Sabinene: A New Green Solvent Used in the Synthesis of Thiazolo[5,4-b]pyridines by Thermal or Microwave Activation

Following the work already carried out in our laboratory on eucalyptol, a new green solvent derived from biomass, we are now looking at sabinene as another new green solvent. Sabinene is also derived from biomass, has no known toxicity and can be recycled by distillation. We have shown that it can be used as it is or distilled to synthesize thiazolo[5,4-b]pyridine heterocycles by thermal activation or microwave irradiation. This new solvent was compared with various conventional and green solvents. The conditions were optimised to enable us to carry out the syntheses in satisfactory yields, and we were able to show that sabinene, a natural bicyclic monoterpene, could be used effectively as a solvent.


Introduction
Organic chemistry mainly uses petroleum-based products or solvents, which have a major impact on the environment. Today, it is important to preserve our non-renewable resources by using new types of solvents derived from biomass and to think about the economy of the atom. In 2019, we were able to highlight a new solvent derived from biomass, eucalyptol [1]. This has been compared with known conventional and green solvents and has shown an undeniable interest in the organic synthesis of numerous nitrogenous and sulphurous oxygenated heterocyclic compounds via, in particular, nucleophilic substitutions, cyclisations, various metal-catalysed couplings and multicomponent reactions [1][2][3][4][5]. Although we are still applying this new solvent in various synthetic processes, within the team we are continuing our efforts to limit our environmental impact and are therefore looking at other biomass-derived solvents. Sabinene 1 (Figure 1) is a compound in the family of unsaturated monoterpene hydrocarbons with the molecular formula C 10 H 16 . Sabinene is its trivial name, while its IUPAC name is 4-methylidene-1-(propan-2-yl)bicyclo[3.1.0]hexane. It is classified as a food additive and as a flavouring agent in the perfume industry. It is known for its anti-inflammatory, antioxidant, antifungal, [6,7], antiseptic, antimicrobial [8] and bactericidal properties [9].
On the one hand, heterocycles are widely present in many agrochemical and pharmaceutical products [22][23][24][25][26][27][28]. To date, the number of pharmaceutical products containing a heterocyclic part in their skeleton, and in particular bicyclic heterocycles, is estimated to be around 70%, hence the importance of mastering synthesis protocols and carrying them out under the safest possible conditions, for the development and production of new environmentally friendly drugs or agrochemical compounds [25,[29][30][31]. On the other hand, thiazolo [5,4-b]pyridine analogues are known for their promising properties and are therefore the subject of various developments [32][33][34][35][36], particularly in oncology, as some analogues show very good inhibition (in the nanomolar range) of phosphoinositide 3-kinase (PI3K) [37]. This is an important target for survival, proliferation and differentiation, and therefore for targeted tumour therapy [38]. These compounds can be synthesised in several ways, depending on the functionalities envisaged, in particular on the 6-membered ring. They can also be synthesised in a single step from a chloronitropyridine and a suitably substituted thioamide or thiourea [39]. We chose to use the one-step method starting from a 3-amino-2-chloropyridine derivative and an isothiocyanate, a synthetic method already used when we investigated laser irradiation as a new activation method in organic synthesis [40]. This reaction was chosen because of the interest of this type of heterocycle, but also because while the reagents are soluble, the product precipitates out of the medium, making it easy to visualise its production.

Optimisation in Various Solvents
On the basis of the results obtained in a previous study [40] involving coupling between 3-amino-2-chloropyridine 2a and phenyl isothiocyanate 3a to obtain N-phenylthiazolo [5,4-b]pyridin-2-amine 4a, the synthesis was first carried out in various conventional solvents before being performed in green solvents. To investigate the ranges and limits, the temperature was maintained at a set point of 110 °C for an internal temperature of 100 °C in a sealed tube for each experiment by conventional heating using a stirring plate. It was found that 4 h were required at this temperature in conventional solvents, and that increasing the reaction time did not provide any significant improvement. It should be noted that the product obtained is the HCl salt product already described by Atland and Molander [41], which exhibits a characteristic NMR spectrum. We tried to carry out the reaction in the presence of a base equivalent such as K2CO3, but the reaction proved inefficient under these conditions. The solution was to proceed in two stages, forming the Sabinene has also been reported as a starting material for advanced biofuels [20,21]. Here, it is used as a green solvent for the synthesis of various thiazolo [5,4-b]pyridines compared to eucalyptol or cyclopentyl methyl ether (CPME), limonene and citral.
On the one hand, heterocycles are widely present in many agrochemical and pharmaceutical products [22][23][24][25][26][27][28]. To date, the number of pharmaceutical products containing a heterocyclic part in their skeleton, and in particular bicyclic heterocycles, is estimated to be around 70%, hence the importance of mastering synthesis protocols and carrying them out under the safest possible conditions, for the development and production of new environmentally friendly drugs or agrochemical compounds [25,[29][30][31]. On the other hand, thiazolo [5,4-b]pyridine analogues are known for their promising properties and are therefore the subject of various developments [32][33][34][35][36], particularly in oncology, as some analogues show very good inhibition (in the nanomolar range) of phosphoinositide 3-kinase (PI3K) [37]. This is an important target for survival, proliferation and differentiation, and therefore for targeted tumour therapy [38]. These compounds can be synthesised in several ways, depending on the functionalities envisaged, in particular on the 6-membered ring. They can also be synthesised in a single step from a chloronitropyridine and a suitably substituted thioamide or thiourea [39]. We chose to use the one-step method starting from a 3-amino-2-chloropyridine derivative and an isothiocyanate, a synthetic method already used when we investigated laser irradiation as a new activation method in organic synthesis [40]. This reaction was chosen because of the interest of this type of heterocycle, but also because while the reagents are soluble, the product precipitates out of the medium, making it easy to visualise its production.

Optimisation in Various Solvents
On the basis of the results obtained in a previous study [40] involving coupling between 3-amino-2-chloropyridine 2a and phenyl isothiocyanate 3a to obtain N-phenylthiazolo [5,4-b]pyridin-2-amine 4a, the synthesis was first carried out in various conventional solvents before being performed in green solvents. To investigate the ranges and limits, the temperature was maintained at a set point of 110 • C for an internal temperature of 100 • C in a sealed tube for each experiment by conventional heating using a stirring plate. It was found that 4 h were required at this temperature in conventional solvents, and that increasing the reaction time did not provide any significant improvement. It should be noted that the product obtained is the HCl salt product already described by Atland and Molander [41], which exhibits a characteristic NMR spectrum. We tried to carry out the reaction in the presence of a base equivalent such as K 2 CO 3 , but the reaction proved inefficient under these conditions. The solution was to proceed in two stages, forming the product in salt form and then neutralising it in the presence of a base. We therefore continued our study without a base and formed the products in salt form.
As the yields obtained were moderate in both conventional and green solvents, we concentrated on the latter to optimise reaction time, using one equivalent of each reagent. While citral only led to a disappointing yield of 21% in 16 h, increasing the reaction time was beneficial for the other green solvents, allowing us to achieve satisfactory yields of 58 to 75% (Table 1, entries 7,9 and 11). Beyond 16 h, we did not observe any improvement in performance. We used sabinene as a new solvent and obtained encouraging results ( Table 1, entries 10 and 11), although not as good as with eucalyptol or CPME (entries 6 to 9). Citral is a compound that does not behave very well at this temperature: the medium blackens as soon as the reaction temperature reaches 95 • C, whereas its boiling point is 229 • C ( Table 1, entry 14). Given this degradation of the medium, we have not studied this solvent in depth, concentrating instead on sabinene and its comparison with eucalyptol, CPME and limonene. product in salt form and then neutralising it in the presence of a base. We therefore continued our study without a base and formed the products in salt form.
As the yields obtained were moderate in both conventional and green solvents, we concentrated on the latter to optimise reaction time, using one equivalent of each reagent. While citral only led to a disappointing yield of 21% in 16 h, increasing the reaction time was beneficial for the other green solvents, allowing us to achieve satisfactory yields of 58 to 75% (Table 1, entries 7,9 and 11). Beyond 16 h, we did not observe any improvement in performance. We used sabinene as a new solvent and obtained encouraging results (Table  1, entries 10 and 11), although not as good as with eucalyptol or CPME (entries 6 to 9). Citral is a compound that does not behave very well at this temperature: the medium blackens as soon as the reaction temperature reaches 95 °C, whereas its boiling point is 229 °C ( Table 1, entry 14). Given this degradation of the medium, we have not studied this solvent in depth, concentrating instead on sabinene and its comparison with eucalyptol, CPME and limonene.

Optimisation in Green Solvents
We therefore continued our optimisation in the previously mentioned green solvents before applying this new solvent (sabinene) to the synthesis of various compounds. The starting 3-amino-2-chloropyridine 2a (1.5 mmol) was heated in 1 mL of solvent in the presence of phenyl isothiocyanate 3a ( Table 2). The yield was improved by increasing the amount of pyridine reagent (

Optimisation in Green Solvents
We therefore continued our optimisation in the previously mentioned green solvents before applying this new solvent (sabinene) to the synthesis of various compounds. The starting 3-amino-2-chloropyridine 2a (1.5 mmol) was heated in 1 mL of solvent in the presence of phenyl isothiocyanate 3a ( Table 2). The yield was improved by increasing the amount of pyridine reagent (Table 2, entries 5, 9 and 12).
Since sabinene is commercially available at 75% purity (Merck, natural sabinene), we distilled it under reduced pressure with a membrane pump at 12 mbar, at 40 • C. However, we found that the reactions carried out in distilled or undistilled sabinene were unaffected and that the yields were equivalent, so we continued our study with commercially available undistilled sabinene.
To visualise the evolution of the reaction, the different phases, using 3-amino-2chloropyridine 2a heated in 1 mL of sabinene in the presence of 4-bromophenyl isothiocyanate, were photographed at different reaction times, starting with the control after mixing the compounds, then during the heating period at 60 • C, after 5 min at 100 • C, after 30 min at 100 • C, then after 4 h at 100 • C, and finally after 24 h of reaction at 100 • C, where complete precipitation of the product could be seen ( Figure 2).  Since sabinene is commercially available at 75% purity (Merck, natural sabinene), we distilled it under reduced pressure with a membrane pump at 12 mbar, at 40 °C. However, we found that the reactions carried out in distilled or undistilled sabinene were unaffected and that the yields were equivalent, so we continued our study with commercially available undistilled sabinene.
To visualise the evolution of the reaction, the different phases, using 3-amino-2-chloropyridine 2a heated in 1 mL of sabinene in the presence of 4-bromophenyl isothiocyanate, were photographed at different reaction times, starting with the control after mixing the compounds, then during the heating period at 60 °C, after 5 min at 100 °C, after 30 min at 100 °C, then after 4h at 100 °C, and finally after 24h of reaction at 100 °C, where complete precipitation of the product could be seen ( Figure 2).  Since sabinene is commercially available at 75% purity (Merck, natural sabinene), we distilled it under reduced pressure with a membrane pump at 12 mbar, at 40 °C. However, we found that the reactions carried out in distilled or undistilled sabinene were unaffected and that the yields were equivalent, so we continued our study with commercially available undistilled sabinene.
To visualise the evolution of the reaction, the different phases, using 3-amino-2-chloropyridine 2a heated in 1 mL of sabinene in the presence of 4-bromophenyl isothiocyanate, were photographed at different reaction times, starting with the control after mixing the compounds, then during the heating period at 60 °C, after 5 min at 100 °C, after 30 min at 100 °C, then after 4h at 100 °C, and finally after 24h of reaction at 100 °C, where complete precipitation of the product could be seen ( Figure 2).

Optimisation of Thiazolo-Pyridine Synthesis in Sabinene Under Microwave Irradiation and Thermal Conditions
Based on these initial results, the reaction was carried out under the conditions described in Table 2, entry 12, in order to obtain the optimum yield. We then tried to reduce the reaction time by using microwave activation. The temperature was also adapted. After completion, the product was filtered and washed with ethyl acetate and diethyl ether, and no further purification was required (Table 3). Under microwave irradiation, the best results were obtained in 1 h at 160 • C, close to the boiling point of sabinene. However, as this temperature is not compatible with all isothiocyanates, we tried to reduce the reaction temperature and found that at 130 • C, the time required was 2 h. As sabinene is not a polar solvent and therefore not the most interesting for microwave reactions, we used 25% of a co-solvent that increases this polarity and chose ethanol or acetonitrile, which has proved very interesting as a co-solvent in previous work under microwave irradiation [42]. It turns out that while ethanol offers no improvement, acetonitrile halves the reaction time at 130 • C, while increasing the yield very slightly (Table 3, entries 4 and 6).
We applied these optimised conditions to a number of isothiocyanates 3 starting with 3-amino-2-chloropyridine 2a under thermal or microwave activation. The results are summarised in Scheme 1. Six new structures were synthesised and obtained in high yields and purity by thermal and microwave activation, the other four having already been obtained in a previous study [40]. Secondly, we investigated the use of other pyridines substituted with an alkyltype electron donor group. Six new structures were synthesised and obtained in high yields and purity by thermal and microwave activation, the other four having already been obtained in a previous study [40]. Secondly, we investigated the use of other pyridines substituted with an alkyltype electron donor group.
Using 3-amino-2-chloro-5-methylpyridine 2b and phenyl isothiocyanate 3a in a sealed tube with 1 mL of sabinene required heating to 160 • C under thermal conditions and took 16 h to achieve a satisfactory result. After the reaction, the mixture was filtered with ethyl acetate and the desired compound was synthesised in a 66% yield (Table 4, entry 5). Six new structures were synthesised and obtained in high yields and purity by thermal and microwave activation, the other four having already been obtained in a previous study [40]. Secondly, we investigated the use of other pyridines substituted with an alkyltype electron donor group.
Using 3-amino-2-chloro-5-methylpyridine 2b and phenyl isothiocyanate 3a in a sealed tube with 1mL of sabinene required heating to 160 °C under thermal conditions and took 16 h to achieve a satisfactory result. After the reaction, the mixture was filtered with ethyl acetate and the desired compound was synthesised in a 66% yield (Table 4, entry 5). In this case, too, we obtained the product in salt form and the reaction temperature was increased due to the donor effect of the para at the chlorine atom, which could explain the lower reactivity of the latter given the mechanism of formation of 2-aminothiazolo [5,4b]pyridine (Scheme 2).
Molecules 2023, 28, x FOR PEER REVIEW 7 of 18 In this case, too, we obtained the product in salt form and the reaction temperature was increased due to the donor effect of the para at the chlorine atom, which could explain the lower reactivity of the latter given the mechanism of formation of 2-aminothiazolo [5,4b]pyridine (Scheme 2). This mechanism was validated by Atland and Molander [41] with the formation of thiourea, in which the tautomeric thione or thiol displaced the chlorine atom.
Under microwave irradiation, the best results were obtained using 1.1 equiv. 3amino-2-chloro-5-methylpyridine 2b and 1 equiv. isothiocyanate 3, in a sealed tube with 1mL sabinene. The reaction was carried out at 130 °C for 2 h to give a yield of 64% (Table  5, entry 7). Again, at the end of the reaction, the mixture was filtered and rinsed with ethyl acetate. This mechanism was validated by Atland and Molander [41] with the formation of thiourea, in which the tautomeric thione or thiol displaced the chlorine atom.
Under microwave irradiation, the best results were obtained using 1.1 equiv. 3-amino-2-chloro-5-methylpyridine 2b and 1 equiv. isothiocyanate 3, in a sealed tube with 1 mL sabinene. The reaction was carried out at 130 • C for 2 h to give a yield of 64% (Table 5, entry  7). Again, at the end of the reaction, the mixture was filtered and rinsed with ethyl acetate. This mechanism was validated by Atland and Molander [41] with the formation of thiourea, in which the tautomeric thione or thiol displaced the chlorine atom.
Under microwave irradiation, the best results were obtained using 1.1 equiv. 3amino-2-chloro-5-methylpyridine 2b and 1 equiv. isothiocyanate 3, in a sealed tube with 1mL sabinene. The reaction was carried out at 130 °C for 2 h to give a yield of 64% (Table  5, entry 7). Again, at the end of the reaction, the mixture was filtered and rinsed with ethyl acetate. We applied these optimised conditions to certain isothiocyanates with 3-amino-2chloro-5-methylpyridine 2b under thermal or microwave activation. The results are summarised in Scheme 3.

Entry
Reaction Time Temperature Yield We applied these optimised conditions to certain isothiocyanates with 3-amino-2chloro-5-methylpyridine 2b under thermal or microwave activation. The results are summarised in Scheme 3. The use of 3-amino-2-chloro-5-methylpyridine 2b gives good yields when activated by conventional heating. The results remain lower under microwave irradiation, but enable the desired products to be generated more quickly. We were able to overcome the deactivating effect of methyl in this reaction by adjusting the conditions.
In parallel, we tested these conditions using phenyl isocyanate 6 to obtain the corresponding oxazolopyridines 8. However, as reported by Sun and co-workers [43], in this case and under our conditions, we also stopped at urea 7 (Scheme 4). The use of 3-amino-2-chloro-5-methylpyridine 2b gives good yields when activated by conventional heating. The results remain lower under microwave irradiation, but enable the desired products to be generated more quickly. We were able to overcome the deactivating effect of methyl in this reaction by adjusting the conditions.
In parallel, we tested these conditions using phenyl isocyanate 6 to obtain the corresponding oxazolopyridines 8. However, as reported by Sun and co-workers [43], in this case and under our conditions, we also stopped at urea 7 (Scheme 4). The use of 3-amino-2-chloro-5-methylpyridine 2b gives good yields when activated by conventional heating. The results remain lower under microwave irradiation, but enable the desired products to be generated more quickly. We were able to overcome the deactivating effect of methyl in this reaction by adjusting the conditions.
In parallel, we tested these conditions using phenyl isocyanate 6 to obtain the corresponding oxazolopyridines 8. However, as reported by Sun and co-workers [43], in this case and under our conditions, we also stopped at urea 7 (Scheme 4).

Scheme 4.
Synthesis of corresponding urea instead of desired oxazolopyridine.

General Information
All reagents were purchased from commercial suppliers and used without further purification. Natural sabinene was purchased from Merck (KGaA, Darmstadt, Germany) with 75% of purity. Unless otherwise specified, sabinene was used in its commercial form. 1

General Information
All reagents were purchased from commercial suppliers and used without further purification. Natural sabinene was purchased from Merck (KGaA, Darmstadt, Germany) with 75% of purity. Unless otherwise specified, sabinene was used in its commercial form. 1 H and 13 C NMR spectra were recorded on a Bruker DPX 250 ( 13 C, 62.9 MHz) (Bruker, Wissembourg, France), Bruker Avance II 250.13 ( 13 C, 63 MHz), Bruker Avance 400.13 ( 13 C, 101 MHz) (Bruker, Wissembourg, France), or on a Bruker Avance III HD nanobay 400.13 ( 13 C, 101 MHz) (Bruker, Wissembourg, France). Chemical shifts are expressed in parts per million (ppm) and were calibrated on deuterated or residual non-deuterated solvent peaks for 1 H and 13 C spectra. The following abbreviations are used for proton spectra multiplicities: b: broad, s: singlet, d: doublet, t: triplet, q: quartet, p: pentuplet, m: multiplet. Microwave-assisted reactions were carried out in a Biotage Initiator microwave synthesis instrument and temperatures were measured using an IR sensor (Biotage, Uppsala, Sweden). Melting points (p.m. ( • C)) were taken on samples placed in open capillary tubes on a Thermo Fisher Melting Point Instrument Digital 9000 Series IA9200X6 and were not corrected. High-resolution mass spectra (HRMS) were performed on a Bruker 4G Maxis UHR-q-TOF mass spectrometer (Bruker, Wissembourg, France), with an electrospray ionization (ESI) mode. The numbering of the atoms on the molecules has been chosen arbitrarily and is indicated on the drawings of the molecules for a better understanding of the NMR spectra.

General Procedure (1)
The substituted 3-amino-2-chloropyridine 2 (1.65 mmol; 1.1 equiv.) and substituted isothiocyanate 3 (1.5 mmol; 1 equiv.) were dissolved in 1.0 mL of sabinene and stirred at 100 • C for 16 h. The mixture was allowed to cool to room temperature. The mixture was then filtered and washed with ethyl acetate followed by diethyl ether. The product was isolated without further purification.

General Procedure (2)
In a sealed tube, the substituted 3-amino-2-chloropyridine 2 (1.65 mmol ; 1.1 equiv.) and substituted isothiocyanate 3 (1.5 mmol ; 1 equiv.) were dissolved in 1.0 mL of sabinene. The mixture was placed under microwave irradiation for 2 h at 130 • C. The mixture was allowed to cool to room temperature. Then, the reaction was filtered and washed with ethyl acetate followed by diethyl ether. The product was isolated without further purification.

General Procedure (3)
In a sealed tube, the substituted 3-amino-2-chloropyridine 2 (1.65 mmol; 1.1 equiv.) and substituted isothiocyanate 3 (1.5 mmol; 1 equiv.) were dissolved in the solvent consisting of 0.75 mL sabinene and 0.25 mL acetonitrile. The mixture was placed under microwave irradiation for 2 h at 130 • C. After cooling to room temperature, the reaction mixture was filtered and washed with ethyl acetate followed by diethyl ether. The product was isolated without further purification.