New Rare Ent-Clerodane Diterpene Peroxides from Egyptian Mountain Tea (Qourtom) and Its Chemosystem as Herbal Remedies and Phytonutrients Agents

Genus Stachys, the largest genera of the family Lamiaceae, and its species are frequently used as herbal teas due to their essential oils. Tubers of some Stachys species are also consumed as important nutrients for humans and animals due to their carbohydrate contents. Three new neo-clerodane diterpene peroxides, named stachaegyptin F-H (1, 2, and 4), together with two known compounds, stachysperoxide (3) and stachaegyptin A (5), were isolated from Stachys aegyptiaca aerial parts. Their structures were determined using a combination of spectroscopic techniques, including HR-FAB-MS and extensive 1D and 2D NMR (1H, 13C NMR, DEPT, 1H-1H COSY, HMQC, HMBC and NOESY) analyses. Additionally, a biosynthetic pathway for the isolated compounds (1–5) was discussed. The chemotaxonomic significance of the isolated diterpenoids of S. aegyptiaca in comparison to the previous reported ones from other Stachys species was also studied.


Introduction
The genus Stachys (woundwort) has about 300 species growing wild in the temperate and tropical regions throughout the world except the continent of Australia and New Zealand [1]. In the Mediterranean region and Iran, Stachys species are known as mountain tea with great medicinal and nutritional values due to their traditional uses as food additives, herbal teas, and medicinal

Results and Discussion
The CH 2 Cl 2 :MeOH (1:1) extract of S. aegyptiaca aerial parts afforded three new ent-neo-clerodane diterpenoids, named stachaegyptin F (1), stachaegyptin G (2), and stachaegyptin H (4), together with two known compounds, stachysperoxide (3) and stachaegyptin A (5) (Figure 1), using chromatographic techniques. Their structures were established using extensive 1D [ 1 H (Table 1), 13 C NMR (Table 2) (Table 2), which was in agreement with the molecular formula. Their multiplicities were deduced from the results of 13 C DEPT NMR analyses as four methyls, five methylenes (two olefinic), six methines (two olefinic and two oxygenated at δ C 73.2 and δ C 83.7), and five quaternary carbons (two olefinic and one keto at δ C 199.7) ( Table 2). With 20 carbons and six degrees of unsaturation; one of them was assigned as a keto group (δ C 199.8) and three were attributed to double bonds, therefore, compound 1 is apparently a bicyclic diterpene. The 1 H NMR analysis of 1 (Table 1) displayed typical signals for two tertiary methyls at δ H 1.02 and 1.39 (each 3H, s), a secondary methyl at δ H 1.09 (3H, d, J = 7.0 Hz) and an olefinic methyl at δ H 1.92 (3H, s), which showed a correlation in the Double Quantum Filtered COSY (DQF-COSY) spectrum with an olefinic proton signal at δ H 5.68 (1H, br s), indicating the presence of a trisubstituted double bond. The spectrum also showed two oxomehine protons at δ H 4.09 (1H, br d, J = 3.4) and δ H 4.66 (1H, dd, J = 7.5 and 2.7 Hz), an ABX spin system at δ H 5.17 (1H, d, J = 11.0 Hz), δ H 5.49 (1H, d, J = 17.0 Hz) and δ H 6.29 (1H, dd, J = 17.0, 11.0 Hz), and two terminal olefinic protons at δ H 5.23 and 5.13 (each 1H, s). The COSY spectrum exhibited four spin systems coupled with ring A, ring B, and the side chain ( Figure 2). All these accumulated data are regular with the plain skeleton of neo-clerodane diterpenes formerly isolated from this genus [27,40,55]. Molecules 2020, x, x FOR PEER REVIEW 3 of 13 Interpretation of the 2D NMR data, including DQF-COSY, HMQC and HMBC, clearly indicated that we are dealing with a structure similar to that of stachaegyptin A (5), previously isolated from this species, and its structure was confirmed by X-ray crystallography [40]. The distinct difference observed in the 1 H NMR spectrum of 1 was the additional oxymethine proton at δH 4.66 (1H, dd, J = 7.5 and 2.7 Hz) (H-12), which showed couplings in the DQF-COSY spectrum with H2-11 at δH 1.64 (1H, dd, J = 16.5, 7.5 Hz) (H-11a) and δH 1.50 (1H, dd, J = 16.5, 2.7 Hz) (H-11b), while in the HMQC spectrum this proton showed a correlation with the oxymethine carbon at δC 83.7. The 13 C NMR data of 1 also revealed similarities with those of stachaegyptin A (5) except that the methylene carbon C-12 in 5 was replaced by the oxomethine carbon at δC 83.7 in 1. The HMBC experiment ( Figure 2) confirmed the presence of 12-oxymethine in 1 by the HMBC connections from H-12 (δH 4.66) to C-9 (δC 39.6), C-11 (δC 41.2), C-14 (δC 134.8) and C-16 (δC 116.5). With four oxygen atoms in 1 (C20H30O4, HR-FAB-MS), three of them were assigned from the 13 C NMR data as two oxomethine carbons [δC 73.2 (C-7) and δC 83.7 (C-12)] and one keto group at δC 199.8 (C-2). Additionally, and due to the lack of an additional oxymethine signal, the remaining oxygen should, therefore, be a part of a hydroperoxyl group instead of a hydroxyl group.   Interpretation of the 2D NMR data, including DQF-COSY, HMQC and HMBC, clearly indicated that we are dealing with a structure similar to that of stachaegyptin A (5), previously isolated from this species, and its structure was confirmed by X-ray crystallography [40]. The distinct difference observed in the 1 H NMR spectrum of 1 was the additional oxymethine proton at δ H 4.66 (1H, dd, J = 7.5 and 2.7 Hz) (H-12), which showed couplings in the DQF-COSY spectrum with H 2 -11 at δ H 1.64 (1H, dd, J = 16.5, 7.5 Hz) (H-11a) and δ H 1.50 (1H, dd, J = 16.5, 2.7 Hz) (H-11b), while in the HMQC spectrum this proton showed a correlation with the oxymethine carbon at δ C 83.7. The 13 C NMR data of 1 also revealed similarities with those of stachaegyptin A (5) except that the methylene carbon C-12 in 5 was replaced by the oxomethine carbon at δ C 83.7 in 1. The HMBC experiment ( Figure 2) confirmed the presence of 12-oxymethine in 1 by the HMBC connections from H-12 (δ H 4.66) to C-9 (δ C 39.6), C-11 (δ C 41.2), C-14 (δ C 134.8) and C-16 (δ C 116.5). With four oxygen atoms in 1 (C 20 H 30 O 4 , HR-FAB-MS), three of them were assigned from the 13 C NMR data as two oxomethine carbons [δ C 73.2 (C-7) and δ C 83.7 (C-12)] and one keto group at δ C 199.8 (C-2). Additionally, and due to the lack of an additional oxymethine signal, the remaining oxygen should, therefore, be a part of a hydroperoxyl group instead of a hydroxyl group. This was supported by the positive TLC spray test for hydroperoxides (N,N-dimethyl-1,4phenylenediammonium chloride) [56] as well as from the unusual downfield chemical shift of 12-oxymethine at δ C 83.6, which was very similar to those reported for related 12-hydroperoxy diterpenes [56,57]. Related 12-hydroxy diterpenes, by contrast, showed a 12-oxymethine between δ C 62.0-64.0 [58][59][60]. Comprehensive assignment of 1 was established from the results of DQF-COSY, HMQC, and HMBC NMR experiments. Therefore, 1 could be elucidated as 12-hydroperoxy derivative of 5. This was supported by the positive TLC spray test for hydroperoxides (N,N-dimethyl-1,4phenylenediammonium chloride) [56] as well as from the unusual downfield chemical shift of 12oxymethine at δC 83.6, which was very similar to those reported for related 12-hydroperoxy diterpenes [56,57]. Related 12-hydroxy diterpenes, by contrast, showed a 12-oxymethine between δC 62.0-64.0 [58][59][60]. Comprehensive assignment of 1 was established from the results of DQF-COSY, HMQC, and HMBC NMR experiments. Therefore, 1 could be elucidated as 12-hydroperoxy derivative of 5. The relative stereochemistry of 1 was determined by the coupling constants, the NOESY experiments ( Figure 3) with inspection of the 3D molecular model, and the biogenetic correlation with stachaegyptin A (5), where its structure and stereochemistry were confirmed by X-ray crystallography [40]. The hydroxyl group configuration at C-7 was assigned to be α (axial), conferring the small coupling constants of H-7 (3.4 Hz), which was similar to those reported for 5 and other neoclerodane diterpenes [27,40]. The NOESY connections between H-7 (δH 4.09) and H-8 (δH 1.90) indicated that these protons are on β-configuration of the B ring. The NOESY correlations observed The relative stereochemistry of 1 was determined by the coupling constants, the NOESY experiments ( Figure 3) with inspection of the 3D molecular model, and the biogenetic correlation with stachaegyptin A (5), where its structure and stereochemistry were confirmed by X-ray crystallography [40]. The hydroxyl group configuration at C-7 was assigned to be α (axial), conferring the small coupling constants of H-7 (3.4 Hz), which was similar to those reported for 5 and other neo-clerodane diterpenes [27,40]. The NOESY connections between H-7 (δ H 4.09) and H-8 (δ H 1.90) indicated that these protons are on β-configuration of the B ring. The NOESY correlations observed between CH 3 -17 (δ H 1.09) and CH 3 -20 (δ H 1.02) and between CH 3 -20 and CH 3 -19 (δ H 1.39) indicated that these methyl groups are all on the same side in an α-configuration. The absence of a NOESY correlation between CH 3 -19α and H-10 revealed that the A/B ring system was trans-diaxially oriented, and the orientation of H-10 was β. All of previous results were well matched with the biogenetic precedent and formerly reported NMR chemical shift data for stachaegyptin 5 and related neo-clerodane diterpenes with the same configurations [27,40]. The C-12 configuration was determined by the NOESY analysis with inspection of the 3D molecular model (Figure 3). The observed correlations between H-12 (δ H 4.66), H-1β (δ H 2.29), and H-10 (δ H 2.14) implied that these protons were in closeness and confirmed that the C-12 stereo center had the R configuration as those reported for (12R) 12-hydroperoxy and 12-hydroxy diterpenes [56][57][58][59][60][61][62]. Therefore, the structure of 1 was established as 12(R)-12-hydroperoxy-7α-hydroxy-neo-cleroda-3,13(16),14-triene-2-one, and was named stachaegyptin F. biogenetic precedent and formerly reported NMR chemical shift data for stachaegyptin 5 and related neo-clerodane diterpenes with the same configurations [27,40]. The C-12 configuration was determined by the NOESY analysis with inspection of the 3D molecular model (Figure 3). The observed correlations between H-12 (δH 4.66), H-1β (δH 2.29), and H-10 (δH 2.14) implied that these protons were in closeness and confirmed that the C-12 stereo center had the R configuration as those reported for (12R) 12-hydroperoxy and 12-hydroxy diterpenes [56][57][58][59][60][61][62]. Therefore, the structure of 1 was established as 12(R)-12-hydroperoxy-7α-hydroxy-neo-cleroda-3,13(16),14-triene-2-one, and was named stachaegyptin F. Compound 2 was isolated as a colorless oil with an optical rotation of [α] 25 D 29 (c, 0.005, MeOH). The FAB-MS spectrum of 2 exhibited the base peak at m/z 357 [M + Na] + , consistent with a molecular formula C20H30O4, which was established by a molecular ion peak at m/z 357.2042[M + Na] + (calcd. for C20H30O4Na, 357.2044) in the HR-FAB-MS analysis. This formula was the same as that reported for 1. The positive reaction on TLC with N,N-dimethyl-1,4-phenylenediammonium chloride) [60] also revealed the presence of a hydroperoxid as in 1. The 1 H and 13 C NMR spectra of 2 (Tables 1 and 2) were almost identical with those reported for 1, except for the upfield chemical shifts of CH3-17 (δH 0.99) as well as H-8 (δH 1.71), in addition to the downfield shift of H-1β (δH 2.60) in 2 comparing with those of 1. The 2D NMR experiments including the DQF-COSY, HMQC, and HMBC exhibited an identical planar structure to that of 1. Additionally, combined NOESY and coupling contacts analysis clearly indicated that 2 is matching the relative stereochemistry of 1 in the bicyclic system. All the above data and differences between 1 and 2 established that 2 should be an epimer of 1 at C-12 (S configuration) as previously shown in related compounds [57,[60][61][62]. This was supported by the NOESY experiment with inspection of the 3D-molecular model ( Figure 3). The strong correlations between H-12, H-10β, and H-8β, together with the absence of a NOESY correlation between H-12 and H-1β, confirmed the S configuration at C-12 in 2 instead of 12R as in 1.    (Tables 1 and 2) were almost identical with those reported for 1, except for the upfield chemical shifts of CH 3 -17 (δ H 0.99) as well as H-8 (δ H 1.71), in addition to the downfield shift of H-1β (δ H 2.60) in 2 comparing with those of 1. The 2D NMR experiments including the DQF-COSY, HMQC, and HMBC exhibited an identical planar structure to that of 1. Additionally, combined NOESY and coupling contacts analysis clearly indicated that 2 is matching the relative stereochemistry of 1 in the bicyclic system. All the above data and differences between 1 and 2 established that 2 should be an epimer of 1 at C-12 (S configuration) as previously shown in related compounds [57,[60][61][62]. This was supported by the NOESY experiment with inspection of the 3D-molecular model (Figure 3). The strong correlations between H-12, H-10β, and H-8β, together with the absence of a NOESY correlation between H-12 and H-1β, confirmed the S configuration at C-12 in 2 instead of 12R as in 1.
Further confirmation was given by the relative downfield shift of H-1β at δ H 2.60 in 2, instead of that at δ H 2.29 in 1, which was attributed to the presence of H-1β in a close proximity to the hydroperoxyl group. By contrast, H-8β and CH 3 -17 were slightly shifted at higher-field (δ H 1.71 and δ H 0.99, respectively), than those of 1 at δ H 1.90 (H-8β) and δ H 1.09 (CH 3 -17) [57,59,61,62]. Accordingly, the structure of 2 was established as 12(S)-12-hydroperoxy-7α-hydroxy-neo-cleroda-3,13(16),14-triene-2-one, and was named stachaegyptin G. Both epimers 1 and 2 have 6 stereocenters, and only one center (C-12) was inverted from 12R to 12S. Therefore, 1 and 2 are diastereomers.  (Table 2), which displayed 20 carbon resonances. Their multiplicities were determined from DEPT analysis as five methyls, four methylenes (one oxygenated at δ C 69.8), six methines (two olefinic and two oxygenated at δ 73.3 and 79.0), and five quaternary carbons (two olefinic and one keto at δ 200.7). The 1 H NMR spectrum of 1 (Table 1)  The 1 H and 13 C NMR spectra as well as the 2D NMR data, including DQF-COSY, HMQC and HMBC (Figure 2), clearly established that we are dealing with a structure almost identical to that of stachyaegyptin C (3), previously isolated from this species [41]. The distinct differences observed in the 1 H NMR spectrum of 4 showed a slightly higher-field position chemical shift of CH 3 -17 (δ H 1.06) in 4 than that in 3 (δ H 1.13), also H-8 was shifted at higher field (δ H 1.69) in 4 than that of 3 (δ H 2.06). In contrary, the chemical shift of H-1β was at lower field value (δ H 2.80) in 4 than 3 (δ H 2.32). The results of the 2D NMR experiments achieved an indistinguishable planar structure to that of 3. The NOESY and coupling contacts analysis clearly indicated that 4 had identical relative stereochemistry with 3 in the bicyclic system. All the above data and differences between 4 and 3 established that compound 4 should be an isomer of 3 epimerized at C-12 (S configuration). This result was supported by the NOESY experiment with inspection of the 3D molecular model (Figure 3).
To the best of our knowledge, these new diterpenes hydroperoxides (1 and 2) and the cyclic peroxide (4) are rare secondary metabolites.

Proposed Biosynthetic Pathway of the Isolated Compounds
Biosynthetically, diterpenoids classes in plant catalyze a proton-initiated cationic cycloisomerization of geranylgeranyl diphosphate (GGPP), generating a labdane-type intermediate [63]. Subsequently, labdane as precursor can undergo a stepwise migration process of methyl and hydride shift, yielding a halimane-type intermediate, which can then progress to either cis or trans clerodanes [31]. Compound 5 is proposed to go through simply enzymatic hydroxylation and oxidation of clerodane-type intermediate [64]. Based on Capon's model for biosynthesis of endoperoxides, compound 5 is subjected to enzymatic hydroperoxidation at C-12 to generate compound 1, which then undergoes oxa-Michael cyclization to produce compound 3 [65]. In addition, both compound 1 and 3 can generate their corresponding epimers 2 and 4, respectively, by further rearrangement and isomerization reactions (Figure 4).

General Procedures
The 1 H NMR (600 MHz, CDCl 3 ), 13 C NMR (150 MHz, CDCl 3 ), and the 2D NMR spectra were recorded on a JEOL JNM-ECA 600 spectrometer (JEOL Ltd., Tokyo, Japan). All chemical shifts (δ) are given in ppm units with reference to TMS as an internal standard, and coupling constants (J) are reported in Hz. The IR spectra were taken on a Shimadzu FT-IR-8100 spectrometer. Specific rotations were measured on a Horiba SEPA-300 digital polarimeter (l = 5 cm).

Plant Material
The aerial parts of S. aegyptiaca were collected from Southern Sinai in Egypt during May 2016. A voucher specimen (SK-1055) has been deposited in the Herbarium of Saint Katherine protectorate, Egypt, with collection permission granted for scientific purposes by the Saint Katherine protectorate.