Synthesis of Carvone-Derived 1,2,3-Triazoles Study of Their Antioxidant Properties and Interaction with Bovine Serum Albumin

Natural L-carvone was utilized as a starting material for an efficient synthesis of some terpenyl-derived 1,2,3-triazoles. Chlorination of carvone, followed by nucleophilic substitution with sodium azide resulted in the preparation of 10-azidocarvone. Subsequent CuAAC click reaction with propargylated derivatives provided an efficient synthetic route to a set of terpenyl-derived conjugates with increased solubility in water. All investigated compounds exhibit high antioxidant activity, which is comparable with that of vitamin C. It was also found that serum albumin and the terpenyl-1,2,3-triazoles hybrids spontaneously undergo reversible binding driven by hydrophobic interactions, suggesting that serum albumin can transport the target triazoles.


Introduction
Natural compounds play a significant role in the design of new drugs and prevention of various diseases [1].Thus, more than 60% of current drugs for the treatment of cancer and infectious diseases are of natural origin [2].Terpenoids are one of the largest classes of chiral natural compounds, which includes more than 23,000 compounds.Many terpene derivatives are widely used in perfumes, playing the role of cosmetic products and food additives [3].However, medicine is not less important area of application of terpenes, since the majority of compounds of this class exhibits pronounced biological activity.For example, the antimalarial drug artemisinin and the anticancer drug paclitaxel (Taxol ® ) are the two most prominent members of the class of terpenes used in medicine (Figure 1).

Introduction
Natural compounds play a significant role in the design of new drugs and prevention of various diseases [1].Thus, more than 60% of current drugs for the treatment of cancer and infectious diseases are of natural origin [2].Terpenoids are one of the largest classes of chiral natural compounds, which includes more than 23,000 compounds.Many terpene derivatives are widely used in perfumes, playing the role of cosmetic products and food additives [3].However, medicine is not less important area of application of terpenes, since the majority of compounds of this class exhibits pronounced biological activity.For example, the antimalarial drug artemisinin and the anticancer drug paclitaxel (Taxol ® ) are the two most prominent members of the class of terpenes used in medicine (Figure 1).Annual sales of terpenic derivatives in the world are estimated at about $20 billion [4].The scope of terpenes is constantly expanding; therefore, terpenoids will play an increasingly important role as therapeutic and prophylactic agents for human diseases.
Monoterpenes-C 10 H 16 compounds and their derivatives are the simplest type of terpenoids.They are the basis of essential oils, floral aromas and perform plant protective functions [5,6].Despite the fact that these are small and fairly simple molecules, a number of monoterpenes have an antitumor effect, demonstrating not only the ability to prevent the formation or progression of cancer, but the ability to reduce existing malignant tumors [7].Most abundant in nature are terpenoids having menthane skeleton (Figure 2).For example, limonene is the most common monocyclic monoterpene found in nature.It can be found in a variety of trees and grasses (for example, Mentha spp.).Limonene is an important component of the peel of oranges and lemons, as well as the essential oil of cumin.The limonene fragment can be found in the structure of many drugs [7][8][9][10][11].Carvone, the main monoterpene of cumin seed.It has been shown that carvone oil prevents chemically induced carcinoma of the lung [12].In addition, carveol has chemoprophylactic activity against breast cancer during the initiation phase [13].Perillyl alcohol, a hydroxylated limonene analog, exhibits chemopreventive activity against chemically induced liver cancer [14] and tumor recurrences in animal models [15].
Molecules 2018, 23, x FOR PEER REVIEW 2 of 13 Annual sales of terpenic derivatives in the world are estimated at about $20 billion [4].The scope of terpenes is constantly expanding; therefore, terpenoids will play an increasingly important role as therapeutic and prophylactic agents for human diseases.
Monoterpenes-C10H16 compounds and their derivatives are the simplest type of terpenoids.They are the basis of essential oils, floral aromas and perform plant protective functions [5,6].Despite the fact that these are small and fairly simple molecules, a number of monoterpenes have an antitumor effect, demonstrating not only the ability to prevent the formation or progression of cancer, but the ability to reduce existing malignant tumors [7].Most abundant in nature are terpenoids having menthane skeleton (Figure 2).For example, limonene is the most common monocyclic monoterpene found in nature.It can be found in a variety of trees and grasses (for example, Mentha spp.).Limonene is an important component of the peel of oranges and lemons, as well as the essential oil of cumin.The limonene fragment can be found in the structure of many drugs [7][8][9][10][11].Carvone, the main monoterpene of cumin seed.It has been shown that carvone oil prevents chemically induced carcinoma of the lung [12].In addition, carveol has chemoprophylactic activity against breast cancer during the initiation phase [13].Perillyl alcohol, a hydroxylated limonene analog, exhibits chemopreventive activity against chemically induced liver cancer [14] and tumor recurrences in animal models [15].Nowadays, one of the most promising synthetic trends for expanding molecular complexity from a given scaffold have been the "click" reactions."Click" reactions are a class of efficient, fast, universal, and selective reactions that are characterized by high yields and simple isolation of products.The standard "click" of chemistry for today is the formation of 1,2,3-triazoles catalyzed by copper salts (I) in the reaction of azides and terminal alkynes (CuAAC) [16][17][18][19][20].Although 1,2,3-triazole fragment is generally absent in natural compounds, synthetic molecules containing 1,2,3-triazole cycles represent a wide class of physiologically active substances exhibiting various types of biological activity, for example, antibacterial, antiallergic, antiviral, antifungal properties [21,22].Apparently, the pharmacological activity of compounds containing 1,2,3-triazole groups is due to the structural and electronic similarity of the 1,2,3-triazole fragment and the amide group.The 1,2,3-triazole fragment can be considered as a conformationally constrained bioisostere of the amide group.Greater stability of the 1,2,3-triazole scaffold under physiological conditions, led to the widespread use of the "click" reaction as an effective method for the synthesis of various biologically active compounds in drug design.The triazole fragment is an important pharmacophoric unit, and a large number of drugs containing this heterocycle are known (Figure 3).Due to the commercial success of some pharmaceutical preparations based on the triazole ring, many pharmaceutical companies and academic groups have shown interest in developing new methods for synthesizing triazole compounds and screening their biological activity [23,24].For example, antifungal drugs containing triazole rings are known: itraconazole, fluconazole, voriconazole [25], antiviral drug ribavirin and murbritinib (used to treat breast, bladder, kidney and prostate cancer).Ribavirin is a drug for the treatment of viral infections such as herpes and hepatitis [26,27].Nowadays, one of the most promising synthetic trends for expanding molecular complexity from a given scaffold have been the "click" reactions."Click" reactions are a class of efficient, fast, universal, and selective reactions that are characterized by high yields and simple isolation of products.The standard "click" of chemistry for today is the formation of 1,2,3-triazoles catalyzed by copper salts (I) in the reaction of azides and terminal alkynes (CuAAC) [16][17][18][19][20].Although 1,2,3-triazole fragment is generally absent in natural compounds, synthetic molecules containing 1,2,3-triazole cycles represent a wide class of physiologically active substances exhibiting various types of biological activity, for example, antibacterial, antiallergic, antiviral, antifungal properties [21,22].Apparently, the pharmacological activity of compounds containing 1,2,3-triazole groups is due to the structural and electronic similarity of the 1,2,3-triazole fragment and the amide group.The 1,2,3-triazole fragment can be considered as a conformationally constrained bioisostere of the amide group.Greater stability of the 1,2,3-triazole scaffold under physiological conditions, led to the widespread use of the "click" reaction as an effective method for the synthesis of various biologically active compounds in drug design.The triazole fragment is an important pharmacophoric unit, and a large number of drugs containing this heterocycle are known (Figure 3).Due to the commercial success of some pharmaceutical preparations based on the triazole ring, many pharmaceutical companies and academic groups have shown interest in developing new methods for synthesizing triazole compounds and screening their biological activity [23,24].For example, antifungal drugs containing triazole rings are known: itraconazole, fluconazole, voriconazole [25], antiviral drug ribavirin and murbritinib (used to treat breast, bladder, kidney and prostate cancer).Ribavirin is a drug for the treatment of viral infections such as herpes and hepatitis [26,27].

Results and Discussion
This study is devoted to the synthesis of carvone-derived 1,2,3-triazoles 4a-i prepared according to the strategy outlined in Scheme 1.We proposed that due to the presence of the enone fragment in the structure, such conjugates would behave as antioxidants.On the other hand, the triazole and amine moieties will play an auxiliary role of improving water solubility of these molecules.10-Azido-carvone was chosen as a key building block to study copper-catalyzed azide alkyne cycloaddition (CuAAC).A number of propargylated amino derivatives 3 were studied as alkyne partners for this reaction.They can be prepared using standard alkylation of NH-derivatives with propargyl bromide (Scheme 1) [28].To achieve this goal, L-carvone (1) was chlorinated using calcium hypochlorite−CO2 system to provide 10-chlorocarvone (2) [29].Subsequent treatment of 2 with sodium azide resulted in synthesis of 10-azidocarvone.We decided to utilize synthetically attractive one-pot protocol to avoid isolation and purification of this intermediate product.For this aim, chloride 2 was treated with sodium azide in DMSO or acetonitrile to yield 10-azido-carvone, which was used without isolation in model CuAAC reaction with N-propargylmorpholine 3a.It was found that the in the case of DMSO as a solvent the yield of model conjugate 4a is moderate.However, with acetonitrile as a

Results and Discussion
This study is devoted to the synthesis of carvone-derived 1,2,3-triazoles 4a-i prepared according to the strategy outlined in Scheme 1.We proposed that due to the presence of the enone fragment in the structure, such conjugates would behave as antioxidants.On the other hand, the triazole and amine moieties will play an auxiliary role of improving water solubility of these molecules.10-Azido-carvone was chosen as a key building block to study copper-catalyzed azide alkyne cycloaddition (CuAAC).A number of propargylated amino derivatives 3 were studied as alkyne partners for this reaction.They can be prepared using standard alkylation of NH-derivatives with propargyl bromide (Scheme 1) [28].

Results and Discussion
This study is devoted to the synthesis of carvone-derived 1,2,3-triazoles 4a-i prepared according to the strategy outlined in Scheme 1.We proposed that due to the presence of the enone fragment in the structure, such conjugates would behave as antioxidants.On the other hand, the triazole and amine moieties will play an auxiliary role of improving water solubility of these molecules.10-Azido-carvone was chosen as a key building block to study copper-catalyzed azide alkyne cycloaddition (CuAAC).A number of propargylated amino derivatives 3 were studied as alkyne partners for this reaction.They can be prepared using standard alkylation of NH-derivatives with propargyl bromide (Scheme 1) [28].To achieve this goal, L-carvone (1) was chlorinated using calcium hypochlorite−CO2 system to provide 10-chlorocarvone (2) [29].Subsequent treatment of 2 with sodium azide resulted in synthesis of 10-azidocarvone.We decided to utilize synthetically attractive one-pot protocol to avoid isolation and purification of this intermediate product.For this aim, chloride 2 was treated with sodium azide in DMSO or acetonitrile to yield 10-azido-carvone, which was used without isolation in model CuAAC reaction with N-propargylmorpholine 3a.It was found that the in the case of DMSO as a solvent the yield of model conjugate 4a is moderate.However, with acetonitrile as a To achieve this goal, L-carvone (1) was chlorinated using calcium hypochlorite−CO 2 system to provide 10-chlorocarvone (2) [29].Subsequent treatment of 2 with sodium azide resulted in synthesis of 10-azidocarvone.We decided to utilize synthetically attractive one-pot protocol to avoid isolation and purification of this intermediate product.For this aim, chloride 2 was treated with sodium azide in DMSO or acetonitrile to yield 10-azido-carvone, which was used without isolation in model CuAAC reaction with N-propargylmorpholine 3a.It was found that the in the case of DMSO as a solvent the yield of model conjugate 4a is moderate.However, with acetonitrile as a solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).solvent and copper iodide as a catalyst, the isolated yield was improved significantly.For example, 4a was obtained in up to 78% yield within a reasonable reaction time (10 h) using only 5 mol % of copper iodide (Table 1).Probably, such observation can be explained by formation of the complex of copper iodide with final products.Next, the reaction with a number of propargylated amines and amides was investigated.To our delight, the corresponding triazole derived carvone conjugates 4 were isolated in up to 84% yield.The efficient procedure provided 4 as crystalline compounds.Their structure was confirmed by combination of spectroscopic methods (see SI).As a result, this part of study gave as a family of carvone derivatives 4 with variable amino-(amido) substituents in the triazole ring (Table 2).With these new compounds in hand, we decided to study the antioxidant properties of the synthesized terpenyl-1,2,3-triazoles 4. For this aim the interaction with HO • radicals in the presence of the competitive acceptor 4-nitroso-N,N-dimethylaniline (PNDMA) was studied [30][31][32].The initiation of HO • radicals was carried out by photolysis of H 2 O 2 (10 −3 mol•L −1 ) under the action of UV radiation (λ = 313 nm with the use of a special filter).The rate of initiation of HO • radicals (Figure 4a-f) was determined by the change in PNDMA absorption (A 440 ).It was found that compounds 4a-e have better water-solubility and their antioxidant properties were investigated.Below are given the kinetic data of the effect of 4a-e different concentrations on the optical density of PNDMA, depending on the time of irradiation (Figure 4a-e).
Molecules 2018, 23, x FOR PEER REVIEW 5 of 13 With these new compounds in hand, we decided to study the antioxidant properties of the synthesized terpenyl-1,2,3-triazoles 4. For this aim the interaction with HO • radicals in the presence of the competitive acceptor 4-nitroso-N,N-dimethylaniline (PNDMA) was studied [30][31][32].The initiation of HO • radicals was carried out by photolysis of H2O2 (10 −3 mol•L −1 ) under the action of UV radiation (λ = 313 nm with the use of a special filter).The rate of initiation of HO • radicals (Figure 4a-f) was determined by the change in PNDMA absorption (А440).It was found that compounds 4ae have better water-solubility and their antioxidant properties were investigated.Below are given the kinetic data of the effect of 4а-e different concentrations on the optical density of PNDMA, depending on the time of irradiation (Figure 4a-e).The constants of the interaction rate of HO • radicals with 4a-e were calculated by the Equation (1) [31,32]: where 1.25 × 10 10 mol −1 s −1 L is the constant of the interaction rate of HO • radicals with PNDMA, [P] is the concentration of 4a-e, W 1 and W 2 are rates of PNDMA discoloring in distilled water and in the presence of 4a-e respectively.The rate constants are shown in Table 3.As one can see from Table 3, all the compounds tested exhibit significant antioxidant activity.Moreover compound 4b is slightly inferior to the known antioxidant ascorbic acid used as a control compound.The transport of active aglycones of drugs is known to be carried out by various interactions with the proteins.The most abundant protein in the blood plasma is the transport protein serum albumin (up to 60%) which can reversibly bind various endogenous and exogenous compounds [33][34][35].To reveal possibility of such binding, fluorescence spectroscopy was used for model compound 4e with the bovine serum albumin (BSA).The thermodynamic parameters (∆H, ∆S and ∆G) can be used to propose the binding mode.For the typical hydrophobic interactions, both ∆H and ∆S are positive, while negative ∆H and ∆S result from the hydrogen bond formation and van der Waals forces, and electrostatic interactions are responsible for the cases when ∆H < 0 and ∆S > 0 [36].The value of binding constant (K b ) for transport protein ligand interaction in the range of10 3 -10 6 M −1 indicate the reversibility of binding [37].The results obtained for BSA-4e system are summarized in Table 4. Negative values of ∆G show that the binding process proceeds spontaneously.The positive values of ∆H and ∆S show that the stability of the BSA-4e system is due mainly to hydrophobic interactions.Table 4.The values of thermodynamic parameters for BSA binding with 4e at 298 and 308 K.The distance between the protein and the ligand can be calculated using the theory of resonance energy transfer (Foerster theory) [38].Average distance between BSA-4e decreases in the range of 2-8 nm and the energy transfer efficiency increases when the temperature was increased from 298 K to 308 K (Table 5).However, such interactions of BSA with 4e are not very strong.Thus, the transport of 4e can be performed by serum albumin-BSA.
Table 5.The values of the overlap integral, the energy transfer efficiency, the Forster radius and the distance between the BSA and 4e.

Table 1 .
Optimization of synthesis of 4a.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 1 .
Optimization of synthesis of 4а.

Table 3 .
The rate constants for the reaction.