Triphenylphosphonium Analogues of Betulin and Betulinic Acid with

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Triphenylphosphonium Analogues of Betulin and Betulinic Acid with Biological Activity: A Comprehensive Review Mirosława Grymel,*,†,‡ Mateusz Zawojak,† and Jakub Adamek†,‡ †

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Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, 44-100 Gliwice, Poland ‡ Biotechnology Center of Silesian University of Technology, 44-100 Gliwice, Poland ABSTRACT: Naturally occurring pentacyclic lupane triterpenoids such as betulin (1) or betulinic acid (2) and their synthetic derivatives display a broad spectrum of biological activities and, therefore, have been the subject of great interest. However, the use of these compounds as potential therapeutic agents is limited by their low bioavailability, high hydrophobicity, and insufficient intracellular accumulation. In this context, research on modifications of the parent structures that will improve their pharmacokinetic properties is particularly important. In the past few years, methods of synthesis as well as cytotoxic and antiparasitic properties of a series of lupane triterpenoids modified by introducing one or two triphenylphosphonium moieties at the C-2, C-3, C-28, or C-30 positions by carbon−carbon or ester bonds have been described. The presence of triphenylphosphonium groups affects not only physical properties but also the mechanism of action of a potential drug. This review summarizes published findings on synthetic methods and biological properties of the triphenylphosphonium derivatives of betulin and betulinic acid.

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INTRODUCTION Betulin (1, 3-lup-20(29)-ene-3β,28-diol) and betulinic acid (2, (3β)-3-hydroxy-lup-20(29)-en-28-oic acid) belong to an important group of naturally occurring pentacyclic lupane-type triterpenoids, which can be isolated from the bark of several species of trees, especially from the white birch (Betula pubescens).1,2 Betulin was first described in 1788 by Lowitz, and betulinic acid in 1902 by Retzlaff; however, it was the discovery in 1995 by Pisha et al. that apoptosis of some cancer cells is induced by betulinic acid that resulted in a significant increase in interest in these types of compounds.3,4 Betulin (1) and betulinic acid (2) show similar effects on living organisms and possess a broad spectrum of biological activities demonstrated by their antitumor,5−20 antibacterial,18,21 antiHIV, 16−20,22,23 anti-inflammatory, 16−21,24−26 antiretroviral,27−29 antimalarial,16,20,21,25 antiobesity,26 hepatoprotective,11,15,30 and immunomodulatory properties.26 In addition, these compounds are antioxidants31 and reduce oxidative stress.20,26 They have been reported to inhibit the activation of TLR/NF-κB, p38, and c-JUN N-terminal kinase pathways, which may cause an anti-inflammatory effect.17−20 Moreover, betulin (1) and betulinic acid (2) have a positive effect on the treatment of atopic dermatitis32 and accelerate wound healing.33 However, their low bioavailability, poor solubility in water, and insufficient intracellular accumulation limit the use of both these pentacyclic lupane-type triterpenoids (1 and 2) as potential therapeutic agents. Therefore, numerous studies on the © XXXX American Chemical Society and American Society of Pharmacognosy

synthesis of their new semisynthetic derivatives with improved pharmacokinetic properties are being performed. Generally, chemical modifications of a triterpene backbone involve the three most reactive functional groups: (i) the secondary hydroxy group at the C-3 position, (ii) the primary hydroxy group (betulin) or carboxylic acid group (betulinic acid) at position C28, and (iii) the isopropenyl side chain. Modifications at position C-2 or in the triterpene ring of the betulin or betulinic acid skeleton are definitely less common (Chart 1).

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SEMISYNTHETIC ANALOGUES OF BETULIN AND BETULINIC ACID The structural modifications of betulin (1) and betulinic acid (2) are aimed not only at improving physical properties but, above all, at increasing the pharmacological activity. In the past decade, the most promising reports concern the antitumor properties of betulin and betulinic acid derivatives, which are based on their mechanism of inducing apoptosis. Being aware of the large number of semisynthetic analogues of betulin and betulinic acid widely described in the literature, in this review, focus is made on selected examples exhibiting high and confirmed biological activity. In 2009, Drag-Zalesinska et al. described the synthesis of the esters of betulin and betulinic acid with L-amino acids. Their Received: October 4, 2018

A

DOI: 10.1021/acs.jnatprod.8b00830 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Review

toward A-549 and human colon carcinoma (HCT-116) cell lines was also exhibited by compounds 15−18, containing a triazole moiety.11 The introduction of sugar residues (Glc, Rha) at position C-3 of betulinic acid resulted in the increased cytotoxicity toward A-549 and DLD-1 cell lines in comparison to the parent structure 2.12 Although the potential anticancer properties of betulin and betulinic acid derivatives are the most widely recognized, research on their antiviral and antibacterial activities is also being conducted. For example, one of the breakthroughs in the search for anti-HIV agents was the synthesis of 3-O-(3′,3′dimethylsuccinyl)betulinic acid (21), also known as bevirimat or DSB (Chart 2). It showed a very strong ability to inhibit

cytotoxic properties toward several cell lines such as human pancreatic carcinoma parental (EPP85−181P), daunorubicinresistant human pancreatic carcinoma (EPP85−181RDB), and human gastric carcinoma parental (EPG85−275P) were investigated. The greatest increase in activity against EPP85− 181P cells relative to that of betulin was observed for derivatives 3 and 4, with alanine or lysine moieties at position C-3 and C-28 (Table 1).13 Chart 1. Chemical Structures of Betulin (1) and Betulinic Acid (2)

Chart 2. Structures of Bevirimat (21) and Betulin 3,28Dioxyme (22)

A few years later, Yang et al. obtained a number of betulin derivatives differing in the type of amine moiety attached to the succinyl linker by modifying the skeleton of betulin at the C-28 position. Cytotoxic activities of selected and most active examples against various human tumor cell lines are shown in Table 1.14 Studies on the cytotoxic properties of betulinic acid derivatives were conducted by several research groups. A brief summary of these is presented in Table 2. As can be seen, the largest increase in cytotoxicity toward MCF-7 cells was achieved for structure 12, which can be related to its high polarity,9 whereas compound 10 showed the most promising cytotoxic properties against human lung carcinoma (A-549). High activity

replication of HIV type 1 in infected H9 lymphocytes and successfully passed the second phase of clinical trials. Unfortunately, in 2010, clinical research was canceled because patients did not have a uniform response to the treatment.34,35 Studies on the activity of semisynthetic betulin and betulinic acid analogues against Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria (Chlamydia pneumoniae, Helicobacter pylori, Enterobacter aerogenes, Escher-

Table 1. Cytotoxic Activities of Betulin (1) and Its Derivatives (3−8) against Pancreatic Carcinoma Parental (EPP85−181P), Human Prostate Carcinoma (PC-3), Human Breast Carcinoma (MCF-7 and Bcap-37), Human Gastric Carcinoma (MGC-803), and Human Malignant Melanoma (A375) Cell Lines14

B



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Table 2. Cytotoxic Activity of Betulinic Acid (2) and Its Derivatives (9−20) against Human Tumor Cell Lines

Chart 3. Chemical Structures of Triphenylphosphonium Derivatives of Betulin (23−31)

ichia coli, Pseudomonas aeruginosa) and fungi (Candida albicans, Candida krusei) showed that the synergistic action of classical drugs in combination with betulin and betulinic acid derivatives allowed the use of smaller drug doses and also reduced the development of drug resistance.36−39

groups in order to use their specific property to act as mitochondrial ligands. In contrast to other cellular organelles, mitochondria have a high negative transmembrane potential (Δψm). This potential is much higher in the case of tumor cells, giving the opportunity for selective delivery and accumulation of anticancer agents in the mitochondria-targeted therapies.40−44 High lipophilicity and large ionic radius of the triphenylphosphonium cation (TPP+) effectively reduce the activation energy required for membrane passage. The presence of a delocalized lipophilic cation such as the triphenylphosphonium ion can accelerate the transport of biologically active molecules across

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TRIPHENYLPHOSPHONIUM ANALOGUES OF BETULIN AND BETULINIC ACID Modifications leading to compounds with precisely targeted biological activity are particularly important. One of the most promising examples in this area is the conjugation of a parent molecule (for example, 1 or 2) with triphenylphosphonium C



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Chart 4. Chemical Structures of Triphenylphosphonium Derivatives of Betulinic Acid (32−57)

Scheme 1. Synthesis of 3β,28-Diacetoxy-30-triphenylphosphonio-lup-20(29)-ene Bromide (23)a

Reagents and conditions: (a) Ac2O, Py, DMAP or AcCl, THF, Py, DMAP, 20 °C; (b) NBS, CCl4; (c) PPh3, CH3CN, reflux, Ar.

a

the mitochondrial membrane by as much as 107−108 times when compared to hydrophilic cations such as Na+.41,45 The strategy of drug modification by conjugating with phosphonium groups has been used successfully for well-known anticancer drugs such as doxorubicin or cisplatin, facilitating their transport and selective accumulation in cancer cells. Many other examples of this methodology, which have been used to target small,

bioactive molecules to the mitochondria can be found in the literature.40−42,46−50 In this section, presented are the structures of pentacyclic triterpenoid conjugates (23−57) with one or two triphenylphosphonium cation(s) (TPP+) (Charts 3, 4), for which the synthesis and biological properties are described in this review. D



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Scheme 2. General Synthesis of Triphenylphosphonium Derivatives of Betulin (24−31)a

a

Reagents and conditions: (a) Br(CH2)nCOOH, DCC, DMAP, DCM, rt, 1−2 h; (b) 2Br(CH2)nCOOH, DCC, DMAP, DCM. rt, 1−2 h; (c) PPh3, CH3CN, reflux, 6−8 h; (d) HCOOH, reflux;62 (e) Ac2O, Py, reflux.62

Scheme 3. General Synthesis of Triphenylphosphonium Derivatives of Betulinic Acid (32−37)a

Reagents and conditions: (a) H2 Pd/C, MeOH/THF (50:50), 20 °C, 94% yield; (b) CH2N2, Et2O, 20 °C, 89% yield or BnCl, DMF, K2CO3, 55 °C, 92% yield; (c) KN(SiMe3), Et3B, C3H5Br, DMF, 20 °C, Ar, 77−79% yield; (d) NaBH4, CeCl3·7H2O, MeOH/THF, −30 → 20 °C, Ar; (e) Lselectride. THF, −78 → 20 °C, Ar; (f) Ac2O, Py, DMAP, 20 °C; (g) BH3·THF, THF, 20 °C, Ar; (h) l2, PPh3, imidazole, THF, 0 °C; (i) MsCl, Py, DCM, DMAP, 20 °C; (j) LiBr, Me2CO, Δ, Ar; (k) PPh3, PhMe, A, Ar; (l) Pd/C, Et2O. a

E



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Scheme 4. Synthesis of Allyl Triphenylphosphonium Salts 38−41a

Reagents and conditions: (a) Ac2O, Py, DMAP or AcCl, THF, Py, DMAP, 20 °C; (b) CH2N2, Et2O, 0 → 20 °C, 2 h; (c) (COCl)2, DCM, 0 → 20 °C, 2 h, GlyOEt or AlaOMe, Et3N, DCM; (d) NBS, CCl4; (e) PPh3, CH3CN, reflux, Ar.

a

equivalents of ω-bromoalkanecarboxylic acid leads to C-3, C-28 betulin diesters (65−67), in over 80% yield. The nucleophilic substitution of halogenated betulin derivatives (60−67) using an excess of triphenylphosphine (molar ratio of 1:4) in CH3CN under reflux for 6−8 h gave phosphonium salts (24−28) or bisphosphonium salts of betulin (29−31) with good or very good yields, as is shown in Scheme 2. Synthesis of Betulinic Acid with TPP+ Substituents. As in the case of betulin (1), the introduction of the triphenylphosphonium group to the betulinic acid molecule has been described several times. In 2013, Spivak et al. developed interesting methods for preparing phosphonium salts 32−37, which can be considered as betulinic acid derivatives modified at position C-2 (see Scheme 3).52,55,56 The crucial step of this synthesis is the formation of dihydrobetulonates (68 and 69) with the allyl group at the C2 position. Compounds 68 and 69 were obtained from betulin (1) using previously described procedures.57 The reduction process of the keto group in ring A with NaBH4 or L-selectride followed by acylation of 70−72 with Ac2O (this step is not necessary but increases the final yield of phosphonium salt; compare yields of 34 and 35 with 32, 33, 36, and 37) and the hydroboration of 70 and 72−73 gave the primary alcohols 76− 80 with good yields. These types of alcohols are transformed readily into iodides 81−83 by their reaction with iodine in the presence of imidazole and triphenylphosphine. Alternatively, alcohols 76−80 can be converted to bromides 84−86 in the

Synthesis of Betulin with TPP+ Substituents. Depending on the position of modification on the native skeleton, several ways of introducing the triphenylphosphonium moiety into the betulin molecule were described. In 2014, Spivak et al. obtained a triphenylphosphonium salt (23) from betulin with a good yield as a result of a three-step synthesis (see Scheme 1).51,52 First, betulin (1) was acetylated with an excess of acetic anhydride or with acetyl chloride in the presence of 4-(dimethylamino)pyridine (DMAP) and pyridine.53 Then, ester 58 was transformed to allyl C-30 bromide (59) by reaction with Nbromosuccinimide (NBS) in CCl4.54 The introduction of the triphenylphosphonium group at position C-30 of the betulin skeleton occurred after refluxing the allyl bromide (59) with an excess of PPh3 in CH3CN under an argon atmosphere, with 92% yield. The Tsepaeva group reported that the betulin scaffold can be also conjugated with the triphenylphosphonium group through an acyl linker at the C-28 or C-3 and C-28 positions.41 It was found that in the Steglich-type esterification with ωbromoalkanecarboxylic acids in dichloromethane in the presence of DMAP and dicyclohexylcarbodiimide (DCC), C3- and C-28-functionalized derivatives of betulin can be obtained with high regioselectivity. In the case of using one equivalent of ω-bromoalkanecarboxylic acid, C-28 betulin esters (60−62) are commonly produced, which may be confirmed by 1 H NMR spectroscopic analysis, whereas the use of two F



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Scheme 5. Synthesis of a Phosphonium Analogue of Betulinic Acid Containing a 1,2-Enone Moiety (56)a

a

Reagents and conditions: (a) DDQ, dioxane, reflux, Ar; (b) NBS, CCl4; (c) PPh3, CH3CN, reflux. Ar.

Scheme 6. Synthesis of Phosphonium (42−46) and Bisphosphonium (51, 52) Analogues of Betulinic Acida

Reagents and conditions: (a) Br(CH2)nBr, K2CO3, CH3CN, DMF, 50 °C; (b) PPh3, CH3CN, reflux, Ar; (c) NBS, CCl4; (d) Cl2CCOOH, DMAP, DCC, DCM, rt, 2−7 h. a

Scheme 7. Synthesis of a Phosphonium Analogue of Betulinic Acid (57)a

Reagents and conditions: (a) Br(CH2)4Br, K2CO3, CH3CN, DMF, 50 °C; (b) PPh3, CH3CN, reflux, Ar.

a

them with an excess of triphenylphosphine in CH3CN under argon. Attempts to introduce a TPP+ cation at position C-30, using a structural analogue of betulinic acid (96) containing a 1,2-enone moiety in ring A, have also been successful (Scheme 5).40 The double bond was formed by dehydrogenation of methyl betulanoate (95) by 2,3-dichloro-5,6-dicyanoquinone (DDQ).60 Then, the phosphonium salt (56) was obtained in a similar way to the phosphonium salt of betulin (23, Scheme 1) or phosphonium analogues of betulinic acid (38−41, Scheme 4). Again, the key step was the nucleophilic substitution of the corresponding bromide, which was conducted in the presence of an excess of triphenylphosphine under reflux in CH3CN, with 80% yield.

two-step procedure as detailed in Scheme 3. Halides 81−86 react smoothly with an excess of triphenylphosphine in toluene under reflux, giving triphenylphosphonium salts 32−37 with good yields. In 2017, Spivak et al. reported the synthesis of allyl triphenylphosphonium salts 38−41 by modification of the C30 position of the triterpene backbone of betulinic acid derivatives 88 and 89; see Scheme 4.40 In order to enhance the cytotoxicity, compounds 88 and 89 were conjugated with Ophthalic ester at the C-3 position58 or with natural amino acids at the C-28 position.59 Following this, bromides 90−92 and 94 were prepared by treating the corresponding triterpenoids with NBS in CCl4. Finally, several triphenylphosphonium salts (38− 41) were obtained from bromides 90−92 and 94 by refluxing G



Review

Scheme 8. Synthesis of Phosphonium Analogues of Betulinic Acid (47−50)a

Reagents and conditions: (a) AcCl, DMAP, Py, THF, 20 °C; (b) tri(ethylene glycol) dibromide, K2CO3, DMF, 50 °C, 3 h; (c) tri(ethylene glycol) ditosylate, K2CO3, CH3CN, 70 °C, 7 h; (d) PPh3, CH3CN, reflux, Ar, 20−35 h; (e) Cl2CCOOH, DMAP, DCC, DCM, rt, 2−7 h. a

Scheme 9. Synthesis of Phosphonium Analogues of Betulinic Acid (53−55)a

a

Reagents and conditions: (a) Br(CH2)4COOH, DMAP, EDC, DCM, rt; (b) PPh3, CH3CN, reflux; (c) Mel or EtBr, K2CO3, DMF, rt.

nium salts (42−46, 51, 52) was based on reaction with an excess of triphenylphosphine under reflux in CH3CN. The above-mentioned method was used to modify position C-28 of the betulinic acid analogue 104, containing a 1,2-enone moiety (Scheme 7), as described in detail by Spivak et al. in 2017.40 The synthesis of triphenylphosphonium derivatives of betulinic acid (47−50) containing a triethylene glycol spacer at the C-28 position has also been described in the literature. A triethylene glycol linker can be introduced by the reaction of 2 with tri(ethylene glycol) dibromide or ditosylate in the presence

The methodology shown in Scheme 6 allows synthesizing both phosphonium lupane triterpenoid derivatives (42−46) and bisphosphonium salts (51 and 52) modified at positions C28 and C-30, with good yields.40 In order to modify the position C-28 by an ester bond, betulinic acid (2) was treated with dibromoalkanes in dimethylformamide (DMF) in the presence of K2CO3. A 10-fold excess of dibromoalkanes avoided the formation of dimers. Moreover, the resulting bromides (98− 103) reacted easily with NBS, which can be useful for the double functionalization of betulinic acid (2) at both positions C-28 and C-30. The transformation of bromides 98−103 to phosphoH



Review

Table 3. Synthesis of Triphenylphosphonium Analogues of Betulin and Betulinic Acid: A Summary Table of the Crucial Steps reaction conditions modification(s) analogues of betulin

analogues of betulinic acid

no. C-30

23

C-28

24−26

C-3 and C-28

26−28 29−31

C-2 C-30

32−37 38−40 41

C-28

42−45 47, 49 48

C-28 and C-30

51, 52

C-30 and ring A

56

C-28 and ring A

57

C-3

53 54, 55

reagent

temp

NBS, CCl4 PPh3, CH3CN Br(CH2)nCOOH,a DCC, DMAP, DCM PPh3, CH3CN PPh3, CH3CN Br(CH2)nCOOH,a DCC, DMAP, DCM PPh3, CH3CN eight-stage synthesis NBS, CCl4 PPh3, CH3CN NBS, CCl4 PPh3, CH3CN Br(CH2)nBr,b K2CO3, CH3CN, DMF PPh3, CH3CN tri(ethylene glycol) dibromide, K2CO3, DMF PPh3, CH3CN tri(ethylene glycol) ditosylate, K2CO3, CH3CN PPh3, CH3CN Br(CH2)nBr,b K2CO3, CH3CN, DMF NBS, CCl4 PPh3, CH3CN NBS, CCl4 PPh3, CH3CN Br(CH2)4Br, K2CO3, CH3CN, DMF PPh3, CH3CN Br(CH2)4COOH, DMAP, EDC, DCM PPh3, CH3CN PPh3, CH3CN

Δ Δ rt Δ Δ rt Δ Δ Δ Δ Δ Δ Δ 50 °C Δ 70 °C Δ Δ rt Δ Δ Δ Δ Δ Δ Δ Δ

yield [%]

ref

60 92 85−95 95 15−37 50−85 80−90 21−94 60−72 66−90 79 70 60−75 62−80 49−61 69−70 60 91 60 54−76 73−75 68 80 60 90 50 28 37−52

51, 55 41 62 41 51, 55 41, 51

40, 61

40

40 40 62

a

n = 1, 3, 4. bn = 4, 8.

Table 4. IC50 (μM) of Compounds 24−26 and 29−31 for Selected Human Cell Lines (in Vitro, MTT Assay)41 IC50, μM no.

PC-3

MCF-7

MCF-7/Vinb

HSF

1 24 25 26 29 30 31 doxorubicin vinblastine

148.6 ± 13.1 6.8 ± 1.9 0.16 ± 0.02 0.12 ± 0.01 6.9 ± 0.76 0.54 ± 0.11 0.47 ± 0.05

227.0 ± 19.4 >10 0.43 ± 0.13 0.70 ± 0.24 8.7 ± 1.5 0.35 ± 0.05 0.48 ± 0.07 0.15 ± 0.02 0.50 ± 0.02

233.4 ± 13.3 >10 0.045 ± 0.01 0.75 ± 0.12 4.3 ± 0.66 0.88 ± 0.22 0.84 ± 0.17 0.30 ± 0.05 9.2 ± 1.1

164.4 ± 12.3 9.9 ± 0.93 2.1 ± 0.57 1.5 ± 0.16 5.6 ± 0.46 0.86 ± 0.16 0.76 ± 0.23

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of K2CO3 at an elevated temperature. This functionalization may be preceded by an acylation step of the OH group at the C-3 position. The resulting bromides or tosylates (106−108) reacted with PPh3 in CH3CN (reflux) under argon to give the expected phosphonium salts (47−49) with good or very good yields (Scheme 8).61 The triphenylphosphonium moiety was also linked at the C-3 position of betulinic acid derivatives. The synthesis involved esterification of 2 with ω-bromoalkanoic acid, followed by the nucleophilic substitution of the bromides obtained (109−111) under standard conditions (Scheme 9).62 A combined summary of the various approaches discussed above for the synthesis of triphenylphosphonium analogues of betulin and betulinic acid is presented in Table 3.

BIOLOGICAL ACTIVITY OF THE TRIPHENYLPHOSPHONIUM ANALOGUES OF BETULIN AND BETULINIC ACID

Biological properties of betulin (1) and betulinic acid (2), in particular, pharmaceutical activities, have been the subject of numerous studies. However, their further development as potential antitumor drugs is conditional on overcoming such limitations as low bioavailability, poor aqueous solubility, and limited intracellular accumulation. In the years 2013−2017, cytotoxic and antiparasitic properties of a number of semisynthetic lupane triterpenoid analogues were described in which the parent skeleton (1 and 2) was modified by introducing the triphenylphosphonium group at the C-2, C-3, C-28, and C-30 positions (Charts 3 and 4). I



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Table 5. IC50 (μM) of Compounds 26−28 and 53−55 for Selected Cancer Cell Lines: K562, HL-60, ECA-109, A549, and HepG262 IC50, μM no.

K562

HL-60

ECA-109

A549

HepG2

HL-7702

1 26 27 28 2 53 54 55

12.9 ± 1.4 0.57 ± 0.03 0.81 ± 0.07 0.62 ± 0.06 22.0 ± 1.2 1.2 ± 0.12 3.6 ± 0.41 4.0 ± 0.91

30.9 ± 0.52 0.60 ± 0.03 0.71 ± 0.06 0.58 ± 0.05 18.5 ± 1.1 0.32 ± 0.09 3.1 ± 0.49 2.8 ± 0.21

26.6 ± 2.6 0.78 ± 0.08 0.95 ± 0.12 1.1 ± 0.07 28.1 ± 3.3 0.92 ± 0.11 4.8 ± 0.13 6.0 ± 0.26

18.2 ± 0.46 0.61 ± 0.07 1.1 ± 0.03 0.83 ± 0.20 17.1 ± 0.25 1.3 ± 0.12 5.0 ± 0.57 4.8 ± 0.05

10.7 ± 0.06 1.1 ± 0.05 1.1 ± 0.05 2.2 ± 0.46 25.3 ± 3.8 1.1 ± 0.02 4.4 ± 0.39 4.5 ± 0.38

26.8 ± 2.3 6.6 ± 0.52 8.3 ± 0.36 7.2 ± 2.4 35.1 ± 3.1 4.9 ± 0.92 6.3 ± 1.1 7.9 ± 1.6

Figure 1. Cytotoxic activity of compound 26 against selected cancer cell lines.

antiproliferative activity toward MCF-7 (IC50 = 0.045 μM; see Table 4) and compounds 26 (a butylene linker) toward prostate adenocarcinoma cell lines (PC-3; IC50 = 0.12 μM; see Table 4). The activity of triterpenoids with two triphenylphosphonium groups (30 and 31) is not significantly different from compounds with one group (24−26). Only in the case of human skin fibroblast cells (HSF) do analogues (29−31) show lower IC50 values compared to compounds (24−26). Studies on the cytotoxicity of other triphenylphosphonium derivatives of lupane-type pentacyclic triterpenoids were performed in 2017 by Ye et al. IC50 values for six compounds, including betulin derivatives (26−28, modification at the C-28 position) and betulinic acid derivatives (53−55, modification at the C-3 position), against five selected human cancer cell lines chronic myeloid leukemia cells (K562), human promyelocytic leukemia (HL-60), esophageal squamous carcinoma (ECA109), human alveolar adenocarcinoma cells (A549), and human hepatoblastoma (HepG2)and one normal human hepatocyte cell line (HL-7702) were determined. In all cases, IC50 values were found to be significantly lower than for the parent compounds (1 and 2).62 The IC50 values of compounds 26−28 and 53−55 for cancer and normal human hepatocyte cell lines indicated that these analogues exhibit some selectivity to tumor cells and more potent cytotoxic effects against them compared with the parent structure (1; see Table 5). In addition, no activity of 4carboxybutyltriphenylphosphonium bromide toward the cell lines examined confirmed that the presence of the triphenylphosphonium group is not sufficient to effect high cytotoxicity. The entire structure of triterpenoids with the triphenylphosphonium moiety plays an important role.62

Cytotoxic Activity of Triphenylphosphonium Derivatives of Betulin and Betulinic Acid. Among all of the interesting properties of lupane-type pentacyclic triterpenoids and their synthetic analogues, the most attention is paid to their potential antitumor activities. In the case of triphenylphosphonium derivatives, the mechanism of biological action involves targeting the mitochondrial apoptotic pathway, promoted by the presence of a positively charged, lipophilic TPP+ group. Moreover, these compounds, while inducing apoptosis of cancer cells, show low toxicity to normal cells.2 In 2017, Tsepaeva et al. described biologically active triphenylphosphonium derivatives of betulin in which the native scaffold is modified at the 28- or 3- and 28-positions. A series of newly synthesized compounds 24−26 and 29−31 were evaluated for cytotoxic effects against prostate adenocarcinoma (PC-3), human breast carcinoma (MCF-7), vinblastineresistant human breast cancer (MCF-7/Vinb), and human skin fibroblast (HSF) cells. The results obtained as IC50 values (half-maximal inhibitory concentrations) are summarized in Table 4. A detailed correlation analysis between structure and activity confirmed the crucial role of the TPP+ group on the biological properties. In general, cytotoxic activity of the conjugates of betulin with TPP+ is much higher in comparison with the native triterpenoids and increases as follows: 24 ∼ 29 < 26 ∼ 30 ∼ 31 < 25.41 The second factor affecting the activity of tested compounds is the length of the alkyl linker in the ester group that connects the TPP+ moiety to the triterpenoid scaffold. The presence of a methylene linker does not increase the biological activity, in contrast to the propylene and butylene ones. For example, compound 25 (a propylene linker) shows the highest J



Review

positions by a carbon−carbon or ester bond. Up to now, methods for the synthesis of over 25 such derivatives have been developed. Moreover, many compounds exhibit some selectivity to tumor cells and higher cytotoxicity in comparison with the parent structures (1 and 2). In some cases, the antiproliferative activity is particularly high and the spectrum of action is very broad (betulin modification at C-28 by −OCO(CH2)4-TPP+ exhibits cytotoxic effect toward PC-3 (IC50 = 0.12 μM), K-562 (IC50 = 0.57 μM), HL-60 (IC50 = 0.60 μM), A-549 (IC50 = 0.61 μM), MCF-7 (IC50 = 0.70 μM), and ECA-109 (IC50 = 0.78 μM)). Furthermore, easy access to the parent structures, relatively simple reaction procedures, and high yields should facilitate research to expand the pool of compounds available for testing as potential antitumor agents. The results of the structure−activity relationship presented by several research groups revealed a significant influence of the TPP+ group on the biological properties of phosphonium derivatives of betulin and betulinic acid. High cytotoxic activity is related to their accumulation in the mitochondria. In this context, lupane triterpenoids with triphenylphosphonium substituents can be considered as mitochondrial targeting agents, promising a new therapeutic strategy for the treatment of cancer.

Among all of the above-described triphenylphosphonium derivatives of lupane-type pentacyclic triterpenoids, compound 26 deserves special attention because of its high cytotoxic activity and selectivity against a wide range of cancer cells (Figure 1). Cytotoxic activity of phosphonium derivatives of betulinic acid was also tested against other cancer cell lines such as a murine tumor Ehrlich ascites carcinoma (P-815) or human breast carcinoma (MCF-7) and esophageal squamous carcinoma (ECA-109). To this end, a series of betulinic analogues with one or two triphenylphosphonium moieties at the C-2, C-28, or C-30 positions were synthesized. In some cases, the ring A was also modified to contain the 1,2-enone moiety. All examined salts display cytotoxic activity, much higher than that of betulinic acid (32−37, 43−52, IC50 = 1.2−2.5 μM in Ehrlich cells ECA109, IC50 = 0.97−1.1 μM in P-815 cells, IC50 = 0.70−2.3 μM in MCF-7 cells, and IC50 = 0.74−1.3 μM in TET21N cells). The key role of the triphenylphosphonium group in the increase of biological activity has also been confirmed in these cases.40,61 However, the presence of various functional groups (OAc-3β, OAc-3α, OH-3β, OH-3α, 3β-O-phthalic ester, 3β-dichloroacetate, COOMe-28, COOH-28) at different positions in the triterpene structure and even the introduction of a double bond to the A ring do not significantly affect the cytotoxicity. The exception was compound 37, COOBn-28, for which an increase in IC50 values was observed for two cell lines: ECA-109 and MCF-7 (IC50 = 4.8 and 4.9 μM, respectively). It seems that the type and length of the spacer linking the triphenylphosphonium group with the betulinic acid skeleton have the relatively greatest influence on the cytotoxic activity. An excellent example is compound 49, containing a hydrophilic triethylene glycol bridge, which showed the highest activity against two cell lines: MCF-7 and TET21N (IC50 = 0.70 and 0.74 μM, respectively). Antischistosomal Activity of Triphenylphosphonium Derivatives of Betulin and Betulinic Acid. One of the greatest problems caused by parasitic infections in the contemporary world is schistosomiasis, a disease caused by the flukes from the species Schistosoma mansoni. The urgent need for new biologically active substances toward flukes is associated with limited access to therapeutic agents for this disease as well as the ability of the parasites to become resistant to existing drugs. In 2014, Spivak et al., inspired by the strong effect of triphenylphosphonium cation on Schistosoma viability, started in vitro and then in vivo studies using triphenylphosphonium derivatives of betulin and betulinic acid against these types of parasites. Tests showed twice higher cytotoxicity to schistosomiasis of betulin and betulinic acid analogues with a triphenylphosphonium group at the C-30 position (23: IC50 = 0.64 μg/mL, 38: IC50 = 0.76 μg/mL) compared to the analogues modified at the C-2 position (32: IC50 = 1.5 μg/mL, 33: IC50 = 1.4 μg/mL).51

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AUTHOR INFORMATION

Corresponding Author

*Phone (M. Grymel): +48 032 237 1873. Fax: +48 032 237 20 94. E-mail: [email protected]. ORCID

Mirosława Grymel: 0000-0003-4649-1194 Jakub Adamek: 0000-0002-2960-6247 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The preparation of this review was supported by grants from the National Science Center, Poland (NCN), under grant no. 2015/ 19/D/ST5/00733.

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REFERENCES

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CONCLUSIONS Betulin (1) and betulinic acid (2) display a broad spectrum of biological activities, but a central problem is their poor solubility in bodily fluids. A proper modification of the parent structure not only improves physical properties, including solubility in bodily fluids, but also targets specific biological activity. One of the most promising transformations is the introduction of one or two triphenylphosphonium groups at selected positions on the parent structures. Usually, the triterpenoid molecules are linked to a triphenylphosphonium moiety at the C-2, C-30, or C-28 K



Review

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Triphenylphosphonium Analogues of Betulin and Betulinic Acid with

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