A Neoclerodane Orthoester and Other New Neoclerodane Diterpenoids From Teucrium Yemense, Which Stimulate the Secretion of Insulin From Pancreatic Islets

Teucrium yemense, a medicinal plant commonly grown in Saudi Arabia and Yemen, is traditionally used to treat infections, kidney diseases, rheumatism, and diabetes. Extraction of the dried aerial parts of the plant with methanol, followed by further extraction with butanol and chromatography, gave twenty novel neoclerodanes. Their structures, relative congurations and some conformations were determined by MS and 1-D and 2-D NMR techniques. Most were fairly conventional but one contained an unusual stable orthoester, one had its (C-16)-(C-13)-(C-14)-(C-15) (tetrahydro)furan unit present as a succinic anhydride and one had a rearranged carbon skeleton resulting from ring-contraction to give a central octahydroindene bicyclic core, rather than the usual decalin. Mechanisms are proposed for the biosynthetic formation of the orthoester and for the ring-contraction. Four novel neoclerodanes increased the glucose-triggered release of insulin from isolated murine pancreatic islets by more than the standard drug tolbutamide, showing that they are potential leads for the development of new anti-diabetic drugs. H-7 H-6. C-6. C-5. CH 2 3.69, 4.36; HO-19 4.07) HSQC Both H-19 strong 3-bond HMBC cross-peaks to quaternary C-4 (δ 68.8), part of a spiro-oxirane. In oxirane, H-18 left resonated as a doublet at δ 2.69 a doublet at 2.89. These diastereotopic protons were distinguished by NOESY from H-18 left to HO-3 (δ 4.66) and one H-2 (δ 1.30) and from H-18 right to H-19 (δ 3.69), H-6 Three-bond HMBC from both H-18 identied C-3 (δ 64.3), from which H-3 4.19) HSQC. HMBC from C-3 identied its HO-3 as a doublet at δ 4.66. The ring completed HMBC and HSQC cross-peaks identifying C-2 (δ 34.1), H-2 1.30, δ 2.05), C-1 22.0) H-1 a strong HMBC linking H-2 (δ with C-10


Introduction
Teucrium is a genus of the Lamiaceae family. Plants in this large genus are perennial herbs, shrubs and subshrubs but present many different appearances. 1 They are widespread in the Middle East, Southeast Asia, Central and South America and countries surrounding the Mediterranean Sea. 2 Saudi Arabia hosts six species of Teucrium and is thought to be one of the original centres in which these plants developed. 3 Various Teucrium species have been used traditionally for millennia as diuretic, diaphoretic, antiseptic and antipyretic agents. 4 In Saudi Arabia, they have been used in folk medicine to treat diabetes but several other therapeutic activities have been reported in different countries. [4][5][6][7] Plants of this genus have been shown to contain diterpenoids, avonoids, iridoids, tannins, saponins, alkaloids, sterols, coumarins and glycosides. 4,8−10 One species, T. yemense (De .), is a medicinal plant commonly grown in Saudi Arabia. It is used traditionally to treat infections, kidney diseases, rheumatism and diabetes. 8,11,12 Moreover, extracts of a related species, T. polium, have recently been shown to have activity in animal models of diabetes. 13,14 We reported previously the isolation and characterisation of six neoclerodanes from an ethyl acetate (EtOAc) extract of T. yemense, of which two stimulated the growth of E. coli but none had antimicrobial or anthelmintic activity. 15 Nine other neoclerodanes had been identi ed from this plant by Sattar et al. without evaluation of their biological activity, 9 whereas other neoclerodanes have been isolated from other Teucrium species. 16,17 Neoclerodanes have been also characterised from Scutellaria species 18,19 and Linaria species, 20 while neoclerodanes from Salvia have been identi ed as inhibitors of HSP90 and as κ-opioid receptor agonists. [21][22][23][24] Here, we disclose the isolation and structures of twenty new neoclerodanes from the butanol (BuOH) extract of T. yemense and report that nine examples enhance the insulin-triggered release of insulin from isolated murine pancreatic islets, indicating potential anti-diabetic activity.
The orthoacetate unit of 1 was more challenging to identify. The methyl protons gave a singlet at δ 1.43, which is inappropriate for an acetate ester, with the 13 CH 3 signal at δ 23.9. 2-Bond HMBC linked this CH 3 to the orthoester carbon signal at δ 107.7 / 107.8, which is inappropriate for an ester carbonyl. Thus this 2-carbon unit was not a conventional acetate ester, which was consistent with no loss of 60 Da (HOAc) in the MS fragmentation. The 1 H NMR spectrum in (CD 3 ) 2 SO showed only one OH resonance (HO-3, δ 5.23, with COSY and NOESY correlations with H-3); thus the oxygens in the lower part of the structure must be ethers. H-18 endo (δ 3.87) and H-18 exo both formed HMBC cross-peaks with the 13 C signal(s) at δ 107.7 / 107.8. Thus one of these signals must have been due to the orthoester carbon (four bonds from H 2 -18) and the other due to acetal carbon C-5 (three bonds from H 2 -18). This con rmed the ring-closure of the (C- showing an acetate ester. Twenty-two discrete 13 C NMR signals were observed (2 ⋅ CH 3 , 6 ⋅ CH 2 , 8 ⋅ CH, 6 ⋅ C q (ester and ketone). IR con rmed these carbonyls, with bands at 1712 cm − 1 and 1796 cm − 1 , respectively. A hydroxy group absorbed at 3478 cm − 1 .
The NMR spectra showed, in addition to the peaks for this major compound, a full set of peaks for a minor component, with similar chemical shifts and multiplicities. This minor set of peaks integrated for ca. 10% of the major compound present. As the sample gave only one pure peak on HPLC, we ascribed these peaks to a minor diastereoisomer in slow equilibrium with the major diastereoisomer. These are likely to be epimers at the lactol hemiacetal C-20. This assignment was supported by the largest differences in chemical shift between the epimers being for H-3 (Δδ 0.1 ppm), H-14 (Δδ 0.05 ppm), and H 2 -19 (Δδ 0.2), as these four protons are close in space to the epimeric C-20. We assign the structure 2 ( Fig. 1) 13 C NMR signals were observed: 2 ⋅ CH 3 , 6 ⋅ CH 2 , 7 ⋅ CH, 6 ⋅ C q , including a carbonyl (δ 172.5). IR showed bands for OH (3513 cm − 1 ) and one carbonyl (1716 cm − 1 ).
The NMR data (Table S1, SI) indicated that 3 was a neoclerodane. The upper part was an aromatic furan, with 1 H NMR signals (Table S1, SI) at δ 6.42 (H-14), δ 7.39 (H-16) and δ 7.40 (H-15). The furan 13 C NMR signals were at δ 108.4 (C-14), δ 130.1 (C-13), δ 138.6 (C-16) and δ 143.9 (C-15), with appropriate HSQC and HMBC connectivities. The chemical shift of H-12 (δ 4.85) showed that it was not part of a lactone or lactol system, con rmed by the lack of a HMBC cross-peak to the signal for carbonyl C-20 (δ 172.5). The identity of H-12 signal was con rmed by HMBC cross-peaks to C-13, C-14, and C-16. Further HMBC crosspeaks were seen from H-12 to C-11 (δ 35.8 or δ 40.0) and to C-9 (δ 48.5), linking this upper side-chain to the main decalin. The con guration at C-12 could not be established. The bridging lactone was established by HMBC cross-peaks between carbonyl C-20 and H 2 19 (δ 4.45 and δ 4.68). The identity of H 2 19 had been con rmed by each signal being a doublet with only geminal coupling 2 J = 12.5 Hz) and by HMBC cross-peaks to the C-6 (δ 110.0) and to C-4 (δ 84.1). C-19 (δ 66.4) was identi ed by HSQC crosspeaks to H 2 -19 and by strong HMBC to H-10 (δ 2.24-2.48 m). The signal at δ 72.8 was assigned to C-3 on the basis of HMBC cross-peaks to both H-2 (δ 1.4 and δ 2.1) and to one of the H-1 signals (δ 2.35). H-3 (δ 3.87) was characterised by a HSQC cross-peak to C-3, COSY cross-peaks to both H-2 and HMBC crosspeaks to C-1 (δ 21.9), C-2 (δ 30.3) and C-4 (δ 84.1). H-3 is axial, as shown by the large 3 J ax−ax (11.6 Hz) to H-2 ax . The H-1 signal at δ 1.29 is a quartet (J = 13.2 Hz); thus this signal is for H-1 ax . NOESY correlations from H-3 to one H-19 (δ 4.45) and to H-1 ax showed that these are on the same face of the decalin and thus that the decalin is trans-fused and that ring A is in the chair conformation. The conformation of ring B is less clear, owing to overlap of 1 H NMR signals but MM2-minimisation suggests that it may be a attened boat. The fused tetrahydrofuran and the acetal were identi ed as follows. Acetal carbon C-6 was characterised by its chemical shift (δ 110.0) and by HMBC cross-peaks to both H-19. Further HMBC correlations were seen to the doublets at δ H 3.90 and δ H 4.45, showing that they were due to H 2 -18 and demonstrating the closure of the tetrahydrofuran ring. The acetal at C-6 was identi ed by observation of an OMe group (δ H 3.40, δ C 48.9), linked by HMBC to C-6. These data characterise the structure of 3, fatimanol G, as shown in Fig. 1 The 1 H NMR spectrum contained only one set of signals, showing that one of the possible hemiacetal diastereoisomers had signi cantly lower energy than the other but it was not possible to determine which from the spectroscopic data. The methyl protons (H-2', δ 2.05) of the acetate showed a strong HMBC to the corresponding ester carbonyl (C-1', δ 170). C-1' also showed a cross-peak to H-3, con rming the location of the ester. We assign structure 5 ( Fig. 1)  spectrum showed discrete signals for 2 ⋅ CH 3 , 4 ⋅ CH 2 , 7 ⋅ CH and 7 ⋅ C q . The 1 H and 13 C NMR spectra (Table S2, SI) (with 2D spectra) were very similar to those for 4, with the addition of signals for a methoxy group (δ H 3.19, δ C 51.0). The protons of this group showed a strong HMBC cross-peak to the 13 C signal for C-6 (δ 150.8), identifying the methoxy group as being part of an acetal at this position. These data demonstrate that 6 (fatimanol J, Fig. 1 . From the down eld H-11 signal, it was then possible to use HMBC to identify the signals for C-9 (δ 50.2), C-10 (δ 43.9) and the saturated lactone carbonyl C-20 (δ 174.6). This lactone was con rmed by HMBC cross-peaks from C-20 to H-19 (δ 4.58, δ 4.64), and the latter were linked on to C-4 (δ 85.6), C-5 (δ 49.7) and C-6 (δ 108.  were seen in the IR spectrum. Combined interpretation of the NMR spectra (Table S3,  3) were identi ed by HSQC. A further HMBC cross-peak from H-11 (δ 1.4) to the 13 C signal at δ 66.5 (CH 2 ) showed that the latter was due to C-20. HSQC con rmed the geminally coupled doublets (δ 3.97 and δ 4.03) (J = 12.0 Hz) as due to the two H-20. These were linked by HMBC to the carbonyl 13  Further strong NOESY cross-peaks from both H-19 to H-3 con rmed the latter as axial down on ring A and a cross-peak to H-1 (δ 2.1) also suggested that this was on the lower face. On the upper face, the down eld H-18 signal (δ 3.21) gave a cross-peak to H-6 (δ 3.78), which also allowed differentiation of the two oxirane proton signals. The relative con guration at C-15 could not be determined. These data show the structure of 8 ( Fig. 1), fatimanol L.  6) and with C-15. HMBC was also useful in linking C-13, C-13, and C-16 with H-12 (δ 5.10 m) and HSQC identi ed C-12 at δ 71.6. A COSY cross-peak from H-12 to the multiplet signal at δ 1.85 identi ed the latter as one H-11 and HSQC led to C-11 (δ 40.  Figure 1, with many fused rings and bridges. Fortunately, these fusions and bridges make the structure fairly rigid and it was straightforward to assign the relative stereochemical con gurations by use of coupling constants (Karplus relationship) and NOESY. Figure 2 shows the key NOESY interactions used in this assignment. Particularly useful was the NOE interaction between the methoxy protons and H-18 exo (δ 3.89), which con rms that the methoxy is on the lower face of the trans-decalin. The name fatimanol O is assigned to the novel compound 11 ( Figure 1).  Completing ring A, the up eld H-1 gave a strong COSY cross-peak with H-10. The structure of 12 is less rigid than 11 but it was still possible to assign its relative con gurations by NOESY (Fig. 2). These data con rm the structure of 12, fatimanol P (Fig. 1).  . HSQC revealed C-7 (δ 37.9) and thence H eq -7 (δ 1.47). Both H-7 gave strong 2-bond HMBC correlations with C-6 (δ 69.4), from which HSQC showed the signal at δ 3.36 as being H-6. The COSY cross-peak from H ax -7 to H-6 was strong, whereas that from H eq -7 to H-6 was much weaker, suggesting that the dihedral angle (H eq -7)-(C-7)-(C-6)-(H-6) was ca. 90° and thus that H-6 was axial.
Establishing ring A was more challenging. A 2-bond HMBC cross-peak from H-10 identi ed C-1 at δ 24.6, from which HSQC showed that the H-1 signals were at δ 1.87 and δ 1.98. A strong 3-bond HMBC linked C-10 with one H-2 signal (δ 1.87), whereas the cross-peak to the other H-2 (δ 1.27) was weaker. A 3-bond HMBC cross-peak was also seen linking H-1 (δ 1.27) to C-5; the 3-bond path between these two nuclei cannot pass through C-1 and C-10, thus ring A must be ve-membered. The only structure consistent with these connectivities is the lactol / cyclic acetal shown. It was not possible to obtain a good NOESY spectrum, so the relative con guration shown in Chart 1 is speculative, except where suggested by 3 J H−H coupling constants. The NMR spectra contained a second (smaller) set of peaks, which we ascribe to the presence of a minor diastereoisomer in slow equilibrium, probably the epimer at the acetal C-18. Thus 13 (fatimanol Q) has the novel structure shown in Fig. 1 and thence H-10 (δ 1.96). From here, C-20 (δ 63.0) was located by HMBC to H-10; the H-20 protons were approximately co-incident at δ 3.28, with HMBC cross-peaks to C-9 (weak, 2-bond), C-10, and C-11. Twobond HMBC linked C-10 to both H-1 (δ 1.75 and δ 2.00), from which C-1 (δ 21.5) was identi ed by HSQC.  (Table S4). Examination of the coupling constants allowed con rmation of the trans-decalin structure and the relative con gurations of most of the substituents, although the relative con guration at C-12 could not be established. We assign the novel structure shown in Fig. 1 to 14  Finally, H-10 is on the upper face, as it shows a trans-diaxial coupling to H-1 ax . Thus the bicycle is a trans-decalin. These spectroscopic assignments were aided partly by 1 H, COSY, and HSQC spectra obtained of a solution in CDCl 3 , which were better resolved, although shortage of sample precluded identi cation of the quaternary carbons (Table S5). Minor differences in chemical shift were seen for H-2 eq , H-3, H-14, H-15, H-18, and H-19, probably re ecting minor changes in hydrogen-bonding and consequent minor changes in conformation. The COSY spectrum con rmed the H-H connectivities within the molecule. We assign the novel structure shown in Fig. 1 to 15  . The larger differences were in the upper part of the decalin and in the lactone, which suggested that the stereochemical difference between 16 and 15 was at C-9 or at C-12. A detailed study of NOESY data in that area was undertaken. Firstly, the equatorial methyl H-17 gave a strong NOESY cross-peak to H-12, suggesting that these were on the same face of the γ-lactone. Secondly, H-12 gave a strong NOESY correlation with one H-11 (δ 2.41) but only weakly with the other H-11 (δ 2.33). Since the furan protons H-14 and H-16 both formed strong NOESY cross-peaks with H-11 (δ 2.33), the furan and this up eld H-11 must be on the same face of the γ-lactone and this face must be opposite to that carrying H-12. These data are consistent with 16 having the opposite con guration at C-12 from 15. Other NOESY interactions, COSY cross-peaks and 1 H-1 H coupling constants in the decalin were consistent with trans-con guration.
The spectroscopic assignments were aided, in part, by 1 H, COSY, and HSQC spectra of a solution in CDCl 3 , which were better resolved, although shortage of sample precluded identi cation of the quaternary carbons and NOESY data could not be obtained. We assign the novel structure shown in Fig. 1 to 16 (Table S6, SI) allowed assignment of all the signals and con rmed that the overall structure was similar to a conventional neoclerodane. However, C-6 was quaternary but not a carbonyl (cf. 2). Two methoxy groups were also evident. Turning to the con guration of 18, the 1 H chemical shift of MeO-6 was unusually low (δ 2.97).
Examination of a molecular model showed that this methyl group, if on the lower face, would be held in the anisotropic shielding zone of the C-20 carbonyl; thus it is likely to be located on the lower face of the fused tricycle. The con guration at the other acetal C-18 could not be determined, although it was clear that only one diastereoisomer was present. A 1 H NMR spectrum of 18 was also obtained in CDCl 3 , which was consistent with the structure determined above. The novel structure shown in Fig. 1  (fatimanol X) was the desacetyl analogue of 19, with the structure shown in Fig. 1.
The occurrence of orthoesters in plant natural products was reviewed by Liao et al. in 2008. 28 Orthoacetates have been reported in only a limited number of frameworks, principally the daphnane diterpenoids, phragmalin limonoids, bufadienolide and ergostanoid steroids. Several of these diterpene orthoacetates have potent biological activities, including systemic toxicity. In each case, the three oxygen atoms of the orthoacetate unit were situated appositely in space for formation of the orthoester and the rigidity of the framework of the diterpene contributed to the stability of this usually highly acid-labile functionality. We propose the mechanism shown in Figure 3 for formation of the orthoacetate in 1.
Proposed intermediate 23 is 19-acetylteulepicin, reported by Savona et al. 16 to be a secondary metabolite in T. buxifolium and the formal oxidation product of 2 at the lactol. It is therefore feasible that 23 is the true biosynthetic precursor of 1 in T. Bohlmann's group suggested that their ring-contracted compounds had arisen from rearrangements involving migration of C-2 to bond with C-4. Mechanisms proposed include protonation of a hydroxy group at C-4 to initiate the rearrangement, 34 trapping the aldehyde formed from C-3 with a hydroxy group at C-10 34,35 and formation of a C-1 = C-10 double bond. 34,36,37 In each case, the C-10 position is oxidised.
However, in the present case, C10 is not oxidised with either an oxygen function or an alkene. We propose the mechanism in Figure 4 for the biosynthetic ring-contraction. In this mechanism, the rearrangement is triggered by protonation and ring-opening of the strained oxirane. The leaving group oxygen is on the lower face of the ring and thus almost antiperiplanar to the C-3-C-2 bond. The oxirane-opening and the migration of C-2 are probably concerted, given this conformation. C-2 will approach C-4 from the opposite side to the leaving group, resulting in stereochemical inversion at C-4. This places C-18 on the upper face and the newly generated carbocation (C-3) on the lower face, where it can readily form a hemiacetal with HO-19. This hemiacetal closes the second 5-membered ring such that the two 5-membered rings are cisfused, an energetically favoured arrangement.
Acid anhydrides are relatively uncommon in natural products, owing to their potential for electrophilic reactivity and hydrolysis. However, we have rmly identi ed 10 as having a succinic anhydride moiety as the upper ring; this is the rst such neoclerodane to be reported. Only one natural product containing a simple (non-fused) succinic anhydride, tubogenic anhydride A, has previously been isolated, from Aspergillus tubingensis. 38 Compounds 2,3,7-9,11,12,14-17,19,20 contain the conventional neoclerodane carbon framework with various differences in oxidation level, acetylation, bridging rings and ring-opening at the C-4/C-18 oxirane, whereas 4-6,18 lack C-19. The C-12-C-20 lactol in 2 has precedent in gnaphalin (isolated from T. gnaphalodes), 39 although the latter lacks HO-3. The C-19-C-20 bridging lactone of 3,7,10 is present in teulepicephin 15 and many other neoclerodanes. Compound 11 contains a related bridging acetal, giving a rigid polycyclic structure. This polycycle is also present in teucrin P 1 (also from T. gnaphalodes), 40 teupyrenone (from T. pyrenaicum), 41 and teupolin III, 42 although the latter do not have the additional lower fused tetrahydrofuran to stiffen the structure further. The upper hydroxyfuranone hemiacetal in 7 is also present in the neoclerodane salvidivin (from Salvia divinorum), 43 whereas the methoxyfuranone acetal moiety in 8,9 has precedent in the labdane 15-methoxyvelutine C (from Marrubium thessalum). 44 Most neoclerodanes have the 12-S con guration, so the identi cation of 16 as a 12-R neoclerodane is noteworthy. Gács-Baitz et al. 42 used NOE NMR spectroscopy to determine con guration at C-12 in neoclerodanes featuring the upper aromatic furan and the spiro-lactone but did not have an exact epimeric pair for their study; 15  Compounds 1-12 were evaluated for their ability to enhance the glucose-triggered secretion of insulin by freshly isolated murine pancreatic islets, using our previous assay. 48 In negative-control islets, insulin secretion was 9.1 ± 0.3 ng islet − 1 h − 1 , triggered by glucose (16.7 mM) (Fig. 5). This release was increased 2.2-fold by the standard drug tolbutamide (20.2 ± 1.3 ng islet − 1 h 1 ). The tested compounds showed a range of activities. Compounds 1,2,12 showed little or no effect on the secretion of insulin. Compounds 3-6 and 11 increased the glucose-triggered release of insulin by approximately the same extent as the positive control tolbutamide. Encouragingly, 7-10 showed strong enhancement of insulin secretion, by 3-4, although these are not as potent as the coumarins cluteolin D and clueolin J (from Clutia lanceolata). 49

Conclusions
We report the isolation and identi cation of twenty new neoclerodanes from the traditional medicinal plant T. yemense. Compound 1 contains an orthoacetate, which is previously unreported in naturallyoccurring neoclerodanes. As shown (Fig. 3), the acetate, oxirane and ketone groups in proposed precursor 23 are appositely located to facilitate formation of the orthoester; precursor 23 is 19-acetylteulepicin, previously identi ed in T. buxifolium. The upper (tetrahydro)furan unit in 10 is a succinic anhydride, a reactive moiety not often found in plants but presumably stable in the arid climate in which T. yemense grows in nature. Compound 13 results from a relatively unusual ring-contracting skeletal rearrangement during biosynthesis. Interestingly, 7-10 were found to enhance the glucose-triggered release of insulin from isolated murine pancreatic islets to a greater extent than the standard anti-diabetic drug tolbutamide; these compounds represent new leads for the development of treatments for this widespread disease.