Pharmaceutical Biology 2004, Vol. 42 No. 1, pp.

6267

Alkaloids and Diterpenes From Croton moritibensis

Vicente Toscano de Arajo-Jnior1, Marcelo Sobral da Silva1, Emdio Vasconcelos Leito da-Cunha1,2, Maria de Ftima Agra1, Raimundo Nonato da Silva-Filho1, Jos Maria Barbosa-Filho1 and Raimundo Braz-Filho3 Laboratrio de Tecnologia Farmacutica, Universidade Federal da Paraba, Brazil; 2Departamento de Farmcia e Bioqumica, Universidade Estadual da Paraba, Brazil; 3Setor de Qumica de Produtos Naturais, Universidade Estadual Norte Fluminense, Brazil
1

Abstract
Three novel compounds, two alkaloids [2-ethoxycarbonyltetrahydroharman] and 6-hydroxy-2-methyltetrahydroharman and a diterpene [12-hydroxy-13-methyl-1,8,11,13podocarpatetraen-3-one], together with other known substances were isolated from Croton moritibensis. Their structures were established by spectroscopic methods, including 2D NMR techniques. Keywords: Croton moritibensis, Euphorbiaceae, alkaloids, diterpenes. known as velame-preto. From the aerial parts, four alkaloids (14) and three diterpenes (57) were isolated. They include the known b-carboline harman (1) and tetrahydroharman (2) alkaloids which have been isolated previously from Passiora incarnata L. (Tsuchiya et al., 1999) and Haloxylon scoparium Pomel. (Benkrief et al., 1990), respectively. 2-Ethoxycarbonyl-tetrahydroharman (3) and 6-hydroxy-2-methyltetrahydroharman (4) are described here for the rst time as natural products. Regarding the diterpenes, the cleistanthene-type sonderianol (5) and the podocarpane-type 12-hydroxy-13-methyl-8,11,13podocarpatrien-3-one (6) have been isolated previously from Croton sonderianus Muell. Arg. (Craveiro & Silveira, 1982) and Croton salutaris Casar (Itokawa et al., 1991), respectively. To the best of our knowledge, the diterpene 12hydroxy-13-methyl-1,8,11,13-podocarpatetraen-3-one (7) has not been reported previously as a natural product. The structures of these natural products were established by spectral analysis, mainly 1H and 13C NMR including homo (1H1 H-COSY) and heteronuclear [1H-13C-COSY-nJCH (n = 1, HMQC; n = 2 and 3, HMBC)] 2D experiments, which were also used to perform the complete and unambiguous 1H and 13 C NMR chemical shift assignments.

Introduction
The Euphorbiaceae family is widespread in Northeastern Brazil, being represented by several species (Webster, 1994). The genus Croton with over 800 species has two centers of diversity: one is in tropical America and the other in Africa (Webster et al., 1996). It is the largest genus of the subfamily Crotonoideae which is characterized by hairs stellate or lepidote, laticifers not articulated with coloured to whitish latex, cyanogenesis via the valine/isoleucine pathway and phorbol esters positive (Seigler, 1994). In our continuing search for new biolocally-active compounds from this genus we isolated previously clerodane and labdane diterpenes from Croton polyandrus Spreng (ArajoJnior et al., 2002) and alkaloids from Croton muscicapa Muell. Arg. (Arajo-Jnior et al., 2004). In this paper we report the chemical investigation of Croton moritibensis Baill, a native shrub from our region, where it is popularly

Material and methods

General experimental procedures
1

H NMR (500 MHz) and 13C NMR (125 MHz) were recorded at room temperature with a Bruker NMR spectrometer (DRX

Accepted: March 4, 2004 Address correspondence to: Jos Maria Barbosa-Filho, Laboratrio de Tecnologia Farmacutica, Universidade Federal da Paraba, Cx. Postal 5009, 58051-970. Joo Pessoa, PB, Brazil. E-mail: jbarbosa@ltf.ufpb.br DOI: 10.1080/13880200490505618 2004 Taylor & Francis Ltd.

Alkaloids and diterpenes from Croton moritibensis 500) with an inverse multinuclear 5 mm probe head equipped with a shielded gradient coil. The spectra were recorded in CDCl3, and the solvent signals (7.27 and 77.0 ppm) were used as reference. The chemical shifts (d) are given in ppm, and the coupling constant (J ) in Hz. All programs used for performing the 2D NMR experiments are part of the Bruker library. Column chromatography was carried out over alumina and silica gel 60 for alkaloidal and non alkaloidal separation respectively. The substances were puried by repeated PTLC (1.25 mm thick, 20 20 cm Si gel PF254 plates). The solvent systems used were different mixtures of hexane, chloroform and methanol. Plant material Croton moritibensis Baill was collected in the caatinga vegetation near the municipality of So Joo do Carir, State of Paraba, Brazil in March 2002. A voucher specimen (AGRA 2332) is deposited in the Herbarium Prof. Lauro Pires Xavier (JPB), Universidade Federal da Paraba, Brazil. Extraction and isolation The dried and powdered aerial parts of C. moritibensis (2 kg) were repeatedly extracted with 95% EtOH at room temperature. The ethanol extract was concentrated under vacuum. The alkaloids and diterpenes obtained were isolated with the use of a series of partitions involving acid/base separation. The procedure was as follows: the crude EtOH extract was stirred in a solution of 3% HCl and ltered over Celite, the precipitate was treated with ethyl ether. The ether extract containing the non basic substances was designated Fraction A. The acidic solution was made alkaline with NH4OH to pH 9 and extracted with CHCl3. The residue of this CHCl3 extract containing the basic substances was called Fraction B. Chromatography of the non alkaloidal portion (Fraction A) on CC Si gel (gradient elution) followed by successive chromatographic PTLC yielded an unseparated mixture of 5 + 6 + 7 (11 mg). A combination of column chromatography on alumina and PTLC Si gel of the alkaloidal portion (Fraction B) yielded pure 1 (76 mg), 2 (41 mg), 3 (6 mg) and 4 (9 mg).

63

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR data for (1) in CDCl3 (d in ppm, J in parentheses in Hz), including results of heteronuclear 2D experiments 1H-13C-COSY-nJCH (n = 1, HMQC; n = 2 and 3, HMBC).* HMQC dC C 1 10 11 12 13 CH 3 4 5 6 7 8 CH3 14 HN dH
2

HMBC JCH
3

JCH

141.84 134.79 128.67 122.20 140.45 138.49 113.15 122.05 120.39 128.54 111.85 20.31

3H-14 8.36 (d, 5.3) 7.84 (d, 5.3) 8.13 (dl, 7.3) 7.31 (m) 7.56 (m) 7.55 (m) 2.85 (s) 8.72 (sl) H-4 H-3

H-3 H-4, 3H-14 H-3, H-5 H-4, H-6 H-5, H-7

H-7 H-8 H-6

* Homonuclear 1H-1H-COSY spectrum was also used for these assignments. Chemical shifts of hydrogen atoms obtained from 1D 1 H NMR spectra. Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD- and DEPT-13C NMR spectra.

Results and Discussion

Comparative analysis of the HBBD- and DEPT-13C NMR spectra was used to attribute signals corresponding to quaternary, methine, methylene and methyl carbon atoms in compounds 17. The major constituents of the crude alkaloid extract were separated, puried and identied as harman (1) and tetrahydroharman (2) by spectral analysis, mainly 1D and 2D 1H and 13 C NMR data (Tables 1 and 2) compared with values described in the literature (Seki et al., 1993; Lee et al.,

1994). The new natural tetrahydroharman derivatives 3 and 4 were characterized on the basis of spectral data, including 2D 1H and 13C-NMR homo and heteronuclear techniques (Tables 2 and 3). The 1H NMR spectra (1D and 2D 1H-1H-COSY) of 3 and 4 showed similarity with 2, revealing as signicant differences the presence of a 2-ethoxycarbonyl group in 3 [dH 4.24 (q, J = 7.0 Hz, 2H-15) and 1.33 (t, J = 7.0 Hz, 3H-16)] and a hydroxyl function located at carbon atom C-6 [dH 6.80 (d, J = 2.1 Hz, H-5), 6.63 (dd, 2.1 and 8.6 Hz, H-7) and 7.12 (d, J = 8.6 Hz, H-8)] and an additional methyl group [dH 2.56 (s, 3H-14)] in 4 (Tables 3 and 4). In fact, the EIMS of 3 showed the molecular ion at m/z 258 ([M]+, 84.0%) and the base peak at m/z 243 ([M-Me.], 3a). The 13C NMR (HBBD and DEPT) spectra of 3 conrmed the presence of the 2-ethoxycarbonyl group by the signals at dC 155.74 (C-14, carbonyl group), 61.71 (CH2-15) and 14.03 (CH3-16) and indicated its location at nitrogen atom N-2 by modication of the chemical shifts observed in the signals of the carbon atoms CH-1 [DdC = 47.33 (3) - 49.45 (2) = -2.12 ppm)] and CH2-3 [DdC = 38.59 (3) - 43.50 (2) = -4.91 ppm)] as a consequence of the g-effects of the oxygen atoms of the 2-ethoxycarbonyl group. These deductions were also conrmed by HMQC and HMBC spectra (Table 3). The presence of the MeN-2 and the

64

V.T. de Arajo-Jnior et al.

Table 2. 1H (500 MHz) and 13C (125 MHz) NMR data for (2) in CDCl3 (d in ppm, J in parentheses in Hz), including results of heteronuclear 2D experiments 1H-13C-COSY-nJCH (n = 1, HMQC; n = 2 and 3, HMBC).* HMQC dC C 9 10 11 12 13 CH 1 5 6 7 8 CH2 3 4 CH3 13 dH
2

HMBC JCH
3

JCH

137.83 137.69 108.17 128.68 137.83 49.45 118.77 119.78 122.07 112.00 43.50 22.84

3.96 (q, 6.8) 7.37 (dl, 7.8) 7.00 (tl, 7.8) 7.07 (tl, 8.0) 7.31 (dl, 8.0) 3.12 (dt, 12.3, 4.3) 2.79 (ddd, 12.3, 9.4, 4.8) 2.66 (ddd, 15.2, 9.4, 4.3) 2.56 (15.2, 4.8) 1.39 (d, 6.8)

2H-4 2H-4 H-1 3H-14 H-7 H-6 H-7 2H-4 2H-3

H-5, H-7 2H-4, 3H-14 H-1, 2H-3, H-5 H-6, H-8 H-5, H-7 2H-3 H-7 H-8 H-5 H-6

20.38

H-1

* Homonuclear 1H-1H-COSY spectrum was also used for these assignments. Chemical shifts of hydrogen atoms obtained from 1D 1H NMR spectra. Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD- and DEPT-13C NMR spectra.

location of the hydroxyl group at C-6 (6-hydroxy-) of 4 were deduced by the 13C NMR (HBBD and DEPT) spectra (Table 4) involving comparison with the spectral data of 2 by: a) chemical shift of the signal corresponding to the additional methyl group at dC 39.23; b) modication in the chemical shifts of the carbons CH-1, CH2-3 and CH2-4 produced by the presence of this additional methyl group, revealing as anticipated a deshielding the CH-1 [DdC = 54.70 (4) - 49.45 (2) = +6.25 ppm] and CH2-3 (DdC = 49.63 (4) - 43.50 (2) = +6.13 ppm] by b-effects and shielding of the CH2-4 (DdC = 18.29 (4) - 22.84 (2) = -4.45 ppm] by g-effect; and c) modication in the chemical shifts of the carbons CH-5, CH-7 and C-9 shielding by ortho-effects [CH-5: DdC = 100.31 (4) -118.77 (2) = -18.46 ppm); CH-7: DdC = 108.88 (4) - 122.07 (2) = -13.19 ppm] and para-mesomeric effects [C-9: DdC = 130.08 (4) - 137.83 (2) = -7.75 ppm]. The HMQC and HMBC spectra were used to conrm these deductions (Table 4). Thus, the two new alkaloids isolated from Croton moritibensis were characterized as 2-ethoxycarbonyltetrahydroharman (3) and 6-hydroxy-2-methyltetrahydroharman (4).

Compounds 5, 6 and 7 were obtained initially as an oily mixture seemingly homogeneous by TLC. However, the analysis of the 13C-NMR (HBBD and DEPT) spectra allowed us to recognize the presence of signals corresponding to three main components. The two major compounds 5 (54.3%) and 6 (33.3%) were identied on the basis of detailed spectral analysis, mainly homo and heteronuclear 1D and 2D 1H and 13 C NMR, and comparison with literature data, as sonderianol (Craveiro et al., 1982) and 12-hydroxy-13-methyl8,11,13-podocarpatrion-3-one (Itokawa, et al., 1991). The homonuclear 1H-1H-COSY and heteronuclear 1H-13C-COSYn JCH (n = 1, HMQC; n = 2 and 3, HMBC) 2D shiftcorrelations were also used to assign unequivocally the chemical shifts of all hydrogen and carbon atoms (Tables 5 and 6). These assignments were facilitated by comparison with values previously reported in the literature for these compounds (Craveiro et al., 1982; Itokawa et al., 1991). The differences of some 13C chemical shifts were also veried and such mistakes may be eliminated on the basis of our data (Tables 5 and 6) obtained by the 2D NMR experiments. After the structural characterization of 5 and 6 it was possible to analyze the additional signals observed in the 1D and 2D 1H

Alkaloids and diterpenes from Croton moritibensis

OH
12

65

R N H 1 Me N
7

5 6 12 11

4 3 1 2

11 20 9 10 5 4 18 8 7 6

13 14

Me
17

13 8

N H

10

N
1 2

R1 O

Me
14

H
19

2 R = R1 = H 3 R = H, R1 = COOEt 4 R = OH, R1 = Me
4

15

16

5 1,2-dihydro, R = CH = CH2 6 1,2-dihydro, R = H 7 D1, R = H

5 6 12 11

7 13 8

N H 3a

10 1

N
2

O O

Figure 1.

Compounds isolated from Croton moritibensis.

Table 3. 1H (500 MHz) and 13C (125 MHz) NMR data for (3) in CDCl3 (d in ppm, J in parentheses in Hz), including results of heteronuclear 2D experiments 1H-13C-COSY-nJCH (n = 1, HMQC; n = 2 and 3, HMBC).* HMQC dC C 10 11 12 13 15 CH 1 5 6 7 8 CH2 3 4 16 CH3 14 17 dH
2

Table 4. 1H (500 MHz) and 13C (125 MHz) NMR data for (4) in CDCl3 (d in ppm, J in parentheses in Hz), including results of heteronuclear 2D experiments 1H-13C-COSY-nJCH (n = 1, HMQC; n = 2 and 3, HMBC).* HMQC HMBC dH
2

HMBC JCH
3

JCH C 6 9 10 11 12 13 CH 1 5 6 7 8 CH2 3 4 CH3 14 15

dC

JCH

JCH

136.26 127.06 127.06 136.26 155.74 47.33 118.17 119.79 122.05 111.04 38.59 21.30 61.71 19.46 14.03

3H-14 H-6, H-8 H-5, H-7

148.28 130.08 134.2 103.77 125.85 130.08 54.70 100.31 108.88 109.39 49.63 18.29

3.69 (q, 6.6) 6.80 (d, 2.1) 6.63 (dd, 8.6, 2.1) 7.12 (d, 8.6) 3.19 (m) 2.80 (m) 2.81 (m) 2.71 (m) 1.51 (d, 6.6) 2.56 (s)

H-5 H-1 2H-4

H-8 H-5, H-7 2H-4, 3H-14 2H-3 H-8 H-5, H-7 2H-3, 3H-15 H-7 H-5

5.41 (sl) and 5.31 (sl) 7.50 (d, 8.0) 7.13 (tl, 8.0) 7.19 (tl, 8.0) 7.34 (dl, 8.0) 4.53 (sl) and 4.39 (sl) 3.21 (sl) 2.83 (sl) 2.76 (dd, 15.2, 2.4) 4.24 (ql, 7.0) 1.52 (d, 5.7) 1.33 (t, 7.0)

3H-14 H-7 H-8 H-5 H-6

3H-14

2H-4 2H-3

3H-15

14.76 39.23

H-1

* Homonuclear 1H-1H-COSY spectrum and comparison with data of 2 (Table 2) were also used for these assignments. Chemical shifts of hydrogen atoms obtained from 1D 1H NMR spectra. Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD- and DEPT-13C NMR spectra.

* Homonuclear 1Hx1H-COSY spectrum and comparison with data of 2 (Table 2) were also used for these assignments. Chemical shifts of hydrogen atoms obtained from 1D 1H NMR spectra. Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD- and DEPT-13C NMR spectra.

66

V.T. de Arajo-Jnior et al.

Table 5. 1H (500 MHz) and 13C (125 MHz) NMR data for (5 + 6 + 7) in CDCl3, compared with 13C chemical shifts described in the literature (in parentheses) for 5 and 6 (Craveiro et al., 1982; Itokawa et al., 1991). 5 dC C 3 4 8 9 10 12 13 14 CH 1 2 5 11 14 15 CH2 1 2 6 7 16 CH3 17 18 19 20 dH dC 6 dH dC 7 dH

218.87 47.58 124.84 145.70 37.55 152.81 120.45 139.37

(219.7) (47.6) (125.7) (140.1) (37.6) (153.4) (120.8) (146.6) 1.89 (m) 6.72 (s) 6.60 (dd, 17.2, 11.4) 2.35 (m) 1.90 (m) 2.67 (m) 2.60 (m) 1.82 (m) 1.74 (m) 2.85 (m) 2.57 (m) 5.55 (dd, 11.4, 1.8) 5.19 (dd, 17.2, 1.8) 2.22 (s) 1.18 (s) 1.14 (s) 1.29 (s)

218.87 47.66 126.59 146.16 36.73 152.76 122.50

(217.3) (47.4) (126.9) (146.5) (37.2) (152.2) (121.8)

205.85 46.41 126.48 142.79 38.87 152.76 125.38

50.40 (50.5) 110.78 (110.2) 135.62 (136.2) 38.12 (38.2) 34.95 (34.9) 20.61 (20.5) 29.61 (29.4) 119.91 (120.5) 13.26 (13.1) 27.07 (26.9) 21.79 (21.2) 24.79 (24.7)

50.87 (50.7) 111.77 (111.5) 131.44 (131.9)

1.89 (m) 6.70 (s) 6.83 (s)

138.86 123.68 50.01 118.47 131.85

7.13 (d, 9.7) 5.92 (d, 9.7) 6.74 (s) 6.90 (s)

37.79 (37.6) 34.90 (34.7) 20.61 (20.4) 30.11 (29.9)

2.35 (m) 1.90 (m) 2.67 (m) 2.60 (m) 1.82 (m) 1.74 (m) 2.85 (m) 2.77 (m)

22.34 33.36 2.85 2.83

15.71 (15.3) 27.16 (24.6) 21.81 (26.9) 24.82 (21.1)

2.21 (s) 1.18 (s) 1.14 (s) 1.26 (s)

15.01 28.62 20.61 23.65

2.30 (s) 1.18 (s) 0.94 (s) 1.24 (s)

* Homonuclear 1H-1H-COSY and heteronuclear HMBC (Table 6) spectra were also used for these assignments. Chemical shifts of hydrogen atoms obtained from 1D 1H NMR spectra. Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD- and DEPT-13C NMR spectra.

and 13C NMR spectra. The remaining signals observed in the 1 H and 13C NMR (Tables 5 and 6) allowed us to postulate the structure of the new diterpene 7, revealing as a major difference in the comparison with the data of 6 the presence of a double bond located between the carbon atoms C-1 and C-2, consistent with the molecular ion at m/z 270 ([M].+, 36.1%, a difference of 2 Daltons when compared with the molecular ion at m/z 272 of 6) observed in the mass spectrum obtained by GC/MS. The presence of a conjugated ketone carbonyl group (a,b-unsaturated system) was deduced by signals at dH 7.13 (d, J = 9.7 Hz, H-1) and 5.92 (d, J = 9.7

Hz, H-2) together with dC 205.85 (C-3, conjugated carbonyl group), 150.70 (CH-1) and 123.68 (CH-2) revealed by the 1H and 13C NMR spectra, respectively (Tables 5 and 6). Thus, the structure of the new diterpene, in a mixture containing 5 and 6, was postulated as 12-hydroxy-13-methyl-1,8,11,13podocarpatetraen-3-one (7). On the basis of the intensity of the signals corresponding to H-15 (dH 6.60, dd, J = 11.4 and 17.9 Hz) of 5, H-14 (dH 6.83, s) of 6 and H-1 (dH 5.92, d, J = 9.7 Hz) of 7 in the 1H NMR spectrum the percentages 54.3, 33.3 and 12.4%, respectively, the concentration of these diterpenes in the mixture were estimated.

Alkaloids and diterpenes from Croton moritibensis

Table 6. Long-range heteronuclear couplings observed in HMBC spectrum of (5 + 6 + 7), in CDCl3 as solvent.* 5
2

67

Acknowledgements
The authors are grateful to CNPq, CAPES, IMSEAR and FAPERJ (Brazil) for grants and fellowships. Sincere thanks are also due to NAPRALERT for a search of Croton species and CENAUREMN/UFC for providing the NMR spectra measurements.

6
3

7
3

JCH

JCH

JCH

JCH

JCH

JCH

C 3

2H-2

H-5 3H-18 3H-19 H-6 H-11 H-15 H-5 2H-7 3H-20 H-11 2H-2 3H-17 H-11 H-15 2H-16 3H-18 3H-19 2H-7

2H-2

4 8

H-1 3H-18 3H-19 H-2

References
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2H-7

H-6 H-11 H-5 H-14 3H-20 H-11 H-14 3H-17 H-11 H-2 H-1 H-1

H-11

H-11

10 12 13 14 CH 1 2 5 11 14 15 CH2 1 2 6 7 16 CH3 17 18 19 20

3H-20 H-11 3H-17 H-15 2H-6

3H-20 H-11

H-14 3H-17

3H-20 2H-1 2H-7 2H-6 H-16t 2H-7

3H-17 3H-20

H-14 H-14

H-14

H-14

H-5 3H-19 H-5 3H-19 2H-1

* Homonuclear 1H-1H-COSY and heteronuclear HMQC (Table 5) spectra were also used for these assignments. Chemical shifts of hydrogen atoms obtained from 1D 1H NMR spectra.