Skeleton Synthesis of a Plant-Derived Radioprotective Alkaloid Born to Produce a Novel Fused Heterocycle

Alkaloids are a material treasure bestowed on humans by nature owing to their numerous biological activities. Orychophragine D, an alkaloid isolated from the seeds of Orychophragmus violaceus was identified as bearing a novel skeleton and proved to have an excellent radioprotective effect. Different from the common alkaloid structure, the main block of orychophragine D is constructed of an oxotriazine and an oxopiperazine, which are connected in parallel by a C-N bond. In this paper, a preparation method for the novel heterocycle skeleton of orychophragine D is proposed for the first time. N-Boc-L-serine was utilized as the original material to complete the preparation with 11 steps in a 13% overall yield. A hydroxyl group was established on the side chain of the skeleton as the reaction site for researchers to conduct further structural modification or derivatization.


Introduction
Alkaloids are the weapons and shields of plants, protecting plants against biotic and abiotic stresses [1]. Biotic stress includes pests, pathogenic microorganisms, and predators. The main functions of alkaloids are to respond to antibacterial, antifungal, and antiherbivory biotic stress [2]. For example, piperine from black pepper and αtomatine from tomatoes have been reported to have antibacterial and antifungal effects [3,4]. Swainsonine from Locoweed has been reported to cause intoxication in livestock due to its inhibition of αmannosidase activity, affecting N-glycan cell membrane synthesis [5]. The abiotic pressures faced by plants mainly come from harsh environments such as drought, salinity, and high temperatures [6]. Alkaloids synthesized by plants are stored in organelles of specific glands, released, and exported to target tissues when stress signals from the environment are sensed [7,8]. In short, alkaloids are essential for plant survival.
Alkaloids are also a material treasure bestowed on humans by nature owing to their numerous biological activities. Novel bioactive alkaloids from plants are constantly being discovered [9]. Alkaloids named orychophragine A-D isolated from the seeds of Orychophragmus violaceus have unique skeletons and excellent antiradiation or antitumor biological activity [10]. In one study, orychophragine D could improve the survival rate of mice to 100% at a radiation dose of 8 Gy, while the survival rate of the vehicle was 0% [11]. Different from the common alkaloid structure, the main block of orychophragine D consists of an oxotriazine and an oxopiperazine, which are connected in parallel by a C-N bond (Figure 1). In this study, a preparation method for this novel heterocycle skeleton of orychophragine D was developed for the first time. N-Boc-L-serine was used as the original material to prepare this heterocycle with 11 steps and a 13% overall yield. The hydroxyl group was established in the side chain of the fused heterocycle for researchers to bond ( Figure 1). In this study, a preparation method for this novel heterocycle skeleton of orychophragine D was developed for the first time. N-Boc-L-serine was used as the original material to prepare this heterocycle with 11 steps and a 13% overall yield. The hydroxyl group was established in the side chain of the fused heterocycle for researchers to conduct further transformation or derivatization of this heterocycle, which might lead to drug candidates with better radioprotective effects.

Results
We began our protocol with N-Boc-L-serine (Boc means t-butyloxy carbonyl, as shown in Scheme 1), with the chiral center being retained until the end product. The hydroxyl in 6 was protected by tetrahydropyran (THP) because THP can exist stably in the presence of a strong base or reducing agent during the synthesis process [12]. Carboxylic acid 6 was converted into an amide under the action of 1,1'-Carbonyldiimidazole (CDI) and ammonia to give 7. Amide 7 was reduced with an excess dose of lithium aluminum hydride (LiAlH4) while the amide and N-Boc were reduced to an amino and a N-methyl, respectively, in a one-step reaction to give 8. The Boc group on amino not only played a protective role, its reduced product, methyl, was also retained in the product as part of the backbone. Amine 8 underwent an ammonolysis with dimethyl oxalate to form dioxypiperazine 9. Scheme 1. Synthesis of the dioxypiperazine intermediate.
Benzyl (Bn) was initially used as the hydroxyl-protecting group in our protocol. Unfortunately, benzyl could not be removed in the synthesis route. Catalytic hydrogenation and other common methods to remove benzyl could not be performed. THP was selected as a qualified protective group due to its stability and ease of operation. However, THP

Results
We began our protocol with N-Boc-L-serine (Boc means t-butyloxy carbonyl, as shown in Scheme 1), with the chiral center being retained until the end product. The hydroxyl in 6 was protected by tetrahydropyran (THP) because THP can exist stably in the presence of a strong base or reducing agent during the synthesis process [12]. Carboxylic acid 6 was converted into an amide under the action of 1,1'-Carbonyldiimidazole (CDI) and ammonia to give 7. Amide 7 was reduced with an excess dose of lithium aluminum hydride (LiAlH 4 ) while the amide and N-Boc were reduced to an amino and a N-methyl, respectively, in a one-step reaction to give 8. The Boc group on amino not only played a protective role, its reduced product, methyl, was also retained in the product as part of the backbone. Amine 8 underwent an ammonolysis with dimethyl oxalate to form dioxypiperazine 9.
bond ( Figure 1). In this study, a preparation method for this novel heterocycle skeleton orychophragine D was developed for the first time. N-Boc-L-serine was used as the or inal material to prepare this heterocycle with 11 steps and a 13% overall yield. The h droxyl group was established in the side chain of the fused heterocycle for researchers conduct further transformation or derivatization of this heterocycle, which might lead drug candidates with better radioprotective effects.

Results
We began our protocol with N-Boc-L-serine (Boc means t-butyloxy carbonyl, shown in Scheme 1), with the chiral center being retained until the end product. The h droxyl in 6 was protected by tetrahydropyran (THP) because THP can exist stably in presence of a strong base or reducing agent during the synthesis process [12]. Carboxy acid 6 was converted into an amide under the action of 1,1'-Carbonyldiimidazole (CD and ammonia to give 7. Amide 7 was reduced with an excess dose of lithium aluminu hydride (LiAlH4) while the amide and N-Boc were reduced to an amino and a N-meth respectively, in a one-step reaction to give 8. The Boc group on amino not only playe protective role, its reduced product, methyl, was also retained in the product as part the backbone. Amine 8 underwent an ammonolysis with dimethyl oxalate to form dio piperazine 9. Scheme 1. Synthesis of the dioxypiperazine intermediate.
Benzyl (Bn) was initially used as the hydroxyl-protecting group in our protocol. U fortunately, benzyl could not be removed in the synthesis route. Catalytic hydrogenat and other common methods to remove benzyl could not be performed. THP was select as a qualified protective group due to its stability and ease of operation. However, TH Scheme 1. Synthesis of the dioxypiperazine intermediate.
Benzyl (Bn) was initially used as the hydroxyl-protecting group in our protocol. Unfortunately, benzyl could not be removed in the synthesis route. Catalytic hydrogenation and other common methods to remove benzyl could not be performed. THP was selected as a qualified protective group due to its stability and ease of operation. However, THP could be selectively removed from the subsequently introduced Boc. We chose to replace the protective group in 9, with tert-butyl diphenyl silyl (TBDPS) being a suitable alternative.
TBDPS could be removed with tetrabutylammonium fluoride (TBAF), which has been proven to not affect the stability of Boc [13]. The removal of THP in 9 and the introduction of TBDPS in 10 were conducted following common methods described in the literature (Scheme 2).
Molecules 2023, 28, x FOR PEER REVIEW 3 could be selectively removed from the subsequently introduced Boc. We chose to repla the protective group in 9, with tert-butyl diphenyl silyl (TBDPS) being a suitable alter tive. TBDPS could be removed with tetrabutylammonium fluoride (TBAF), which h been proven to not affect the stability of Boc [13]. The removal of THP in 9 and the int duction of TBDPS in 10 were conducted following common methods described in the erature (Scheme 2).

Scheme 2.
Replacement of the protecting group.
Two amide carbonyls in 11 (Scheme 3) were chemically selective for Lawesson r gent. No thiolation occurred on the carbonyl adjacent to the tertiary amine when equivalent of the Lawesson reagent was controlled below 0.5, and sulfide 12 was therefo prepared efficiently. In the initial attempt to prepare 13, we obtained a low yield becau there were many factors that affected the reaction, such as the Lewis acid, solvent, te perature, and even the order in which the reactants were added. We found that if 12 a 1-Boc-guanidine were mixed before Lewis acid was added, this reaction had a bet chemical selectivity. This addition order was applied to investigate the relationship tween the reactants and yield (Table 1). We found that when mercuric chloride served the Lewis acid and the solvent was N, N-dimethylformamide (DMF), 13 could be synt sized in a satisfactory yield. In the closed-loop reaction of 13, CDI was proven to hav better performance than did triphosgene both in terms of operation and yield. The TBD and Boc in compound 14 were removed with TBAF and trifluoroacetic acid (TFA), resp tively. The removal of TBDPS did not affect the stability of Boc to give 15, which facilita the further transformation and derivatization of this heterocycle.  Two amide carbonyls in 11 (Scheme 3) were chemically selective for Lawesson reagent. No thiolation occurred on the carbonyl adjacent to the tertiary amine when the equivalent of the Lawesson reagent was controlled below 0.5, and sulfide 12 was therefore prepared efficiently. In the initial attempt to prepare 13, we obtained a low yield because there were many factors that affected the reaction, such as the Lewis acid, solvent, temperature, and even the order in which the reactants were added. We found that if 12 and 1-Bocguanidine were mixed before Lewis acid was added, this reaction had a better chemical selectivity. This addition order was applied to investigate the relationship between the reactants and yield (Table 1). We found that when mercuric chloride served as the Lewis acid and the solvent was N, N-dimethylformamide (DMF), 13 could be synthesized in a satisfactory yield. In the closed-loop reaction of 13, CDI was proven to have a better performance than did triphosgene both in terms of operation and yield. The TBDPS and Boc in compound 14 were removed with TBAF and trifluoroacetic acid (TFA), respectively. The removal of TBDPS did not affect the stability of Boc to give 15, which facilitated the further transformation and derivatization of this heterocycle. could be selectively removed from the subsequently introduced Boc. We chose to replace the protective group in 9, with tert-butyl diphenyl silyl (TBDPS) being a suitable alternative. TBDPS could be removed with tetrabutylammonium fluoride (TBAF), which has been proven to not affect the stability of Boc [13]. The removal of THP in 9 and the introduction of TBDPS in 10 were conducted following common methods described in the literature (Scheme 2). Scheme 2. Replacement of the protecting group.
Two amide carbonyls in 11 (Scheme 3) were chemically selective for Lawesson reagent. No thiolation occurred on the carbonyl adjacent to the tertiary amine when the equivalent of the Lawesson reagent was controlled below 0.5, and sulfide 12 was therefore prepared efficiently. In the initial attempt to prepare 13, we obtained a low yield because there were many factors that affected the reaction, such as the Lewis acid, solvent, temperature, and even the order in which the reactants were added. We found that if 12 and 1-Boc-guanidine were mixed before Lewis acid was added, this reaction had a better chemical selectivity. This addition order was applied to investigate the relationship between the reactants and yield (Table 1). We found that when mercuric chloride served as the Lewis acid and the solvent was N, N-dimethylformamide (DMF), 13 could be synthesized in a satisfactory yield. In the closed-loop reaction of 13, CDI was proven to have a better performance than did triphosgene both in terms of operation and yield. The TBDPS and Boc in compound 14 were removed with TBAF and trifluoroacetic acid (TFA), respectively. The removal of TBDPS did not affect the stability of Boc to give 15, which facilitated the further transformation and derivatization of this heterocycle.

Discussion
We exerted considerable effort in choosing and substituting the protecting groups in the synthesis route to achieve the chemoselectivity of reactions and selective removal of the protecting groups. A single exposed functional group could facilitate further modification or derivatization of the heterocycle by investigators.
During the preparation of 13, it was found that the addition order of reactants had a significant impact on the yield. When 12 and Lewis acid were mixed in absence of 1-Boc-guanidine, many byproducts appeared; meanwhile, when 12 and 1-Boc-guanidine were mixed before Lewis acid was added, this reaction had a better chemical selectivity. According to this phenomenon, a prediction of the reaction mechanism was promoted, as shown in Scheme 4. When mercury chloride was mixed with 12 before 1-Boc-guanidine was added, the sulfur negative ions attacked the mercury ions to form a carbocation intermediate 12a, and the carbocation ions of the intermediate 12a attacked the negative electric groups within molecule 12, resulting in the formation of by-products. If mercury chloride was added to the mixture of 1-Boc-guanidine and 12, the intermediate 12a preferentially attacked the more nucleophilic guanidine to form intermediate 12b, in which the mercury sulfide left with a pair of electrons and two protons was removed with a base to form compound 13.

Discussion
We exerted considerable effort in choosing and substituting the protecting groups in the synthesis route to achieve the chemoselectivity of reactions and selective removal of the protecting groups. A single exposed functional group could facilitate further modification or derivatization of the heterocycle by investigators.
During the preparation of 13, it was found that the addition order of reactants had a significant impact on the yield. When 12 and Lewis acid were mixed in absence of 1-Bocguanidine, many byproducts appeared; meanwhile, when 12 and 1-Boc-guanidine were mixed before Lewis acid was added, this reaction had a better chemical selectivity. According to this phenomenon, a prediction of the reaction mechanism was promoted, as shown in Scheme 4. When mercury chloride was mixed with 12 before 1-Boc-guanidine was added, the sulfur negative ions attacked the mercury ions to form a carbocation intermediate 12a, and the carbocation ions of the intermediate 12a attacked the negative electric groups within molecule 12, resulting in the formation of by-products. If mercury chloride was added to the mixture of 1-Boc-guanidine and 12, the intermediate 12a preferentially attacked the more nucleophilic guanidine to form intermediate 12b, in which the mercury sulfide left with a pair of electrons and two protons was removed with a base to form compound 13.

Reagents and Instruments
All chemicals were obtained from a supplier (Sigma-Adrich, St. Louis, MO, USA, TCI, Ark). The NMR spectra were recorded with a JNM-ECA-400 spectrometer at 300K. Mass spectra were recorded with a Thermo Finnigan LCQ Advantage spectrometer. Silica gel chromatography was performed using 200-300 mesh silica gel. (6) Compound 6 was prepared following the methods described in the literature [14].

Conclusions
The novel heterocycle skeleton in orychophragine D, a promising radioprotective alkaloid derived from the seeds of Orychophragmus violaceus was prepared. The synthesis was started with N-Boc-L-serine and completed in 11 steps and a 13% overall yield. The hydroxyl group was established on the side chain of the skeleton as the reaction site for researchers to conduct further structural modification or derivatization. We hope that this study could contribute to the discovery of new molecules with excellent radiation protective activity.