An efficient one-pot synthesis and biological evaluation of novel ( E )-2-aroyl-4-arylidene-5-oxotetrahydrofuran derivatives

Abstract

An efficient one-pot base-mediated approach to (E)-2-aroyl-4-arylidene-5-oxotetrahydro-furans is developed. Nine (E)-2-aroyl-4-arylidene-5-oxotetrahydrofurans are synthesized in good yields via tandem Passerini and cyclization reactions, starting from Baylis–Hillman acids, aryl glyoxals, and isocyanides at room temperature in the presence of Cs₂CO₃. In addition, the MTT assay is used to evaluate their cytotoxicities toward the cervical cancer cell lines C-33A, CaSki, and SiHa and the hepatocarcinoma cell line HepG2. The results show that some of the compounds inhibit the proliferation of cancer cells significantly.

Keywords

cancer one-pot proliferation synthesis

Introduction

Heterocyclic compounds, especially pyrroles, pyridines, furans, and β-lactams, are used extensively in medicinal chemistry, industry, and pesticides due to their useful properties. The structural modification, synthesis, and bioactivity of heterocyclic compounds are important research fields in organic and pharmaceutical chemistry. Derivatives of heterocycles containing a tetrahydrofuranone moiety are widely found in plants.^1,2 They have significant anticancer³ and anti–Toxoplasma gondii activity⁴ and can also be used as seed germination stimulants,⁵ in dermatology, and in capillary drug preparation.⁶

Studies have shown that the alkaloids rhopaladins A-D, which are present in marine cysts, have remarkable cytotoxicity against human tumor cell lines,⁷ especially rhopaladin B (Scheme 1).⁸ We have previously synthesized 2-aroyl-4-arylidene-5-oxopyrrolidines via tandem Ugi and cyclization reactions from Baylis–Hillman acids, primary amines, aryl glyoxals, and isonitriles⁹ and found that they are bioisosteres of rhopaladins because of the presence of five-membered nitrogen heterocycles with arylidene and aroyl groups. Furthermore, (E)-2-aroyl-4-arylidene-5-oxopyrro-lidines have significant cytotoxicities toward cervical cancer cells.⁹ In order to find improved or novel heterocyclic cores, we used the Baylis–Hillman and Passerini reactions to synthesize a series of tetrahydrofuranone derivatives that are bioisosteres of 2-aroyl-4-arylidene-5-oxopyrrolidines. The strategy for the preparation of (E)-2-aroyl-4-arylidene-5-oxotetrahydrofurans is shown in Scheme 1. Meanwhile, the effects of these novel compounds on the proliferation of cervical cancer cells and hepatoma cells were studied by the MTT assay.¹⁰

Scheme 1.

The strategy for the choice of (E)-2-aroyl-4-arylidene-5-oxotetrahydrofurans as targets.

Results and discussion

Initially, the Passerini reaction condensation intermediate (4a) was prepared from α-bromomethyl cinnamic acid (1a), 4-nitrophenylglyoxal (2a), and tert-butyl isocyanide (3). When the reactants were stirred at room temperature in methanol or ether for 15 days, the reaction was incomplete. In acetonitrile, the reaction did not proceed at room temperature. When the temperature was raised to 40 °C, the reaction was complete in 4 h, but the quantity of side-products increased and the yield of intermediate (4a) was reduced. In CH₂Cl₂, the condensation reaction was complete in 48 h at room temperature (Scheme 2), and the presence of the condensation intermediate (4a) and compound (5a) was verified by ¹H NMR spectroscopy. Finally, intermediate (4a) was completely converted into compound (5a) via a base-mediated approach.

Scheme 2.

Synthesis of intermediate 4a.

The basicity plays an important role in the base-mediated approach. In order to avoid side reactions caused by too high a pH, a base was added to adjust the pH of the solution to 8 during the reaction (Table 1). The best result was obtained when solid cesium carbonate was used as the base (Table 1, entry 4). With optimized condition, we synthesized (E)-2-aroyl-4-arylidene-5-oxotetrahydrofuran derivatives 5 from various Baylis–Hillman acids 1, aryl glyoxals 2, and isocyanide 3 (Scheme 3). All the reactions occurred smoothly to give compounds 5 in yields of 66%–80% (Table 2). It was found that the yields of compounds 5 were mainly related to the nature of the Ar² substituent on aryl glyoxals 2. The stronger the electron-withdrawing effect of Ar² substituent, the higher the yield of compounds 5. The Ar¹ substituent on Baylis–Hillman acids 1 had little impact on the reaction yields (Table 3).

Table 1.

Optimization of the synthesis of compound 5a.

Entry	Base	Reaction conditions	Yield (%)
1	K₂CO₃	rt/12 h	52
2	NaHCO₃	rt/12 h	38
3	NEt₃	rt/12 h	–
4	Cs₂CO₃	rt/12 h	73

Scheme 3.

Synthesis of (E)-2-aroyl-4-arylidene-5-oxotetrahydrofuran derivatives 5.

Table 2.

Synthesis of compounds 5.

Product	Ar¹	Ar²	R	Yield (%)
5a	Ph	4-O₂NC₆H₄	t-Bu	73
5b	4-ClC₆H₄	4-O₂NC₆H₄	t-Bu	78
5c	Ph	4-BrC₆H₄	n-Bu	66
5d	Ph	2,4-Cl₂C₆H₄	t-Bu	75
5e	4-ClC₆H₄	4-BrC₆H₄	t-Bu	80
5f	4-F₃CC₆H₄	4-BrC₆H₄	t-Bu	72
5g	4-ClC₆H₄	4-ClC₆H₄	t-Bu	79
5h	4-CH₃OC₆H₄	4-ClC₆H₄	t-Bu	80
5i	Ph	4-BrC₆H₄	t-Bu	73

Table 3.

Cytotoxicity of compounds 5 in vitro (IC₅₀, μM).

Compound	C-33A	CaSki	SiHa	HepG2	L02
5a	>100	>100	>100	>100	>100
5b	>100	>100	>100	>100	>100
5c	>100	>100	>100	>100	>100
5d	35.2	46.7	57.8	89.4	>100
5e	>100	>100	>100	>100	>100
5f	>100	>100	>100	>100	>100
5g	49	>100	78	>100	>100
5h	75	89	>100	>100	>100
5i	>100	>100	>100	>100	>100
Cisplatin	13.16	28.6	>100	19.27	21.34

The structures of compounds 5a–i were confirmed by their spectroscopic data. The ¹H NMR spectra of compounds 5 showed chemical shifts for the aromatic hydrogens generally between 8.39 and 6.90 ppm, the alkene hydrogen is generally around 7.60 ppm, and the NH is around 6.60 ppm. When the electron-withdrawing effect of Ar² is enhanced, the chemical shift moves to a lower field. The signal for the CH₂ of the furan ring occurs as two doublets at around 4.44 and 3.34 ppm, respectively. The ¹³C NMR spectral data of compounds 5 show the characteristic chemical shift of a carbonyl carbon around 189 ppm, and the amide and ester carbonyls around 169 and 167 ppm, respectively. The characteristic quaternary carbon on the furan ring occurs around 86 ppm. The products are racemates, but the configuration of the double bond of the Baylis–Hillman acid is known to be retained after the condensation and cyclization reactions.^9,11

The cytotoxicities of compounds 5 were tested by the MTT assay; meanwhile, cisplatin was used as the positive control. The results indicated that there were three compounds, 5d, 5g, and 5h, which could inhibit the proliferation of most of the cells. Other compounds were hardly cytotoxic to cancer cells. The IC₅₀ of 5d to C-33A cells was 32.5 μmol, which can significantly inhibit cancer cell proliferation.

Conclusion

By combining the Baylis–Hillman reaction with the Passerini reaction, nine (E)-2-aroyl-4-arylidene-5-oxotetrahydrofuran derivatives have been synthesized via tandem Passerini and cyclization reactions starting from Baylis–Hillman acids. None of the compounds have previously been reported in the literature. The products are easy to obtain under mild conditions, via a simple procedure and in high yields. In the experimental process, the Passerini reaction intermediates do not need to be separated and purified, but are directly used in the subsequent step. The results of cytotoxicity studies indicated that compound 5d had a significant inhibitory effect on cancer cells.

Experimental section

General information

Melting points were measured with an X-4 melting point instrument (uncorrected thermometer) produced by Beijing Ruili Analytical Instrument Co., Ltd. Mass spectrometry was performed with a Finnigan trace MS analyzer (direct injection method). Elemental analysis was performed using a Vario EL III analyzer. ¹H NMR and ¹³C NMR spectra were measured at 600 or 400 MHz using spectrometers. The solvent was CDCl₃ or DMSO-d₆ with tetramethylsilane (TMS) as the internal standard.

Experimental procedures

A solution of dried Baylis–Hillman acid 1 (1 mmol) and dichloromethane (10 mL) was stirred in a 25-mL round-bottom flask. The aryl glyoxal 2 (1 mmol) and isocyanide 3 (1 mmol) were added consecutively to the solution, and the mixture was stirred at room temperature for 48 h. The pH value of the reaction system was adjusted to about 8 by adding solid cesium carbonate intermittently during the reaction process. Dichloromethane was removed under reduced pressure, and the target compounds 5 were recrystallized from ether.

(E)-4-Benzylidene-N-tert-butyl-2-(4-nitrobenzoyl)-5-oxotetrahydrofuran-2-carboxamide (5a)

White crystals (0.31 g, 73%), m.p. 210–211 °C; ¹H NMR (CDCl₃, 600 MHz): δ 8.31 (d, J = 8.4 Hz, 2H, Ar-H), 8.23 (d, J = 8.4 Hz, 2H, Ar-H), 7.64–7.48 (m, 6H, Ar-H and =CH), 6.58 (s, 1H, NH), 4.44 (d, J = 18.6 Hz, 1H, ${CH}_{2}^{a}$ ), 3.38 (d, J = 18.0 Hz, 1H, ${CH}_{2}^{b}$ ), 1.36 (s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 150 MHz): δ 189.4, 169.3, 167.1, 150.5, 140.3, 140.3, 138.4, 133.6, 130.6, 129.2, 123.6, 123.5, 119.9, 85.8, 52.5, 29.0, 28.4. MS (EI): m/z (%) = 422 (M⁺), (4), 322 (69), 272 (100), 216 (86), 150 (25), 113 (69), 57 (43). Anal. calcd for C₂₃H₂₂N₂O₆: C, 65.39; H, 5.25; N, 6.63. Found: C, 65.33; H, 5.33; N, 6.78.

(E)-N-tert-butyl-4-(4-chlorobenzylidene)-2-(4-nitrobenzoyl)-5-oxotetrahydrofuran-2-carboxamide (5b)

White crystals (0.35 g, 78%), m.p. 143–144 °C; ¹H NMR (CDCl₃, 600 MHz): δ 8.31 (d, J = 8.4 Hz, 2H, Ar-H), 8.22 (d, J = 7.8 Hz, 2H, Ar-H), 7.58–7.46 (m, 5H, Ar-H and =CH), 6.55 (s, 1H, NH), 4.40 (d, J = 18.6 Hz, 1H, ${CH}_{2}^{a}$ ), 3.40 (d, J = 18.0 Hz, 1H, ${CH}_{2}^{b}$ ), 1.36 (s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 150 MHz): δ 189.2, 169.0, 167.0, 150.5, 138.7, 138.2, 137.1, 132.0, 131.7, 130.6, 129.4,123.6, 120.5, 85.7, 52.6, 29. 0, 28.4. MS (EI): m/z (%) = 456 (M⁺), (4), 356 (66), 306 (95), 250 (100), 150 (66), 57 (74). Anal. calcd for C₂₃H₂₁ClN₂O₆: C, 60.46; H, 4.63; N, 6.13. Found: C, 60.21; H, 4.68; N, 6.19.

(E)-2-(4-Bromobenzoyl)-N-butyl-4-benzylidene-5-oxotetrahydrofuran-2-carboxamide (5c)

White crystals (0.30 g, 66%), m.p. 186–187 °C; ¹H NMR (CDCl₃, 400 MHz): δ 7.96 (d, J = 7.7 Hz, 2H, Ar-H), 7.60–7.46 (m, 7H, Ar-H and =CH), 6.86 (s, 1H, NH), 4.38 (d, J = 18.4 Hz, 1H, ${CH}_{2}^{a}$ ), 3.48 (d, J = 17.9 Hz, 1H, ${CH}_{2}^{b}$ ), 3.40–3.23 (m, 2H, CH₂), 1.50–1.28 (m, 4H, 2CH₂), 0.89 (t, J = 6.2 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 100 MHz): δ 190.0, 169.8, 167.7, 139.8, 133.7, 132.3, 132.2, 131.9, 131.2, 130.8, 130.6, 129.1, 120.4, 86.5, 39.5, 31.2, 19.9, 13.6, 7.9. MS (EI): m/z (%) = 455 (M⁺), (2), 355 (14), 272 (100), 183 (94), 172 (21), 57 (31). Anal. calcd for C₂₃H₂₂BrNO₄: C, 60.54; H, 4.86; N, 3.07. Found: C, 60.45; H, 4.92; N, 3.02.

(E)-4-Benzylidene-N-tert-butyl-2-(2,4-dichlorobenzoyl)-5-oxotetrahydrofuran-2-carboxamide (5d)

White crystals (0.33 g, 75%), m.p. 199–200 °C; ¹H NMR (CDCl₃, 600 MHz): δ 7.60–7.30 (m, 8H, Ar-H and =CH), 6.44 (s, 1H, NH), 4.18 (d, J = 18.6 Hz, 1H, ${CH}_{2}^{a}$ ), 3.51 (d, J = 18.0 Hz, 1H, ${CH}_{2}^{b}$ ), 1.32 (s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 150 MHz): δ 194.1, 169.2, 165.6, 139.7, 137.2, 134.4, 133.5, 132.5, 130.7, 130.5, 130.2, 129.0, 128.3, 126.6, 120.3, 87.1, 52.3, 34.0, 28.2. MS (EI): m/z (%) = 445(M⁺), (2), 345 (15), 272 (100), 216 (67), 143 (23), 57 (18). Anal. calcd for C₂₃H₂₁Cl₂NO₄: C, 61.89; H,4.74; N, 3.14. Found: C, 61.85; H, 4.93; N, 3.32.

(E)-2-(4-Bromobenzoyl)-N-tert-butyl-4-(4-chlorobenzylidene)-5-oxotetrahydrofuran-2–carboxamide (5e)

White crystals (0.39 g, 80%), m.p. 186–187 °C; ¹H NMR (CDCl₃, 400 MHz): δ 7.94 (d, J = 7.6 Hz, 2H, Ar-H), 7.61–7.44 (m, 7H, Ar-H and =CH), 6.52 (s, 1H, NH), 4.34 (d, J = 18.2 Hz, 1H, ${CH}_{2}^{a}$ ), 3.35 (d, J = 18.3 Hz, 1H, ${CH}_{2}^{b}$ ), 1.34 (s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 100 MHz): δ 190.0, 169.8, 167.7, 139.8, 133.7, 132.3, 132.2, 131.9, 131.2, 130.8, 130.6, 129.1, 120.4, 86.5, 39.5, 31.2, 19.9, 13.6, 7.9. MS (EI): m/z (%) = 489 (M⁺), (4), 389 (31), 306 (78), 206 (31), 183 (100), 57 (38). Anal. calcd for C₂₃H₂₁BrClNO₄: C, 56.29; H, 4.31; N, 2.85. Found: C, 56.16; H, 4.38; N, 2.81.

(E)-2-(4-Bromobenzoyl)-N-tert-butyl-4-(4-trifloromethyl benzylidene)-5-oxotetrahydrofuran-2-carboxamide (5f)

White crystals (0.38 g, 72%), m.p. 244–245 °C; ¹H NMR (CDCl₃, 400 MHz): δ 7.94 (d, J = 7.4 Hz, 2H, Ar-H), 7.74–7.63 (m, 7H, Ar-H and =CH), 6.44 (s, 1H, NH), 4.38 (d, J = 18.5 Hz, 1H, ${CH}_{2}^{a}$ ), 3.44 (d, J = 18.4 Hz, 1H, ${CH}_{2}^{b}$ ), 1.35(s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 100 MHz): δ 189.5, 168.9, 166.9, 137.7, 137.0, 135.0, 132.0, 131.1, 130.5, 129.5, 126.1, 126.0, 126.0, 123.5, 86.2, 52.5, 34.0, 28.4. MS (EI): m/z (%) = 523 (M⁺), (3), 423 (17), 340 (89), 183 (100), 240 (27), 57 (27). Anal. calcd for C₂₄H₂₁BrF₃NO₄: C, 54.98; H, 4.04; N, 2.67. Found: C, 54.88; H, 4.09; N, 2.63.

(E)-4-Benzylidene-N-tert-butyl-2-(4-chlorobenzoyl)-5-oxotetrahydrofuran-2-carboxamide (5g)

White crystals (0.35 g, 79%), m.p. 218–219 °C; ¹H NMR (CDCl₃, 600 MHz): δ 8.03 (d, J = 8.4 Hz, 2H, Ar-H), 7.53–7.42 (m, 7H, Ar-H and =CH), 6.58 (s, 1H, NH), 4.37 (d, J = 18.0 Hz, 1H, ${CH}_{2}^{a}$ ), 3.60 (d, J = 18.0 Hz, 1H, ${CH}_{2}^{b}$ ), 1.35 (s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 150 MHz): δ 189.2, 169.3, 167.1, 140. 5, 138.0, 136.8, 132.1, 131.6, 131.1, 130.9, 129.3, 128.8, 121.2, 86.1, 52.3, 33.9,28.3. MS (EI): m/z (%) = 445 (M⁺), (10), 345 (26), 306 (33), 250 (38), 139 (100), 57 (30). Anal. calcd for C₂₃H₂₁Cl₂NO₄: C, 61.89; H, 4.74; N, 3.14. Found: C, 61.72; H, 4.79; N, 3.24.

(E)-N-tert-butyl-2-(4-chlorobenzoyl)-4-(4-methoxybenzylidene)-5-oxotetrahydrofuran-2–carboxamide (5h)

White crystals (0.35 g, 80%), m.p. 186–187 °C; ¹H NMR (CDCl₃, 600 MHz): δ 8.04 (d, J = 8.4 Hz, 2H, Ar-H), 7.53–7.42 (m, 5H, Ar-H and =CH), 6.96 (d, J = 8.4 Hz, 2H, Ar-H), 6.66 (s, 1H, NH), 4.38 (d, J = 18.6 Hz, 1H, ${CH}_{2}^{a}$ ), 3.86 (s, 3H, CH₃), 3.34 (d, J = 18.0 Hz, 1H, CH₂^b), 1.35 (s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 150 MHz): δ = 189.4, 169.9, 167.5, 161.5, 140.3, 139.3, 132.4, 131.7, 130.9,128.7, 126.4, 117.3, 114.4, 86.0, 55.5, 52.2, 33.9, 28.3. MS (EI): m/z (%) = 441 (M⁺), (10), 341 (42), 302 (80), 246 (100), 139 (77), 57 (20). Anal. calcd for C₂₄H₂₄ClNO₅: C, 65.23; H, 5.47; N, 3.17. Found: C, 65.28; H, 5.49; N, 3.43.

(E)-2-(4-Bromobenzoyl)-N-tert-butyl-4-benzylidene-5-oxotetrahydrofuran-2-carboxamide (5i)

White crystals (0.33 g, 73%), m.p. 174–176 °C; ¹H NMR (CDCl₃, 400 MHz): δ 7.94 (s, 2H, Ar-H), 7.61–7.45 (m, 7H, Ar-H and =CH), 6.50 (s, 1H, NH), 4.38 (d, J = 18.2 Hz, 1H, ${CH}_{2}^{a}$ ), 3.40 (d, J = 18.2 Hz, 1H, ${CH}_{2}^{b}$ ), 1.35 (s, 9H, 3CH₃); ¹³C NMR (CDCl₃, 100 MHz): δ 189.6, 169.5, 167.2, 139.7, 133.7, 132.2, 131.8, 131.1, 130.7, 130.5, 129.0, 120.5, 86.1, 52.3, 34.0, 28.4. MS (EI): m/z (%) = 455 (M⁺), (6), 355 (27), 272 (69), 183 (100), 172 (28), 57 (35). Anal. calcd for C₂₃H₂₂BrNO₄: C, 60.54; H, 4.86; N, 3.07. Found: C, 60.62; H, 4.82; N, 3.11.

¹H NMR (CDCl₃, 600 MHz) of 4a: δ 8.36–7.48 (m, 9H, Ar-H and =CH), 6.63 (s, 1H, NH), 6.41 (s, 1H, OCH), 4.47 (dd, J₁ = 67.2 Hz, J₂ = 10.2 Hz, 1H, CH₂^a), 1.40 (s, 9H, 3CH₃).

MTT assay

Cell proliferation was evaluated using the MTT assay. Cervical cancer cell lines C-33A, SiHa, and CaSki were cultured in DMEM/MEM/1640 medium; HepG2 and L02 were cultured in 1640 medium, which contained 10% fetal bovine serum (FBS). The cells were grown in an incubator with a humidified atmosphere of 5% CO₂ at 37 °C. The cells were incubated for 24 h in 96-well plates at a density of 5 × 10³ cells/well. The cells were treated with varying concentrations of compounds 5 and cisplatin for 48 h, and cultured in the same medium containing 5 mg/mL MTT for 4 h. The crystal was then dissolved in DMSO (150 μL). The absorbance at 490 nm (A490 nm) was measured using an enzyme-labeled meter (BioTEK Inc., Biotek MQX200). The effect of treatment with compounds 5 and cisplatin on cell viability was evaluated relative to the readings of the control cells. A490 nm represents the number of viable cells.

Supplemental Material

CHL_958626_Supplementary_information_003 – Supplemental material for An efficient one-pot synthesis and biological evaluation of novel (E)-2-aroyl-4-arylidene-5-oxotetrahydrofuran derivatives

Supplemental material, CHL_958626_Supplementary_information_003 for An efficient one-pot synthesis and biological evaluation of novel (E)-2-aroyl-4-arylidene-5-oxotetrahydrofuran derivatives by Hong-Mei Wang, Xiu-Lian Zhu, Qin-Hua Chen, Ming-Wu Ding and Xiao-Hua Zeng in Journal of Chemical Research

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: We gratefully acknowledge financial support of this work by the National Natural Science Foundation of China (81872509), the Hubei Provincial Technology Innovation Project (2017ACA176), the Shiyan Municipal Science and Technology Bureau Science and Technology Project (Grant No. 18K79), the Scientific Research Project of Educational Commission of Hubei Province of China (Grant No. B2018111), and the Postgraduate Innovation Project of Hubei University of Medicine (Grant No. YC2020042).

ORCID iD

Xiao-Hua Zeng

Supplemental material

Supplemental material for this article is available online.

References

Wang

Fan

Sajan

, et al. Org Lett 2017; 19: 2182–2185.

Guo

Pei

Chen

, et al. J Asian Nat Prod Res 2012; 14: 210–215

Chen

Han

Zou

, et al. J Biol Chem 2014; 289: 17184–17194.

Zhang

Shen

Wang

, et al. Eur J Med Chem 2018; 15: 414–427.

Yokota

Nomura

Yoneyama

, et al. J Pestic Sci 2017; 42: 58–61.

Santos

Legendre

Villis

PCM

Jr , et al. Acta Crystallographica 2012; 68: o294–297.

Poli

Giambastiani

Malacria

, et al. Tetrahedron Lett 2001; 42: 6287–6289.

Sá

Fernandes

Ferreira

, et al. Tetrahedron Lett 2008; 49: 1228–1232.

Wang

Zhu

Deng

, et al. J Chem Res 2020; 0: 1–4.

10.

Imtara

Kmail

Touzani

, et al. Evid-Based Compl Alt 2019; 2019: 8768210.

11.

Zeng

Wang

, et al. Tetrahedron 2013: 3823–3828.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

2.06 MB

0.00 MB