Design, Synthesis, and in Vitro Biological Evaluation of 14-Hydroxytylophorine-dichloroacetate Co-drugs as Antiproliferative Agents

Abstract

Co-drug, or mutual-prodrug, is a drug design approach consisting of covalently linking two active drugs so as to improve the pharmacokinetics and/or pharmacodynamics properties of one or both drugs. Co-drug strategy has proven good success in overcoming undesirable properties such as absorption, poor bioavailability, nonspecificity, and gastrointestine tract (GIT) side effects. In this work, we successfully developed a co-drug of 14-hydroxytylophorine, a phenanthroindolizidine derivative with remarkable antiproliferative activity, and dichloroacetate, a known inhibitor of pyruvate dehydrogenase kinase. Dichloroacetate steers tumour cell metabolism from glycolysis back to glucose oxidation, which in turn reverses the Warburg effect and renders tumour cells with a proliferative disadvantage. The obtained co-drugs retained the cytotoxicity of 14-hydroxytylophorine. However, they showed similar unselectivity towards normal cells.

Introduction

Cancer is a complex disease characterized by uncontrolled cellular division. It is the second-leading cause of mortality after cardiovascular diseases. Chemotherapy exerts reasonable success against cancer. However, these drugs’ lack of selectivity can cause severe side effects, and the emergence of drug resistance are two major drawbacks of the effective use of chemotherapeutic agents clinically.¹⁾

Natural products are a major source of currently available drugs. Approximately half U.S. Food and Drug Administration (FDA)-approved small molecule anticancer drugs emerging between the early 1940 s till the present decade were either natural products or directly derived from them.²⁾ Phenanthroindolizidines are a family of alkaloids isolated from different species of Asclepiadaceas. Phenanthroindolizidines display very interesting biological activities, such as excellent cytotoxicity against different human cancerous cell lines. Prototype compounds, such as tylocrebrine (1) and tylophorine (2) (Fig. 1), not only exhibit low-nanomolar half-maximal growth inhibition (GI₅₀) values but also are effective against multidrug-resistant (MDR) human cancerous cell lines.³⁾ Notwithstanding their therapeutic potential, no compound in this class was able to pass clinical trials. Their main drawbacks are severe central nervous system (CNS) toxicity and significant loss of anti-cancer activity when administered in vivo.⁴⁾

Fig. 1. General Chemical Structure of Phenanthroindolizidines Alkaloids

Co-drug, or mutual-prodrug, is a drug design approach consisting of covalently linking two active drugs so as to improve the pharmacokinetics and/or pharmacodynamics properties of one or both drugs. Delivering the two therapeutic compounds simultaneously to their site of action is another advantage of this technique, given that both drugs are released concomitantly and quantitatively after absorption.⁵⁾ Co-drug strategy has proven success in overcoming undesirable properties such as absorption, poor bioavailability, nonspecificity, and gastrointestine tract (GIT) side effects.⁶⁾ Sulfasalazine, a salicylic acid derivative coupled to sulfapyridine via an azo linkage, was the first approved co-drug for the treatment of rheumatoid arthritis.⁷⁾ Another example co-drug is benorylate, which is acetylsalicylic acid and paracetamol linked to each other by an ester group. Benorylate is an effective anti-inflammatory agent with good gastric tolerance since it is absorbed intact.⁸⁾ However, the need for a specific functional group to link the two drugs conveniently is the main disadvantage of co-drug technique.⁵⁾

In this study we developed a co-drug of 14-hydroxytylophorine and dichloroacetate. The two molecules are combined together through an ester linkage. Dichloroacetate, a known inhibitor of pyruvate dehydrogenase kinase (PDK), steers tumour cell metabolism from glycolysis back to glucose oxidation, which in turn reverses the Warburg effect and renders tumour cells with a proliferative disadvantage.^9–12)

Results and Discussion

14-Hydroxytylophorine (3) was chosen for this study because it has an alcohol function that can be esterified. Optically pure (13aS,14S)-14-hydroxytylophorine (3a) and its enantiomer (13aR,14R)-14-hydroxytylophorine (3b) were synthesized as described previously.¹³⁾ Both compounds (3a–b) were then esterified by dichloroacylchloride to yield the desired co-drugs (4a–b), Fig. 2.

Fig. 2. Chemical Synthesis of 14-Hydroxytylophorindichloroacetate Co-drugs

The physicochemical properties of co-drug (4a) were then evaluated and compared with those of the parent alkaloid (3a) (Table 1). The water solubility of (S)-14-hydroxytylophorine (3a) at pH 7.4 was slightly increased following transformation of its alcohol function into dichloroester; the log D was increased by more than one unit. Blood–brain barrier parallel artificial membrane permeability assay (BBB-PAMPA) predicted that the co-drug (4a) would have lower BBB permeability than the free alcohol (3a).

Table 1. Physicochemical Properties of (13aS,14S)-14-Hydroxytylophorine (3a) and Its Dichloroacetate Co-drug (4a)

Compound	PBS solubility µM mean ± S.E.	Log D mean ± S.E.	Permeability log[10−6 cm/s] mean ± S.E.
3a	44 ± 1	2.69 ± 0.03	−4.9 ± 0.07
4a	138 ± 5	4.16 ± 0.4	−5.3 ± 0.00

Values are average of 2 independent experiments.

The cytotoxicities of 14-hydroxytylophorines (3a–b), the developed co-drugs (4a–b), and doxorubicin, as reference, were evaluated in resazurin-based assay¹⁴⁾ against two reference cell lines: human embryonic kidney cells (HEK293) and hamster Chinese ovary cells (HEK293) and against four cancerous cell lines: neuroblastoma (SK-N-DZ), Burkitt’s lymphoma (Ramos), hepatocarcinoma (HepG2), and colorectal adenocarcinoma (DLD-1) (Table 2).

Table 2. Cytotoxicity of 14-Hydroxytylophorines (3a–b) and Their Dichloroacetate Co-drugs (4a–b)

Compound	IC50 (µM) cell lines
	HEK293	CHO-K1	SK-N-DZ	Ramos	HepG2	DLD-1
Doxorubicin	0.16	1.21	1.33	2.6	2.9	>15
3a	6.0	2.0	2.0	6.0	5.0	>15
3b	2.6	0.5	0.6	13.8	4.7	5.5
4a	0.6	0.6	0.5	7.0	6.0	2.6
4b	4.2	2.0	4.7	9.7	6.0	12.0

(Values are average of 4 independent experiments.)

Although co-drugs (4a–b) displayed strong cytotoxicity attested by IC₅₀s in the low micromolar range, comparable to those obtained for doxorubicin; the co-drug strategy, in the case of 14-hydroxytylophorine and dichloroacetate, did not show significant improvement in the antiproliferative activity or selectivity against normal cells.

Conclusion

We successfully synthesized co-drugs of 14-hydroxytylophorine. The developed co-drugs retained the cytotoxicity of the parent alkaloids. However, the co-drug strategy did not enhance their activity or selectivity.

Experimental

(13aS,14S)-2,3,6,7-Tetramethoxy-9,11,12,13,13a,14-hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-14-yl 2,2-dichloroacetate (4a)

A 100 mL Schlenk flask was charged with (13aS,14S)-14-hydroxytylophorine (3a) (0.4 g, 0.98 mmol) in 20 mL anhydrous dichloromethane and (270 µL, 2.0 mmol) triethylamine. To the solution was added dichloroacetyl chloride (0.216 g, 1.47 mmol) in 10 mL CH₂Cl₂ dropwise through a constant pressure funnel at 0°C under an atmosphere of argon overnight. The white suspension was then turned to a yellow clean solution. After evaporating the solvent under reduced pressure, the residue was purified by column chromatography on silica gel (PE : EA = 1 : 1) to give compound 4a (0.26 g, 52%) as a yellow solid. mp: 134–135°C; purity: 97.1% (SHIMADZU, VP-ODS, 250 × 4.6 mm, eluent MeOH : H₂O = 90 : 10, flow speed: 1 mL/min, wave: 254 nm, t_R = 4.134 min); purity 98.1%, dr about 1 : 1 (by HPLC, CHIRALPAK AD-H, 250 × 4.6 mm, eluent: i-PrOH : Hexane : Et₃N = 25 : 75 : 0.1, flow speed: 1 mL/min, wave: 254 nm, t_R = 15.600 min (containing both (13aS,14S)- and (13aS,14R)-7); ¹H-NMR (400 MHz, CDCl₃) δ: 7.70 (s, 2H), 7.17 (s, 1H), 7.05 (s, 1H), 6.66 (s, 1H), 6.05 (s, 1H), 4.67 (d, J = 15.2 Hz, 1H), 4.07 (s, 6H), 3.99 (s, 6H), 3.58–3.41 (m, 2H), 2.70–2.60 (m, 1H), 2.46–2.32 (m, 1H), 2.09–1.67 (m, 4H).¹³C-NMR (100 MHz, CDCl₃) δ: 165.8, 149.6, 149.3, 148.9, 148.8, 129.9, 125.1, 124.8, 123.8, 123.1, 122.4, 103.4, 103.3, 103.2, 103.0, 70.5, 64.5, 63.3, 56.0, 55.9, 54.8, 53.6, 29.7, 24.8, 21.7. High resolution (HR)MS electrospray ionization (ESI) calcd for C₂₆H₂₈Cl₂NO₆ (M + H)⁺ 520.1294, found 520.1282.

(13aR,14R)-2,3,6,7-Tetramethoxy-9,11,12,13,13a,14-hexahydrodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-14-yl 2,2-dichloroacetate (4b)

The synthetic procedure was similar to that of compound 4a. Compound 4b was obtained as a yellow solid (55%). mp: 133–135°C; purity 88.1% (SHIMADZU, VP-ODS, 250 × 4.6 mm, eluent MeOH : H₂O = 90 : 10, flow speed: 1 mL/min, wave: 254 nm, t_R = 6.021 min); purity 98.2%, enantiomeric excess (ee) 90.2% (by HPLC, CHIRALPAK AD-H, 250 × 4.6 mm, eluent i-PrOH : Hexane : Et₃N = 25 : 75 : 0.1, flow speed: 1 mL/min, wave: 254 nm, t_R = 8.181 min); ¹H-NMR (400 MHz, CDCl₃) δ: 7.70 (s, 2H), 7.17 (s, 1H), 7.05 (s, 1H), 6.66 (s, 1H), 6.05 (s, 1H), 4.67 (d, J = 15.2 Hz, 1H), 4.07 (s, 6H), 3.99 (s, 6H), 3.58–3.41 (m, 2H), 2.70–2.60 (m, 1H), 2.46–2.32 (m, 1H), 2.09–1.67 (m, 4H); ¹³C-NMR (100 MHz, CDCl₃) δ: 165.8, 149.6, 149.3, 148.9, 148.8, 129.9, 125.1, 124.8, 123.8, 123.1, 122.4, 103.4, 103.3, 103.2, 103.0, 70.5, 64.5, 63.3, 56.0, 55.9, 54.8, 53.6, 29.7, 24.8, 21.7; HR-MS ESI calcd for C₂₆H₂₈Cl₂NO₆ (M + H)⁺ 520.1294, found 520.1282.

Acknowledgment

The authors would like to acknowledge the financial support provided by King Abdulaziz City for Science and Technology (KACST), Grant No. 13-MED2515-10.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials (Spectroscopy data for compounds (4a, b), and water solubility, Log D, PAMPA-BBB and cytotoxicity assays protocols).

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