A₃ Adenosine Receptor Antagonists with Nucleoside Structures and Their Anticancer Activity

Abstract

The overexpression of the A₃ adenosine receptor (AR) in a number of cancer cell types makes it an attractive target for tumor diagnosis and therapy. Hence, in the search for new A₃AR ligands, a series of novel 2,N⁶-disubstituted adenosines (Ados) was synthesized and tested in radioligand binding and functional assays at ARs. Derivatives bearing a 2-phenethylamino group in the N⁶-position were found to exert higher A₃AR affinity and selectivity than the corresponding N⁶-(2,2-diphenylethyl) analogues. 2-Chloro-N⁶-phenylethylAdo (15) was found to be a potent full A₃AR agonist with a Ki of 0.024 nM and an EC₅₀ of 14 nM, in a cAMP accumulation assay. Unlike 15, the other ligands behaved as A₃AR antagonists, which concentration-dependently reduced cell growth and exerted cytostatic activity on the prostate cancer cell line PC3, showing comparable and even more pronounced effects with respect to the ones elicited by the reference full agonist Cl-IB-MECA. In particular, the N⁶-(2,2-diphenylethyl)-2-phenylethynylAdo (12: GI₅₀ = 14 µM, TGI = 29 µM, and LC₅₀ = 59 µM) showed the highest activity proving to be a potential antitumor agent. The cytostatic effect of both A₃AR agonist (Cl-IB-MECA) and antagonists (12 and other newly synthesized compounds) confirm previous observations according to which, in addition to the involvement of A₃ARs, other cellular mechanisms are responsible for the anticancer effects of these ligands.

1. Introduction

Ubiquitous nucleosides and nucleotides are involved in several biological functions and they play a crucial role in some mechanisms, such as cell growth, migration, differentiation, bacterial-induced inflammation and growth factor secretion [1,2,3,4]. The nucleoside adenosine (Ado, 1, Figure 1) performs its function by interacting with the four A₁, A_2A, A_2B and A₃ adenosine receptors (ARs) belonging to the superfamily of G protein-coupled receptors (GPCRs). The activation of A₁AR and A₃AR mainly produces a decrease of the intracellular cyclic adenosine monophosphate (cAMP) concentration, while stimulation of the A_2AAR and A_2BAR leads to an opposite effect.

In physiological conditions, this peculiar nucleoside is present in almost all cells and tissues in low concentrations (nM range), while in stress conditions such as hypoxia, which characterizes tumors, its concentration increases (µM range) [2]. A₃ARs overexpression in various types of cancer cell has been reported in numerous studies [5] and has been demonstrated in lung, breast, melanoma, prostate, pancreatic, and liver cancers, as well as malignant pleural mesothelioma (MPM), glioblastoma, and lymphoma [2,6,7].

However, the particular issue is focused on the role of the A₃AR in regulating cell proliferation and death, as this receptor acts differently depending on the type of tissue in which it is expressed [8]. On this basis, the A₃AR is an attractive target for cancer diagnosis or for its ability to counteract the tumor grow, although there is evidence that, in some cancer types, activation of the A₃AR promotes cell proliferation and survival, while in others it activates cytostatic and apoptotic pathways [9,10].

This dual behavior seems to be due to the different regulation/dysregulation of the Wnt pathway induced by A₃AR activation in different tumor types [10]. In this regard, the mechanisms that influence the cancer cells’ proliferation or death could also be due to the modulation of Ado levels, which can be modified by affecting the pathways of Ado generation, degradation, and elimination [11,12]. Furthermore, A₃AR ligands conjugated with metallacarborans, containing iron and cobalt, protect cells from cross-resistance showed by anticancer drugs such as cisplatin, carboplatin, and doxorubicin [13,14].

The known full A₃AR agonist, 2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA, also called CF-102 or Namodenoson, 2, Figure 1), has been extensively studied on tumor cell proliferation and this compound has been reported to inhibit cancer cell proliferation in in vitro and in vivo tumor models [15,16]. In a randomized, placebo-controlled phase II trial for hepatocellular carcinoma and moderate hepatic dysfunction, Cl-IB-MECA did not meet its primary end-point even though it showed a favorable safety profile and preliminary efficacy [17].

Recently, we reported the ability of Cl-IB-MECA analogues and Ado derivatives to inhibit cell growth of human prostate (PC3), colon (Caco-2) and hepatocyte (Hep G2) carcinomas, using Cl-IB-MECA as the reference compound [18]. The results showed a good anticancer effect in all three tumor cell lines by Cl-IB-MECA, while its analogue N⁶-methyl-2-phenylethynyladenosine-5′-N-methyluronamide (3, Figure 1) was almost inactive. On the contrary, a very good antitumor activity, comparable to that of Cl-IB-MECA itself, was exerted by the two potent and selective A₃AR ligands, N⁶-(2,2-diphenylethyl)-2-hexynylAdo (4) and N⁶-(2-phenylethyl)-2-(2-phenylethylamino)Ado (5), which are Ado derivatives, bearing steric hindering aromatic groups in N⁶- and 2-positions, the synthesis of which has not yet been published (Figure 1) [18].

It is worthwhile to note that compound 3 behaves as a full A₃AR agonist endowed with higher A₃AR affinity and selectivity compared to Cl-IB-MECA [18,19]. The lack of anticancer activity of compound 3 indicated that the active compounds exerted their cytotoxic activity not only through the interaction with A₃ARs and suggested the possible involvement of other cellular mechanisms. Indeed, further experiments led to hypothesize that the anticancer activity of compound 4 was due to its ability to induce apoptosis and to raise the level of reactive oxygen species (ROS) [18].

Starting from these observations, the purpose of this work was to identify new potential anticancer agents and to verify that the antitumor activity of these molecules is not closely associated with the A₃AR binding affinity, confirming the possible intervention of other mechanisms. Hence, taking into account the structure of the two Ado derivatives 4 and 5, which showed cytotoxic activity in the three tumor cell lines mentioned above, in the present work, the synthesis of a new series of 2,N⁶ di-substituted Ado derivatives bearing the same substituents of 4 and 5 at the N⁶-position and a chlorine atom, different alkynyl chains and a phenethylthio group at the 2-position, was undertaken (Figure 2).

The new compounds were tested in radioligand binding and functional assays, to assess their affinity (A₁, A_2A, and A₃ARs) and potency (A_2BAR) at human ARs. Furthermore, they were tested in a PC3 prostate cell line, to establish their antiproliferative and cytotoxic activities, and in a functional cAMP assay to evaluate their ability to activate the A₃AR. Since the lack of sugar modification can lead to A₃AR antagonists [19], this last experiment was aimed at verifying whether these compounds are able or not to activate the A₃AR and, therefore, if they behave like Cl-IB-MECA.

2. Results and Discussion

2.1. Chemistry

Here, we report the synthesis of the newly nucleosides 10–13, 15, and 16, together with that of the previously reported compounds 4 and 5, which was carried out using a divergent approach starting from commercial guanosine (Scheme 1 and Scheme 2).

Commercial guanosine was converted in the protected 2-amino-6-chloropurineriboside 6, in two steps, as already described (Scheme 1) [20]. By a modification of the Sandmeyer reaction, consisting in a diazotization with isoamyl nitrite and diiodomethane, the 2-iodo derivative 7 was prepared from 6, using dry THF as a solvent, without iodine as previously reported [21]. The 6-chloro-2-iodopurineriboside 7 was then reacted with 2-phenethylamine or 2,2-diphenylethylamine, at r. t., to selectively displace the 6-chlorine atom. Then, methanolic ammonia was added at r. t. for the complete removal of protecting groups at the sugar moiety. The corresponding 2-iodo-N⁶-substituted Ados 8 and 9 were obtained as white powders after chromatography, with very good yields (Scheme 1).

The reaction of 7 to give 2-phenethylamino-N⁶-phenethylAdo (5) was performed in two steps. First, 7 was reacted with phenethylamine at 120 °C in a sealed vial, using DMF and potassium carbonate as a solvent and catalyst, respectively. Since these conditions led to the substitution of both the halogens in the 2- and 6-positions and the partial deprotection of the sugar moiety, the mixture, in a second step, was treated with methanolic ammonia at r. t. to have the complete sugar deprotection and to obtain 8 (Scheme 2).

Reaction of 2-iodo-N⁶-phenylethylAdo (8) with phenylethylthiol, in dry DMF and in the presence of potassium carbonate at 120 °C, in a steel vial, furnished the 2-thioderivative 13, with good yield after chromatography (Scheme 1).

The 2-alkynylderivatives 10–12 and 4 were prepared by reacting the intermediates 8 or 9 under Sonogashira cross-coupling conditions, using the suitable 1-alkyne and bis(triphenylphosphine)palladium dichloride, copper iodide, and triethylamine as catalysts, in DMF dry as solvent (Scheme 1).

The 2-chloroadenosine derivatives 15 and 16 were synthesized starting from 6, which was reacted with isoamyl nitrite and antimony chloride in DCM, at 0 °C, to get the 2,6-dichloropurineriboside 14. This compound was, in turn, treated with 2-phenethylamine and 2,2-diphenylethylamine, using ethanol as solvent and potassium carbonate as catalyst, to obtain the desired nucleosides 15 and 16 (Scheme 2).

2.2. Binding Assay at A₁, A_2A, and A₃ ARs and Functional Studies at A_2BARs

The new compounds 10–13 and 15–16, together with the 2-iodo nucleoside intermediates 8 and 9, were tested in radioligand binding assay at human recombinant ARs, expressed in Chinese hamster ovary (CHO) cells, to evaluate their affinity for A₁, A_2A, and A₃ AR subtypes. [³H]CCPA (2-chloro-N⁶-cyclopentylAdo), [³H]NECA (5′-N-ethylcarboxamidoAdo), and [³H]HEMADO (2-hexynyl-N⁶-methylAdo) were used as respective radioligands [22,23]. The results are reported as Ki values in nM (± standard errors) (

Table 1. Affinity (Ki, nM) of compounds 4, 5, 8–13, 15, and 16 in radioligand binding assays at human A₁, A_2A, and A₃ ARs subtypes.

Cmp	R2	hA1R a (Ki nM)	hA2AR b (Ki nM)	hA3R c (Ki nM)	A1/A3	A2A/A3
Cl-IB-MECA [24]		1240	5360	1.4	886	3829
5 [18]	NHCH2CH2Ph	357	1368	0.33	1082	4145
8	I	144 ± 32	4119 ± 998	5 ± 1.2	29	824
15	Cl	1.64 ± 0.37	660 ± 129	0.024 ± 0.005	68	27,500
13	SCH2CH2Ph	263 ± 19	3359 ± 752	30 ± 7.1	9	112
10	C≡C(CH2)3CH3	129 ± 8.2	146 ± 48	1.5 ± 0.3	86	97
11	C≡CPh	809 ± 117	2983 ± 271	3.8 ± 0.6	213	785
9	I	182 ± 25.4	1243 ± 253.1	11 ± 1.8	17	113
16	Cl	0.76 ± 0.2	266 ± 35	0.13 ± 0.01	6	2046
4 [18]	C≡C(CH2)3CH3	984	153	27	36	6
12	C≡CPh	211 ± 53	450 ± 84	106 ± 25	2	4

^a Displacement of specific [³H]CCPA binding in membranes from CHO cells, stably transfected with human recombinant A₁AR. ^b Displacement of specific [³H]NECA binding in membranes from CHO cells, stably transfected with human recombinant A_2AAR. ^c Displacement of specific [³H]HEMADO binding in membranes from CHO cells, stably transfected with human recombinant A₃AR. Data (n = 3–5) are expressed as means ± standard errors.

). In the case of A_2BAR, the potency of selected compounds was determined through a functional GloSensor cAMP assay [22]. Since their EC₅₀ values resulted > 30 µM, these data are not shown in Table 1.

Cl-IB-MECA and the already known A₃AR ligands 4 and 5 are reported as reference compounds. Cl-IB-MECA is an A₃AR full agonist endowed with high affinity (Ki = 1.4 nM) and a very good A₃AR selectivity of 886 and 3829 fold vs. the A₁ and A_2A ARs, respectively. The newly synthesized compounds behave, in general, as A₃AR ligands with high affinity (Ki in the nM and sub-nM range) and various degrees of selectivity. In particular, the already reported 2-phenethylamino-N⁶-phenethylAdo (5) possesses a very high A₃AR affinity with a KiA₃AR = 0.33 nM and a selectivity of thousands of times versus both the other AR subtypes. This compound was the A₃AR ligand endowed with the most balanced affinity and selectivity values for the A₃AR, better also than the reference Cl-IB-MECA.

The replacement of the 2-phenethylamino group of 5 with halogens led to an increase of affinity compared to the A₁AR subtype, but different results were found on the other subtypes. In fact, the 2-iodo derivative 8 showed a reduced affinity for both A_2A and A₃ ARs, compared to 5. On the contrary, for the 2-chloro derivative 15 there was an increase in affinity for these subtypes. It is worthwhile to note that the 2-chloro-N⁶-phenethylAdo (15) possesses an A₃AR Ki in the pM range and a very high selectivity vs. the A_2A subtype (27,500 fold), although it is less selective for the A₁AR with respect to the parent compound 5 and Cl-IB-MECA.

Substitution of the phenethylamino group at the 2-position of 5 with different alkynyl chains, as in compounds 10 and 11, or with the isosteric phenethylthio group, as in compound 13, has resulted in derivatives that maintain high A₃AR affinity, although with lower A₃AR selectivity. The presence of a bulky 2,2-diphenylethyl group in the N⁶-position, as in compounds 9, 16, 4, and 12, caused a general decrease in A₃AR affinity and selectivity (see 9 vs. 8, 16 vs. 15, 4 vs. 10, and 12 vs. 11). It should be noted that, also in this series of compounds, the 2-chloro derivative showed the highest A₃AR affinity (16; KiA₃AR = 0.13 nM).

2.3. Antiproliferative and Cytotoxic Assays

To assess the antitumor activity of all the synthesized compounds 4, 5, 8–13, 15, 16, their antiproliferative and cytotoxic effects were evaluated in the PC3 cell line by the sulforhodamine B (SRB) assay, according to the National Cancer Institute protocol [25]. The reference A₃AR agonist Cl-IB-MECA was tested with the same protocol for comparison, in order. The compounds were used at concentrations of 1, 10, 25, 50, and 100 μM for 48 h at 37 °C. The antitumor activity was estimated by measurements of three parameters: Growth Inhibition 50 (GI₅₀), the compound concentration (µM) required to inhibit 50% net of cell growth; Total Growth Inhibition (TGI), the compound concentration (µM) required to inhibit 100% of cell growth; Lethal Concentration 50 (LC₅₀), the compound concentration (µM) required to kill 50% of the initial cell number. The results are shown in

Table 2. Cytostatic and cytotoxic activity of compounds 4, 5, 8–13, 15, and 16 determined with the sulforhodamine B assay. The evaluated parameters GI₅₀, TGI, and LC₅₀ are reported in comparison of A₃AR affinity of tested compounds.

Cpd	R2	GI50 (μM)	TGI (μM)	LC50 (μM)	A3AR (nM)
Cl-IB-MECA		18 ± 0.4	44 ± 3.3	110 ± 8.4	1.4
5	NHCH2CH2Ph	237 ± 9.4	>500	>500	0.33
8	I	42 ± 2.7	113 ± 6.7	301 ± 13.4	5.0
15	Cl	51 ± 5.2	262 ± 8.3	>500	0.024
13	SCH2CH2Ph	41 ± 4.2	>500	>500	30
10	C≡C(CH2)3CH3	13 ± 1.6	77 ± 2.9	452 ± 10.5	1.5
11	C≡CPh	2.5 ± 0.8	19 ± 6.1	151 ± 8.9	3.8
9	I	24 ± 1.2	48 ± 3.5	94 ± 3.5	11
16	Cl	35 ± 3.1	94 ± 4.1	253 ± 11.5	0.13
4	C≡C(CH2)3CH3	16 ± 0.5	35 ± 1.5	80 ± 9.8	27
12	C≡CPh	14 ± 0.9	29 ± 4.9	59 ± 10.1	106

LC₅₀, GI₅₀ and TGI values are shown as mean ± standard deviation (SD) of three different experiments calculated using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

and Figure S1, along with A₃AR affinity.

The compounds demonstrated to concentration-dependently reduce cell growth of the PC3 prostate cancer cell line. Most of them showed a significant inhibitory effect on cell proliferation and a pronounced cytotoxic activity comparable to the one elicited by Cl-IB-MECA, after 48 h exposure. Compounds 5, 8, 13, and 15, bearing a phenethyl chain at the N⁶-position, exhibited a lower ability to inhibit cell proliferation and cell survival than Cl-IB-MECA, which showed a GI₅₀ = 18 µM, TGI = 44 µM, and LC₅₀ = 110 µM. On the contrary, compounds 10 and 11, bearing the same N⁶-phenethyl group and an alkynyl chain at the 2-position, exhibited a higher cytostatic effect than Cl-IB-MECA (10: GI₅₀ = 13 µM and 11: GI₅₀ = 2.5 µM and TGI = 19 µM).

It should be noted that the N⁶-phenethyl-2-phenylethynylAdo (11) is endowed with the best cytostatic activity, among all the tested derivatives. Furthermore, two of these compounds, 13 and 15, at a concentration of 500 µM, were not able to kill all the cells, in fact their LC₅₀ was higher than 500 µM. It should be noted that, also in the series of the N⁶-(2,2-diphenylethyl) derivatives 4, 9, 12, and 16, the compounds bearing an alkynyl chain at the 2-position showed a pronounced cytostatic activity, being more active than Cl-IB-MECA. These two compounds were found to be more potent than the reference also for their cytotoxic effects (4 and 12: LC₅₀ of 80 and 59 µM, respectively, compared to Cl-IB-MECA: LC₅₀ = 110 µM). The N⁶-(2,2-diphenylethyl)-2-phenylethynylAdo (12) resulted as the most active compound with a GI₅₀ = 14 µM, TGI = 29 µM, and LC₅₀ = 59 µM. The observation that the A₃AR ligands 5 (Ki = 0.33 nM), 15 (Ki = 0.024 nM), and 16 (Ki = 0.13 nM), were not those with the highest cytostatic and cytotoxic effects, despite their remarkable A₃AR affinity, seems to demonstrate that the antitumor activity of the tested 2,N⁶-disubstitued Ados is not strictly correlated to their affinity for the A₃AR subtype.

2.4. Functional Activity at Human A₃AR

Among the four AR subtypes, A₃AR appears to be the most sensitive to small chemical changes in its ligands. Indeed, various Ado derivatives, which were previously claimed as full or partial A₃AR agonists, behaved subsequently as A₃AR antagonists [24]. In several papers we have reported that the MECA and NECA derivatives substituted in 2-position with alkynyl chains and bearing a methyl or a methoxy group at the N⁶-position behave as full A₃AR agonists like Cl-IB-MECA [19,26]. Conversely, the corresponding Ado derivatives lose efficacy, proving to act as partial agonists or antagonists on this receptor subtype [19,26]. Since the here presented newly synthesized A₃AR ligands possess an intact ribose moiety in their structure, a functional assay was performed to measure their ability to activate the A₃AR subtype. Then, the intrinsic activity of the selected compounds 4, 10–12, and 15, chosen on the basis of their very high A₃AR affinity or antitumor activity, was evaluated. In particular, the ligands were analyzed in a functional experiment to assess their ability to inhibit forskolin-stimulated cAMP production through the human A₃AR, compared to the full agonist Cl-IBMECA [22].

Results showed that the reference compound Cl-IB-MECA and the 2-chloro-N⁶-phenethylAdo (15) were able to completely counteract the stimulation of adenylyl cyclase induced by 10 µM forskolin, so behaving as A₃AR full agonists. On the contrary, compounds 4 and 10–12 did not affect cAMP levels induced by forskolin, when tested alone, but were able, to a different extent, to lower the effect of the full agonist, demonstrating behavior as A₃AR antagonists. For all the compounds, the EC₅₀ or IC₅₀ values were calculated and the results are reported in

Table 3. Activity of 4, 10–12, and 15, compared to Cl-IBMECA, in a cAMP accumulation assay at the human A₃AR stably transfected in CHO cells.

Cpd	A3AR CHO Cells
	EC50, nM	IC50, nM
Cl-IB-MECA	2.8 ± 1.4
15	14 ± 3.4
10		31 ± 6
11		79 ± 15
4		380 ± 80
12		153 ± 12

The EC₅₀ or IC₅₀ values are calculated from concentration–response curves fitted by a nonlinear regression with the Prism program (GraphPAD Software, San Diego, CA, USA). Each concentration was tested three–five times in duplicate and the values are given as the mean ± S.E.

and Figure S2. The EC₅₀ of Cl-IB-MECA was in a low nM range, according to the literature data [27], while the full agonist 15, which showed the best A₃AR affinity (KiA₃AR = 0.024 nM), exhibited an EC₅₀ of 14 nM. Among the four antagonists, 10 (IC₅₀ = 31 nM) and 11 (IC₅₀ = 79 nM), bearing a phenethyl group at the N⁶-position, showed a greater potency than 4 (IC₅₀ = 380 nM) and 12 (IC₅₀ = 153 nM), which present a 2,2-diphenylethyl chain in the same position. This order of potency was in close agreement with the compounds’ A₃AR binding affinities. These data confirm that the efficacy of the A₃AR ligands is closely related to the nature of the substituents present at the different positions of the nucleoside structure [19]. It is worth noting that the presence of a chlorine atom in 2-position of N⁶-phenethylAdo furnished the full A₃AR agonist 15 like Cl-IB-MECA but, unlike the latter, 15 showed no cytotoxic activity at concentrations up to 500 µM. On the other hand, compounds 4 and 10–12, which behaved as A₃AR antagonists, demonstrated comparable and even greater antitumor activity than Cl-IB-MECA in PC3 prostate cells, at least in the cAMP accumulation assay. These inconsistencies, due to the antitumor activity of both the full agonist Cl-IB-MECA and the A₃AR antagonists 4 and 10–12 on the same tumor cell type, cannot be explained by the different role played by the A₃AR in the regulation of cell proliferation and death, depending on the tissue type in which it is expressed, but they suggest the possible involvement of other cellular mechanisms, as we have previously hypothesized [18]. On the other hand, this is not the first report in which A₃AR antagonists have been found to exert anticancer activity [28,29].

3. Materials and Methods

3.1. Chemical Synthesis

3.1.1. General Methods

Melting points were determined with a Büchi apparatus and are uncorrected. ¹H NMR spectra were obtained with a Bruker Ascend 500 MHz spectrometer; δ values are in ppm, J values are in Hz. All exchangeable protons were confirmed by the addition of D₂O. Mass spectra were recorded on an HP 1100-MSD series instrument. Thin-layer chromatography (TLC) was carried out on pre-coated TLC plates with silica gel 60 F₂₅₄ (Fluka). For column chromatography, silica gel 60 (Merck) was used. Elemental analyses were determined on Fisons Instruments Model EA 1108 CHNS-O model analyzer and are within 0.4% of theoretical values. Purity of the compounds is ≥98%, according to elemental analysis data.

3.1.2. Synthesis of 6-Chloro-2-iodo-2′,3′,5′-tri-O-acetyladenosine (7)

Compound 6 (2.0 g, 4.68 mmol) was dissolved in anhydrous THF (5 mL), and diiodomethane (5.7 mL, 70.20 mmol), copper iodide (935 mg, 91 mmol) and isoamyl nitrite (2 mL, 14.51 mmol) were added. The mixture was refluxed for 5 h at 70 °C. The solvent was evaporated to dryness and the mixture was made into a slurry and purified by flash chromatography eluting with CHCl₃-c-Hex (70:30) to obtain 7 as a white solid after recrystallization with MeOH. Yield: 79%; m.p.: 181–183 °C. ¹H-NMR (DMSO-d6) δ: 1.99 (s, 3H, CH₃), 2.04 (s, 3H, CH₃), 2.09 (s, 3H, CH₃), 4.27 (m, 2H, CH₂-5′), 4.39 (m, 1H, H-4′), 5.60 (t, 1H, J = 11.0 Hz, H-3′), 5.86 (t, 1H, J = 10 Hz, H-2′), 6.27 (d, 1H, J = 5.2 Hz, H-1′), 8.80 (s, 1H, H-8). ESI-MS: positive mode m/z 539.0 [M+H]⁺, 561.0 [M+Na]⁺.

3.1.3. Synthesis of 2-Iodo-N⁶-(2-phenylethyl)adenosine (8)

2-phenylethylamine (0.49 mL, 3.89 mmol) and K₂CO₃ (2.56 g, 18.55 mmol) were added to a solution of 7 (2.0 g, 3.71 mmol) in dry DMF (19 mL), and the mixture was left to react for 16 h under nitrogen atmosphere at r.t. After that, methanol saturated by ammonia (NH₃/MeOH, 10 mL) was added and the mixture left under stirring for 30 min. After the removal of all volatiles under vacuum, the crude was purified by flash column chromatography eluting with DCM-MeOH (100% DCM to 95:5) and recrystallization from DCM/n-Hex mixture to obtain 8 as a white powder. Yield: 67%, m.p.: 120–122 °C. ¹H-NMR (DMSO-d₆) δ: 3.67 (m, 2H, CH₂Ph) 4.35 (m, 1H, HCH-5′), 4.40 (m, 1H, HCH-5′), 4.44 (m, 2H, NHCH₂), 4.71 (q, 1H, J = 3.6 Hz, H-4′), 4.9 (q, 1H, J = 3.2 Hz, H-3′), 5.3 (m, 1H, H-2′), 5.84 (d, 1H, J = 4.4 Hz, OH), 6.01 (m, 1H, OH), 6.26 (d, 1H, J = 6.4 Hz, OH), 6.59 (d, 1H, J = 6.0 Hz, H-1′), 7.98 (m, 1H, H-Ph), 8.08 (m, 5H, H-Ph and N⁶-H), 9.10 (s, 1H, H-8). ESI-MS: positive mode m/z 598.3 [M+H]⁺, 520.3 [M+Na]⁺. Anal (C₁₈H₂₀IN₅O₄) C, H, N.

3.1.4. Synthesis of 2-Iodo-N⁶-(2,2-diphenylethyl)adenosine (9)

Compound 7 was suspended in CH₃CN (4 mL) and 2,2-diphenylethylamine (0.62 mmol, 122 mg) was added, followed by Et₃N (2.24 mmol, 312 µL). The solution was stirred at r. t. for 8 hrs. NH₃/MeOH (5 mL) was added to the mixture and stirred for 1 h at r. t. All volatiles were removed under vacuum and the crude was made into slurry, the mixture was chromatographed by flash column eluting with CHCl₃-MeOH (99:1–96:4) to obtain 9 as a white powder. Yield 98%, m.p.: 170–172 °C. ¹H-NMR (DMSO-d6) δ: 3.51 (m, 1H, HCH-5′), 3.61 (m, 1H, HCH-5′), 3.89 (t, 1H, J = 6.0 Hz, H-4′), 4.01 (t, 2H, CH₂-Ph) 4.08 (m, 2H, H-3′), 4.45 (m, 1H, H-2′), 4.55 (t, 1H, J = 6.2 Hz, NH-CH₂), 5.00 (d, 1H, J = 5.0 Hz, OH), 5.18 (d, 1H, J = 5.0 Hz, OH), 5.45 (d, 1H, J = 5.0 Hz, OH), 5.77 (d, 1H, J = 5.0 Hz, H-1′), 7.17 (m, 6H, H-Ph), 7.29 (m, 4H, H-Ph), 8.22 (s, 1H, H-8), 8.26 (t, 1H, J = 5.5 Hz, NH). ESI-MS: positive mode m/z 574.0 [M+H]⁺, 596.0 [M+Na]⁺. Anal (C₂₄H₂₄IN₅O₄) C, H, N.

3.1.5. 2-Phenylethylamino-N⁶-(2-phenylethyl)adenosine (5)

2-phenylethylamine (0.41 mL, 3.25 mmol) and K₂CO₃ (127.85 mg, 0.92 mmol) were added to a solution of 7 (100 mg, 0.185) in DMF in a sealed steel bomb, and set at 120 °C for 16 hrs. Then, the reaction was cooled to r.t. and NH₃/CH₃OH was added and the reaction left for 30 min under stirring. After the removal of volatiles, the crude mixture was purified by flash column chromatography eluting with DCM-MeOH (100% DCM to 96:4) and recrystallization from DCM/n-Hex to obtain 5 as a light-brown powder. Yield 81%, m.p.: 135–137 °C. ¹H-NMR (DMSO-d₆) δ: 2.87 (m, 4H, 2 x NHCH₂CH₂-Ph), 3.48 (m, 4H, 2 x NHCH₂CH₂Ph), 3.51 (m, 1H, HCH-5′), 3.63 (m, 1H, HCH-5′), 3.87 (q, 1H, J = 3.6 Hz, H-4′), 4.10 (q, 1H, J = 3.2 Hz, H-3′), 4.58 (m, 1H, H-2′), 5.10 (d, J = 4.4 Hz, 2H, 2 x OH), 5.36 (d, J = 6.4 Hz, 1H, OH), 5.73 (d, J = 6.0 Hz, 1H, H-1′), 6.32 (m, 1H, N²-H), 7.17 (m, 1H, H-Ph), 7.26 (m, 5H, H-Ph and N⁶-H), 7.89 (s, 1H, H-8). ESI-MS: positive mode m/z 490.9 [M+H]⁺, 512.8 [M+Na]⁺. Anal (C₂₆H₃₀N₆O₄) C, H, N.

3.1.6. Synthesis of N⁶-(2-Phenylethyl)-2-phenylethylthioadenosine (13)

A mixture of 8 (395 mg, 0.24 mmol) in anhydrous DMF (6 mL), anhydrous K₂CO₃ (552 mg, 4 mmol), and 2-phenylethylthiol (500 μL, 4 mmol) was heated in a sealed steel bomb at 120 °C for 16 h. After removal of volatiles, the crude was purified via flash column chromatography eluting with CHCl₃-MeOH (96:4) to obtain 13 as a white solid. Yield: 75%; m.p.: 126–128 °C; ¹H-NMR (DMSO-d₆) δ: 2.91 (m, 2H, PhCH₂CH₂NH), 2.98 (t, 2H, J = 7.6 Hz, SCH₂CH₂-Ph), 3.68 (m, 2H, NHCH₂CH₂Ph), 3.31 (m, 2H, SCH₂), 3.53 (m, 1H, HCH-5′), 3.61 (m, 1H, HCH-5′), 3.92 (q, 1H, J = 4.0 Hz, H-4′), 4.11 (q, 1H, J = 4.0 Hz, H-3′), 4.55 (q, 1H, J = 6.0 Hz, H-2′), 5.07 (t, 1H, J = 5.2 Hz, OH), 5.19 (d, 1H, J = 4.8 Hz, OH), 5.43 (d, 1H, J = 6.0 Hz, OH), 5.84 (d, 1H, J = 6.4 Hz, H-1′), 7.20 (m, 2H, 2 x H-Ph), 7.30 (m, 8H, 2 x H-Ph), 8.05 (m, 1H, N⁶-H), 8.22 (s, 1H, H-8). ESI-MS: positive mode m/z 507.9 [M+H]⁺, 529.8 [M+Na]⁺, 1037.0 [2M+Na]⁺. Anal (C₂₆H₂₉N₅O₄S) C, H, N.

3.1.7. General Procedure for the Synthesis of 2-Alkynyl-N⁶-substituted Adenosines 10, 11, 4, and 12

To a solution of 8 or 9 (0.37 mmol), in dry DMF (2.0 mL), triethylamine (1.5 mL, 11.1 mmol), bis(triphenylphosphine)palladium dichloride (5.4 mg, 0.0074 mmol), CuI (0.32 mg, 0.0018 mmol), and the suitable 1-alkyne (250 μL, 2.22 mmol) were added. The mixture was stirred under nitrogen at r. t. for 16 h, then the mixture was evaporated to dryness and the residue purified through silica gel flash column chromatography eluting with the suitable eluent.

2-Hexynyl-N⁶-(2-phenylethyl)adenosine (10)

Compound 10 was prepared from 8 by reaction with 1-hexyne. The mixture residue was purified through silica gel flash column chromatography eluting with CHCl₃-MeOH (97:3) to acquire 10 as white solid. Yield: 38%; m.p.: 164–166 °C. ¹H-NMR (DMSO-d₆) δ: 0.90 (t, J = 7.2 Hz, 3H, CH₃), 1.42 (m, 2H, CH₂CH₃), 1.52 (m, 2H, CH₂CH₂CH₃), 2.42 (m, 2H, CH₂-C≡C), 2.87 (m, 2H, CH₂CH₂NH), 3.33 (m, 2H, CH₂CH₂NH), 3.53 (m, 1H, HCH-5′), 3.64 (m, 1H, HCH-5′), 3.93 (q, J = 3.2 Hz, 1H, H-4′), 4.10 (m, 1H, H-3′), 4.50 (m, 1H, H-2′), 5.18 (m, 2H, 2 x OH), 5.44 (m, 1H, OH), 5.84 (d, J = 6.4 Hz, 1H, H-1′), 7.18 (m, 3H, H-Ph), 7.25 (m, 2H, H-Ph), 7.96 (m, 1H, NH), 8.37 (s, 1H, H-8). ESI-MS: positive mode m/z 451.9 [M+H]⁺, 473.9 [M+Na]⁺, 925.1 [2M+Na]⁺. Anal (C₂₄H₂₉N₅O₄) C, H, N.

2-Phenylethynyl-N⁶-(2-phenethyl)adenosine (11)

Compound 11 was prepared from 8 by reaction with 1-phenylacetylene. The mixture residue was purified through silica gel flash column chromatography eluting with CHCl₃-MeOH (96:4) to acquire 11 as white solid. Yield: 70%; m.p.: 230–232 °C. ¹H-NMR (DMSO-d₆) δ: 2.93 (m, 2H, CH₂Ph), 3.57 (m, 2H, CH₂NH), 3.65 (m, 1H, HCH-5′), 3.69 (m, 1H, HCH-5′), 3.95 (q, J = 3.2 Hz, 1H, H-4′), 4.12 (q, J = 3.2 Hz, 1H, H-3′), 4.53 (q, J = 5.2 Hz, 1H, H-2′), 5.21 (d, J = 4.8 Hz, 2H, 2 x OH), 5.49 (d, J = 6.0 Hz, 1H, OH), 5.89 (d, J = 6.0 Hz, 1H, H-1′), 7.46 (m, 3H, H-Ph), 7.62 (m, 2H, H-Ph), 7.19 (m, 3H, H-Ph), 7.28 (m, 2H, H-Ph), 8.1 (m, 1H, N⁶-H), 8.44 (s, 1H, H-8). ESI-MS: positive mode m/z 471.9 [M+H]+, 493.8 [M+Na]+, 965.0 [2M+Na]⁺. Anal (C₂₆H₂₅N₅O₄) C, H, N.

2-Hexynyl-N⁶-(2,2-diphenylethyl)adenosine (4)

Compound 4 was prepared from 9 by reaction with 1-hexyne. The mixture residue was purified through silica gel flash column chromatography eluting with CHCl₃-MeOH (97:3) to acquire 4 as white solid. Yield: 38%; m.p.: 164–166 °C. ¹H NMR (DMSO-d6) δ: 0.93 (t, 3H, CH₃), 1.47 (m, 2H, CH₂CH₃), 1.54 (m, 2H, CH₂CH₂CH₃), 2.43 (m, 2H, CH₂-C≡C), 3.53 (m, 1H, H-CH-5′), 3.62 (m, 1H, H-CH-5′), 3.92 (brs, 1H, H-4′), 4.06 (m, 1H, CH-Ph₂), 4.09 (m, 1H, H-3′), 4.49 (m, 2H, CH₂-NH), 4.55 (t, J = 7.6 Hz, 1H, H-2′), 5,17 (m, 2H, 2 x OH), 5.42 (d, J = 6 Hz, 1H, OH), 5.82 (d, J = 6 Hz, 1H, H-1′), 7,17 (m, 2H, H-Ph), 7.28 (m, 8H, H-Ph), 7.89 (app s, 1H, NH), 8.32 (s, 1H, H-8). ESI-MS: positive mode m/z 528.0 [M+H]⁺, 549.9 [M+Na]⁺. Anal (C₃₀H₃₃N₅O₄) C, H, N.

N⁶-(2,2-Diphenylethyl)-2-phenylethynyladenosine (12)

Compound 12 was obtained from 5 by reaction with phenylacetylene. The desired compound was obtained after purification through silica gel flash column chromatography eluting with DCM-MeOH (94: 6) as a yellow solid, yield: 51%; m.p.: 121–125 °C. ¹H-NMR: (DMSO-d6) δ: 3.64 (m, 3H, H-5′), 3.97 (s, 1H, H-4′), 4.13 (s, 2H, NH-CH₂), 4.55 (d, 2H, J = 4.5 Hz, H-3′), 4.61 (d, 1H, J = 7.5 Hz, H-2′), 5.22 (brt, 2H, OH), 5.47 (d, 1H, J = 5.5 Hz, OH), 5.89 (d, 1H, J = 5.5 Hz, H-1′), 7.59 (m, 15H, H-Ph), 8.00 (s, 1H, NH), 8.41 (s, 1H, H-8). ESI-MS: positive m/z 547.9 [M+H]⁺, 569.8 [M+Na]⁺. Anal (C₃₂H₂₉N₅O₄) C, H, N.

3.1.8. Synthesis of 2,6-Dichloro-2′,3′,5′-tri-O-acetyl-(β-D-ribofuranosyl)purine (14)

Compound 6 (1.0 g, 2.3 mmol) was dissolved in dry DCM (40 mL). Then, SbCl₃ (746.5 mg, 3.27 mmol) and isoamyl nitrite (1.4 mL, 9.89 mmol) were added to the solution and it was left to react at 0 °C for 5 h. After this time, volatiles were removed under vacuum, the mixture was then extracted with DCM, the organic layer collected and dried over Na₂SO₄. The solvent was filtered and evaporated and the mixture was made in to slurry and purified by flash chromatography eluting with DCM/MeOH (99.5:0.5). Compound 14 was obtained as a white solid after crystallization with MeOH. Yield: 73%; m.p. 132–134 °C; ¹H-NMR (DMSO-d6) δ: 2.00 (s, 3H, CH₃), 2.04 (s, 3H, CH₃), 2.11 (s, 3H, CH₃), 4.29 (d, 2H, J = 3.4 Hz, CH-5′), 4.31 (d, 1H, J = 2.4 Hz, H-4′), 5.63 (t, 1H, J = 4.0 Hz, H-3′), 5.91 (t, 1H, J = 5.4 Hz, H-2′), 6.32 (d, 1H, J = 5.5 Hz, H-1′), 8.92 (s, 1H, H-8). ESI-MS: positive mode m/z 468.7 [M+Na]⁺. Anal C₁₆H₁₆Cl₂N₄O₇) C, H, N.

3.1.9. General Procedure for the Synthesis of 2-Chloro-N⁶-substituted adenosines 15 and 16

Compound 14 (120 mg, 0.26 mmol) was added, in turn, to a solution of phenylethylamine (0.1 mL, 0.79 mmol) and Et₃N (0.1 mL) in absolute ethanol (10 mL) or a solution of 2,2-diphenylethylamine (122 mg, 0.79 mmol) and Et₃N (0.1 mL) in absolute ethanol (10 mL). The reaction was left under stirring 15 h at r. t., then, NH₃/MeOH (10 mL) was added and the reaction left under stirring 1 h. Volatiles were removed under vacuum, the mixture made into slurry and purified by flash chromatography eluting with the suitable solvent.

3.1.10. 2-Chloro-N⁶-(2-phenylethyl)adenosine (15)

Compound 15 was obtained by reaction of 14 with 2-phenethylamine. The final compound was obtained after chromatography eluting with CH₂Cl₂-MeOH (97:3) and crystallization with EtOAc/n-Hex/diethyl ether as a white powder. Yield: 47%; m.p.: 153–155 °C. ¹H-NMR (DMSO-d6) δ: 2.90 (t, 2H, CH₂-Ph), 3.59 (m, 3H, H-5′), 3.94 (d, 1H, J = 4.5 Hz, H-4′), 4.05 (s, 1H, NH-CH₂), 4.11 (d, 1H, J = 5.0 Hz, H-3′), 4.49 (m, 1H, H-2′), 5.05 (d, 1H, J = 6.5 Hz, OH), 5.22 (d, 1H, J = 4.5 Hz, OH), 5.48 (d, 1H, J = 8.0 Hz, OH), 5.80 (d, 1H, J = 7.0 Hz, N-H’), 7.32 (m, 5H, Ph), 8.37 (s, 1H, N-H-8), 8.45 (d, 1H, J = 7.5 Hz, N-H). ESI-MS: positive mode m/z 427.8 [M+Na]⁺. Anal (C₁₈H₂₀ClN₅O₄) C, H, N.

3.1.11. 2-Chloro-N⁶-(2,2-diphenylethyl)-adenosine (16)

Compound 16 was obtained by reaction of 14 with 2,2-diphenylethylamine. The final compound was obtained after chromatography eluting with CH₂Cl_2/MeOH (95:5) and crystallization with ethyl acetate/n-Hex/diethyl ether as white powder. Yield: 67%; m.p.: 110–113 °C; ¹H-NMR (DMSO-d₆) δ: 3.55 (m, 3H, H-5′), 3.94 (t, 1H, H-4′), 3.96 (brs, 2H, CH₂-Ph), 4.1 (m, 2H, H-3′), 4.48 (brs, 1H, NH-CH₂), 4.52 (m, 1H, H-2′), 4.55 (d, 1H, J = 5 Hz, OH), 5.06 (d, 1H, J = 5 Hz, OH), 5.21 (d, 1H, J = 5 Hz, OH), 5.81 (d, 1H, J = 5 Hz, H-1′), 7.19 (m, 10H, Ph), 8.33 (s, 1H, H-8), 8.51 (s, 1H, NH). ESI-MS: positive mode m/z 482.8 [M+H]⁺, 504.1 [M+Na]⁺. Anal (C₂₄H₂₄ClN₅O₄) C, H, N.

3.2. Biological Assays at Human Adenosine Receptors

3.2.1. Cell Culture

Chinese hamster ovary (CHO) cells stably expressing human ARs were grown adherently and maintained in Dulbecco’s Modified Eagles Medium with nutrient mixture F12 (DMEM/F12), supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin, 1mM Sodium pyruvate and 0.1 mg/mL Geneticin (G418) at 37 °C and aerated with 5% CO₂: 95% O₂.

3.2.2. Membrane Preparation

All pharmacological methods followed the procedures as described earlier [22]. In brief, membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtypes through two centrifugations at different speeds. The first low-speed (1000 g) centrifugation allowed the removing of cell fragments and nuclei, while the second, performed at high speed (100,000 g), allowed the precipitation of the crude membrane fractions.

The resulting membrane pellet was resuspended in the buffer used for the respective binding experiments, frozen in liquid nitrogen and stored in aliquots at −80 °C.

3.2.3. Binding Assay

The binding affinity of the novel compounds was evaluated using radioligand competition experiments in CHO cells stably expressing hA₁AR, hA_2AAR, and hA₃AR subtypes. The radioligands used were 1.0 nM [³H]CCPA (K_D = 1.1 nM) for hA₁, 10 nM [³H]NECA (K_D = 20 nM) for hA_2A, and 1.0 nM [³H]HEMADO (K_D = 1.5 nM) for hA₃ receptors. Results were expressed as K_i values (dissociation constants), which were calculated with the program GraphPad (GraphPAD Software, San Diego, CA, USA). Each concentration was tested three–five times in duplicate and the values are given as the mean ± standard error (S.E.).

The potency of compounds at the hA_2B receptor (expressed on CHO cells) was determined through GloSensor cAMP Assay.

3.2.4. Functional Agonism or Antagonism at A_2B or A₃ ARs in GloSensor cAMP Assay

CHO cells stable expressing A_2B or A₃ARs and the plasmid encoding the biosensor were used to study functional agonism and antagonism of understudy ligands. The desired cell number was incubated in equilibration medium containing a 3% v/v GloSensor cAMP reagent stock solution, 10% FBS and 87% of CO₂ independent media. After 2 h of incubation, cells were dispensed in wells of 384 well plate and when a steady-state basal signal was obtained the agonism profile was studied, adding to wells the reference agonist NECA or compounds at different concentrations. In the case of the G_i coupled receptor A₃AR Forskolin (FSK) 10 μM was added 10 min after the agonists and various luminescence readings were performed at different incubation times.

The antagonist profile of the compounds was evaluated by assessing their ability to counteract an agonist-induced increase or decrease (A_2B and A₃ARs, respectively) of cAMP accumulation [22]. The cells were incubated with different antagonist concentrations and then treated with a fixed dose of NECA (10 μM for A_2BAR and 1 μM for A₃AR). FSK 10 μM was added 10 min after the agonists and various luminescence reads were performed at different incubation times.

Responses were expressed as percentage of the maximal relative luminescence units (RLU) and concentration–response curves were fitted by a nonlinear regression with the Prism program (GraphPAD Software, San Diego, CA, USA). The agonist or antagonist profile of compounds was expressed as EC₅₀ or IC₅₀, respectively [30]. Each concentration was tested three–five times in duplicate and the values are given as the mean ± S.E.

3.3. Biological Studies on Cancer Cell Line

3.3.1. Cell Lines

The PC3 prostate cancer cell line used is from Type Culture Collection, ATCC, (Rockville, MD, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg streptomycin at 37 °C in a humidified incubator containing 5% CO₂.

3.3.2. Reagents

DMSO (used as vehicle), sulforhodamine B (SRB), trichloroacetic acid (TCA), and acetic acid were from Sigma Aldrich (Sant Louis, MO, USA).

3.3.3. Cell Growth Inhibition Assay

To determine the effects of compounds 4, 5, 8–13, 15, 16, and Cl-IB-MECA on PC3 cell line, an SRB assay was performed. Cells were seeded at 4 × 10³ cells/well in a 96-well microplate. Twenty-four hours later, cultures were treated with increasing concentrations of compounds, 1, 10, 25, 50, 100 μm, for 48 h at 37 °C in a 5% CO₂ atmosphere and 95% relative humidity. At the same time (t = 0), and after drug treatments, 100 μL of 10% (w/v) TCA were added to each well, incubated for 1 h at 4 °C, washed with deionized water, and dried at room temperature. One hundred microliters of SRB solution were added to each well, incubated for 10 min at room temperature, rinsed four times with 1% (v/v) acetic acid, and allowed to dry at room temperature. Finally, 100 μL of 10 mM Tris base solution (pH 10.5) was added to each well, and the absorbance was measured at 515 nm in a microplate reader (BioTek Instruments, Winooski, VT, USA). The absorbance at t = 0 was compared with the absorbance at the end of the experiment to determine cell growth in treated cells compared with control cells. The antitumor activity was estimated by measurements of three parameters: Growth Inhibition 50 (GI₅₀), the drug concentration (µM) required to inhibit 50% net of cell growth; Total Growth Inhibition (TGI), the drug concentration (µM) required to inhibit 100% of cell growth; and Lethal Concentration 50 (LC₅₀), the drug concentration (µM) required to kill 50% of the initial cell number. LC₅₀, GI₅₀ and TGI values are shown as mean ± standard deviation (SD) of three different experiments calculated using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

3.3.4. Statistical Analysis

The statistical significance was determined by Student’s t-test by using, as control, the reference compound 2-Cl-IB-MECA. * p < 0.05.

4. Conclusions

Novel di-substituted Ado derivatives bearing a N⁶-phenethyl or a more steric hindered N⁶-(2,2-diphenylethyl) group combined with halogens, alkynes or a phenylethylthio chain in 2-position were synthesized and tested in radioligand binding assays at A₁, A_2A, and A₃ ARs. Selected compounds were also tested in a functional assay at A_2BARs and, in most cases, were found not able to activate the receptor at concentrations up to 30 µM. In the binding studies, all the compounds were found to be A₃AR ligands with high affinity (Ki in the nM and sub-nM range) and different degree of selectivity. In general, compounds bearing a 2-phenethylamino group in N⁶-position resulted endowed with higher affinity and selectivity than the corresponding analogues bearing a more hindered 2,2-diphenylethyl group in the same position. Furthermore, in the PC3 prostate cancer cell line, all the compounds concentration-dependently reduced the cell growth and most of them showed a significant inhibitory effect on proliferation and a pronounced cytotoxic activity comparable to that of Cl-IB-MECA. In particular, the ligands with the best cytostatic properties were those bearing a N⁶-(2,2-diphenylethyl) group, indicating that the antitumor activity is not closely related to the affinity for the A₃AR subtype. Finally, functional cAMP assays demonstrated that compounds endowed with the best cytotoxic activity behave as A₃AR antagonists, confirming the hypothesis that other cellular mechanisms are involved in the anticancer properties of these A₃AR ligands. Therefore, further experiments will be needed to explain the precise pathways responsible for their anticancer effects.