Characterization of Sesquiterpene Dimers from Resina Commiphora

Feb 12, 2018 - The remaining NMR data and the results of 2D NMR studies show the existence of a structural unit in 4 that is very similar to that of t...
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Article Cite This: J. Org. Chem. 2018, 83, 2725−2733

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Characterization of Sesquiterpene Dimers from Resina Commiphora That Promote Adipose-Derived Stem Cell Proliferation and Differentiation Jia-Wang Liu,†,|| Ming-Yu Zhang,‡ Yong-Ming Yan,‡ Xiao-Yi Wei,⊥ Lu Dong,† Yan-Xia Zhu,*,‡ and Yong-Xian Cheng*,†,‡,§ †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China ‡ Guangdong Key Laboratory for Genome Stability & Disease Prevention, School of Pharmaceutical Sciences, Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Department of Cell Biology and Molecular Genetics, Shenzhen University Health Science Center, Shenzhen, 518060, PR China § Henan University of Chinese Medicine, Zhengzhou 450008, PR China || University of Chinese Academy of Sciences, Beijing 100049, PR China ⊥ Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Guangzhou 510650, PR China S Supporting Information *

ABSTRACT: The new sesquiterpene dimers commiphoroids A−D (1−4) were isolated from Resina Commiphora, and their structures were assigned by spectroscopic methods and X-ray diffraction analysis. Compounds 1 and 2 are stereoisomers of putative [2 + 4]-cycloaddition reactions, and 3 is a trinorsesquiterpene dimer containing a 6/6/5/6/6/6 hexacyclic framework, while 4 possesses a 8-oxabicyclo[3.2.1]oct-6-ene skeletal core. Plausible biosynthetic pathways for 1−4 are proposed. Biochemical studies show that compound 1 promotes ca. 60% expression of keratinocyte-specific markers in adiposederived stem cells at 10 μM.

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INTRODUCTION Skin, which is the largest organ of the human body, acts as a barrier and plays immunologic, sensorial, and protective roles. Several major therapeutic approaches exist for treatment of skin lesions or damage that occur when humans interact with the environment. One of these is the use of drugs that promote epidermal regeneration,1 and another involves stem cell transplantation on the injured area in order to promote repair and avoid or reduce scar formation.2 Another strategy to induce skin regeneration involves the promotion of stem cell proliferation and differentiation. In this regard, efforts have been made to develop functional biopolymer materials that promote stem cell proliferation and differentiation.2 A great interest exists in finding small molecules that can serve as useful tools in regenerative medicine.3 Several small molecules have been identified in recent years that influence different stages of stem cells maturation, as exemplified by PD98059,3 SB203580,3 sinomenine,3 valproic acid,4 and kenpaulone.4 These findings demonstrate the future potential of using small molecules to affect stem cell modulation. Many plants produce resins in response to injury, a process that to some extent resembles scaring that occurs in humans as part of wound healing. These resins have long been used as © 2018 American Chemical Society

medicinal agents in indigenous and tribal cultures across the world. For example, Resina Commiphora, an exudate of the genus Commiphora, such as Commiphora myrrha (T. Nees) Engl. (Burseraceae), which has been used for the treatment of pain, swelling, trauma, arthritis, ulcer, and sores, has been found to have pronounced effects on activating blood circulation and promoting granulation.5 More than 300 structurally characterized organic compounds have been isolated from this exudate.6 In previous efforts, we isolated four new terpenoids from Resina Commiphora and showed that these substances have intriguing antifibrotic activities.7 In continuing investigations of this exudate, we isolated four new sesquiterpene dimers, called commiphoroids A−D (1−4) (Figure 1). The structural elucidation and proposed biosynthesis of these natural products, along with their activities against adiposederived stem cell proliferation and differentiation, are described below. Received: December 18, 2017 Published: February 12, 2018 2725

DOI: 10.1021/acs.joc.7b03182 J. Org. Chem. 2018, 83, 2725−2733

Article

The Journal of Organic Chemistry

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of 1 and 2 in CDCl3 (δ in ppm, J in Hz) 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′

Figure 1. Structures of 1−4.

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RESULTS AND DISCUSSION The dried myrrha was extracted with EtOH; after removal of the solvent, the extract was suspended in warm water followed by extraction with EtOAc to afford an EtOAc-soluble portion. A combination of chromatography on this extract led to the isolation of compounds 1−4 (Figure 1). Commiphoroid A (1), obtained as colorless needles by crystallization from methanol, was found to have the molecular formula C33H48O6 (10 degrees of unsaturation) by using (+)-HRESIMS, 13C NMR, and DEPT spectroscopy. The 1H NMR spectrum shows the signals of nine methyl groups. Analysis of the 13C NMR and DEPT spectra of 1 (Table 1) reveals that it possesses 33 carbons in the form of 9 methyl (1 methoxyl, 1 acetoxyl), 6 methylene, 9 methine (2 olefinic, 7 aliphatic with 4 oxygenated), 1 quaternary carbon, 1 ketone, 1 carbonyl, 4 olefinic, and 2 oxygenated alkyl carbons. Consideration of the chemical structures of natural products already derived from the genus Commiphora6 led us to speculate that 1 might be a sesquiterpene dimer. The structural architecture of 1 was first elucidated by using 2D NMR. The 1 H−1H COSY spectrum of 1 shows that the following correlations exist: H2-1/H2-2/H-3, H2-5/H2-6, H-8/H-9, and H3-12/H-11/H3-13 (Figure 2). On the basis of these spin systems; the observed HMBC correlations of H3-15/C-3, C-4, C-5, H-11/C-6, C-7, C-8, H-8 (δH 5.05)/C-6, C-7, C-9, H-9/C10, C-1, H3-14/C-10, C-1 (Figure 2); the chemical shifts of C-3 (δC 62.1), C-4 (δC 62.2), C-7, and C-8 (δC 121.1); and finally by considering biogenic relationships, we established that part A of 1 has the structure shown in red lines in Figure 1. Inspection of the remaining carbon signals in the 13C NMR spectrum of 1 showed that they resemble those reported for the related furanogermacren derivatives,8 which does not contain the bridged dihydrofuran moiety. Analysis of the HMBC correlations of H-12′ (δH 4.06)/C-7′ (δC 152.6), C-11′ (δC 143.2) led to the assignment of the structure of part B of 1 shown in blue lines in Figure 1. That parts A and B are connected via C-9-C-8′ and C-10-C-12′ is supported by HMBC correlations of H-9/C-8′ and H-12′/C-9, C-10. The relative configurations at the stereogenic centers in 1 were deduced by utilizing ROESY spectroscopy (Figure 2). The analysis shows that the correlation of H-9/Hb-1 exists, indicating that H-9 and H3-14 are located on opposite faces. Furthermore, ROESY correlations of H-3/Hb-1, H-3/Ha-1, and H3-15/H3-14 indicate that H3-15 and H-3 are trans. Likewise, ROESY correlations of H3-14′/H-2′, H3-14′/H-5′,

4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16 17 18 a

2

δH

δC

Ha: 1.67 m Hb: 1.20 m Ha: 1.87 m Hb: 1.39 m 2.76 d (10.2)

36.8

no.

Ha: 2.08 m Hb: 1.49 m Ha: 2.71 m Hb: 2.01 m 5.05 d (11.7) 2.67 d (11.7) 2.28 m 1.12 overlap 1.05 overlap 1.06 s 1.20 s 4.80 d (10.0) 3.93 m Ha: 1.94 dd (14.4, 8.7) Hb: 1.73 m 2.45 m 5.12 d (6.9)

Ha: 2.44 d (13.9) Hb: 2.09 d (13.9)

4.06 2.25 1.11 1.70 3.18

s s d (8.3) s s

2.15 s

23.7 62.1a 62.2a 36.1 25.6 146.5 121.1 48.5 49.0 32.5 20.8 23.3 22.0 20.8 132.3 74.8 35.1 32.9 81.4 197.7 152.6 100.7 39.4 138.1 143.2 92.7 16.1 16.9 18.1 55.6 170.6 20.7

δH 1.53 1.07 2.12 1.36 2.80

m m m m dd (8.3, 6.3)

2.06 1.01 2.74 1.96

m m m m

5.09 d (11.7) 2.45 d (11.7) 2.24 1.06 1.02 1.16 1.15 4.82 3.94 1.93 1.72 2.45 5.01

m d (6.8) d (6.8) s s d (10.0) m dd (12.3, 5.8) m m d (7.0)

2.49 d (14.0) 2.07d (14.0)

4.10 2.22 1.12 1.69 3.19

s s d (7.5) s s

2.09 s

δC 33.9 26.4 59.1 60.3 38.4 24.8 149.1 119.8 49.8 48.8 35.0 22.3 23.1 21.7 16.2 132.1 74.8 35.3 32.6 81.8 198.2 152.5 100.0 39.7 138.2 143.5 92.6 16.1 17.3 18.0 55.6 170.6 20.5

Signals might be interchangeable.

and H-1′/H-4′ suggest that the relative configurations at C-2′, C-4′, and C-5′ are those shown in Figure 1. In addition, the ROESY correlations of H-8/H-11 and H-2′/H3-15′ indicate that the Δ7(8) and Δ1′(10′) double bonds both have the E configuration. In addition, the correlation of Hb-1/H3-13′ implies that CH3-14 is bonded to a bridge-head atom. In contrast, it is difficult to assign the stereochemistry of the ring C bridge-head atom because of the flexible nature of this macrocyclic ring. Finally, single-crystal X-ray diffraction analysis with Cu Kα radiation (Figure 3)9 was utilized to assign the structure and absolute configuration at the stereogenic centers of 1 as 3S,4S,9S,10S,2′S,4′S,5′R,8′S,12′R. Commiphoroid B (2) has the same molecular formula as 1. Inspection of NMR data suggests that 1 and 2 possess the same planar structure and differ only in their relative stereochemistry. ROESY correlations (Figure 2) of H3-14′/H-2′, H3-14′/H-5′, and H-1′/H-4′ show that the relative configurations of the 2726

DOI: 10.1021/acs.joc.7b03182 J. Org. Chem. 2018, 83, 2725−2733

Article

The Journal of Organic Chemistry

and H3-13/C-7, C-11, C-12 suggest that a dihydrofuran moiety serves as ring C. The tricyclic backbone comprising rings A−C actually resembles that of agarsenone, which was previously isolated from Commiphora erythraea.11 Apart from 2 methyl groups, the remaining 10 olefinic carbon resonances along with a consideration of the biogenesis of sesquiterpenes, prompted us to suggest that the remaining D−F rings system in 3 is the same as that present in cadinane-type sesquiterpenes, except for the absence of an isopropyl group in 3. The observed 1H−1H COSY correlation of H-2′/H-3′ and HMBC correlations of H312′/C-3′, C-4′, C-5′, H-3′/C-5′, H-2′/C-3′, C-4′, C-1′, C-6′, H3-11′/C-1′, C-10′, C-9′, H-9′/C-7′, C-8′, and OH/C-7′, C-8′ (Figure 4) support this proposal. HMBC correlations of H-12/ C-6′, C-7′, C-8′ indicate that one of the connections of the Cand D-rings in 3 involves C-12−C-7′. The lack of a 5′-OH group, the chemical shift of C-11 (δC 82.7), and the requirement of one additional degree of unsaturation suggest that C-11 is bonded to C-5′, which, via an O-atom bridge, is connected to the D-ring. This conclusion is also supported by the observed ROESY correlation of H3-13/H3-12′. The hexacyclic ring system in 3 makes this trinorsesquiterpene dimer an unprecedented carbon skeleton. The relative configurations at the stereogenic centers in 3 were assigned by analysis of the ROESY spectrum, which shows correlations between H3-13/H-12, H3-14/Hb-9, H-12 (weak), H-12/Ha-9 (weak), Hb-9/H-12 (weak), indicating that H-12, H3-13, H3-14 are located on the same face. Furthermore, the observed ROESY correlations of H3-13/H-4 (weak) and H312′/H3-13, H3-15 (weak) suggest that H-4 has the same orientation as that of H3-13. As such, the relative configuration of 3 was assigned as 4R*,10R*,11S*,12S*. To gain further support for assignment of the absolute configuration at centers in 3, ECD (electronic circular dichroism) calculations were performed on (4R,10R,11S,12S)-3. The results show that the calculated, weighted ECD spectrum of (4R,10R,11S,12S)-3 is opposite to that of the natural product (Figure 5), suggesting that the absolute configuration of 3 is 4S,10S,11R,12R. Commiphoroid D (4) has the molecular formula C33H48O6 (10 degrees of unsaturation) as determined using (+)-HRESIMS, 13C NMR, and DEPT spectroscopy. The 1H NMR spectrum shows that 4 contains eight methyl groups and two olefinic/aromatic protons (Table 2). The 13C NMR and DEPT spectra indicate that the 33 carbon resonances can be ascribed to 8 methyl, 6 methylene, 11 methine (9 aliphatic with 3 oxygenated and 2 olefinic), 1 ketone, 1 carbonyl, 4 olefinic, and 2 oxygenated aliphatic carbons. Taking into account the chemical constituents of Commiphora, these data suggested that 4 is a sesquiterpene dimer. Inspection of the NMR data of 4 showed that a number of the signals resemble those reported

Figure 2. Key 2D NMR correlations of 1 and 2.

stereogenic centers in ring C of 2 are same as those in 1. Thus, the difference between 1 and 2 arises from configurational differences at one or more ring A centers. In the same manner as that utilized to analyze 1, a correlation of H-9/Hb-1 indicates that H-9 and H3-14 are on the opposite face of 2. However, the observed ROESY correlations of H-9/H3-15 and H3-14/H-3 indicate that the relative configurations at C-3 and C-4 of 2 are opposite to those in 1. Fortunately, compound 2 was obtained as colorless needles (in methanol) and it was subjected to X-ray diffraction analysis using Cu Kα radiation. This enabled assignment of the absolute configurations of the stereogenic centers in 2 as 3R,4R,9S,10S,2′S,4′S,5′R,8′S,12′R.10 Commiphoroid C (3) has the molecular formula C27H28O4 (14 degrees of unsaturation), deduced by analysis of its (−)-HRESIMS, 13C NMR, and DEPT spectra. The 1H NMR spectrum of 3 shows the presence of five methyl and three olefinic/aromatic protons. The 13C NMR and DEPT spectra (Table 2) indicate that 3 is composed of 5 methyl, 3 methylene, 6 methine (3 aliphatic with 1 oxygenated), 1 ketone, 11 olefinic, with 3 oxygenated, and 1 oxygenated aliphatic carbons. The structure of 3 was elucidated using detailed 2D NMR experiments. The 1H−1H COSY spectrum displays correlations of H2-3/H-4/H2-5, H-4/H3-15, and H3-14/H-10/H-9. The HMBC spectrum shows correlations of H2-3/C-2 (δC 196.4), C-1 (δC 128.5), H2-5/C-6 (δC 149.6), C-7, C-1, H-10/C-1, C-6, H-9/C-7 (δC 113.7), and C-8 (δC 167.6) (Figure 4). These observations enable the structural assignment of rings A and B (Figure 1). HMBC correlations of H-12 (δH 5.56)/C-11, C-13,

Figure 3. X-ray structures of 1 and 2 showing absolute configurations. Displacement ellipsoids are drawn at the 30% probability level. 2727

DOI: 10.1021/acs.joc.7b03182 J. Org. Chem. 2018, 83, 2725−2733

Article

The Journal of Organic Chemistry

Table 2. 1H (800 MHz) and 13C (200 MHz) NMR Data of 3 in CDCl3 and 1H (600 MHz) and 13C (150 MHz) NMR Data of 4 in CDCl3 (δ in ppm, J in Hz) 3 1 2 3 4 5

10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16 17 18 8′-OH 10′-OHa a

δC 128.5 196.4

Ha: 2.54 dd (16.1, 2.3) Hb: 2.15 m 2.31 m Ha: 3.19 d (16.4) Hb: 2.36 overlap

6 7 8 9

4

δH

no.

46.1

1.26 m Ha: 2.43 m Hb: 2.20 m 5.55 d (5.7)

30.6 34.6

2.36 m

149.6 113.7 167.6 Ha: 2.67 d (17.8) Hb: 2.26 d (17.8) 3.29 m

5.56 1.62 1.02 1.17

s s d (7.1) d (6.4)

δH

29.8 25.5 82.7

7.45 d (8.4) 7.22 d (8.4)

81.0 22.8 18.3 21.8 138.7 116.3 127.3

6.98 s

121.3 145.3 120.4 102.8 151.9 117.8

2.65 s 2.38 overlap

127.0 19.6 15.6

1.23 m 1.14 m Ha: 1.65 m Hb: 1.15 m Ha: 1.80 m Hb: 1.44 m 2.42 d (11.1) 2.20 d (11.1) 2.25 m 0.87 d (6.9) 0.99 d (6.7) 1.07 s 4.86 d (9.8) 3.96 m Ha: 1.92 m Hb: 1.65 m 2.43 m 5.19 d (7.9)

Ha: 2.60 d (13.5) Hb: 2.26 d (13.5)

4.65 s 2.18 s 1.11d (7.4) 1.71 s 3.19 s 2.12 s

δC 48.6 23.6 124.9 132.7 47.3 36.8 52.3 21.9 42.3 72.7 45.3 26.1 16.0 21.9 20.9 132.2 75.0 36.3 32.3 80.9 197.0 140.3 97.4 43.9 137.5 149.5 86.3 15.6 18.3 18.3 55.7 170.3 20.7

5.53 s 5.49 s

In pyridine-d5

for cadinane derivatives.12 The difference is that CH3-11 and a methylene group in cadinane derivatives are respectively replaced by a methylene and a methine in 4. This alteration is supported by the observed 1H−1H COSY correlations of H29/H2-8/H-7/H-6/H-5 (δH 2.36) and HMBC correlations of H2-11/C-3, C-4, C-5 (Figure 4). The remaining NMR data and the results of 2D NMR studies show the existence of a structural unit in 4 that is very similar to that of the blue-lined portion of the structure of 1. That the red and blue partial structures (Figure 1) are connected via C-11−C-8′ and C-5−C12′ gains support from the observed HMBC interactions between H-5/C-12′, H-6/C-12′, H-12′/C-4, and H2-11/C-7′,

C-8′, C-9′. As a result, the planar structure of 4 was established to be that shown in Figure 1. An oxygen bridge as part of a rigid 8-oxabicyclo[3.2.1]oct-6ene moiety exists in the structure of 4. In contrast to this structural core, ROESY correlations of H-6/H3-15, H-6/H3-13′, and H3-15/H3-13′ (weak) indicate an α orientation of H-6 and H3-15. Likewise, ROESY correlations of H-1/H-5, H-1/H-7, H5/H-7, H3-13′/H3-13 as well as a weak correlation of H3-13′/ Hb-2 suggest β orientations of H-1, H-5, and H-7. This conclusion is supported by the observed ROESY correlations of 10-OH (in pyridine-d5)/H-1, Ha-2 [Figures S29 and S30, Supporting Information (SI)]. The 10-membered ring E in 4 contains 3 stereogenic centers. ROESY correlations of H3-14′/ 2728

DOI: 10.1021/acs.joc.7b03182 J. Org. Chem. 2018, 83, 2725−2733

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The Journal of Organic Chemistry

Figure 6. Comparison of the experimental ECD spectrum of 4 in MeOH with the M06-2X/TZVP/IEFPCM-calculated spectra of 2′S,4′S,5′R- and 2′R,4′R,5′S-isomers of (1S,5S,6S,7R,10S,8′S,12′R)-4. σ = 0.42, shift = +3 nm, scaling factor = 0.42 for both isomers, respectively.

Figure 4. Key 2D NMR correlations of 3 and 4.

Scheme 1. Plausible Pathway for the Biogenesis of 1−4

Figure 5. Comparison between the experimental and the PBE0/ TZVP/IEFPCM-calculated ECD spectra of 3 in MeOH. σ = 0.40, shift = +3 nm, scaling factor = 0.16.

H-2′, Hb-3′, H-5′, and Ha-3′/OMe-16 indicate that H3-14′, H2′, and H-5′ have the same orientations. Further interpretation of ROESY correlations between H3-15′/H-12′, H-5′ (weak), H3-14′ (weak), H-1′/Hb-3′, H-4′, Ha-9′, and H3-13′/H-5′, H314′ (weak) by using a molecular model enabled us to determine the relative configurations of the blue part of 4 (Figure 4). To assign the absolute configuration of 4, ECD calculations on (1S,5S,6S,7R,10S,2′S,4′S,5′R,8′S,12′R)-4 and (1S,5S,6S,7R,10S,2′R,4′R,5′S,8′S,12′R)-4 were conducted. The results show that the weighted ECD spectrum of the former enantiomer agrees well with that experimentally determined (Figure 6). It is evident that the absolute configurations in the 10-membered ring of 4 are same as those of 2. As mentioned above, 1-4 are dimeric sesquiterpenes. Plausible pathways for the biosynthesis of these natural products involve the intermediacy of trans/cis-farnesyl pyrophosphate (FPP) (Scheme 1). In the routes, two monomeric germacrane intermediates I and II are generated through multiple step sequences, which include oxidation− reduction, epoxidation, esterification, and methoxylation. The Δ9(10) double bond in I serves as a dienophile in [4 + 2]cycloaddition reactions with the furan ring in II, resulting in the formation of cycloadducts 1 and 2. The stereochemical differences between the two isomeric natural products is a consequence of opposite facial orientations of the Δ3(4) double bond in the epoxidation reaction. Likewise, three monomeric

cadinene intermediates III, IV, and V are derived from I through formation of a 10-membered ring followed by evolution into 2 6-membered rings. Subsequent cyclization between the Δ11(12) double bond in III and an α,β-unsaturated ketone in IV via a key [4 + 2]-hetero Diels−Alder cycloaddition forms the possible precursor of 3. Further loss of an isopropyl moiety through oxidative cleavage would result in production of 3. A [4 + 2]-cycloaddition reaction of this type has been utilized in the total synthesis of (+)-ainsliadimer A.13 Moreover, the hydroxyl derivative of intermediate V would give rise to an allyl alcohol, which upon dehydrogenation would generate an oxyallylic cation that undergoes addition to the electron-rich furan ring. In this way, a naturally infrequent [4 + 3]cycloaddition reaction could be involved in the formation of 4. 2729

DOI: 10.1021/acs.joc.7b03182 J. Org. Chem. 2018, 83, 2725−2733

Article

The Journal of Organic Chemistry It is interesting to note that natural products containing seven-membered rings are important leads in the drug development process.14 [4 + 3]-Cycloaddition of allylic cations to dienes is a powerful method for the direct synthesis of sevenmembered rings.14 To our knowledge, the present finding of 4 represents one of few examples of such [4 + 3]-cycloaddition products occurring in nature. Adipose-derived stem cells (ADSCs) have a high proliferation and differentiation potential and, as a result, are beneficial for tissue regeneration.15 Considering the fact that Resina Commiphora has been used in traditional medicine, commiphoroids A−D were evaluated for their ADSC proliferation and differentiation activities using nontoxic concentrations (Figure S31, SI). We found that compounds 1−4 at 10 μM promote morphology changes in ADSCs (Figure S32, SI) consistent with the growth curve shown in Figure S33 (SI). In the first 3 d, 2 and 3 show little toxicity to ADSCs; however, 1 and 4 promote proliferation of ADSCs following 1-week cultivation. Besides the effect on proliferation, we also examined the differentiation-promoting effects of 1 and 4. To demonstrate the progressive epithelial determination of ADSCs, multiple specific markers for keratinocytes (keratins, involucrin, stratifin, and P63) were selected and evaluated using q-PCR, immunocytochemistry, and Western blot analysis. As shown in Figure 7,

Figure 8. Keratinocyte-specific protein expression. After coculturing with 1 or 4, there exists more cytokeratin-5 and stratifin expression in differentiated ADSC. ADSC cocultured with HaCat without compounds was used as the coculture group, and ADSCs only served as the control group. Compound 1 or 4 (10 μM) was added when coculturing.

only 10−20% trans-differentiation.16 Overall, 1 and 4 not only promote phenotypic changes but they also induce genotypic changes in ADSCs, which demonstrates the capacity of these substances to trans-differentiate ADSCs into KLCs and possibly to form a stratified epidermis-like structure.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2401PC spectrometer. CD spectra were obtained on a Chirascan instrument. NMR spectra were measured on a Bruker AV-600 or a AV-800 spectrometer, with TMS as an internal standard. ESIMS and HRESIMS were measured on an API QSTAR Pulsar 1 spectrometer. Agilent 1290 UPLC/6540 Q-TOF were used for HRMS measurements. Silica gel GF254 (Qingdao Marine Chemical Inc.) was used for preparative TLC. Silica gel (200−300 mesh, Qingdao Marine Chemical Inc.), C-18 silica gel (40−60 μm, Daiso Co.), MCI gel CHP 20P (75−150 μm, Mitsubishi Chemical Industries), and Sephadex LH-20 (Amersham Pharmacia) were used for column chromatography. Semipreparative HPLC was carried out using an Agilent 1200 liquid chromatograph equipped with an Agilent Zorbax SB-C18 column (250 mm × 9.4 mm, i.d., 5 μm). Plant Resins. The medicinal material Resina Commiphora (myrrha) was obtained from Juhuacun Market of Material Medica, Kunming, Yunnan Province, PR China, in July 2013. The material was identified by Mr. Bin Qiu at Yunnan Institute of Materia Medica, and a voucher specimen (CHYX-0585-2) was deposited at the State Key Laboratory of Photochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, PR China. Extraction and Isolation. The dried myrrha (50 kg) was ground and soaked with 95% EtOH (180 L × 48 h × 3) to give a crude extract, which was suspended in warm water followed by extraction with EtOAc to afford an EtOAc-soluble extract (8 kg). This extract was divided into six parts (FrA−FrF) by silica gel column chromatography eluting with petroleum ether−acetone (100:0, 100:1, 60:1, 40:1, 20:1, 5:1, 3:1, 1:1, 0:100). FrB (2.4 kg) was further separated via a silica gel column washed with petroleum ether−EtOAc (100:0, 100:1, 60:1, 40:1, 20:1, 5:1, 3:1, 1:1) and petroleum ether−acetone (5:1, 3:1, 1:1) to provide six portions (FrB.1−FrB.6). FrB.5 (186.6 g) was separated via MCI gel CHP 20P eluted with aqueous MeOH (55%−100%) to provide nine portions (FrB.5.1−FrB.5.9). FrB.5.6 (21.2 g) was submitted to a RP-18 column eluted with aqueous MeOH (50%− 100%) to yield six fractions (FrB.5.6.1−FrB.5.6.6). FrB.5.6.5 (5.71 g) was subjected to a MCI gel CHP 20P column washed with aqueous MeOH (70%−100%) to provide four portions (FrB.5.6.5.1− FrB.5.6.5.4). FrB.5.6.5.2 (2.60 g) was passed through Sephadex LH20 (MeOH) to yield three fractions (FrB.5.6.5.2.1−FrB.5.6.5.2.3). FrB.5.6.5.2.1 (1.26 g) was passed through Sephadex LH-20 (MeOH) to yield two fractions (FrB.5.6.5.2.1.1 and FrB.5.6.5.2.1.2).

Figure 7. Keratinocyte-specific gene expression after coculturing with 1 or 4. *p < 0.01, #p < 0.05 versus control. ADSC cocultured with HaCat without compounds was used as the coculture group, and ADSCs only served as the control group. Compound 1 or 4 (10 μM) was added when coculturing.

ADSCs do not express any keratinocyte differentiation markers. Interestingly, after coculturing with 1 or 4, differentiated ADSCs express cytokeratin-5, cytokeratin-10, P63, stratifin, and involucrin at mRNA levels when compared to untreated ADSCs. Likewise, ADSCs cocultured with 1 lead to almost a 4fold up-regulation of keratinocyte markers when compared to that of untreated ADSCs (Figure 7). Overall, gene expression profiles demonstrate that keratinocyte-like cells (KLCs) express keratinocyte-defining markers. To confirm the mRNA results, immuno-cytochemistry and Western blot analyses were used to probe for keratinocyte-specific markers. As the images in Figure 8 show, cytokeratin-5 and stratifin are expressed in KLCs after coculturing with 1 or 4 but not in untreated ADSCs. Furthermore, we evaluated and quantified the percentage of ADSCs trans-differentiating into KLCs. Surprisingly, 1 and 4 promote expression of almost 60% and 40% keratinocytespecific markers, respectively (Figure 9), values that are superior to those previously reported that are in the range of 2730

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The Journal of Organic Chemistry

Figure 9. Semiquantitative analysis of keratinocyte-specific protein expression. *p < 0.01 versus control. ADSC cocultured with HaCat without compounds was used as the coculture group, ADSCs only served as the control group. Compound 1 or 4 (10 μM) was added when coculturing. dipolar and rotational strengths. The equilibrium population of each conformer at 298.15 K was calculated from its ΔG using Boltzmann statistics. The calculated spectra of compounds were generated from the low-energy conformers according to the Boltzmann weighting of each conformer in MeOH solution. Biological Assays of Compounds 1−4. Cell Culture. In view of the fact that ADSCs from different donor tissues have similar biocharacteristics, one human subcutaneous adipose tissue sample weighing about 500 mg was used in this study from a subject undergoing a surgical excision procedure with informed patient consent. We isolated ADSCs according to the method we improved. ADSCs at passage 1 were transfected with PGMIV-CR/IV-zsGreen1MCs-PGK-Puro (IBS, Shanghai, PR China) to express green fluorescence protein permanently. Cell pellets were then suspended in a complete medium [Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS)] and plated in a T flask. Cells were cultured at 37 °C in 5% CO2 in humidified incubators, with 100% of the media replaced every 3 days. Purified ADSCs over passage 4 were used for the following experiments. Human epidermal keratinocyte (HaCaT cells) was purchased from ATCC and cultured with complete medium. Medium was changed every 2 days. Cell Viability Assay. The cell toxicities of compounds 1−4 were examined. Briefly, ADSCs of passage 6 at 5 × 104 cells/mL were plated in 96-well plates with complete medium and incubated in 5% CO2 at 37 °C. Compounds were dissolved in DMSO. After 24 h, the medium was changed to media containing different compounds at various concentrations (5, 10, and 20 μg/mL). The final concentration of DMSO was 0.25%, and the same concentration of DMSO was added into the negative control. Cell viability was assessed by cell counting kit-8 (cck-8), and ADSCs of passage 6 at 5 × 104 cells/mL were plated to develop growth curves by adding complete medium containing different compounds at a concentration of 10 μM, according to the results of the compound’s concentration screening. Cells to which was added complete medium without any compound were used as a control, 10 μL of cck-8 reagent was added to each well, and plates were incubated for 3 h at 37 °C. The percentage of viable cells was determined everyday for 7 days. The optical density values were determined at least in triplicate against a reagent blank at a test wavelength of 450 nm and reference wavelength of 630 nm. The cell morphology was observed under a phase-contrast microscope. Trans-Differentiation of ADSCs into Keratinocyte-like Cells (KLCs). ADSCs and HaCaT cells were inoculated at the ratio of 1:1 in the two chambers separated by cell culture inserts (BD). The semipermeable membrane of the inset (pore size 0.4 μm) allows the diffusion of secreted factors but prevents the cells from transporting from one chamber to the other. There was no cell contact between the two sides of the two chambers. HaCaT cells were in the upper chamber and ADSCs in the lower. Complete medium with different compounds (1 or 4) was added to different groups for 2 weeks, complete media without compounds was used as the coculture group, and ADSCs only served as the control group.

Fr.B.5.6.5.2.1.1 (0.87 g) underwent preparative TLC developed with CHCl3−EtOAc (7:1) to produce eight portions (FrB.5.6.5.2.1.1.1− FrB.5.6.5.2.1.1.8). Further purification of FrB.5.6.5.2.1.1.5 (60.0 mg) by semipreparative HPLC eluted with aqueous MeCN (85%) afforded compounds 1 (3.9 mg, tR = 20.3 min) and 2 (100.8 mg, tR = 22.4 min). Purification of FrB.5.6.5.2.1.1.7 (42 mg) by semipreparative HPLC eluted with aqueous MeCN (85%) afforded compound 4 (4.2 mg, tR = 15.8 min). FrB.5.6.5.3 (1.72 g) was divided into six parts (FrB.5.6.5.3.1−FrB.5.6.5.3.6) by Sephadex LH-20 (MeOH). FrB.5.6.5.3.5 (20.1 mg) was purified by semipreparative HPLC (aqueous MeOH, 85%) to give three portions (FrB.5.6.5.3.5.1− FrB.5.6.5.3.5.3). Compound 3 (1.2 mg, tR = 14.3 min) was obtained from FrB.5.6.5.3.5.2 by HPLC separation (aqueous MeCN, 75%). Commiphoroid A (1). Colorless needles (MeOH); [α]22 D −76.5 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 249 (3.24), 204 (3.59) nm; CD (MeOH) Δε210 +17.70, Δε233 −6.93, Δε312 −2.03; ESIMS (positive) m/z 563 [M + Na]+; (+)-HRESIMS m/z 563.3334 [M + Na]+ (calcd for C33H48NaO6 563.3343); 1H and 13C NMR data, see Table 1. Commiphoroid B (2). Colorless needles (MeOH); [α]22 D −260.5 (c 0.66, MeOH); UV (MeOH) λmax (log ε) 252 (3.32), 204 (3.72); CD (MeOH) Δε213 +23.75, Δε236 −10.03, Δε308 −3.20; ESIMS (positive) m/z 563 [M + Na]+; (+)-HRESIMS m/z 563.3343 [M + Na]+ (calcd for C33H48NaO6 563.3343); 1H and 13C NMR data, see Table 1. Commiphoroid C (3). White powders; [α]25 D +37.9 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 344 (3.36), 310 (3.29) 243 (3.76), 218 (3.77) nm; CD (MeOH) Δε214 −7.25, Δε251 +4.99, Δε289 +0.86; ESIMS (negative) m/z 415 [M − H]−; (−)-HRESIMS m/z 415.1922 [M − H]− (calcd for C27H27O4 415.1915); 1H and 13C NMR data, see Table 2. Commiphoroid D (4). White powders; [α]22 D −190.7 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 249 (3.46), 203 (3.66) nm; CD (MeOH) Δε197 −14.54, Δε251 −9.40; ESIMS (positive) m/z 563 [M + Na] +; (+)-HRESIMS m/z 563.3342 [M + Na] + (calcd for C33H48NaO6 563.3343); 1H and 13C NMR data, see Table 2. Computational Methods. Molecular Merck force field (MMFF) calculations were done using the Spartan’14 program (Wavefunction Inc., Irvine, CA). Density functional theory (DFT) and timedependent density functional theory (TDDFT) calculations were performed with the Gaussian09 program package.17 For conformational analysis, the conformers generated by a MMFF conformational search in an energy window of 10 kcal/mol were subjected to geometry optimization using the DFT method at the B3LYP/SVP level.18,19 Frequency calculations were run at the same level to estimate their relative thermal (ΔE) and free energies (ΔG) at 298.15 K. Energies of the low-energy conformers in MeOH were recalculated at the M06-2X/def2-TZVP level.18,19 Solvent effects were taken into account by using the polarizable continuum model (IEFPCM). The TDDFT calculations were performed using the hybrid PBE1PBE20 and M06-2X21 functionals and Ahlrichs’ basis set TZVP (triple-ζ valence plus polarization).22 The number of excited states is 60 for compound 3 and 30 for 4. The ECD spectra were generated by the program SpecDis23 using a Gaussian band shape from dipole-length 2731

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The Journal of Organic Chemistry Immuno-Cytofluorescence. Using a tissue slide chamber, ADSCs were cocultured with HaCat cells and treated with different compounds as described above for 2 weeks; cells were fixed in 4% paraformaldehyde for 10 min at room temperature and then washed with PBS. Nonspecific bindings were avoided by using blocking solution [phosphate-buffered saline (PBS) solution containing 10% goat serum and 5% bovine serum albumin; Sigma]. For immunofluorescence microscopy, staining was performed using primary rabbit anti-human cytokeratin-5 and primary rabbit anti-human stratifin antibody (Abcam) at 1:1000 dilution, and samples were incubated overnight at 4 °C. After washing with PBS three times for 5 min each, samples were incubated with horseradish peroxidase (HRP)conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Cells were rinsed with PBS and developed using a DAB kit (Thermo). Samples were visualized under a Nikon Eclipse TiE microscope (Tokyo, Japan). Western Blot Analysis. After 2 weeks of coculturing with compounds, cells were lysed with RIPA lysis buffer (Beyotime) and centrifuged at 12 000g for 5 min at 4 °C. Ten milligrams were loaded into SDS−PAGE. An anti-stratifin mAb (1:1000) and anti-cytokeratin5 polyAb (1:1000) (Abcam) were used as primary antibodies. An antiα actin mAb (1:30 000) (Abcam) was used as a loading control. The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Abcam) (1:2500). Immunoreactive proteins were then visualized using ECL Western blotting detection reagent for 15 min at room temperature. Samples were visualized under a Nikon Eclipse Ti-E microscope (Tokyo, Japan) and analyzed using NIS-Elements software (Nikon Corp., Tokyo, Japan). RT-PCR Analysis. After 2 weeks of coculturing with HaCat cells and treatment with compounds, the total RNA was extracted from cell samples using Trizol (Invitrogen). The RNA was reverse-transcribed to complementary DNA (cDNA) using a First Strand cDNA kit (Takara) following the manufacturer’s protocol. Quantitative polymerase chain reaction (qPCR) analysis was then performed using a Quantitect SYBR Green PCR master mix (Takara). For the amplification process, the sense and antisense primers shown in Table S1 (SI) were used. Target gene expression was normalized to βactin levels, and the comparative cycle threshold (CT) method (using the formula 2−ΔΔCT) was used to calculate relative quantification of target mRNAs. Each assay was performed in triplicate with n = 3−5 independent experiments.

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calculations, L.D. contributed to extraction, and Y.X.Z. and Y.X.C. designed the experiments, analyzed the data, and Y.X.C. wrote the paper. Notes

The authors declare no competing financial interest. Crystallographic data of commiphoroid A (1) (deposition no. CCDC 1576223) and commiphoroid B (2) (deposition no. CCDC 1576224) have been deposited at the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/ retrieving.html.

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ACKNOWLEDGMENTS This study was supported by the National Science Fund for Distinguished Young Scholars (81525026). We also gratefully acknowledge support from the Guangzhou Branch of the Supercomputing Center of Chinese Academy of Sciences.

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(1) Li, W.; Chen, W. J.; Liu, W.; Liang, L.; Zhang, M. C. Int. J. Ophthalmol. 2012, 5, 555−561. (2) Tartarini, D.; Mele, E. Front. Bioeng. Biotechnol. 2016, 3, 206. (3) Lukaszewicz, A. I.; Mcmillan, M. K.; Kahn, M. J. Med. Chem. 2010, 53, 3439−3453. (4) Allsopp, T. E.; Bunnage, M. K.; Fish, P. V. MedChemComm 2010, 1, 16−29. (5) Haffor, A.-S. A. J. Immunotoxicol. 2010, 7, 68−75. (6) Hanus, L. O.; Rezanka, T.; Dembitsky, V. M.; Moussaieff, A. Biomed. Pap. 2005, 149, 3−28. (7) Dong, L.; Cheng, L. Z.; Yan, Y. M.; Wang, S. M.; Cheng, Y. X. Org. Lett. 2017, 19, 286−289. (8) Brieskorn, C. H.; Noble, P. Tetrahedron Lett. 1980, 21, 1511− 1514. (9) Crystallographic data of commiphoroid A (1) have been deposited at the Cambridge Crystallographic Data Centre (deposition no. CCDC 1576223). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html. (10) Crystallographic data of commiphoroid B (2) have been deposited at the Cambridge Crystallographic Data Centre (deposition no. CCDC 1576224). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html. (11) Santoro, S.; Superchi, S.; Messina, F.; Santoro, E.; Rosati, O.; Santi, C.; Marcotullio, M. C. J. Nat. Prod. 2013, 76, 1254−1259. (12) Queiroga, C. L.; Ferracini, V. L.; Marsaioli, A. J. Phytochemistry 1996, 42, 1097−1103. (13) Li, C.; Yu, X. L.; Lei, X. G. Org. Lett. 2010, 12, 4284−4287. (14) Harmata, M. Chem. Commun. 2010, 46, 8886−8903. (15) Bailey, A. M.; Kapur, S.; Katz, A. J. Curr. Stem Cell Res. Ther. 2010, 5, 95−102. (16) Stocum, D. Science 2002, 298, 1901−1902. (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision C.01; Gaussian, Inc.: Wallingford, CT, 2010.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b03182. 1D and 2D NMR spectra and HR-ESIMS data of 1−4, crystallographic data of 1 and 2, computational data of 3 and 4, biological figures and tables (PDF) X-ray crystallographic data of 1 in CIF format (CIF) X-ray crystallographic data of 2 in CIF format (CIF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Y.X.Z.: phone/fax, +86-871-65223048; e-mail, yanxiazhu@ szu.edu.cn. *Y.X.C.: phone/fax, +86-871-65223048; e-mail, yxcheng@szu. edu.cn. ORCID

Jia-Wang Liu: 0000-0003-4009-0541 Xiao-Yi Wei: 0000-0002-4053-6999 Yong-Xian Cheng: 0000-0002-1343-0806 Author Contributions

J.W.L. and M.Y.Z. performed the experiments, Y.M.Y. gave some advice on structural identification, X.Y.W. finished ECD 2732

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DOI: 10.1021/acs.joc.7b03182 J. Org. Chem. 2018, 83, 2725−2733