, 551.1 μmol) in anhydrous DCM was precooled to 4°C, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCI (93.75 mg, 604.1 μmol) was added. The mixture was degassed by vacuum sonication and then stirred at room temperature under argon for about 40 hours. Water (5 ml) was added to the reaction mixture and extracted three times with amixture of EtOAc and hexane (2:1, v/v). The combined organic phase was washed with saturated ammonium chloride (NH4Cl) and brine and then dried over anhydrousNa2SO4. The solvent was evaporated, and t