Modified diterpenoids from the tuber of Icacina oliviformis as protein tyrosine phosphatase 1B inhibitors

Junfei Zhou *a, Zhenlong Wu ab, Brian Guo a, Meng Sun a, Monday M. Onakpa c, Guangmin Yao d, Ming Zhao e and Chun-Tao Che a
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA. E-mail: junfei19901203@gmail.com
bInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China
cDepartment of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Abuja, Abuja 920001, Nigeria
dHubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People's Republic of China
eJiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People's Republic of China

Received 30th October 2019 , Accepted 9th December 2019

First published on 10th December 2019


Seven new diterpenoids (1–7) and five known analogs (8–12) were isolated from the tuber of Icacina oliviformis. Their chemical structures were elucidated by spectroscopic analyses, with the spatial configurations defined based on calculated 13C NMR-DP4 analysis and electronic circular dichroism methods. Oliviformislactones A (1) and B (2) are the first examples of rearranged 3,4-seco-pimarane possessing a 6/6/5/5 tetracyclic ring system featuring an unprecedented 4,12-dioxatetracyclo[8.6.0.02,7.010,14]hexadecane core; secopimaranlactone A (3) and secocleistanthone A (4) are the first examples of 3,4-seco-pimarane and 3,4-seco-cleistanthane type diterpenoids, respectively, obtained from the Icacinaceae family. The plausible biosynthetic routes for 1–3 are proposed. Compounds 1–4 and 12 exhibited PTP1B inhibitory activity with IC50 values ranging from 3.24 to 58.05 μM. Among them, 1 (IC50 = 6.78 μM, Ki = 1.51 μM) and 3 (IC50 = 3.24 μM, Ki = 1.25 μM) displayed uncompetitive inhibition responses. In addition, the 16-p-bromobenzoyl derivative of oliviformislactone A (1a) displayed higher potency (IC50 = 87.51 nM, Ki = 0.44 μM) than its natural precursor.


Introduction

Highly oxygenated diterpenoids with complex carbon skeletons, such as paclitaxel and ingenol mebutate, are a viable source for new drug discovery.1 Among the diterpenes, pimaranes represent a sub-group consisting of over two hundred chemical structures reported from plants, fungi, and marine organisms, and many of them possess interesting biological activities.2–4 The genus Icacina (Icacinaceae) is known to be rich in pimarane-type diterpenes, which may serve as chemotaxonomic markers of the taxon.4 In previous studies, our group has isolated a series of pimarane derivatives from Icacina trichantha.5–9 They can be classified into several biogenetically related carbon skeletons (Fig. 1), i.e. pimarane, 16-nor-pimarane, 17-nor-pimarane, 16,17-di-nor-pimarane, 17,19-di-nor-pimarane, 15,16,17-tri-nor-pimarane, and rearranged 17-nor-pimarane.4 Some of these compounds exhibited biological activities such as seed germination inhibition and plant growth inhibition, as well as antifungal and cytotoxic activities.4
image file: c9qo01320b-f1.tif
Fig. 1 Pimarane related diterpenoid carbon skeletons and their proposed biogenetic relationships in Icacinaceae family.

Icacina oliviformis (Poir.) J. Raynal (syn. Icacina senegalensis Juss.), known as “false yam”, is a perennial savannah shrub indigenous in western and Central Africa.10 Its large tuber can be used as an architectural material in the local communities, and the fruits and seeds are often consumed as fresh snacks.11–13 The decoction of I. oliviformis is considered to be a panacea by local people for use in the treatment of fever, malaria, snake bite, eye infection, diarrhea, back pain, and other common ailments.14 However, our knowledge about its chemical composition is lacking. In the literature, only two pimaranes (icacinol and icacinone),15 two steroidal saponins,15 eight clerodane diterpenoids,16 two flavones C-glycosides,17 and trans-clovamide17 have been reported from this species.

In the course of continuing efforts to identify structurally unique and biologically interesting diterpenoids, the dried tuber of I. oliviformis was investigated to afford seven novel diterpenoids (1–7) and five known analogs (8–12) (Fig. 2 and S1). Compounds 1 and 3 exhibited uncompetitive protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. In addition, a more potent PTP1B inhibitor, 16-p-bromobenzoyl oliviformislactone A (1a) (IC50 = 87.51 nM, Ki = 0.44 μM), was prepared. This paper describes the isolation, structural elucidation, biogenetic pathway, PTP1B inhibitory activity, molecular docking, and molecular dynamics simulation of these compounds.


image file: c9qo01320b-f2.tif
Fig. 2 Structures of compounds 1–12 and 1a.

Results and discussion

Oliviformislactone A (1) was obtained as a white amorphous powder, [α]25D +7.5 (c 0.1, MeOH). Its molecular formula was established to be C21H28O7 by an HRESIMS ion at m/z 415.1732 [M + Na]+ (calcd for C21H28O7Na, 415.1733) and 13C NMR data, corresponding to eight indices of hydrogen deficiency. The 1H NMR spectrum of 1 (Table S1) exhibited resonances for two methyls (δH 1.09 and 1.36), a methoxy (δH 3.67), two olefinic methines (δH 5.65 and 5.92), and five oxygenated protons (δH 3.85, 3.88, 3.91, 4.31, and 4.50). The 13C NMR spectrum (Table S1) displayed 21 carbon resonance signals, assignable with the aid of DEPT and HSQC spectra to be three methyls (including one methoxy) (δC 16.9, 21.1, and 52.1), six methylenes, six methines [including one oxygenated and two olefinic methines (δC 85.9, 122.4, and 131.5)] as well as six quaternary carbons [including three carbonyl groups (δC 173.3, 174.4, and 179.1)]. A double bond and three carbonyl groups accounted for four degrees of unsaturation, the rest of four degrees of unsaturation suggested the existence of a tetracyclic system in 1.

The planar structure of 1 was established by careful analysis of the 1H–1H COSY, HSQC, and HMBC data (Fig. 3). Thus, its 1H–1H COSY correlations revealed the presence of four partial structures: C(1)H2–C(2)H2, C(18)H3–C(4)H–C(5)H–C(6)H–C(7)H, C(9)H–C(11)H2–C(12)H2, and C(15)H–C(16)H2. The HMBC correlations from H3-18 to C-4/C-5/C-19 indicated direct linkages of C-5/C-18/C-19 toward C-4. The connections of C-1/C-5/C-9/C-20 through C-10 were constructed based on the HMBC correlations from H2-20 to C-1/C-5/C-10 and from H-9 to C-20. In addition, the HMBC correlations from H3-17 to C-8/C-12/C-13/C-15 led to the assignment of bonding of C-8/C-12/C-15/C-17 towards C-13. Attachments of C-7/C-9/C-14 to C-8 were also assigned by the HMBC correlations from H-7 to C-8/C-9/C-14 and from H-9 to C-8/C-14. Moreover, both OCH3 and H2-2 displayed long-ranged correlations to the carbonyl carbon at δC 173.3 (C-3), revealing the connectivity between C-2 and OCH3via C-3. To fulfill the eight degrees of unsaturation, an additional ring was required in the structure of 1. Given that seven oxygen atoms including three ester carbonyl groups in the molecular formula accounted for four oxygenated carbons and that C-15 (δC 85.9) was dramatically shifted downfield compared to a typical hydroxylated methine carbon, a γ-lactone ring connecting C-14 and C-15 was likely present. The above evidence led to the proposed planer structure of 1.


image file: c9qo01320b-f3.tif
Fig. 3 1H–1H COSY, HMBC, and NOESY correlations of 1.

The relative configuration of 1 was determined by NOESY analysis (Fig. 3). Up to now, all diterpenoids obtained from Icacina plants are (9βH)-pimarane derivatives.4 Therefore, a β-orientation of H-9 was assumed. The NOESY correlation between H-4 and H-9 indicated H-4 is also β-oriented. In addition, the NOESY correlations of H-4β/H-20β, H-20α/H-5, and H-2a/H-5 suggested that H-5 is α-oriented. Moreover, the α-orientations of H-15 and CH3-17 could be defined from the NOESY correlations of H-20α/H2-2, H2-1/CH3-17, CH3-17/H-15, H-7/H-15, and H-7/CH3-17. On the other hand, the relative configuration of C-8 was assigned on the basis of NOESY correlations observed between H-7 and H-15/H3-17. Thus, H-7, H-15, and CH3-17 are cofacial, leading to a cis-fused cyclopentane/γ-lactone (C/D) ring system in 1. The lack of NOE between H-7 and CH2-16 supported such an assignment.

It is noteworthy that the stereochemistry of C-4 and C-5, being located in a δ-lactone ring which is known to exist in a combination of puckered conformations, requires special attention. In order to confirm the relative configurations of C-4 and C-5, theoretical calculations of 13C NMR shifts of the four possible isomers (4R*,5S*)-1, (4R*,5R*)-1, (4S*,5R*)-1, and (4S*,5S*)-1 were performed using the GIAO method with the Gaussian 09 software at the mPW1PW91/6-311G(d,p) level.18,19 Comparison of the experimental and calculated 13C NMR data revealed that the relative configuration of 1 is 4S*,5S* with a DP4 probability of approximately 99.75% (Fig. 4 and 5, Table 1).


image file: c9qo01320b-f4.tif
Fig. 4 Linear correlation plots of calculated-experimental 13C NMR chemical shift values for potential configurations of 1.

image file: c9qo01320b-f5.tif
Fig. 5 Relative errors between the calculated 13C NMR chemical shifts of four potential structures and recorded 13C NMR data of 1.
Table 1 DP4 probability of 13C NMR chemical shifts of 1
Plausible isomers DP4 probability of 13C NMR data of 1
4R*,5S* 4R*,5R* 4S*,5R* 4S*,5S*
Product of probabilities 3.15 × 10−15 4.38 × 10−20 5.72 × 10−21 1.26 × 10−12
Bayes's theorem probability 0.25% 0% 0% 99.75%


Assignment of the absolute configuration was then attempted by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra using time dependent density functional theory (TDDFT).20 The positive Cotton effect at 225.8 nm in the experimental ECD spectrum of 1 was in good agreement with that (224.7 nm) in the calculated ECD spectrum of (4S,5S,8S,9S,10R,13R,15S)-1 (Fig. 9). Therefore, the structure of 1 was determined as depicted and given a trivial name oliviformislactone A.

Oliviformislactone B (2) was obtained as a white amorphous powder, [α]25D +45.7 (c 0.1, MeOH). The HRESIMS ion at m/z 415.1729 [M + Na]+ (calcd for C21H28O7Na, 415.1733) and 13C NMR data indicated it was an isomer of 1. Indeed, the NMR data of the two compounds were quite similar (Table S1), with the exception that H-4 (δH 3.02, qd, J = 7.0, 6.7 Hz) in 2 has now shifted downfield in comparison with that (δH 2.57, qd, J = 7.1, 7.0 Hz) in 1, while C-4 (δC 34.5) in 2 shifted upfield from that (δC 40.4) in 1. Thus, 2 appeared to be the 4-epimer of 1. This conclusion was supported by the observation of NOESY correlation (Fig. 6) between H-4 and H-1b in 2, instead of that between H-4 and H-9/H-20β in 1.


image file: c9qo01320b-f6.tif
Fig. 6 1H–1H COSY, HMBC, and NOESY correlations of 2.

The relative configurations of C-4 and C-5 in 2 were further assigned by the theoretical calculations of 13C NMR shifts with the GIAO method.18,19 The calculated 13C NMR data of (4R*,5S*)-2 accorded perfectly with the experimental results with a DP4 probability of approximately 98.03% (Fig. 7 and 8, Table 2). The experimental ECD spectrum of 2 also matched well with the Boltzmann-averaged ECD spectrum of (4R,5S,8S,9S,10R,13R,15S)-2 (Fig. 9). All in all, the structure of 2 was defined as depicted and it was given a trivial name oliviformislactone B.


image file: c9qo01320b-f7.tif
Fig. 7 Linear correlation plots of calculated-experimental 13C NMR chemical shift values for potential configurations of 2.

image file: c9qo01320b-f8.tif
Fig. 8 Relative errors between the calculated 13C NMR chemical shifts of four potential structures and recorded 13C NMR data of 2.

image file: c9qo01320b-f9.tif
Fig. 9 Experimental and calculated ECD spectra of 1–4.
Table 2 DP4 probability of 13C NMR chemical shifts of 2
Plausible isomers DP4 probability of 13C NMR data of 2
4R*,5S* 4R*,5R* 4S*,5R* 4S*,5S*
Product of probabilities 1.36 × 10−12 3.30 × 10−22 2.81 × 10−21 2.73 × 10−14
Bayes's theorem probability 98.03% 0% 0% 1.97%


The molecular formula of secopimaranlactone A (3) was assigned to be C21H26O6 with nine degrees of unsaturation based on HRESIMS and NMR data. The NMR spectra (Table S1) disclosed the presence of 21 carbon signals belonging to three methyls (including a methoxy), six methylenes (including two oxygenated), four methines (including two olefinic and one oxygenated), and eight quaternary carbons (including two carbonyls and four olefinic carbons). These functional groups accounted for five degrees of unsaturation, indicating that 3 is a tetracyclic diterpenoid. Four coupled spin systems were revealed by the 1H–1H COSY correlations (Fig. S2): C(1)H2–C(2)H2, C(6)H–C(7)H, C(9)H–C(11)H2–C(12)H2, and C(15)H–C(16)H2. The above evidence indicated that 3 featured a seco-pimaratriene structure.

In the HMBC spectrum, the methyl singlet H3-18 correlated with C-4/C-5/C-19 leading to the connections of C-5/C-18/C-19 towards C-4. The HMBC correlations between H2-20 and C-1/C-5/C-9/C-10 helped define the bonding of C-1/C-5/C-9/C-20 to C-10. Furthermore, the observation of HMBC correlations between H-6 and C-4/C-5/C-10 revealed the linkage between C-5 and C-6. The connections of C-12/C-14/C-15/C-17 to C-13 were established by the strong HMBC correlations between H3-17 and C-12/C-13/C-14/C-15. In addition, H-7 was observed to correlate to C-8/C-9/C-14, leading to the connections of C-7/C-9/C-14 to C-8. Moreover, the HMBC cross-peaks observed between OCH3/H-2 and the carbonyl group at δC 174.1 (C-3) pointed to the connectivity between C-2 and OCH3via C-3. Finally, the presences of two oxygen bridges could be confirmed by HMBC correlations between H-20α and C-19 (δC 166.6), and between H-16β and C-14 (δC 162.6), respectively. The above evidence led to the proposal of the planar structure of 3 as a 3,4-seco-pimarane. The NOESY correlations (Fig. S2) of H-9β/H-12β and H-12β/H-15 indicated β-orientation of H-15. The calculated ECD spectrum of (9S,10S,13S,15S)-3 was in good agreement with the experimental results (Fig. 9). Thus, the stereo-structure of 3 was determined and it was given a trivial name secopimaranlactone A. It is the first example of 3,4-seco-pimarane found in the Icacinaceae family.

Secocleistanthanlactone A (4), a white amorphous powder, [α]25D −36.4 (c 0.1, MeOH), was assigned a molecular formula of C21H24O4 based on the HRESIMS ion at m/z 363.1594 [M + Na]+ (calcd for C21H24O4Na, 363.1572) and 13C NMR data. Comparison of the 1H and 13C NMR data (Table 2) of 4 with those of 3 revealed that the major difference was the presence of a phenyl and an ethyl group in 4, replacing the cyclohexene moiety, an oxygenated methine, and an oxygenated methylene in 3. In the HMBC spectrum of 4 (Fig. S3), correlations from H3-16 (δH 1.15, t) to C-14 (δC 140.7) and from H2-15 (δH 2.79, 2.76, each dq) to C-8/C-13/C-14 suggested the location of the ethyl group (CH3-16 and CH2-15) at C-14 (δC 140.7). The attachment of methyl group CH3-17 (δH 2.32, s) at C-13 (δC 135.9) was supported by HMBC correlations between CH13-17 and C-12/C-13/C-14. With the aid of ECD analysis, the experimental result obtained for 4 was in good agreement with the calculated values for (10S)-4 (Fig. 9). The absolute configuration of 4 was thus assigned and it was given a trivial name secocleistanthanlactone A, which represents the first 3,4-seco-cleistanthane diterpenoid bearing a 19,20-lactone.

3-O-Methylhumirianthol (5) was isolated as a white amorphous solid. The molecular formula of C21H28O6 was established by the HRESIMS ion at m/z 399.1784 [M + Na]+ (calcd for C21H28O6Na, 399.1784) and 13C NMR data. The 1H and 13C NMR spectroscopic data (Table S2) of 5 were found to be comparable to those of humirianthol (8),21 an obvious difference being the presence of an additional methoxy group (δH 3.30, s; δC 50.1; 3-OCH3) in 5. It was observed that C-3 (δC 100.9) in 5 was shifted downfield by 3.3 ppm compared to that in 8 (δC 97.6, C-3), suggesting the location of the methoxy group on C-3. This deduction was supported by the HMBC correlation (Fig. S4) observed between OCH3 and C-3. The NOESY correlations (Fig. S4) of H-9β/H-1β, H-1α/H-5, H-5/H3-18 as well as H3-18/H-6 suggested both H-5 and H-6 were α-orientated. Furthermore, α-orientation of H-14 and β-orientation of H-15 were defined on the basis of NOESY correlations of H-14/H-16α and H-15/H-16β. Finally, the experimental ECD data of 5 were in good agreement with the calculated values for (3S,4R,5R,6R,9S,10S,13S,14S,15S)-5 (Fig. S7). The above evidence led to the determination of 5 to be 3-O-methylhumirianthol.

The molecular formula of 3-O-methyl-14-hydroxy-humirianthol (6), a white amorphous solid, was established to be C21H28O7 based on the 13C NMR data and its HRESIMS sodium adduct ion at m/z 415.1732 (calcd for C21H28O7Na, 415.1733). The 1H and 13C NMR data of 6 (Table S2) closely resembled those of 5, with the exception that a hemiacetal quaternary carbon at δC 107.1 (C-14) is now present in 6 instead of an oxygenated methine at δC 87.5 (C-14) in 5. Thus, compound 6 was assumed to be the 14-hydroxyl derivative of 3-O-methylhumirianthol (5). Indeed, this could be verified by the HMBC correlations (Fig. S5) observed between H3-17 (δH 0.99) and C-12/C-13/C-14/C-15. The relative configuration of 6 was shown to be the same as 5 by the NOESY data (Fig. S4 and S5). Thus, the cross-peaks of H-9β/H-1β, H-1α/H-5, H-5/H3-18, H3-18/H-6 as well as H2-12/H-15 indicated that H-5 and H-6 were both α-oriented, whereas H-15 was β-oriented. The experimental ECD spectrum of 6 (Fig. S7) also resembled that of 5, suggesting the same absolute configuration. Consequently, 6 was established to be 3-O-methyl-14-hydroxy-humirianthol.

3-O-Methyl-14-methoxyhumirianthol (7), obtained as an amorphous powder, was determined to have the molecular formula C22H30O7 by the HRESIMS sodium adduct ion at m/z 429.1895 [M + Na]+ (calcd for C22H30O7Na, 429.1889) and 13C NMR data. A comparison of its NMR data of 7 (Table S2) with those of 6 suggested that the former differed from the latter by the presence of an additional methoxyl signal (δH 3.29, s; δC 50.1; OCH3). The chemical shift of C-14 (δC 110.0) in 7 shifted downfield by 2.9 ppm compared to the hemiacetal quaternary carbon C-14 (δC 107.1) in 6, indicating that a methoxy group has replaced the hydroxyl at C-14. This deduction was further supported by the HMBC correlation (Fig. S6) observed between OCH3 (δH 3.29, s) and C-14 (δC 110.0). The NOESY spectrum (Fig. S6) revealed the relative configurations of H-5α, H-6α, H-9β, and H-15β. Finally, the absolute configuration of 7 was determined to be the same as that of 6 by an ECD experiment (Fig. S7). Compound 7 was thus identified to be 3-O-methyl-14-methoxy-humirianthol.

In addition to the new structures (1–7), five known pimarane derivatives (8–12) were identified to be humirianthol (8),21 icacinol (9),5 14α-methoxyhumirianthol (10),6 icacinlactone I (11),6 and 12-hydroxyicacinlactone A (12)6 on the basis of NMR data analyses and comparison of their spectroscopic properties with literature values. Compounds 8 and 10–12 are reported from I. oliviformis for the first time.

Thus far, all pimarane diterpenoids isolated from Icacina plants bear a 9β-H, instead of the more common 9α-H configuration.4 Therefore, compounds 1–3 are highly possible to originate biogenetically from the same (9βH)-pimarane precursor. The plausible biogenetic pathways for 1–3 are proposed as shown in Fig. 10. To start with, icacinol (9)5 could be derived from I by a series of enzymatic oxidation reactions.6 Intermediate IV could be obtained from III by an enzymatic Baeyer–Villiger oxidation reaction,22 followed by the formation of intermediates VII and IX along two different routes. In one route, a carbocation center is formed at C-6 in X by protonation of 6-OH in IX leading to interchangeable intermediates X and XI. The migration of the C-13 alkyl to C-8 is accomplished by an enzymatic Wagner–Meerwein rearrangement in XI,23 which is the key step to form a new carbon–carbon bond between C-8 and C-13 in XII. In this manner, oliviformislactones A (1) and B (2) could be generated by successive nucleophilic and S-adenosylmethionine (SAM)-mediated methylation reactions24 of XII. In the other route, secopimaranlactone A (3) could be formed by repeated dehydration and the SAM-mediated methylation reactions24 of VII. LC-HRMS analysis of the crude extract of I. oliviformis revealed that compounds 1–3 are indeed natural products rather than artifacts (Fig. S8 and S9).


image file: c9qo01320b-f10.tif
Fig. 10 Proposed biosynthetic pathways for 1–3.

Protein tyrosine phosphatase 1B (PTP1B) plays a pivotal role in the negative control of insulin signaling pathway through dephosphorylating the insulin receptor and insulin receptors substrate-1 and -2.25 Previous studies have shown that PTP1B levels are often elevated in insulin-resistant diabetes patients and mice, deleted PTP1B would lead to stronger insulin sensitivity.26 PTP1B inhibitors are thus considered as promising leads to enhance insulin signaling and contribute to insulin-stimulated glucose uptake.27 In addition, PTP1B is recognized as a target of the leptin signaling pathway, which could regulate adipose tissue mass by central hypothalamus-mediated effects on hunger, physical exercise, energy expenditure, and body weight.28 Thus, PTP1B has emerged as one of the potential targets for the treatment of diabetes and obesity, opening a new avenue for drug discovery. Reversible inhibitors can be classified into competitive, noncompetitive, uncompetitive, and mixed types. Among them, uncompetitive inhibition is the most favorable mechanism for drug action due to higher potency in the presence of a wide range of substrate concentrations. However, uncompetitive inhibitors constitute less than 5% of reversible inhibitors. Thus, it is of great interest to look for agents with uncompetitive inhibition responses.

Natural PTP1B inhibitors including terpenoids, flavonoids, phenols, alkaloids, steroids, coumarins, and lignans have been reported in recent years.30,31 Among them, five ent-pimarane analogs exhibited significant PTP1B inhibitory activity.32,33 In this study, all isolates were evaluated for PTP1B inhibitory activity in vitro (Table 3, Fig. S10 and S11).34 Compounds 1 and 3 exhibited enzyme inhibition with IC50 values of 6.78 and 3.24 μM, respectively, whereas the activities of 2, 4, and 12 were moderate. More interestingly, kinetics study revealed that both 1 (Ki = 1.51 μM) and 3 (Ki = 1.25 μM) displayed uncompetitive responses (Table 3, Fig. S13–S18). The kinetic constant Kik/Kiv ratios (Tables S3 and S4) of inhibition for 1 and 3 are in the range of 1.001–1.144, consistent with the uncompetitive mode of inhibition.35

Table 3 PTP1B inhibitory activities of compounds 1–12 and 1a
Compounds IC50a Inhibition typeb (Ki, μM)
a The values presented are the means ± SD of triplicate experiments. b The values of inhibition constant. c Positive control: oleanolic acid.
1 6.78 ± 0.41 μM Uncompetitive (1.51 ± 0.26)
1a 87.51 ± 6.49 nM Uncompetitive (0.44 ± 0.025)
2 32.20 ± 0.59 μM
3 3.24 ± 0.14 μM Uncompetitive (1.25 ± 0.35)
4 12.72 ± 0.53 μM
5 >80
6 >80
7 >80
8 >80
9 >80
10 >80
11 >80
12 58.05 ± 2.51 μM
OAc 4.32 ± 0.20 μM Mix (6.0 ± 1.9)29


Molecular docking was then performed to further understand the possible interactions with the active sites of the PTP1B enzyme (Table S6,Fig. 11 and S22–S34). Compounds 1 and 3 could be well docked into the catalytic site. The ester group of them interacted through several hydrogen bonding with the highly catalytic PTP-loop (Ser216-Arg221), which is responsible for executing the nucleophilic attack on the substrate phosphate moiety.36 In addition, 1 and 3 displayed hydrogen bonding with Tyr46 in the pTyr-loop and Phe182 in the WPD-loop, respectively, which are not only important for substrate binding and enzyme-phosphate formation but also crucial for the absolute specificity of phosphotyrosine substrates.37–40


image file: c9qo01320b-f11.tif
Fig. 11 Low-energy binding conformations of 1–3 and 1a bound to PTP1B enzyme. Dotted green and orange lines indicate hydrogen bonds and σ–π interactions, respectively.

The enzyme–ligand interaction was further investigated using molecular dynamics simulation by estimating the system potential energy, interaction energy, distances, and RMSD (root-mean-square deviation) values (Fig. 12).41,42 Both complexes showed similar potential energy values, but PTP1B-3 reached equilibrium a little earlier than PTP1B-1 (Fig. 12A). On the other hand, the interaction energy and distances between the PTP1B enzyme residues and the ligands demonstrated that the binding affinity of PTP1B-3 was much stronger than that of PTP1B-1 (Fig. 12B and C). In addition, the RMSD values suggested that both complexes are stabilized at 0.2–0.3 nm from 8 ns (Fig. 12D). These results indicate that both 1 and 3 could bind to the high-affinity catalytic areas of the PTP1B enzyme directly and yield stable enzyme–ligand complexes in the saline condition.


image file: c9qo01320b-f12.tif
Fig. 12 Molecular dynamics trajectory analysis of two complexes PTP1B-1 and PTP1B-3. (A) The potential energy of PTP1B-1 and PTP1B-3, (B) The interaction energy of PTP1B-1 and PTP1B-3, (C) The distances between the Cys215 residue of PTP1B enzyme and compounds 1/3, (D) RMSD values of PTP1B-1 and PTP1B-3.

It is known that the presence of appropriate hydrophobic groups in the molecule could enhance the selectivity of PTP1B inhibitory activity.43 Along this line of thought, the 16-p-bromobenzoate ester of 1 (1a) was prepared by treating 1 with p-bromobenzoyl chloride in anhydrous pyridine (Fig. 13).44 Docking simulation (Table S6, Fig. 11 and S35) revealed that 1a could exhibit tighter binding to PTP1B through stronger hydrogen bond interactions between the bromine atom and the high-affinity catalytic PTP-Loop (Ser216-Arg221) and WPD-loop (Phe182) areas, as well as between the lactone group and the Q-loop (Gln262). These loops in the PTP1B catalytic domain act in coordination during the catalytic process to hydrolyze the substrate phosphate moiety.45 In our hands, the docking results suggested that 1a might display more significant PTP1B inhibitory activity than 1. Indeed, 1a exhibited potent PTP1B inhibitory activity with an IC50 value of 87.51 ± 6.49 nM, which is 77.5-fold stronger than its natural precursor 1. As shown in Fig. S19–S21, the Ki value of 1a is 0.44 ± 0.025 μM, and the new kinetic constant Kik/Kiv ratio (Table S5) is in the ranges of 1.003–1.024, which points to uncompetitive inhibition.35


image file: c9qo01320b-f13.tif
Fig. 13 Preparation of 16-p-bromobenzoate ester of 1 (1a).

Based on the biological results obtained so far, preliminary observations can be drawn regarding the structure–activity relationship among the tested compounds. First, the results showed that 3,4-seco-pimarane derivatives with a δ-lactone (as in 1–4) appear to be essential for the activity, whereas pimaranes bearing a cyclohexane A-ring (as in 5–12) displayed dramatically lower PTP1B inhibitory activity. Second, the conformation of the δ-lactone in 1 and 2 seems to affect the interaction with the enzyme. Thus, the binding energy of 1 and 2 to PTP1B were calculated to be −8.63 and −6.41 kcal mol−1, respectively. In addition, molecular docking models revealed that the δ-lactone and the chain (C-1/C-2/C-3) in 1 are closer to the high-affinity catalytic areas and thus creating stronger hydrogen bond interactions, compared with 2. It is therefore not surprising to observe a 4.7-fold increase in potency for 1 (IC50 = 6.78 μM) over that of 2 (IC50 = 32.20 μM). Third, the conjugated system in 3 and 4 might contribute to their inhibitory activity, in line with other PTP1B inhibitors previously reported.46–48 Fourth, among the pimaranes (5–12), only 12 exhibited weak PTP1B inhibitory activity (IC50 = 58.05 μM), suggesting that the phenolic group might contribute to the activity, such as in the case of trivaric acid which is active in a mouse model.49 Finally, the p-bromobenzoyl moiety in 1a could clearly enhance the PTP1B inhibitory activity (IC50 = 87.51 nM), supporting the observations in other studies when hydrophobic groups (such as dithiolan, dithian, phenylpyrrolidine, diphenyl ether, and halogen substituents) are included in the structures.50–53

Further studies are warranted to establish the detailed mechanism of action of these compounds and related derivatives.

Conclusions

Chemical investigation on the tuber of I. oliviformis resulted in the isolation of seven new pimarane derivatives (1–7) and five known analogs (8–12). Oliviformislactones A (1) and B (2) are the first two examples of rearranged 3,4-seco-pimarane possessing a 6/6/5/5 tetracyclic ring system featuring an unprecedented 4,12-dioxatetracyclo[8.6.0.02,7.010,14]hexadecane core. Secopimaranlactone A (3) and secocleistanthone A (4) are the first examples of 3,4-seco-pimarane and 3,4-seco-cleistanthane type diterpenoids, respectively, isolated from the Icacinaceae family. Among them, 1 and 3 exhibited PTP1B inhibitory activity with uncompetitive inhibition responses. The 16-p-bromobenzoate ester of 1 (1a) was shown to be 77.5-fold more potent than 1. These findings not only enrich our knowledge about the chemical diversity of Icacinaceae diterpenoids but also provide useful clues for further design of PTP1B inhibitors.

Experimental section

General experimental procedures

Optical rotations were obtained by a PerkinElmer 241 digital polarimeter at room temperature with a quartz cell with a path length of 100 mm in methanol. A JASCO J-810 spectrometer was applied to measure the CD spectra of all compounds. The UV spectra were recorded on a Shimadzu UFLC system with a PDA detector. IR spectra were measured on a Thermo Scientific Nicolet 6700 FT-IR spectrometer loaded with an OMNIC software. NMR spectra were recorded on a Bruker AV-400 spectrometer, and the 1H and 13C NMR chemical shifts were referenced to the solvent residual peaks for the internal standard (methanol-d4: δH 3.31, δC 49.15; chloroform-d: δH 7.26, δC 77.16; pyridine-d5: δH 8.74, 7.58, 7.22, δC 150.35, 135.96, 123.96). HRESIMS data were acquired on a Bruker micrOTOF II spectrometer in positive ion mode. Samples were purified by semi-preparative HPLC using a Shimadzu UFLC system with a semi-preparative column (Phenomenex C18 column, 250 × 10 mm, 5 μm).

Plant material

The fresh tubers of I. oliviformis were collected by Mr A. Ozioko, botanist at the BDCP laboratories, Nsukka, Nigeria, in June 2011 from the Orba village in Nsukka of the Enugu State, Nigeria. The plant material was authenticated by Prof. B. O. Olorede of the Botany Department, University of Abuja, Nigeria. A voucher specimen (No. 20110610) has been deposited there.

Extraction and isolation

The ground dried tubers of I. oliviformis (8.32 kg) were powered, then extracted by percolation with MeOH at room temperature. The combined MeOH solution was concentrated under reduced pressure to obtain a crude extract (372.1 g), which was dissolved in distilled water (800 mL), and partitioned with EtOAc (1.6 L × 3) and n-BuOH (1.6 L × 3), successively, to afford the EtOAc fraction (74.1 g), BuOH fraction (77.8 g), and water fraction (220.2 g).

The EtOAc fraction was subjected to further separation by a silica gel column chromatography. The elution started with 100% petroleum ether, then mixtures of petroleum ether and acetone, followed by 100% acetone. Monitored by TLC, fractions showing similar profiles were combined into ten combined subfractions (1-1 to 1-10). Subfraction 1-3 was loaded on a silica gel column eluted with petroleum ether and acetone, obtaining six subfractions (2-1 to 2-6). Then, subfraction 2-3 was further separated by a silica gel column eluted with petroleum ether and EtOAc to give ten parts (6-1 to 6-10). The subfraction 6-3 was subjected to a Sephadex LH-20 column to afford three parts (14-1 to 14-3). Subsequently, 14-1 was purified by semi-preparative HPLC on a RP C18 column (MeOH/H2O, 65[thin space (1/6-em)]:[thin space (1/6-em)]35) to yield compounds 8 (21.8 mg, tR = 39.4 min, 0.000262%) and 9 (28.5 mg, tR = 32.6 min, 0.000343%). On the other hand, the combined sample of 6-4 to 6-6 was loaded on an MCI gel column to get two fractions 7-1 (MeOH/H2O, 90[thin space (1/6-em)]:[thin space (1/6-em)]10) and 7-2 (100% acetone). Fraction 7-1 was separated using a Sephadex LH-20 column to yield four fractions (8-1 to 8-4). Fraction 8-1 was further separated to obtain seven parts (9-1 to 9-7) on a silica gel column eluted with CH2Cl2 and MeOH. Subfractions 9-2 and 9-3 were separated on an RP C18 column (MeOH/H2O, 10[thin space (1/6-em)]:[thin space (1/6-em)]90 to 100[thin space (1/6-em)]:[thin space (1/6-em)]0) to obtain seven parts (10-1 to 10-7) and (12-1 to 12-7), respectively. Finally, subfractions 10-1 to 10-3 and 12-1 to 12-3 were purified by semi-preparative HPLC on a RP C18 column, respectively, to afford 6 (3.2 mg, tR = 24.6 min, 0.0000385%) and 10 (11.8 mg, tR = 27.9 min, 0.000142%) from 10-1 (MeOH/H2O, 65[thin space (1/6-em)]:[thin space (1/6-em)]35), compound 3 (6.6 mg, tR = 30.3 min, 0.0000793%) from 10-2 (MeOH/H2O, 75[thin space (1/6-em)]:[thin space (1/6-em)]25), compounds 1 (5.4 mg, tR = 32.4 min, 0.0000649%) and 2 (5.8 mg, tR = 35.8 min, 0.0000697%) from 10-3 (ACN/H2O, 35[thin space (1/6-em)]:[thin space (1/6-em)]65), compounds 11 (12.5 mg, tR = 33.1 min, 0.000150%) and 12 (4.7 mg, tR = 22.4 min, 0.0000565%) from 12-1 (ACN/H2O, 30[thin space (1/6-em)]:[thin space (1/6-em)]70), compounds 5 (2.5 mg, tR = 38.4 min, 0.0000300%) and 7 (14.5 mg, tR = 42.6 min, 0.000174%) from 12-2 (MeOH/H2O, 60[thin space (1/6-em)]:[thin space (1/6-em)]40), as well as compound 4 (8.3 mg, tR = 40.0 min, 0.0000998%) from 12-3 (ACN/H2O, 70[thin space (1/6-em)]:[thin space (1/6-em)]30).

Oliviformislactone A (1). White amorphous powder; [α]25D +7.5 (c 0.1, MeOH); UV (MeOH) λmax (log[thin space (1/6-em)]ε) 200 (3.44) nm; IR νmax 3403, 2955, 2919, 2851, 1731, 1656, 1564, 1461, 1408, 1314, 1256, 1176, 1097, 1054, 1030, 894, 720 cm−1; ECD (MeOH) λε) 226 (+16.74) nm; 1H and 13C NMR data see Table S1; (+)-HRESIMS m/z 415.1732 [M + Na]+ (calcd for C21H28O7Na, 415.1733).
Oliviformislactone B (2). White amorphous powder; [α]25D +45.7 (c 0.1, MeOH); UV (MeOH) λmax (log[thin space (1/6-em)]ε) 200 (3.48) nm; IR νmax 3396, 2919, 2852, 1729, 1650, 1563, 1458, 1403, 1316, 1253, 1176, 1097, 1030, 892, 717 cm−1; ECD (MeOH) λε) 224 (+15.69) nm; 1H and 13C NMR data see Table S1; (+)-HRESIMS m/z 415.1729 [M + Na]+ (calcd for C21H28O7Na, 415.1733).
Secopimaranlactone A (3). White amorphous powder; [α]25D −52.6 (c 0.1, MeOH); UV (MeOH) λmax (log[thin space (1/6-em)]ε) 202 (4.14), 243 (3.96), 280 (3.99), 367 (4.21) nm; IR νmax 3434, 2958, 2927, 2863, 1727, 1659, 1595, 1556, 1460, 1380, 1272, 1125, 1073, 1042, 991, 742 cm−1; ECD (MeOH) λε) 217 (−5.29), 236 (−5.68), 271 (+2.60), 352 (−21.78) nm; 1H and 13C NMR data see Table S1; (+)-HRESIMS m/z 397.1634 [M + Na]+ (calcd for C21H26O6Na, 397.1627).
Secocleistanthanlactone A (4). White amorphous powder; [α]25D −36.4 (c 0.1, MeOH); UV (MeOH) λmax (log[thin space (1/6-em)]ε) 214 (4.96), 242 (4.73), 354 (4.70) nm; IR νmax 3391, 2952, 2921, 2871, 2851, 1733, 1701, 1613, 1565, 1435, 1372, 1335, 1294, 1193, 1162, 1136, 1097, 1060, 1047, 810, 760, 711, 670 cm−1; ECD (MeOH) λε) 215 (+30.54), 243 (+24.07), 352 (−26.57) nm; 1H and 13C NMR data see Table S2; (+)-HRESIMS m/z 363.1594 [M + Na]+ (calcd for C21H24O4Na, 363.1572).
3-O-Methylhumirianthol (5). white amorphous powder; [α]25D −28.2 (c 0.1, MeOH); UV (MeOH) λmax (log[thin space (1/6-em)]ε) 229 (3.20) nm; IR νmax 3481, 3422, 3178, 2878, 1757, 1669, 1568, 1456, 1377, 1300, 1202, 1150, 1082, 1039, 949, 728 cm−1; ECD (MeOH) λε) 200 (−12.40) nm; 1H and 13C NMR data see Table S2; (+)-HRESIMS m/z 399.1784 [M + Na]+ (calcd for C21H28O6Na, 399.1784).
3-O-Methyl-14-hydroxyhumirianthol (6). White amorphous powder; [α]25D −23.5 (c 0.1, MeOH); UV (MeOH) λmax (log[thin space (1/6-em)]ε) 200 (3.23) nm; IR νmax 3523, 3388, 2921, 2855, 1748, 1671, 1584, 1456, 1383, 1300, 1274, 1202, 1146, 1083, 1028, 940, 718 cm−1; ECD (MeOH) λε) 200 (−12.41) nm; 1H and 13C NMR data see Table S2; (+)-HRESIMS m/z 415.1732 [M + Na]+ (calcd for C21H28O7Na, 415.1733).
3-O-Methyl-14-methoxyhumirianthol (7). White amorphous powder; [α]25D −26.8 (c 0.1, MeOH); UV (MeOH) λmax (log[thin space (1/6-em)]ε) 200 (3.27) nm; IR νmax 3602, 3523, 2934, 2884, 1755, 1629, 1454, 1375, 1338, 1299, 1262, 1198, 1149, 1078, 1038, 953, 882, 759, 686 cm−1; ECD (MeOH) λε) 200 (−12.38) nm; 1H and 13C NMR data see Table S2; (+)-HRESIMS m/z 429.1895 [M + Na]+ (calcd for C22H30O7Na, 429.1889).

Preparation of 16-p-bromobenzoate ester of 1 (1a)

Oliviformislactone A (1) (3.0 mg) and p-bromobenzoyl chloride (15.0 mg) were transferred into a vial, to which 1 mL of anhydrous pyridine was immediately added. The mixture was stirred for 4 hours at room temperature.44 After completion of the reaction, the solution was separated by semi-preparative HPLC using ACN[thin space (1/6-em)]:[thin space (1/6-em)]H2O (90[thin space (1/6-em)]:[thin space (1/6-em)]10) as the mobile phase to afford 1a (3.9 mg, 88.8%; 1H and 13C NMR data see Table S1).

Computational methods

The systematic random conformational analyses of all plausible stereoisomers of 1–5 were performed using the SYBYL-X-2.1.1 program with MMFF94s molecular force field. The energy of optimized structures at the B3LYP/6-31+G(d) level in the Gaussian 09 software was applied to screen stable conformers.19 The ECD and NMR calculations of all the selected conformers were carried out with the TD/B3LYP/6-31+G(d) mode in the gas phase and the GIAO/mPW1PW91/6-311G(d,p) method in chloroform, respectively. The overall ECD curves were weighed by Boltzmann distribution of each conformer and compared with the experimental results using the SpecDis 1.71 software with UV correction.54 The overall theoretical NMR data were analysed by using linear regression and DP4 probability to further confirm the relative configurations of C-4/C-5 in 1 and 2.18,55 Consequently, the simulated ECD curves of all plausible stereoisomers of 1–5 were utilized for the absolute configuration determination.

PTP1B inhibitory bioassay

Protein tyrosine phosphatase 1B (PTP1B) activity was defined by measuring the rate of hydrolysis of a surrogate substrate, p-nitrophenyl phosphate (pNPP).34,39 The PTP1B protein (Sigma SRP0212, human recombinant) was dissolved in enzyme dilution buffer containing 50 mmol L−1 Mops, 1 mM BSA, 1 mM EDTA, and 1 mM DTT, pH 6.5 and pre-incubated with individual inhibitor at 37 °C for 15 min. The bio-reaction was initiated by adding a substrate pNPP, which could be dephosphorylated to afford p-nitrophenol (pNP) during the bio-reaction. The absorbance of the solution mixture was monitored at 405 nm every 5 min for half an hour. Consequently, these bio-reactions were quenched by 1 M NaOH solvent. Oleanolic acid was used as a positive control. An analogous experiment without PTP1B and sample was performed as a blank control, and a parallel run without sample served as negative control. The enzyme inhibition rate was estimated by the slope of the kinetic curve in each well. The inhibition percentage (%) was calculated as: slope (OD405 negative/min − OD405 sample/min)/slope (OD405 negative/min − OD405 blank/min) × 100%, where OD405 (negative/min, sample/min, and blank/min) represents the reduction of 405 nm absorbance for 5 min with the test sample and substrate, respectively.

The IC50 values (50% percentage inhibition concentration) were calculated by Origin 9.1 software with the non-linear curve fitting of the percentage of inhibition (%) versus the logarithms of inhibitor concentrations i using the following equation: inhibition (%) = 100/(1 + IC50/10i)k, where k is the Hill coefficient.

Enzyme kinetic bioassay

The enzymatic kinetic assay was performed in a similar manner. Phosphatase activities were measured at a fixed PTP1B concentration (1 μg mL−1 final), while concentrations of the substrate (pNPP: 0.5, 1, 2, 4, and 8 mM) and inhibitors (1, 2, 4, 8, 16, and 32 μM for 1 and 3; 5, 20, 80, 320, 1280, and 5120 nM for 1a) varied. In the time-independent inhibition experiment, the PTP1B enzyme was dissolved in 50 mM Mops buffer (pH 6.5) containing 1 mM DTT, 1 mM EDTA as well as 1 mM BSA. Subsequently, various concentrations of 1, 1a, and 3 were added into the enzyme buffer solution in a 96-well plate, respectively. The mixture of inhibitors and PTP1B enzyme were pre-incubated at 37 °C for 15 minutes. The reactions started by the addition of pNPP and stopped by 1 M NaOH after a 30 min incubation at 37 °C. The absorbance of pNP generated from the surrogate substrate pNPP was monitored at 405 nm every 5 min for half an hour by a microplate reader.56

In order to study the PTP1B inhibition mode of 1, 3, and 1a, two complementary kinetic methods were employed: i.e. Lineweaver–Burk and [S]/V against [I] plots. In the presence of the uncompetitive inhibitor, the Michaelis–Menten equation is described as 1/ν = Km/(Vmax[S]) + [I]/(KiVmax) + 1/Vmax, where ν is the initial rate, Vmax is the maximum rate, and [S] is the substrate concentration. The dissociation constants (Ki) of enzyme–inhibitor–substrate complex were obtained from the plots of [S]/V against [I]. Data were represented as the means ± standard deviations of three replicates.

Molecular docking study

Molecular docking simulations of 1–12, 1a, and oleanolic acid to human PTP1B enzyme were performed by the CDOCKER protocol of Discovery Studio 4.5 (BIOVIA, San Diego, CaA). PTP1B X-ray crystal structure at 1.5 Å resolution (PDB ID: 2hb1) was obtained from RCSB Protein Data Bank (http://www.rcsb.org). The PTP1B protein was protonated and deleted water at pH 7 using the Clean Protein tool, and the all-atom CHARMm forcefield was used to optimize their positions.57 The 3D structures of the inhibitors were prepared by Sybyl-X 2.0 (Tripos Inc., St Louis, MO) software, and the Tripos force field was applied for energy minimization of the molecules with a root-mean-square (RMS) gradient of 0.01 kcal mol−1 Å−1. The implicit solvent model was specified as the Generalized Born with Molecular Volume (GBMV) method. A docking site was defined as all residues within 20 Å from the sulfur atom of the catalytic cysteine residue (Cys215). All other parameters were set as default. Then, energy minimization of the PTP1B protein in complex was operated to remove energetically unfavorable contacts using the same parameters as mentioned above. The binding energies between the enzyme and all inhibitors were calculated by Autodock 4.2.6 software with the Genetic Algorithm method.58,59

Molecular dynamics simulation

Molecular dynamics simulation was performed for two complexes, PTP1B-1 and PTP1B-3, using Discovery Studio 4.5 (BIOVIA, San Diego, CaA).60 These complexes were chosen based on the results of the molecular docking study. The CHARMm force field was selected for parameterization of these complexes.57 Then, the complexes were placed at the center of a cubic box solvated with the aqueous environment of the Extended Simple Point Charge (SPC/E) water model.61 Energy minimization was carried out for 0.1 ns using a maximum force ≥10.0 kJ mol−1 to attain the stable state of the system.62 The entire system was equilibrated in two phases under isothermal and isochoric ensemble (NVT) at a constant 300 K temperature and under the NPT (isothermal-isobaric) ensemble to maintain the stabilized pressure inside the system in a sequential manner.63 All hydrogen bonds were constrained during equilibration by applying LINC algorithms.64 Besides, the Particle Mesh Ewald (PME) module was applied for long range ionic interaction.65 Finally, the entire trajectories were saved for analysis at a frequency of 1 ps during the simulation run.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

B. Guo acknowledges the NIH Office of the Director and the National Center for Complementary and Integrative Health for a trainee fellowship (T32AT007533). M. M. Onakpa acknowledges an award from the Fulbright Junior Development Exchange Program (No. 15120356) to conduct research at the UIC. The use of the high-performance computing platform of Jinan University, Guangzhou, China is acknowledged.

Notes and references

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Footnote

Electronic supplementary information (ESI) available: 1H and 13C NMR spectroscopic data, molecular docking models, Cartesian coordinates, HRESIMS, UV, IR, and 1D/2D NMR spectra. See DOI: 10.1039/c9qo01320b

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