Limonoids From Cultivated Citrus × Sphaerocarpa Y. Nakaj. ex H. Ohba

Abstract

Background

The chemical constituents of Citrus × sphaerocarpa Y. Nakaj. ex H. Ohba, a cultivated Citrus species, remain insufficiently characterized despite its agricultural relevance. To expand knowledge of bioactive metabolites in Citrus plants, a methanol extract of C. × sphaerocarpa peels was investigated.

Methods

The methanol extract from C. × sphaerocarpa peels was repeatedly separated and purified, and the structures of the isolated compounds were elucidated using nuclear magnetic resonance and mass spectrometry analyses. The anti-proliferative activities of the isolated compounds were assessed using the WST-8 assay.

Results

A novel limonoid derivative, 21,23-dihydro-21-oxodeacetylnomilin, was isolated from the peels, along with two known limonoids, limonexic acid and shihulimonin A. None of the compounds showed significant anti-proliferative activity against human glioblastoma cell lines T98G and U-251 MG.

Conclusions

This study provides the first detailed characterization of limonoids from C. × sphaerocarpa, contributing to a deeper chemotaxonomic understanding of this cultivated Citrus species.

Keywords

rutaceae peels limonoids anti-proliferative activity

1. Introduction

Citrus fruits are recognized as rich sources of diverse secondary metabolites, including carotenoids, coumarins, flavonoids, and limonoids.¹ Many of these constituents exhibit notable biological activities, such as anticancer, anti-inflammatory, antidiabetic, and antimutagenic properties,^2-5 making Citrus species important targets for phytochemical and pharmacological studies.⁶ Limonoids are characteristic tetranortriterpenoids predominantly found in the Rutaceae and Meliaceae families and serve as valuable chemotaxonomic markers due to their structurally conserved biosynthetic origins.⁷ C. × sphaerocarpa Y. Nakaj. ex H. Ohba, commonly known as “Kabosu,” is a cultivated Citrus species widely used in Japan as a seasoning fruit.⁸ Kabosu is one of the major sour citrus cultivars traditionally cultivated in Japan, particularly in Oita Prefecture, where it represents a regionally important agricultural product. It has been cultivated for several centuries and is widely consumed as a fresh seasoning fruit or processed into vinegars, dressings, and beverages due to its characteristic refreshing aroma and strong acidity.⁸ Annual production of Kabosu reaches several thousand tons, underscoring its commercial importance among Japanese citrus crops.⁸ Genetic and chemotaxonomic studies have suggested that C. × sphaerocarpa is a natural hybrid of C. reticulata and C. junos, both of which are known to accumulate structurally diverse limonoids.^9,10 Previous phytochemical investigations of related Citrus species, including C. junos, C. hassaku, and C. × sphaerocarpa,^11-13 revealed the presence of nomilin-type and limonin-type limonoids, some of which exhibit cytotoxic, anti-inflammatory, or insect antifeedant activities.¹⁴ However, despite the agricultural and commercial relevance of C. × sphaerocarpa, its limonoid composition remains insufficiently characterized. In addition, no comprehensive study has yet focused on its fruit peels.¹⁵ In an attempt to fill this gap, we here report the isolation and structural elucidation of a novel limonoid, 21,23-dihydro-21-oxodeacetylnomilin (1), together with those of two known limonoids (2 and 3), from the methanol extract of C. × sphaerocarpa peels and evaluate their biological properties.¹³ Furthermore, the anti-proliferative activities of these compounds are assessed using human glioblastoma cell lines.

2. Materials and Methods

2.1. General Experimental Procedures

Specific rotations were measured using a P-2200 digital polarimeter (l= 5 cm; JASCO, Tokyo, Japan). Fourier-transform infrared (FTIR) spectra were recorded on a JASCO FT/IR-4600 spectrometer. Ultraviolet–visible (UV-vis) spectra were acquired on a Shimadzu UV-1850 spectrophotometer (Shimadzu, Kyoto, Japan). High-resolution electrospray-ionization mass spectrometry (HR-ESI-MS) was performed using a JMS-T100LP AccuTOFLC-Plus 4G instrument (JEOL, Tokyo, Japan). ¹H (600 MHz), ¹³C (150 MHz), and 2D nuclear magnetic resonance (NMR) spectra were recorded using a JEOL JNM-ECZ 600R spectrometer. High-performance liquid chromatography (HPLC) was performed using an SPD-10AvpUV-vis detector (Shimadzu, Kyoto, Japan), an LC-10ADvp pump (Shimadzu), an SCL-10Avp system controller (Shimadzu), and LabSolutions LC software (ver. 1.25, Shimadzu) along with two COSMOSIL 5C₁₈-AR-II columns (Nacalai Tesque), 250 × 4.6 mm i.d., and 250 × 10 mm i.d., for analytical and preparative purposes, respectively. Solvent ratios are based on volume. IR, UV, and MS spectra were recorded using HPLC or spectroscopic-grade methanol (Wako Pure Chemical, Japan).

2.2. Plant Material

The fruits of C. sphaerocarpa¹⁶ were collected in Oita Prefecture, Japan, in August 2022. The present study employed the same batch of plant material (voucher no. SOCU-CS-11) as that used in our previous study.¹³ Voucher specimens have been deposited in the herbarium of the Sanyo-Onoda City University, Yamaguchi, Japan. The plant material was examined and identified by Prof. Hiroyuki Tanaka (PhD), Department of Pharmacognosy and Kampo, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University. Total DNA was extracted from plant samples, and their PCR amplification and sequencing were performed by Rizo Inc. (Sample number: PD0166, Tsukuba, Japan).¹⁷ The obtained nucleotide sequences were analyzed using the BLAST tool available at the National Center for Biotechnology Information (NCBI) website. BLAST analysis was also conducted by Rizo Inc. Sequences were queried against the NCBI nucleotide collection (nr/nt) database using default parameters. In this study, BLAST results for the same voucher specimen (SOCU-CS-11) showed the highest sequence similarity to C. junos Siebold ex Tanaka and C. reticulata Blanco. This result is consistent with the known hybrid origin and taxonomic complexity within the Citrus genus,^9,10,16 supporting the identification of the material as C. sphaerocarpa (Figure S1.1.1–5).

2.3. Extraction and Isolation

The extraction and fractionation procedures for the preparation of fraction CSEA8 followed our previously reported method.¹⁸ Briefly, dried peels (22.0 kg) were extracted with MeOH, and the MeOH extract was partitioned into EtOAc-, n-BuOH-, and H₂O-soluble fractions. The EtOAc-soluble fraction was subjected to successive normal-phase and reversed-phase silica gel chromatography to obtain fractions CSEA8-1–12. Fraction CSEA8-6 was further separated by repeated HPLC purification to obtain subfractions CSEA8-6-1–17.

In the present study, compound 3 (retention time: 40.0 min, 2.4 mg) was obtained as a white amorphous solid from subfraction CSEA8-6-16 (22.7 mg) by HPLC [H₂O–MeOH–AcOH (75:25:0.3)]. Compound 2 (retention time: 24.9 min, 9.9 mg) was isolated as a white amorphous solid from fraction CSEA8-7 (1459.9 mg) by HPLC [H₂O–MeCN–AcOH (75:25:0.3)], while compound 1 (retention time: 22.6 min, 8.0 mg) was obtained as a white amorphous solid from subfraction CSEA8-7-9 (13.4 mg) following HPLC [H₂O–MeOH–AcOH (60:40:0.3)]. See Figure 1 for the structures of the isolated compounds (1–3). The purity of compounds 1–3 was determined by HPLC and estimated to be 80–89%, presumably due to trace-level co-elution components with weak UV absorption.

Figure 1.

Skeletal (shorthand) structures of compounds isolated from Citrus × sphaerocarpa Y. Nakaj. ex H. Ohba peels

2.4. 21,23-Dihydro-21-oxodeacetylnomilin (1)

White amorphous solid; [α]_D²⁵−51.4 (c 0.04, MeOH); FTIR (ATR) v_max 1702, 2861, 2941, and 3678 cm⁻¹; UV (MeOH) λ_max (logε) 208.5 nm (4.49); CD (MeOH) λ_max nm (Δε): 203.1 (33.8), 231.1 (–78.1), 264.7 (–8.4), and 302.2 nm (–33.3); ¹H NMR (chloroform-d, 600 MHz) and ¹³C NMR (chloroform-d, 150 MHz) (Table 1); HR-ESI-MS m/z: 511.1959 (Calcd. for C₂₆H₃₂O₉Na [M+Na]⁺ m/z: 511.1939.

Table 1.

¹³C (150 MHz) and ¹H (600 MHz) NMR Data for 21,23-Dihydro-21-oxodeacetylnomilin (1) and 21,23-Dihydro-21-oxolimonin¹⁹ and 21,23-Dihydro-23-hydroxy-21-oxodeacetylnomilin.²⁰

Position	1*		21,23-Dihydro-21-oxolimonin¹⁹ ^**		21,23-Dihydro-23-hydroxy-21-oxodeacetylnomilin²⁰ ^***
Position	δ _C	δ _H (J in Hz)	δ _C	δ _H (J in Hz)	δ _C	δ _H (J in Hz)
1	69.4	3.91 (br d, 7.2)	80.1	4.27 (br d, 4.1)	70.0	3.87 (m)
2	39.3	α 2.95 (m)	36.5	α 2.87 (dd, 1.5, 16.7)	40.1	α 3.43 (d, 15.4)
2	39.3	β 3.31 (m)	36.5	β 2.73 (dd, 4.1, 16.7)	40.1	β 2.90 (m)
3	170.5		170.0		174.3
4	84.6		80.7		86.3
5	49.5	2.64 (dd, 3.6, 14.4)	59.8	2.60 (dd, 3.6, 15.2)	51.2	2.61 (dd, 3.2, 14.2)
6	39.0	α 2.54 (dd, 4.2, 14.4)	37.1	α 2.40 (dd, 3.6, 15.2)	40.1	α 2.50 (dd, 3.2, 14.2)
6	39.0	β 2.74 (t-like, 14.4)	37.1	β 3.16 (t, 15.2)	40.1	β 3.03 (t, 14.2)
7	207.4		208.2		210.3
8	52.6		51.7		54.0
9	43.8	2.79 (m)	48.2	2.83 (m)	45.5	2.86 (m)
10	44.7		46.7		46.1
11	17.3	α 1.33 (m)	18.8	α 1.96 (m)	18.1	α 1.84 (m)
11	17.3	β 1.77 (m)	18.8	β 2.07 (m)	18.1	β 1.66 (m)
12	29.5	α 1.26 (m)	29.0	α 1.44 (m)	30.9	α 1.40 (m)
12	29.5	β 2.25 (m)	29.0	β 2.06 (m)	30.9	β 2.12 (dd, 7.3, 13.5)
13	38.2		39.9		39.3
14	65.7		67.6		67.1
15	53.4	3.83 (s)	55.2	4.19 (s)	54.6	3.86 (s)
16	166.7		167.3		168.6
17	76.0	5.44 (s)	76.6	5.35 (d, 1.2)	77.1	5.40 (s)
18	19.9	1.13 (s)	19.7	1.27 (s)	20.3	1.19 (s)
19	16.8	1.24 (s)	65.7	α 4.65 (d, 13.5)	16.9	1.26 (s)
19	16.8	1.24 (s)	65.7	β 4.97 (d, 13.5)	16.9	1.26 (s)
20	130.3		129.6		133.8
21	175.5		173.1		172.0
22	152.4	7.63 (d, 1.8)	154.4	7.85 (dd, 1.7, 2.9)	153.3	7.42 (br s)
23	70.8	4.91 (br s)	71.9	4.99 (dd, 1.7, 3.5)	99.1	6.23 (br s)
23	70.8	4.92 (br s)	71.9	4.99 (dd, 1.7, 3.5)	99.1	6.23 (br s)
28	33.3	1.45 (s)	21.8	1.13 (s)	33.7	1.44 (s)
29	23.5	1.53 (s)	30.3	1.24 (s)	23.6	1.60 (s)
30	17.3	1.18 (s)	18.2	1.18 (s)	17.3	1.26 (s)

*in chloroform-d.

**in acetone-d₆.

***in methanol-d₄.

2.5. Cell Culture

T98G (RCB1954) and U-251 MG (IFO50288) cells were maintained as reported previously.¹¹

2.6. WST-8 Assay

Cell proliferation was determined using a cell counting kit 8 (CCK-8: Wako Pure Chemical Industries) according to the manufacturer’s instructions and by modifying a previously described method.²¹ Cells were seeded at a density of 3.0×10³ cells/100 µL per well in 96-well cell culture plates (Coster 3596; Corning, NY, USA).²¹ After approximately 24 h, the cells were treated with Adriamycin (Wako Pure Chemical Industries) or the isolated compounds (30 and 60 µM) for 24 h. Next, CCK-8 solution (10 µL) was added to the plates and incubated in a CO₂ incubator for 2 h¹⁹. Absorbance was measured at 450 and 620 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific, MA, USA).²¹

2.7. Statistical Analysis

Statistical analyses were performed with GraphPad Prism software (version 8.43; GraphPad Software, San Diego, CA, USA) using the Dunnett’s test to analyze differences between treatment groups. Differences were considered significant at ^*P< 0.001 when compared against DMSO-treated cells.

3. Results

3.1. Isolating Limonoids From C. × Sphaerocarpa

The extraction and fractionation procedures for obtaining the EtOAc-soluble fraction and fraction CSEA8 followed our previously reported method.¹³ In the present study, repeated HPLC purification afforded a new limonoid: 21,23-dihydro-21-oxodeacetylnomilin (1, 8.0 mg, 0.36 ppm), together with two known limonoids: limonexic acid (2, 9.9 mg, 0.45 ppm)²² and shihulimonin A (3, 2.4 mg, 0.11 ppm)²² (see Figure 1 for compound structures).

3.2. Structure of 21,23-Dihydro-21-oxodeacetylnomilin (1)

21,23-Dihydro-21-oxodeacetylnomilin (1) was isolated as a white amorphous solid with a negative optical rotation ([α]²³_D−51.4 in MeOH). Its molecular formula (C₂₆H₃₂O₉) was determined by HR-ESI-MS; m/z: 511.1959 (Calcd. for C₂₆H₃₂O₉Na [M+Na]⁺ m/z: 511.1939) and ¹³C NMR spectroscopy. The 1D NMR data (Table 1) showed that 1 possesses a carbon skeleton similar to those of deacetylnomilin,²⁴ 21,23-dihydro-23-hydroxy-21-oxodeacetylnomilin,¹⁹ and 21,23-dihydro-23-hydroxy-21-oxodeacetylnomilin.²³ The ¹H and ¹³C NMR spectra (chloroform-d) exhibit characteristic signals for a limonoid group (Table 1), including five methyl groups [δ_H 1.13 (s, H-18), 1.24 (s, H-19), 1.45 (s, H-28), 1.53 (s, H-29), and 1.18 (s, H-30)], five methylene carbons [δ_C 39.3 (C-2), 39.0 (C-6), 17.3 (C-11), 29.5 (C-12), and 70.8 (C-23)], one methine carbon bearing a hydroxyl group [δ_C 69.4 (C-1)], one epoxide methine carbon [δ_C 53.4 (C-15)], and one oxygenated methine carbon [δ_C 76.0 (C-17)] integrated into a lactone moiety, three ester carbonyl carbons [δ_C 170.5 (C-3), 166.7 (C-16), and 175.5 (C-21)], and one ketone carbonyl carbon [δ_C 207.4 (C-7)]. Further studies reported a carbonyl group, including compounds isolated from Citrus species.¹⁹ To further support the proposed structure of compound 1, we compared the ¹³C NMR chemical shifts of C-16 and C-17 with those of structurally related limonoids bearing either intact or cleaved D-rings.²⁰ In compound 1, C-16 and C-17 resonate at δ_C 166.7 and 76.0, respectively (Table 1). In contrast, seco-limonoids^19,20,23 exhibit downfield-shifted C-16 signals (δ_C 171.5) and upfield-shifted C-17 signals (δ_C 72.5), consistent with ring cleavage and altered electronic environments. These comparative data (Table 1) provide additional evidence for the closed-ring structure of compound 1, independent of HMBC correlations. The position of each functional group was determined using correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) NMR spectroscopy (Figure 2). In particular, long-range correlations were observed between the following proton and carbon pairs in 1: H-1/C-3; H-2/C-1, C-3, and C-10; H-6/C-7; H-9/C-19 and C-30; H-15/C-16; H-17/C-20; H-18/C-12, C-13, C-14, and C-17; H-19/C-1, C-5, C-9, and C-10; H-28/C-4, C-5, and C-29; H-29/C-4, C-5, and C-28; and H-30/C-7, C-8, C-9, and C-14. Several limonoids bearing the same α-substituted butenolide group, such as 21,23-dihydro-21-oxolimonin,¹⁹ have been reported. The relative configuration of 1 was determined by analyzing its NOESY spectrum (Figure 2). NOESY cross peaks between H-1 and H-11β; H-1 and H-19; H-2β and H-19; H-2β and H-29; H-5 and H-28; H-6α and H-28; H-6β and H-19; H-6β and H-30; H-9 and H-6α; H-9 and H-18; H-11β and H-19; H-15 and H-18; H-17 and H-12β; H-28 and H-12α; H-29 and H-19 indicate that 1-OH, H-2α, H-5, H-6α, H-9, H-11α, H-12α, H-15, H-18, and H-28 are on one side of the molecule and H-1, H-2β, H-6β, H-11β, H-12β, H-17, H-19, H-29, and H-30 are on the opposite side. Although NOESY analysis supports this relative configuration, the absolute stereochemistry of compound 1 (1S, 5R, 8R, 9R, 10R, 13S, 14R, 15S, 17R) was established by running a comparison with those of structurally related limonoids (e.g., 21,23-dihydro-23-hydroxy-21-oxodeacetylnomilin;¹⁹ [α]²³_D –87.9 in MeOH) and supported by its observed optical rotation ([α]²³_D –51.4 in MeOH). In addition, the CD spectrum of 1 showed characteristic Cotton effects [231.1 nm (Δε –78.1) and 264.7 nm (Δε –8.4)] comparable to those of deacetylnomilin²⁵ [228.5 nm (Δε –2.33) and 266.8 nm (Δε +0.78)], supporting the same absolute configuration. The chemical structure of 1 was established based on the evidence provided above (Figure 1). In addition to NMR analysis, the IR spectra of 1 exhibit characteristic absorptions for hydroxyl (broad band around 3678 cm^-1) and carbonyl (bands around 1702 cm^-1) groups, further supporting the presence of hydroxyl moieties and carbonyl functionalities (Figures S1.9).

Figure 2.

Important 2D NMR and NOESY correlations for 21,23-dihydro-21-oxodeacetylnomilin (1)

3.3. Evaluating Anti-proliferative Effects

The anti-proliferative activities of the isolated compounds (1–3) were evaluated using the WST-8 assay in T98G and U-251 MG human glioma cells. None of the tested compounds exhibited significant anti-proliferative effects at 30 and 60 µM for 24 h (Figures S1.11 and S1.12). In contrast, Adriamycin (80 or 10 µM), as a positive control, reduced T98G and U-251 MG cell proliferation to 72.4% and 60.5%, respectively (Figures S1.12 and S1.13).

4. Discussion

Citrus species are known to produce a wide variety of secondary metabolites, including flavonoids, limonoids, and coumarins.¹ Limonoids are tetranortriterpenoids characteristic to plants belonging to the Rutaceae and Meliaceae families and hold significant chemotaxonomic importance.⁷ C. × sphaerocarpa is suggested to be a hybrid of C. reticulata and C. junos.^9,10 Therefore, its limonoid profile may reflect its phylogenetic background. In this study, one new limonoid (1) and two known limonoids (2 and 3) were isolated from the peels of C. × sphaerocarpa, and their chemical structures and biological activities were elucidated. The new compound 1 is characterized by a C-30 methyl shift, oxidative modification of the D-ring—both typical of nomilin-type limonoids—,and biosynthetically-conserved loss of a C4 carbon side chain originating from the protolimonoid scaffold.²⁶ Compound 1 also possesses an α-substituted butenolide group as a side chain, featuring a ketone at C-21 and methylene group at C-23. Based on a recent elucidation of limonoid biosynthesis in Rutaceae species, compound 1 appears to arise from a melianol-type protolimonoid via a conserved pre-furan intermediate. The presence of a C-30 methyl shift, loss of a C4 carbon side chain, and formation of an α-substituted butenolide moiety are all consistent with established nomilin-type limonoid biosynthetic pathways.²⁵ Therefore, a plausible biosynthetic route for compound 1 is proposed within the framework of known limonoid scaffold remodeling processes (Figures S1.14).

This structural feature is consistent with 21,23-dihydro-21-oxolimonin¹⁹ obtained from the peels of C. reticulata and junosterpene¹¹ isolated from the seeds of C. junos. Hence, it can be suggested that C. × sphaerocarpa retains the limonoid biosynthetic pathways characteristic of Rutaceae. Furthermore, the limonoid profile of C. × sphaerocarpa is notable for its higher diversity than those of C. limon²⁷ and C. hassaku.¹² Such differences may reflect variations in fruit maturation stage, genetic background, or enzyme activities involved in the redox balance of limonoid biosynthesis.^7,26 In the biological activity assays, compounds 1–3 did not exhibit significant antiproliferative effects against T98G or U-251 MG cells. This observation is consistent with the weak antitumor activities reported for limonoids derived from C. junos,¹¹ C. hassaku,¹² and C. × sphaerocarpa.¹³ It suggests that the antitumor activity of limonoids is highly structure-dependent, with the furan ring and A-ring architecture likely contributing to activity.²⁸ A limitation of this study is that the small quantities of isolated compounds precluded detailed structure–activity relationship analysis and additional biological evaluations such as antioxidant or anti-inflammatory assays. Nevertheless, the present work provides the first comprehensive characterization of the limonoid profile of C. × sphaerocarpa—an important contribution to the chemotaxonomic understanding of the Citrus genus. Future studies examining variations in limonoid composition across fruit maturation stages and anatomical parts (peel, pulp, seeds), as well as gene expression analyses related to limonoid biosynthesis, will further clarify the chemical characteristics and phylogenetic positioning of C. sphaerocarpa.

5. Conclusion

21,23-Dihydro-21-oxodeacetylnomilin (1) was isolated as a novel limonoid from the peels of C. × sphaerocarpa. Its chemical structure was elucidated through various spectroscopic techniques. Compounds 2 and 3 were identified as limonexic acid and shihulimonin A, respectively. Although none of the isolated compounds exhibited significant anti-proliferative activity against T98G or U-251 MG cells, the present work provides the first comprehensive account of limonoids from C. × sphaerocarpa. These findings expand the phytochemical knowledge of this cultivated Citrus species and offer a foundation for future studies on its chemotaxonomic significance and potential biological properties.

Supplemental Material

Supplemental Material - Limonoids from cultivated Citrus × sphaerocarpa Y. Nakaj. ex H. Ohba

Supplemental Material for Limonoids from cultivated Citrus × sphaerocarpa Y. Nakaj. ex H. Ohba by Daisuke Imahori, Takuya Muraoka, Yuya Nakao and Hiroyuki Tanaka in Natural Product Communications.

Footnotes

Acknowledgements

This work was supported by the Japan Society for the Promotion of Science (JSPS): KAKENHI Grant Numbers 22K20720 and 23K16308. We thank Ms. Runa Kono (Division of Pharmaceutics, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University) for assisting with sample preparation. The authors gratefully acknowledge the use of the Joint Research Equipment Network at Hiroshima University and thank Dr. Takehiro Hirao for his technical support.

ORCID iDs

Daisuke Imahori

Takuya Muraoka

Hiroyuki Tanaka

Ethical Considerations

Ethical Approval is not applicable for this article.

Consent to Participate

There are no human subjects in this article and informed consent is not applicable.

Author Contributions

Daisuke Imahori: Writing–original draft, Project administration, Conceptualization. Takuya Muraoka: Data curation, Formal analysis. Yuya Nakao: Data curation. Hiroyuki Tanaka: Writing – review & editing, Supervision, Conceptualization.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Japan Society for the Promotion of Science (JSPS): KAKENHI Grant Numbers 22K20720 and 23K16308.

Declaration of Conflicting Interests

The authors declare no potential conflicts of interest with respect to the research, authorship, or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental Material

Supplemental material for this article is available online. ¹H, ¹³C, 2D NMR, CD, IR, UV, and MS spectra of new compound 1.

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