Amino-Acid-Conjugated Natural Compounds: Aims, Designs and Results

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Citation:Arifian, H.; Maharani, R.;

Megantara, S.; Gazzali, A.M.;

Muchtaridi, M.

Amino-Acid-Conjugated Natural Compounds: Aims, Designs and Results. Molecules 2022, 27, 7631.

https://doi.org/10.3390/

molecules27217631

Academic Editors: Claudiu N. Lungu and Ionel Mangalagiu

Received: 28 September 2022 Accepted: 3 November 2022 Published: 7 November 2022

Publisher’s Note:MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations.

Licensee MDPI, Basel, Switzerland.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

molecules

Review

Amino-Acid-Conjugated Natural Compounds: Aims, Designs and Results

Hanggara Arifian^1,2, Rani Maharani^3,4, Sandra Megantara^1,3, Amirah Mohd Gazzali⁵ and Muchtaridi Muchtaridi^1,3,*

1 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Sumedang 45363, Indonesia

2 Department of Pharmacochemistry, Faculty of Pharmacy, Universitas Mulawarman, Samarinda 75119, Indonesia

3 Research Collaboration Centre for Theranostic Radiopharmaceuticals, National Research and Innovation Agency (BRIN), Jakarta 10340, Indonesia

4 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor 45363, Indonesia

5 School of Pharmaceutical Sciences, Universiti Saisn Malaysia, Penang 11800, Malaysia

* Correspondence: [email protected]

Abstract:Protein is one of the essential macronutrients required by all living things. The breakdown of protein produces monomers known as amino acids. The concept of conjugating natural compounds with amino acids for therapeutic applications emerged from the fact that amino acids are important building blocks of life and are abundantly available; thus, a greater shift can result in structural modification, since amino acids contain a variety of sidechains. This review discusses the data available on amino acid–natural compound conjugates that were reported with respect to their backgrounds, the synthetic approach and their bioactivity. Several amino acid–natural compound conjugates have shown enhanced pharmacokinetic characteristics, including absorption and distribution properties, reduced toxicity and increased physiological effects. This approach could offer a potentially effective system of drug discovery that can enable the development of pharmacologically active and pharmacokinetically acceptable molecules.

Keywords:natural compounds; amino acid; conjugation; chemical synthesis

1. Introduction

With the passage of time, the need for more effective therapeutics is increasing, with more diseases being discovered and more health-related problems being understood through advanced research and studies [1]. Often, natural resources from plants, ani- mals and microorganisms are described as suitable candidates [2], and many established medicines originating from natural products were inspired by the chemical synthesis of biologically active materials from natural products. The molecular framework of natural compound synthesis is recognised as an abundant resource for medicinal chemistry and drug development [3–5].

Not all natural products are unsuitable for use as medications [6–8]. Some of them may be considered as potentially useful compounds if they are proven to have certain favourable pharmacological and pharmacokinetic characteristics [9,10]. Many natural compounds, however, require structural modifications through chemical synthesis approaches to meet the required criteria of effective and safe therapeutics [11–13].

Conjugation with amino acids is one of the means of remodelling the structure of natural compounds [14]. Amino acids are chosen based on the premise that they are the most quintessential monomers of living systems [15,16]. Amino acids are also the primary construction blocks for proteins, as well as the substance that sustains biological activities,

Molecules 2022, 27, 7631. https://doi.org/10.3390/molecules27217631 https://www.mdpi.com/journal/molecules

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Molecules 2022, 27, 7631 2 of 34

in addition to playing integral roles in living organisms [17,18]. Moreover, as amino acids have various sidechains, structural alteration is attainable, with a broader shift [19].

The manipulation of amino acid is relatively simple, with a myriad of possible targeted pharmacological activities. To date, several amino acid–natural compound conjugates have been reported that involve curcumin, astaxanthin and quercetin, among others [20–22]. The conjugations aimed to enhance the pharmacological activities, lessen the toxicity, improve the target specificity and increase absorption via peptide transporters [23–25].

This review examines the available literature on amino acid–natural compound conjugations. The method of conjugation is discussed and analysed to gain insightful information on the production of amino acid–natural compound conjugates for future medical applications.

2. Natural Compounds Conjugated with Amino Acids

Various natural compounds have undergone structural modifications and been evaluated as potential therapeutics. This review explores all the relevant studies on this subject, with a specific interest in semisynthetic modifications of the natural compounds. The purpose, synthetic strategies and biological outcomes are also discussed.

2.1. Alkaloids

Alkaloids have a wide range of structural variations. The presence of a basic nitrogen atom is the common denominator. The nitrogen can be a primary, secondary or tertiary amine [26]. Analgesic (codeine), central nervous system depressant (morphine), anti-hypotensive (ephedrine), anticholinergic (atropine), antiemetic (scopolamine) and antimalarial (quinine) products are among the examples of natural-based pharmacological agents in this class [27].

However, alkaloids have unfavourable physical and chemical characteristics, such as a low solubility, low stability at physiological pH, low oral absorption and low overall bioavailability, in addition to having a quick clearance from the body. These problems reduce the efficacies of alkaloids [28,29]. To solve these issues, a number of alkaloid derivatives conjugated with amino acids have been developed, such as piperine, camptothecin and quinine.

2.1.1. Piperine

Piperine (Figure1) is an alkaloid found in various Piperaceae family, including Piper nigrum, Piper longum, Piper chaba, Piper guineense and Piper sarmentosum [30,31]. Piperine is responsible for the peculiar biting feeling associated with black pepper. Black pepper is traditionally used in therapeutics and preservatives and for its scent, in addition to being widely used in human diets [32,33].

Molecules 2022, 27, x FOR PEER REVIEW 2 of 35

construction blocks for proteins, as well as the substance that sustains biological activities, in addition to playing integral roles in living organisms [17,18]. Moreover, as amino acids have various sidechains, structural alteration is attainable, with a broader shift [19].

The manipulation of amino acid is relatively simple, with a myriad of possible tar- geted pharmacological activities. To date, several amino acid–natural compound conju- gates have been reported that involve curcumin, astaxanthin and quercetin, among others [20–22]. The conjugations aimed to enhance the pharmacological activities, lessen the tox- icity, improve the target specificity and increase absorption via peptide transporters [23–

25].

This review examines the available literature on amino acid–natural compound con- jugations. The method of conjugation is discussed and analysed to gain insightful infor- mation on the production of amino acid–natural compound conjugates for future medical applications.

2. Natural Compounds Conjugated with Amino Acids

Various natural compounds have undergone structural modifications and been eval- uated as potential therapeutics. This review explores all the relevant studies on this sub- ject, with a specific interest in semisynthetic modifications of the natural compounds. The purpose, synthetic strategies and biological outcomes are also discussed.

2.1. Alkaloids

Alkaloids have a wide range of structural variations. The presence of a basic nitrogen atom is the common denominator. The nitrogen can be a primary, secondary or tertiary amine [26]. Analgesic (codeine), central nervous system depressant (morphine), anti-hy- potensive (ephedrine), anticholinergic (atropine), antiemetic (scopolamine) and antima- larial (quinine) products are among the examples of natural-based pharmacological agents in this class [27].

However, alkaloids have unfavourable physical and chemical characteristics, such as a low solubility, low stability at physiological pH, low oral absorption and low overall bioavailability, in addition to having a quick clearance from the body. These problems reduce the efficacies of alkaloids [28,29]. To solve these issues, a number of alkaloid de- rivatives conjugated with amino acids have been developed, such as piperine, camptoth- ecin and quinine.

2.1.1. Piperine

Piperine (Figure 1) is an alkaloid found in various Piperaceae family, including Piper nigrum, Piper longum, Piper chaba, Piper guineense and Piper sarmentosum [30,31]. Piperine is responsible for the peculiar biting feeling associated with black pepper. Black pepper is traditionally used in therapeutics and preservatives and for its scent, in addition to being widely used in human diets [32,33].

Figure 1. Piperine.

The synthesis of amino acid–piperine conjugates has been described as having the potential to produce antileishmanial [34] and anticancer agents [35]. As piperine is natu- rally abundant, this compound is a favourable choice for manipulation via this approach.

Furthermore, the chemical reactions and processes involved are simple and straightfor- ward, making the scaled-up manufacturing of piperine derivatives highly possible [35].

Figure 1.Piperine.

The synthesis of amino acid–piperine conjugates has been described as having the potential to produce antileishmanial [34] and anticancer agents [35]. As piperine is natu- rally abundant, this compound is a favourable choice for manipulation via this approach.

Furthermore, the chemical reactions and processes involved are simple and straightforward, making the scaled-up manufacturing of piperine derivatives highly possible [35].

The inhibitory activity and the mode of action of piperine against Leishmania donovani [36] can be enhanced if the piperine is in the form of oil-in-water emulsion [37] or mannose-coated liposomes [38]. In addition, amino acid esters have also demonstrated

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Molecules 2022, 27, 7631 3 of 34

inhibitory actions against Leishmania amazonensis and Leishmania mexicana following their accumulation in the phagolysosomes of these parasites [39].

The synthesis of amino acid–piperine conjugate was divided into three series (Scheme1).

First of all, piperine was converted to piperic acid via the hydrolysis of its amide bond.

The carboxyl group in piperic acid provides a place for the formation of a covalent bond with the amino acids through the formation of a new amide bond. Piperic acid was then conjugated to a protected amino acid using menthane-sulfonyl chloride, CH3SO2Cl, in CH₂Cl₂at 0 ^◦C to produce piperoyl–amino acid methyl ester conjugates. The desired piperoyl–amino acid methyl ester conjugates (3–7) were reported to be in the range of 40–75% yields. Later, deprotection was conducted using Al2O3 in microwave-assisted solid phase until the ester group was converted into free carboxyl groups (8–12) with 70–80% yields. The other analogues are the saturated derivatives of the conjugate of the first series. The approach used for the synthesis of these analogues was carried out in two ways, namely, (i) the direct hydrogenation of the first series’ conjugate, and (ii) the initial hydrogenation of piperic acid to give tetrahydropiperic acid, followed by the conjugation of the saturated piperic acid with amino acid methyl esters [34] (Scheme2).

Molecules 2022, 27, x FOR PEER REVIEW 3 of 35

The inhibitory activity and the mode of action of piperine against Leishmania donovani [36] can be enhanced if the piperine is in the form of oil-in-water emulsion [37] or man- nose-coated liposomes [38]. In addition, amino acid esters have also demonstrated inhib- itory actions against Leishmania amazonensis and Leishmania mexicana following their accu- mulation in the phagolysosomes of these parasites [39].

The synthesis of amino acid–piperine conjugate was divided into three series (Scheme 1). First of all, piperine was converted to piperic acid via the hydrolysis of its amide bond. The carboxyl group in piperic acid provides a place for the formation of a covalent bond with the amino acids through the formation of a new amide bond. Piperic acid was then conjugated to a protected amino acid using menthane-sulfonyl chloride, CH

³

SO

²

Cl, in CH

²

Cl

²

at 0 °C to produce piperoyl–amino acid methyl ester conjugates. The desired piperoyl–amino acid methyl ester conjugates (3–7) were reported to be in the range of 40–75% yields. Later, deprotection was conducted using Al

²

O

³

in microwave- assisted solid phase until the ester group was converted into free carboxyl groups (8–12) with 70–80% yields. The other analogues are the saturated derivatives of the conjugate of the first series. The approach used for the synthesis of these analogues was carried out in two ways, namely, (i) the direct hydrogenation of the first series’ conjugate, and (ii) the initial hydrogenation of piperic acid to give tetrahydropiperic acid, followed by the con- jugation of the saturated piperic acid with amino acid methyl esters [34] (Scheme 2).

Scheme 1. Reagents and conditions: (a) 20% KOH, CH3OH, 80°C, 3 days, 88%; (b) CH3SO2Cl, Et3N, DCM, 0 °C, 30 min, 85%; (c) amino acid methyl ester, Et3N, DCM, RT, 40–75%; (d) KF-Al2O3 (40%), microwave, 1000 W, 3–4 min, 70–80%. R in order: L-phenylalanine; L-tyrosine; L-valine; L-methionine; L-tryptophan.

Scheme 2. Reagents and conditions: type i (a) 5% Pd/C, H2 (40 Psi), MeOH, 30 min; type ii (b) 5%

Pd/C, H2 (40 Psi), MeOH, 30 min; (c) CH3SO2Cl, Et3N, DCM, 0 °C, 30 min, 85%; (d) amino acid methyl ester, Et3N, DCM, RT.

All of the conjugates showed potential against both the amastigote and the pro- mastigote forms of the parasite (Table 1). The IC

⁵⁰

values of piperine against the amastigotes and promastigotes were 0.7 and 2.5 mM, respectively. When compared with piperine or amino acid esters, the piperoyl–amino acid ester conjugates (3–7) exhibited a significant increase in their activity.

Table 1. Antileishmanial activity and cytotoxicity of piperoyl–amino acid conjugates. (1) Piperine;

(2) piperic acid; (3) piperoyl-L-phenylalanine methyl ester; (4) piperoyl-L-tyrosine methyl ester; (5) piperoyl-L-valine methyl ester; (6) piperoyl-L-methionine methyl ester; (7) piperoyl-L-tryptophan methyl ester; (8) piperoyl-L-phenylalanine; (9) piperoyl-L-tyrosine; (10) piperoyl-L-valine; (11) pip- eroyl-L-methionine; (12) piperoyl-L-tryptophan; (13) tetrahydropiperoyl-L-phenylalanine methyl Scheme 1.Reagents and conditions: (a) 20% KOH, CH3OH, 80^◦C, 3 days, 88%; (b) CH3SO2Cl, Et3N, DCM, 0^◦C, 30 min, 85%; (c) amino acid methyl ester, Et₃N, DCM, RT, 40–75%; (d) KF-Al₂O₃(40%), microwave, 1000 W, 3–4 min, 70–80%. R in order: L-phenylalanine; L-tyrosine; L-valine; L-methionine;

L-tryptophan.

The inhibitory activity and the mode of action of piperine against Leishmania donovani [36] can be enhanced if the piperine is in the form of oil-in-water emulsion [37] or man- nose-coated liposomes [38]. In addition, amino acid esters have also demonstrated inhib- itory actions against Leishmania amazonensis and Leishmania mexicana following their accu- mulation in the phagolysosomes of these parasites [39].

The synthesis of amino acid–piperine conjugate was divided into three series (Scheme 1). First of all, piperine was converted to piperic acid via the hydrolysis of its amide bond. The carboxyl group in piperic acid provides a place for the formation of a covalent bond with the amino acids through the formation of a new amide bond. Piperic acid was then conjugated to a protected amino acid using menthane-sulfonyl chloride, CH

3

SO

2

Cl, in CH

2

Cl

2

at 0 °C to produce piperoyl–amino acid methyl ester conjugates. The desired piperoyl–amino acid methyl ester conjugates (3–7) were reported to be in the range of 40–75% yields. Later, deprotection was conducted using Al

2

O

3

in microwave- assisted solid phase until the ester group was converted into free carboxyl groups (8–12) with 70–80% yields. The other analogues are the saturated derivatives of the conjugate of the first series. The approach used for the synthesis of these analogues was carried out in two ways, namely, (i) the direct hydrogenation of the first series’ conjugate, and (ii) the initial hydrogenation of piperic acid to give tetrahydropiperic acid, followed by the con- jugation of the saturated piperic acid with amino acid methyl esters [34] (Scheme 2).

Scheme 1. Reagents and conditions: (a) 20% KOH, CH³OH, 80°C, 3 days, 88%; (b) CH³SO²Cl, Et³N, DCM, 0 °C, 30 min, 85%; (c) amino acid methyl ester, Et³N, DCM, RT, 40–75%; (d) KF-Al²O³ (40%), microwave, 1000 W, 3–4 min, 70–80%. R in order: L-phenylalanine; L-tyrosine; L-valine; L-methionine; L-tryptophan.

Scheme 2. Reagents and conditions: type i (a) 5% Pd/C, H² (40 Psi), MeOH, 30 min; type ii (b) 5%

Pd/C, H² (40 Psi), MeOH, 30 min; (c) CH³SO²Cl, Et³N, DCM, 0 °C, 30 min, 85%; (d) amino acid me- thyl ester, Et3N, DCM, RT.

All of the conjugates showed potential against both the amastigote and the pro- mastigote forms of the parasite (Table 1). The IC

50

values of piperine against the amastigotes and promastigotes were 0.7 and 2.5 mM, respectively. When compared with piperine or amino acid esters, the piperoyl–amino acid ester conjugates (3–7) exhibited a significant increase in their activity.

Table 1. Antileishmanial activity and cytotoxicity of piperoyl–amino acid conjugates. (1) Piperine;

(2) piperic acid; (3) piperoyl-L-phenylalanine methyl ester; (4) piperoyl-L-tyrosine methyl ester; (5) piperoyl-L-valine methyl ester; (6) piperoyl-L-methionine methyl ester; (7) piperoyl-L-tryptophan methyl ester; (8) piperoyl-L-phenylalanine; (9) piperoyl-L-tyrosine; (10) piperoyl-L-valine; (11) pip- eroyl-L-methionine; (12) piperoyl-L-tryptophan; (13) tetrahydropiperoyl-L-phenylalanine methyl Scheme 2. Reagents and conditions: type i (a) 5% Pd/C, H₂ (40 Psi), MeOH, 30 min; type ii (b) 5% Pd/C, H2(40 Psi), MeOH, 30 min; (c) CH3SO2Cl, Et3N, DCM, 0^◦C, 30 min, 85%; (d) amino acid methyl ester, Et₃N, DCM, RT.

All of the conjugates showed potential against both the amastigote and the promastig- ote forms of the parasite (Table1). The IC₅₀values of piperine against the amastigotes and promastigotes were 0.7 and 2.5 mM, respectively. When compared with piperine or amino acid esters, the piperoyl–amino acid ester conjugates (3–7) exhibited a significant increase in their activity.

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Table 1.Antileishmanial activity and cytotoxicity of piperoyl–amino acid conjugates. (1) Piperine;

(2) piperic acid; (3) piperoyl-L-phenylalanine methyl ester; (4) piperoyl-L-tyrosine methyl ester;

(5) piperoyl-L-valine methyl ester; (6) piperoyl-L-methionine methyl ester; (7) piperoyl-L- tryptophan methyl ester; (8) piperoyl-L-phenylalanine; (9) piperoyl-L-tyrosine; (10) piperoyl-L-valine;

(11) piperoyl-L-methionine; (12) piperoyl-L-tryptophan; (13) tetrahydropiperoyl-L-phenylalanine methyl ester; (14) tetrahydropiperoyl-L-tyrosine methyl ester; (15) tetrahydropiperoyl-L-valine methyl ester; (16) tetrahydropiperoyl-L-methionine methyl ester; (17) tetrahydropiperoyl-L- tryptophan methyl ester.

Compound IC₅₀(mM)

Amastigotes Promastigotes

1 0.75 2.56

2 0.39 1.76

3 0.19 0.83

4 0.21 0.79

5 0.07 0.88

6 0.17 0.82

7 0.12 0.86

8 0.26 0.93

9 0.25 0.76

10 0.31 1.03

11 0.24 1.31

12 0.24 1.03

13 0.21 0.67

14 0.20 0.54

15 0.24 0.72

16 0.22 0.61

17 0.18 0.47

The combination of piperic acid and valine methyl ester (Figure2) was shown to be the most efficient against amastigotes, with an IC50value of 0.07 mM (compound 5) [34]. The effectiveness of valine may be related to the strong need of valine to produce NADH in the procyclic phase of Leishmania donovani [40]. On the other hand, the antileishmanial activity against amastigotes was reduced when the carboxyl group of amino acid functionality was deprotected in compounds 8–12 (Table1) compared to compounds 3–7 (with the protected amino acid group). The antileishmanial activity of the conjugates 13–17 against the amastigotes decreased when the piperine subunit’s conjugated double bonds were reduced. However, the antileishmanial activity of the conjugates against promastigotes was boosted. The most active compound against the promastigotes was tetrahydropiperoyl tryptophan methyl ester (compound 17), with an IC₅₀value of 0.47 mM.

Figure 2. (a) Piperoyl-L-valine methyl ester; (b) tetrahydropiperoyl-L-phenylalanine methyl ester;

(c) tetrahydropiperoyl-L-tyrosine methyl ester; (d) tetrahydropiperoyl-L-valine methyl ester; (e) tet- rahydropiperoyl-L-methionine methyl ester; (f) tetrahydropiperoyl-L-tryptophan methyl ester.

2.1.2. Camptothecin

Camptothecin (Figure 3), a quinolone type of alkaloid, is a potent antitumor agent isolated from Camptotheca acuminate [41,42]. However, it has a low solubility and some adverse effects [43,44]. One of the strategies used to overcome this problem is the conjugation of camptothecin with poly-α-L-glutamic acid [45].

Figure 3. Camptothecin.

The conjugation of 50 kDa poly-R-(L-glutamic acid) (PG) and L-camptothecin was achieved by means of the esterification of the hydroxyl group at the C-20 position with amino acid as a linker (Scheme 3). Amino acid was added to a solution of camptothecin, DMAP and DIPC in DMF at room temperature to form amino acid–camptothecin. Then, 50% TFA was used to deprotect the amino acid linker. Thereafter, an amino-acid-linked camptothecin solution in anhydrous DMF was added to the poly-R-(L-glutamic acid) solution. The DMAP and DIPC in DMF were gradually added to the combination and chilled in an ice bath. The mixture was stirred for two days at room temperature [45].

Figure 2. Cont.

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2.1.2. Camptothecin

Camptothecin (Figure 3), a quinolone type of alkaloid, is a potent antitumor agent isolated from Camptotheca acuminate [41,42]. However, it has a low solubility and some adverse effects [43,44]. One of the strategies used to overcome this problem is the conjugation of camptothecin with poly-α-L-glutamic acid [45].

The conjugation of 50 kDa poly-R-(L-glutamic acid) (PG) and L-camptothecin was achieved by means of the esterification of the hydroxyl group at the C-20 position with amino acid as a linker (Scheme 3). Amino acid was added to a solution of camptothecin, DMAP and DIPC in DMF at room temperature to form amino acid–camptothecin. Then, 50% TFA was used to deprotect the amino acid linker. Thereafter, an amino-acid-linked camptothecin solution in anhydrous DMF was added to the poly-R-(L-glutamic acid) solution. The DMAP and DIPC in DMF were gradually added to the combination and chilled in an ice bath. The mixture was stirred for two days at room temperature [45].

Figure 2. (a) Piperoyl-L-valine methyl ester; (b) tetrahydropiperoyl-L-phenylalanine methyl es- ter; (c) tetrahydropiperoyl-L-tyrosine methyl ester; (d) tetrahydropiperoyl-L-valine methyl ester;

(e) tetrahydropiperoyl-L-methionine methyl ester; (f) tetrahydropiperoyl-L-tryptophan methyl ester.

2.1.2. Camptothecin

Camptothecin (Figure3), a quinolone type of alkaloid, is a potent antitumor agent isolated from Camptotheca acuminate [41,42]. However, it has a low solubility and some adverse effects [43,44]. One of the strategies used to overcome this problem is the conjugation of camptothecin with poly-α-L-glutamic acid [45].

2.1.2. Camptothecin

Camptothecin (Figure 3), a quinolone type of alkaloid, is a potent antitumor agent isolated from Camptotheca acuminate [41,42]. However, it has a low solubility and some adverse effects [43,44]. One of the strategies used to overcome this problem is the conju- gation of camptothecin with poly-α-L-glutamic acid [45].

The conjugation of 50 kDa poly-R-(L-glutamic acid) (PG) and L-camptothecin was achieved by means of the esterification of the hydroxyl group at the C-20 position with amino acid as a linker (Scheme 3). Amino acid was added to a solution of camptothecin, DMAP and DIPC in DMF at room temperature to form amino acid–camptothecin. Then, 50% TFA was used to deprotect the amino acid linker. Thereafter, an amino-acid-linked camptothecin solution in anhydrous DMF was added to the poly-R-(L-glutamic acid) so- lution. The DMAP and DIPC in DMF were gradually added to the combination and chilled in an ice bath. The mixture was stirred for two days at room temperature [45].

Figure 3.Camptothecin.

The conjugation of 50 kDa poly-R-(L-glutamic acid) (PG) and L-camptothecin was achieved by means of the esterification of the hydroxyl group at the C-20 position with amino acid as a linker (Scheme3). Amino acid was added to a solution of camptothecin, DMAP and DIPC in DMF at room temperature to form amino acid–camptothecin. Then, 50% TFA was used to deprotect the amino acid linker. Thereafter, an amino-acid-linked camptothecin solution in anhydrous DMF was added to the poly-R-(L-glutamic acid) solution. The DMAP and DIPC in DMF were gradually added to the combination and chilled in an ice bath. The mixture was stirred for two days at room temperature [45].

Scheme 3. Synthesis of the PG conjugates of S-camptothecin. Reagents and conditions: (a) amino acid linker (R), DIPC, DMAP, DMF; (b) 50% TFA-CH²Cl²; (c) poly-R-(L-glutamic acid) (PG), DIPC, DMAP, DMF. R is the following amino acids: conjugate no. (2): L-glycine; (3) L-alanine; (4) β-ala- nine; (5) 4-NH-butyryl; (6) 2-O-acetyl; (7) 4-O-butyryl; (8) γ-glutamic acid.

The in vivo activity of the conjugates was evaluated against B-16 melanomas (Table 2). When compared to a similar camptothecin dose level, poly-R-(L-glutamic acid)-gly- cine-camptothecin (Figure 4) was found to have the best antitumor efficacy. This conju- gate was able to suppress the growth of B-16 tumour cells following 48 h of treatment at a lower dose in comparison to the poly-R-(L-glutamic acid)-camptothecin and the other poly-R-(L-glutamic acid)-linker-camptothecin conjugates.

Figure 4. Poly-R-(L-glutamic acid)-L-glycine-camptothecin.

Table 2. Effects of poly-R-(L-glutamic acid) conjugates of camptothecin on the in vivo growth of subcutaneous B-16 melanomas. TGD: tumour growth delays. (1) Poly-R-(L-glutamic acid)-camp- tothecin; (2) poly-R-(L-glutamic acid)-glycine-camptothecin; (3) poly-R-(L-glutamic acid)-alanine- camptothecin; (4) poly-R-(L-glutamic acid)-(β-alanine)-camptothecin; (5) poly-R-(L-glutamic acid)- (4-NH-butyryl)-camptothecin; (6) poly-R-(L-glutamic acid)-(2-O-acetyl)-camptothecin; (7) poly-R- (L-glutamic acid)-(4-O-butyryl)-camptothecin; (8) poly-R-(L-glutamic acid)-(γ-glu)-camptothecin.

Compound Dose

(mg) B-16

TGD

(Days)

1

48 4

2

35 2

3

62 2

4

67 1

5

60 1

6

75 4

7

35 3

8

41 1

2.1.3. Quinine

Quinine (Figure 5) was isolated from Cinchona bark for the first time in France in 1820, and it was recognised as a major component of the bark [46,47]. Given that most antimalarial medications reveal parasite resistance within a few years after their introduc- tion to the market, quinine has a relatively stronger track record [48,49]. A low parasite resistance towards quinine has made the structure favourable for overcoming infections by this parasite [50].

Scheme 3.Synthesis of the PG conjugates of S-camptothecin. Reagents and conditions: (a) amino acid linker (R), DIPC, DMAP, DMF; (b) 50% TFA-CH₂Cl₂; (c) poly-R-(L-glutamic acid) (PG), DIPC, DMAP, DMF. R is the following amino acids: conjugate no. (2): L-glycine; (3) L-alanine; (4) β-alanine;

(5) 4-NH-butyryl; (6) 2-O-acetyl; (7) 4-O-butyryl; (8) γ-glutamic acid.

The in vivo activity of the conjugates was evaluated against B-16 melanomas (Table2).

When compared to a similar camptothecin dose level, poly-R-(L-glutamic acid)-glycine- camptothecin (Figure4) was found to have the best antitumor efficacy. This conjugate was able to suppress the growth of B-16 tumour cells following 48 h of treatment at a lower dose in comparison to the poly-R-(L-glutamic acid)-camptothecin and the other poly-R-(L-glutamic acid)-linker-camptothecin conjugates.

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Molecules 2022, 27, 7631 6 of 34

Table 2. Effects of poly-R-(L-glutamic acid) conjugates of camptothecin on the in vivo growth of subcutaneous B-16 melanomas. TGD: tumour growth delays. (1) Poly-R-(L-glutamic acid)- camptothecin; (2) poly-R-(L-glutamic acid)-glycine-camptothecin; (3) poly-R-(L-glutamic acid)- alanine-camptothecin; (4) poly-R-(L-glutamic acid)-(β-alanine)-camptothecin; (5) poly-R-(L-glutamic acid)-(4-NH-butyryl)-camptothecin; (6) poly-R-(L-glutamic acid)-(2-O-acetyl)-camptothecin; (7) poly- R-(L-glutamic acid)-(4-O-butyryl)-camptothecin; (8) poly-R-(L-glutamic acid)-(γ-glu)-camptothecin.

Compound Dose (mg) B-16 TGD (Days)

1 48 4

2 35 2

3 62 2

4 67 1

5 60 1

6 75 4

7 35 3

8 41 1

Scheme 3. Synthesis of the PG conjugates of S-camptothecin. Reagents and conditions: (a) amino acid linker (R), DIPC, DMAP, DMF; (b) 50% TFA-CH²Cl²; (c) poly-R-(L-glutamic acid) (PG), DIPC, DMAP, DMF. R is the following amino acids: conjugate no. (2): L-glycine; (3) L-alanine; (4) β-ala- nine; (5) 4-NH-butyryl; (6) 2-O-acetyl; (7) 4-O-butyryl; (8) γ-glutamic acid.

The in vivo activity of the conjugates was evaluated against B-16 melanomas (Table 2). When compared to a similar camptothecin dose level, poly-R-(L-glutamic acid)-gly- cine-camptothecin (Figure 4) was found to have the best antitumor efficacy. This conju- gate was able to suppress the growth of B-16 tumour cells following 48 h of treatment at a lower dose in comparison to the poly-R-(L-glutamic acid)-camptothecin and the other poly-R-(L-glutamic acid)-linker-camptothecin conjugates.

Figure 4. Poly-R-(L-glutamic acid)-L-glycine-camptothecin.

Table 2. Effects of poly-R-(L-glutamic acid) conjugates of camptothecin on the in vivo growth of subcutaneous B-16 melanomas. TGD: tumour growth delays. (1) Poly-R-(L-glutamic acid)-camp- tothecin; (2) poly-R-(L-glutamic acid)-glycine-camptothecin; (3) poly-R-(L-glutamic acid)-alanine- camptothecin; (4) poly-R-(L-glutamic acid)-(β-alanine)-camptothecin; (5) poly-R-(L-glutamic acid)- (4-NH-butyryl)-camptothecin; (6) poly-R-(L-glutamic acid)-(2-O-acetyl)-camptothecin; (7) poly-R- (L-glutamic acid)-(4-O-butyryl)-camptothecin; (8) poly-R-(L-glutamic acid)-(γ-glu)-camptothecin.

Compound Dose

(mg) B-16

TGD

(Days)

1

48 4

2

35 2

3

62 2

4

67 1

5

60 1

6

75 4

7

35 3

8

41 1

2.1.3. Quinine

Quinine (Figure 5) was isolated from Cinchona bark for the first time in France in 1820, and it was recognised as a major component of the bark [46,47]. Given that most antimalarial medications reveal parasite resistance within a few years after their introduc- tion to the market, quinine has a relatively stronger track record [48,49]. A low parasite resistance towards quinine has made the structure favourable for overcoming infections by this parasite [50].

Figure 4.Poly-R-(L-glutamic acid)-L-glycine-camptothecin.

2.1.3. Quinine

Quinine (Figure 5) was isolated from Cinchona bark for the first time in France in 1820, and it was recognised as a major component of the bark [46,47]. Given that most antimalarial medications reveal parasite resistance within a few years after their introduction to the market, quinine has a relatively stronger track record [48,49]. A low parasite resistance towards quinine has made the structure favourable for overcoming infections by this parasite [50].

Figure 5. Quinine.

Amino acid conjugates of quinine were synthesised to produce new compounds us- ing a different synthetic strategy from the development of the previous quinine deriva- tives, such as chloroquine. The conjugates’ synthesis also aimed to overcome the re- sistance problems, increase the drug delivery efficiency and improve the performance of quinine as an antimalarial agent [51,52].

The synthetic strategy used was to form an ester bond between the quinine and amino acid or peptide. The amino acid or peptide was selected based on the difference in the amino acid polarity. Quinine was O-acylated with N-protected acylbenzotriazoles in the presence of anhydrous potassium carbonate in anhydrous DMF for 10 to 30 min at 50

°C to produce amino acid and peptide conjugates of quinine (Scheme 4). The reaction time was shortened by utilising microwave irradiation, which is important for minimising the formation of epimers during the conjugation process [51].

Scheme 4. Synthesis of quinine conjugates. PG: protected groups. a: K

²

CO

³

, DMF. R is the following amino acids: conjugate no. (2) R

¹

: L-glycine; (3) R

¹

: L-alanine; (4) R

¹

: L-phenylalanine; (5) R

¹

: L-iso- leucine; (6) R

1

: L-histidine; (7) R

1

: L-serine; (8) R

1

: L-glutamic acid; (9) R

1

: L-lysine; (10) R

1

: L-aspartic acid; (11) R

1

: L-cysteine; (12) R

1

: L-alanine, R

2

: L-phenylalanine; (13) R

1

: L-valine, R

2

: L-leucine; (14) R

1

: L-isoleucine, R

2

: L-glycine.

In vitro tests were performed on the quinine–amino acid/peptide conjugates, as well as on quinine itself, against the blood stage of the P. falciparum strain 3D7 (Table 3). Qui- nine was highly effective, having an IC

⁵⁰

value of 18 nM. The highest IC

⁵⁰

conjugates were acylbenzotriazoles-aspartic acid-quinine (compound 10) at 17 nM and acylbenzotriazoles- L-isoleucine-glycine-quinine (compound 14) at 23 nM (Figure 6), which have a compara- ble antimalarial activity to that of quinine. These findings suggest that the conjugation of short peptides with the hydroxyl group of quinine does not affect its antimalarial proper- ties [51].

Figure 5.Quinine.

Amino acid conjugates of quinine were synthesised to produce new compounds using a different synthetic strategy from the development of the previous quinine derivatives, such as chloroquine. The conjugates’ synthesis also aimed to overcome the resistance problems, increase the drug delivery efficiency and improve the performance of quinine as an antimalarial agent [51,52].

The synthetic strategy used was to form an ester bond between the quinine and amino acid or peptide. The amino acid or peptide was selected based on the difference in the amino acid polarity. Quinine was O-acylated with N-protected acylbenzotriazoles in the

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Molecules 2022, 27, 7631 7 of 34

presence of anhydrous potassium carbonate in anhydrous DMF for 10 to 30 min at 50^◦C to produce amino acid and peptide conjugates of quinine (Scheme4). The reaction time was shortened by utilising microwave irradiation, which is important for minimising the formation of epimers during the conjugation process [51].

Figure 5. Quinine.

Amino acid conjugates of quinine were synthesised to produce new compounds us- ing a different synthetic strategy from the development of the previous quinine deriva- tives, such as chloroquine. The conjugates’ synthesis also aimed to overcome the re- sistance problems, increase the drug delivery efficiency and improve the performance of quinine as an antimalarial agent [51,52].

The synthetic strategy used was to form an ester bond between the quinine and amino acid or peptide. The amino acid or peptide was selected based on the difference in the amino acid polarity. Quinine was O-acylated with N-protected acylbenzotriazoles in the presence of anhydrous potassium carbonate in anhydrous DMF for 10 to 30 min at 50

°C to produce amino acid and peptide conjugates of quinine (Scheme 4). The reaction time was shortened by utilising microwave irradiation, which is important for minimising the formation of epimers during the conjugation process [51].

Scheme 4. Synthesis of quinine conjugates. PG: protected groups. a: K²CO³, DMF. R is the following amino acids: conjugate no. (2) R¹: L-glycine; (3) R¹: L-alanine; (4) R¹: L-phenylalanine; (5) R¹: L-iso- leucine; (6) R¹: L-histidine; (7) R¹: L-serine; (8) R¹: L-glutamic acid; (9) R¹: L-lysine; (10) R¹: L-aspartic acid; (11) R¹: L-cysteine; (12) R¹: L-alanine, R²: L-phenylalanine; (13) R¹: L-valine, R²: L-leucine; (14) R¹: L-isoleucine, R²: L-glycine.

In vitro tests were performed on the quinine–amino acid/peptide conjugates, as well as on quinine itself, against the blood stage of the P. falciparum strain 3D7 (Table 3). Qui- nine was highly effective, having an IC

⁵⁰

value of 18 nM. The highest IC

⁵⁰

conjugates were acylbenzotriazoles-aspartic acid-quinine (compound 10) at 17 nM and acylbenzotriazoles- L-isoleucine-glycine-quinine (compound 14) at 23 nM (Figure 6), which have a compara- ble antimalarial activity to that of quinine. These findings suggest that the conjugation of short peptides with the hydroxyl group of quinine does not affect its antimalarial proper- ties [51].

Scheme 4. Synthesis of quinine conjugates. PG: protected groups. (a): K₂CO₃, DMF. R is the following amino acids: conjugate no. (2) R₁: L-glycine; (3) R₁: L-alanine; (4) R₁: L-phenylalanine;

(5) R₁: L-isoleucine; (6) R₁: L-histidine; (7) R₁: L-serine; (8) R₁: L-glutamic acid; (9) R₁: L-lysine;

(10) R1: L-aspartic acid; (11) R1: L-cysteine; (12) R1: L-alanine, R2: L-phenylalanine; (13) R1: L-valine, R₂: L-leucine; (14) R₁: L-isoleucine, R₂: L-glycine.

In vitro tests were performed on the quinine–amino acid/peptide conjugates, as well as on quinine itself, against the blood stage of the P. falciparum strain 3D7 (Table3). Quinine was highly effective, having an IC50 value of 18 nM. The highest IC50 conjugates were acylbenzotriazoles-aspartic acid-quinine (compound 10) at 17 nM and acylbenzotriazoles- L-isoleucine-glycine-quinine (compound 14) at 23 nM (Figure6), which have a comparable antimalarial activity to that of quinine. These findings suggest that the conjugation of short peptides with the hydroxyl group of quinine does not affect its antimalarial properties [51].

Table 3.In vitro antimalarial activities of compounds against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum. (1) Quinine; (2) boc-glycine-quinine; (3) boc-L-alanine-quinine; (4) boc-L- phenylalanine-quinine; (5) boc-L-isoleucine-quinine; (6) boc-L-histidine-quinine; (7) boc-L-serine- quinine; (8) boc-L-glutamic acid-quinine; (9) Z-L-lysine-quinine; (10) Z-L-aspartic acid-quinine;

(11) Z-L-cysteine-quinine; (12) Z-L-alanine-L-phenylalanine-quinine; (13) Z-L-valine-L-leucine- quinine; (14) Z-L-isoleucine-glycine-quinine.

Compound IC50(nM)

1 18

2 27

3 36

4 38

5 550

6 42

7 95

8 76

9 71

10 17

11 40

12 120

13 74

14 23

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Molecules 2022, 27, 7631 8 of 34

(a) (b)

Figure 6. (a) Acylbenzotriazoles-L-aspartic acid-quinine; (b) acylbenzotriazoles-L-isoleucine-gly- cine-quinine.

Table 3. In vitro antimalarial activities of compounds against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum. (1) Quinine; (2) boc-glycine-quinine; (3) boc-L-alanine-quinine; (4) boc-L- phenylalanine-quinine; (5) boc-L-isoleucine-quinine; (6) boc-L-histidine-quinine; (7) boc-L-serine- quinine; (8) boc-L-glutamic acid-quinine; (9) Z-L-lysine-quinine; (10) Z-L-aspartic acid-quinine; (11) Z-L-cysteine-quinine; (12) Z-L-alanine-L-phenylalanine-quinine; (13) Z-L-valine-L-leucine-quinine;

(14) Z-L-isoleucine-glycine-quinine.

Compound IC⁵⁰ (nM)

1

18

2

27

3

36

4

38

5

550

6

42

7

95

8

76

9

71

10

17

11

40

12

120

13

74

14

23

2.2. Flavonoids

Flavonoids are found in vegetables and fruits and have health-promoting properties, without causing substantial adverse effects [53,54]. Flavonoids have been discovered to have a wide range of biological properties, including antioxidant, antibacterial, antima- larial, antiviral and anticancer properties [55–57]. A typical phenylchromen-4-one scaffold is generally found in flavonoids. [58].

Despite the fact that flavonoids have positive health effects, the therapeutic results still depend on the quality of these substances’ pharmacokinetic profiles following admin- istration. Even though they are in the form of glycosides, flavonoids are poorly bioavail- able, with a low water solubility, and are rapidly altered by environmental elements, in- cluding temperature, pH and light. The methods by which flavonoids are absorbed via the gastrointestinal tract are intricate, and they are not well-absorbed in the intestine in their native state [59].

Figure 6.(a) Acylbenzotriazoles-L-aspartic acid-quinine; (b) acylbenzotriazoles-L-isoleucine-glycine- quinine.

2.2. Flavonoids

Flavonoids are found in vegetables and fruits and have health-promoting properties, without causing substantial adverse effects [53,54]. Flavonoids have been discovered to have a wide range of biological properties, including antioxidant, antibacterial, antimalarial, antiviral and anticancer properties [55–57]. A typical phenylchromen-4-one scaffold is generally found in flavonoids. [58].

Despite the fact that flavonoids have positive health effects, the therapeutic results still depend on the quality of these substances’ pharmacokinetic profiles following adminis- tration. Even though they are in the form of glycosides, flavonoids are poorly bioavailable, with a low water solubility, and are rapidly altered by environmental elements, including temperature, pH and light. The methods by which flavonoids are absorbed via the gastrointestinal tract are intricate, and they are not well-absorbed in the intestine in their native state [59].

Therefore, the modification of their structures is a potentially useful option that can be used to improve their pharmacokinetic profile and also construct derivatives that are more biologically potent than their predecessor compounds. Herein, the actuality of the modifications of quercetin, curcumin and icaritin is described. The novel compounds’

preparation involves the formation of linkages between these flavonoids and specific amino acids.

2.2.1. Quercetin

Quercetin (Figure7) is one of the compounds from natural ingredients that shows several effective biological activities [60,61]. Quercetin belongs to the flavanol group and is widely found in daily drinks, such as grapes, onions, tea and so on [62,63]. The bioavailability of quercetin is quite low in the blood plasma. In addition, the pure form of quercetin is easily excreted [64,65].

Therefore, the modification of their structures is a potentially useful option that can be used to improve their pharmacokinetic profile and also construct derivatives that are more biologically potent than their predecessor compounds. Herein, the actuality of the modifications of quercetin, curcumin and icaritin is described. The novel compounds’

preparation involves the formation of linkages between these flavonoids and specific amino acids.

2.2.1. Quercetin

Quercetin (Figure 7) is one of the compounds from natural ingredients that shows several effective biological activities [60,61]. Quercetin belongs to the flavanol group and is widely found in daily drinks, such as grapes, onions, tea and so on [62,63]. The bioa- vailability of quercetin is quite low in the blood plasma. In addition, the pure form of quercetin is easily excreted [64,65].

Figure 7. Quercetin.

One way to overcome this problem is to synthesise quercetin–amino acid conjugates in the hope that the prodrug, quercetin, will have an improved water solubility and re- duced rate of hydrolysis [66]. The conjugation of amino acids with quercetin not only serves to improve its physicochemical properties but also to overcome the resistance to the use of anticancer drugs, or what is commonly called cancer multidrug resistance (MDR). The inhibition of the drug efflux by Pgp and Pgp ATPase assays showed that amino acid–quercetin conjugates interact with the drug-binding site of Pgp to stimulate its ATPase activity [67].

Quercetin–amino acid conjugates are produced through the synthesis of quercetin with nonpolar amino acids (alanine, valine, phenylalanine and methionine), which are positively charged (lysine) and negatively charged (aspartic acid, glutamine) by binding the amino acid to the B-ring of quercetin via a carbamate linker. Due to the acidity, high oxidability and reactivity of the catechol moiety, the protected amino acids’ interaction with excess quercetin primarily caused the esterification of the B-ring hydroxyls [68]. The series of quercetin–amino acid conjugates were produced as follows: synthetic amino acid carbamate derivatives of quercetin were produced through the conversion of amino acids to the corresponding activated urethanes, followed by alcoholysis with quercetin. At room temperature, amino acids, bis(4-nitrophenyl) carbonate and N, N-diisopropylethylamine were added to the solution. The mixture was agitated for a further 12 h at room tempera- ture after quercetin was added. To produce the required quercetin prodrugs, the tert-bu- tyl-amino acid quercetin carbamates were deprotected with TFA at 0 °C (Scheme 5) [66].

O O

OH

HO OH

OH O

O

OH

HO O

OH

O N

H R

OH O a,b

Scheme 5. Syntheses of the quercetin analogues. (a): (4-NO²-PhO)²CO, DIPEA, DMF, 0 °C to rt; (b):

TFA, CH²Cl², 0 °C, rt. R in order: (2) L-alanine; (3) L-valine; (4) L-lysine; (5) L-phenylalanine; (6) L- Figure 7.Quercetin.

One way to overcome this problem is to synthesise quercetin–amino acid conjugates in the hope that the prodrug, quercetin, will have an improved water solubility and

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Molecules 2022, 27, 7631 9 of 34

reduced rate of hydrolysis [66]. The conjugation of amino acids with quercetin not only serves to improve its physicochemical properties but also to overcome the resistance to the use of anticancer drugs, or what is commonly called cancer multidrug resistance (MDR). The inhibition of the drug efflux by Pgp and Pgp ATPase assays showed that amino acid–quercetin conjugates interact with the drug-binding site of Pgp to stimulate its ATPase activity [67].

Quercetin–amino acid conjugates are produced through the synthesis of quercetin with nonpolar amino acids (alanine, valine, phenylalanine and methionine), which are positively charged (lysine) and negatively charged (aspartic acid, glutamine) by binding the amino acid to the B-ring of quercetin via a carbamate linker. Due to the acidity, high oxidability and reactivity of the catechol moiety, the protected amino acids’ interaction with excess quercetin primarily caused the esterification of the B-ring hydroxyls [68]. The series of quercetin–amino acid conjugates were produced as follows: synthetic amino acid carbamate derivatives of quercetin were produced through the conversion of amino acids to the corresponding activated urethanes, followed by alcoholysis with quercetin. At room temperature, amino acids, bis(4-nitrophenyl) carbonate and N, N-diisopropylethylamine were added to the solution. The mixture was agitated for a further 12 h at room temperature after quercetin was added. To produce the required quercetin prodrugs, the tert-butyl- amino acid quercetin carbamates were deprotected with TFA at 0^◦C (Scheme5) [66].

Therefore, the modification of their structures is a potentially useful option that can be used to improve their pharmacokinetic profile and also construct derivatives that are more biologically potent than their predecessor compounds. Herein, the actuality of the modifications of quercetin, curcumin and icaritin is described. The novel compounds’

preparation involves the formation of linkages between these flavonoids and specific amino acids.

2.2.1. Quercetin

Quercetin (Figure 7) is one of the compounds from natural ingredients that shows several effective biological activities [60,61]. Quercetin belongs to the flavanol group and is widely found in daily drinks, such as grapes, onions, tea and so on [62,63]. The bioa- vailability of quercetin is quite low in the blood plasma. In addition, the pure form of quercetin is easily excreted [64,65].

Figure 7. Quercetin.

One way to overcome this problem is to synthesise quercetin–amino acid conjugates in the hope that the prodrug, quercetin, will have an improved water solubility and re- duced rate of hydrolysis [66]. The conjugation of amino acids with quercetin not only serves to improve its physicochemical properties but also to overcome the resistance to the use of anticancer drugs, or what is commonly called cancer multidrug resistance (MDR). The inhibition of the drug efflux by Pgp and Pgp ATPase assays showed that amino acid–quercetin conjugates interact with the drug-binding site of Pgp to stimulate its ATPase activity [67].

Quercetin–amino acid conjugates are produced through the synthesis of quercetin with nonpolar amino acids (alanine, valine, phenylalanine and methionine), which are positively charged (lysine) and negatively charged (aspartic acid, glutamine) by binding the amino acid to the B-ring of quercetin via a carbamate linker. Due to the acidity, high oxidability and reactivity of the catechol moiety, the protected amino acids’ interaction with excess quercetin primarily caused the esterification of the B-ring hydroxyls [68]. The series of quercetin–amino acid conjugates were produced as follows: synthetic amino acid carbamate derivatives of quercetin were produced through the conversion of amino acids to the corresponding activated urethanes, followed by alcoholysis with quercetin. At room temperature, amino acids, bis(4-nitrophenyl) carbonate and N, N-diisopropylethylamine were added to the solution. The mixture was agitated for a further 12 h at room tempera- ture after quercetin was added. To produce the required quercetin prodrugs, the tert-bu- tyl-amino acid quercetin carbamates were deprotected with TFA at 0 °C (Scheme 5) [66].

O O

OH

HO OH

OH O

O

OH

HO O

OH

O N

H R

OH O a,b

Scheme 5. Syntheses of the quercetin analogues. (a): (4-NO²-PhO)²CO, DIPEA, DMF, 0 °C to rt; (b):

TFA, CH²Cl², 0 °C, rt. R in order: (2) L-alanine; (3) L-valine; (4) L-lysine; (5) L-phenylalanine; (6) L- Scheme 5. Syntheses of the quercetin analogues. (a): (4-NO₂-PhO)₂CO, DIPEA, DMF, 0^◦C to rt;

(b): TFA, CH2Cl2, 0^◦C, rt. R in order: (2) L-alanine; (3) L-valine; (4) L-lysine; (5) L-phenylalanine;

(6) L-aspartic acid; (7) L-methionine; (8) L-glutamic acid; (9) L-alanine-L-aspartic acid; (10) L-alanine- L-glutamic acid.

The water solubilities of the quercetin prodrugs increased dramatically (6.8–53.0 times) in comparison to quercetin (Table4). Amino acids such as aspartic acid, glutamic acid and alanine-glutamic acid enhanced the water solubilities of their corresponding quercetin conjugates by 45.2-, 53.0- and 52.6-fold, respectively.

Table 4. Solubility of quercetin and quercetin–amino acid conjugates in PBS buffer. (1) Quercetin;

(2) quercetin-alanine; (3) quercetin-valine; (4) quercetin-lysine; (5) quercetin-phenylalanine;

(6) quercetin-aspartic acid; (7) quercetin-methionine; (8) quercetin-glutamic acid; (9) quercetin-alanine- aspartic acid; (10) quercetin-alanine-glutamic acid.

Compound Water Solubility (µM) Fold Increase

1 50 1.0

2 1290 25.8

3 767 15.3

4 338 6.8

5 1766 35.3

6 2262 45.2

7 675 13.5

8 2649 53.0

9 2093 41.9

10 2628 52.6

In PBS buffer (t1/2 > 17 h), the quercetin prodrugs were stable, albeit that they were vulnerable to enzymatic hydrolysis in cell lysate containing different activated hydrolysing

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Molecules 2022, 27, 7631 10 of 34

enzymes. The quercetin–glutamic acid conjugate (Figure8b) demonstrated a remarkable resistance to hydrolases, resulting in a much longer half-life (180 min). In comparison to quercetin, the quercetin–aspartic acid and quercetin–glutamic acid conjugates showed an increased intestinal permeability in MDCK cells, which suggests that the quercetin–amino acid conjugates may be recognised and transported by the human peptide transporter hPepT1 [66].

aspartic acid; (7) L-methionine; (8) L-glutamic acid; (9) L-alanine-L-aspartic acid; (10) L-alanine-L- glutamic acid.

The water solubilities of the quercetin prodrugs increased dramatically (6.8–53.0 times) in comparison to quercetin (Table 4). Amino acids such as aspartic acid, glutamic acid and alanine-glutamic acid enhanced the water solubilities of their corresponding quercetin conjugates by 45.2-, 53.0- and 52.6-fold, respectively.

Table 4. Solubility of quercetin and quercetin–amino acid conjugates in PBS buffer. (1) Quercetin;

(2) quercetin-alanine; (3) quercetin-valine; (4) quercetin-lysine; (5) quercetin-phenylalanine; (6) quercetin-aspartic acid; (7) quercetin-methionine; (8) quercetin-glutamic acid; (9) quercetin-alanine- aspartic acid; (10) quercetin-alanine-glutamic acid.

Compound Water Solubility

(μM) Fold

Increase

1

50 1.0

2

1290 25.8

3

767 15.3

4

338 6.8

5

1766 35.3

6

2262 45.2

7

675 13.5

8

2649 53.0

9

2093 41.9

10

2628 52.6

In PBS buffer (t1/2 > 17 h), the quercetin prodrugs were stable, albeit that they were vul- nerable to enzymatic hydrolysis in cell lysate containing different activated hydrolysing en- zymes. The quercetin–glutamic acid conjugate (Figure 8b) demonstrated a remarkable re- sistance to hydrolases, resulting in a much longer half-life (180 min). In comparison to querce- tin, the quercetin–aspartic acid and quercetin–glutamic acid conjugates showed an increased intestinal permeability in MDCK cells, which suggests that the quercetin–amino acid conju- gates may be recognised and transported by the human peptide transporter hPepT1 [66].

(a) (b)

Figure 8. (a) Quercetin–aspartic acid conjugate; (b) quercetin–glutamic acid conjugate.

2.2.2. Icaritin

Icaritin (Figure 9), an active prenyl flavone derived from Epimedium plants, has a wide range of pharmacological and biological properties, including those that can aid in cancer and osteoporosis treatments [69,70].

Figure 8.(a) Quercetin–aspartic acid conjugate; (b) quercetin–glutamic acid conjugate.

2.2.2. Icaritin

Icaritin (Figure9), an active prenyl flavone derived from Epimedium plants, has a wide range of pharmacological and biological properties, including those that can aid in cancer and osteoporosis treatments [69,70].

aspartic acid; (7) L-methionine; (8) L-glutamic acid; (9) L-alanine-L-aspartic acid; (10) L-alanine-L- glutamic acid.

The water solubilities of the quercetin prodrugs increased dramatically (6.8–53.0 times) in comparison to quercetin (Table 4). Amino acids such as aspartic acid, glutamic acid and alanine-glutamic acid enhanced the water solubilities of their corresponding quercetin conjugates by 45.2-, 53.0- and 52.6-fold, respectively.

Table 4. Solubility of quercetin and quercetin–amino acid conjugates in PBS buffer. (1) Quercetin;

(2) quercetin-alanine; (3) quercetin-valine; (4) quercetin-lysine; (5) quercetin-phenylalanine; (6) quercetin-aspartic acid; (7) quercetin-methionine; (8) quercetin-glutamic acid; (9) quercetin-alanine- aspartic acid; (10) quercetin-alanine-glutamic acid.

Compound Water Solubility

(μM) Fold

Increase

1

50 1.0

2

1290 25.8

3

767 15.3

4

338 6.8

5

1766 35.3

6

2262 45.2

7

675 13.5

8

2649 53.0

9

2093 41.9

10

2628 52.6

In PBS buffer (t1/2 > 17 h), the quercetin prodrugs were stable, albeit that they were vul- nerable to enzymatic hydrolysis in cell lysate containing different activated hydrolysing en- zymes. The quercetin–glutamic acid conjugate (Figure 8b) demonstrated a remarkable re- sistance to hydrolases, resulting in a much longer half-life (180 min). In comparison to querce- tin, the quercetin–aspartic acid and quercetin–glutamic acid conjugates showed an increased intestinal permeability in MDCK cells, which suggests that the quercetin–amino acid conju- gates may be recognised and transported by the human peptide transporter hPepT1 [66].

(a) (b)

Figure 8. (a) Quercetin–aspartic acid conjugate; (b) quercetin–glutamic acid conjugate.

2.2.2. Icaritin

Icaritin (Figure 9), an active prenyl flavone derived from Epimedium plants, has a wide range of pharmacological and biological properties, including those that can aid in cancer and osteoporosis treatments [69,70].

Figure 9.Icaritin.

As icaritin has a low toxicity and a high safety profile, it is a good natural chemical entity for further modification, aiming to produce molecules with an improved activity. The icaritin flavone scaffold was first alkylated with ethyl iodoacetate to integrate cationic moieties, which led to the production of flavone amphiphiles and robust bacterial membrane disruption. The key acid was then linked to the appropriate basic amino acids using 2-(3H-[1–3]triazolo [4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexaflu- orophosphate (HATU) or N,N’-diisopropylcarbodiimide (DIC) in combination with HOBt to form icaritin–amino acid conjugates (Scheme6) [71,72].

Figure 9. Icaritin.

As icaritin has a low toxicity and a high safety profile, it is a good natural chemical entity for further modification, aiming to produce molecules with an improved activity.

The icaritin flavone scaffold was first alkylated with ethyl iodoacetate to integrate cationic moieties, which led to the production of flavone amphiphiles and robust bacterial mem- brane disruption. The key acid was then linked to the appropriate basic amino acids using 2-(3H-[1–3]triazolo [4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophos- phate (HATU) or N,N’-diisopropylcarbodiimide (DIC) in combination with HOBt to form icaritin–amino acid conjugates (Scheme 6) [71,72].

Scheme 6. Synthesis of amino-acid-conjugated flavone compounds. (a) Ethyl iodoacetate, K²CO³, acetone, reflux, 12 h; (b) LiOH, THF, H²O, RT, 1.5 h; (c) corresponding basic amino acid, DIC, HOBt, anhydrous DMF, RT, overnight; (d) corresponding basic amino acid, HATU, DIPEA, anhydrous DMF, RT, overnight. R in order: (2) L-histidine; (3) L-arginine; (4) L-lysine.

Cationic moieties should have an impact on the cLogP values of icaritin–amino acid conjugates. The hydrophobicity decreases as the total charge increases. Icaritin–arginine’s cLogP value is 1.65. These findings show that this conjugate’s charge–hydrophobicity bal- ance is a key determinant of its antibacterial activity. If the total charge is too high or too low, the chemical will disrupt the charge–hydrophobicity balance, in addition to decreas- ing the affinity for the bacterial membranes and overall antimicrobial activity. Higher overall charges improve the antibacterial activity.

Of all the substances examined, the icaritin–arginine (Figure 10) conjugate was the most effective. Icaritin–arginine was the only studied flavone derivative that was water- soluble. The strong electrostatic contact between icaritin–arginine and the negatively charged bacterial membrane was thought to be responsible for the greatest enhancement in its antibacterial activity. The guanidinium group on arginine has a more evenly distrib- uted positive charge than the tertiary amine and -NH

2

groups, which significantly im- proves icaritin–arginine’s electrostatic contact with the bacterial membrane.

Figure 10. Icaritin–arginine conjugate.

Scheme 6.Synthesis of amino-acid-conjugated flavone compounds. (a) Ethyl iodoacetate, K₂CO₃, acetone, reflux, 12 h; (b) LiOH, THF, H2O, RT, 1.5 h; (c) corresponding basic amino acid, DIC, HOBt, anhydrous DMF, RT, overnight; (d) corresponding basic amino acid, HATU, DIPEA, anhydrous DMF, RT, overnight. R in order: (2) L-histidine; (3) L-arginine; (4) L-lysine.

Cationic moieties should have an impact on the cLogP values of icaritin–amino acid conjugates. The hydrophobicity decreases as the total charge increases. Icaritin–arginine’s