1H and 13C NMR spectral assignments for 24 novel naphthalenylphenylpyrazolines

¹H

and

¹³C

NMR

spectral

assignments

for

twenty-four

novel

naphthalenylphenylpyrazolines

Dongsoo Koh,^1,+Soon Young Shin^2,+, Jiha Sung¹, Seunghyun Ahn,^3,*Yoongho Lim^3,*

¹Department of Applied Chemistry, Dongduk Women’s University, Seoul 02748, Korea

²Department of Biological Sciences, Konkuk University, Seoul 05029, Korea

³Division of Bioscience and Biotechnology, BMIC, Konkuk University, Seoul 05029, Korea

Short title: NMR spectral assignments of naphthalenylphenylpyrazolines

⁺D Koh and SY Shin contributed equally to this work.

*Corresponding authors:

Prof. Yoongho Lim

Division of Bioscience and Biotechnology

Konkuk University

Hwayang-Dong 1, Kwangjin-Gu

Seoul, Korea 05029

Email: yoongho@konkuk.ac.kr

Tel: 82-2-450-3760

Fax: 82-2-3437-6106

Dr. Seunghyun Ahn

Email: mistahn321@naver.com

Phone: 82-2-453-3760

Keywords: NMR; ¹H NMR; ¹³C NMR; naphthalenylphenylpyrazoline; chalcone

This article has been accepted for publication and undergone full peer review but has not b

een through the copyediting, typesetting, pagination and proofreading process which may lea

d to differences between this version and the Version of Record. Please cite this article as

doi: 10.1002/mrc.4603

Introduction

Compounds bearing the pyrazoline scaffold have been known to show various biological

activities, including antimicrobial, antiviral, anti-tubercular, cardiovascular, anti-

inflammatory, anticancer, antidiabetic, analgesic, and monoamine oxidase inhibitory

activities.^1-9Pyrazolines are five-membered heterocycles with two nitrogens. The biological

activities mentioned above vary based on the type and position of the substituents. For

example, pyrazoline derivatives containing 1,3-diphenyl (Fig. 1A), 3,5-difuranyl (Fig. 1B),

and 1,3,5-triphenyl (Fig. 1C) groups show anti-inflammatory, antimicrobial, and anti-

tubercular activities, respectively.^10-12We designed pyrazolines containing 3-phenyl and 5-

naphthalenyl (Fig. 1D) groups and synthesized twenty-four novel derivatives. Six of these

derivatives have the naphthalen-2-yl group and eighteen have the naphthalen-1-yl group, as

1

listed in Table 1. Herein, complete assignment of the H and ¹³C NMR spectral data is

presented along with high-resolution mass spectrometric data. The data compiled in this

report can be used as a reference for identifying newly synthesized pyrazoline derivatives.

Experimental

Syntheses

The general procedure for the synthesis of compound 16 as a representative pyrazoline (1–

18) is presented.¹³The plant-derived secondary metabolite chalcone is 1,3-diphenyl-prop-2-

en-1-one. If the α,β-unsaturated carbonyl group of chalcone is substituted with pyrazoline,

3,5-diphenyl-pyrazoline can be prepared. Therefore, the previously reported synthetic

approach for preparing chalcone derivatives was applied.¹⁴A solution of (E)-3-(2,3-

dimethoxynaphthalen-1-yl)-1-(2-hydroxy-4,5-dimethoxyphenyl)prop-2-en-1-one (1 mmol,

394 mg) was combined with (6.5 mmol, 0.6 ml) hydrazine monohydrate in 10 ml of

anhydrous ethanol by addition of the latter without a catalyst and then refluxed for 4 h at 358

K. After cooling to room temperature, the reaction mixture produced a precipitate which was

filtered and washed with cold ethanol. The crude solid was purified by recrystallization from

hot ethanol to provide a pure pyrazoline compound (16, m.p. 130−136 °C, 64% yield). The

general methods described above were applied to the synthesis of pyrazolines (19-24) for

synthetic chalcone derived from 2-naphthylaldehyde. The synthesis protocol for the

naphthalenylphenylpyrazolines is illustrated in Scheme 1.

NMR spectra

All naphthalenylphenylpyrazolines were dissolved in deuterated dimethyl sulfoxide (DMSO-

d₆). Because the NH proton peaks of derivatives 1, 3, 5, 6, and 13 were not observed in the ¹H

1

NMR spectra, the H NMR data for these species dissolved in deuterated chloroform were

also collected. The NMR samples were prepared in 2.5 mm NMR tubes at a concentration of

approximately 50 mM. All NMR data were collected on an Avance 400 spectrometer system

(9.4 T; Bruker, Karlsruhe, Germany) at room temperature, and the chemical shifts were

referenced to tetramethylsilane (TMS). The relaxation delay, 90° pulse, spectral width,

1

number of data points, and digital resolution for the H NMR experiments were 1 s, 11.8 µs,

13

5,500 Hz, 32 K, and 0.34 Hz/point, respectively. The corresponding parameters for the C

NMR and distortionless enhancement by polarization transfer (DEPT) experiments were 3 s,

15.0 µs, 21,000 Hz, 64 K, and 0.64 Hz/point, respectively. Two-dimensional experiments,

including correlation spectroscopy (COSY), heteronuclear multiple quantum correlation

(HMQC), and heteronuclear multiple bond correlation (HMBC), were performed with a data

set of 2 K × 256 (t₂× t₁). The long-range coupling time for the HMBC was 70 ms. The NMR

data were processed and analyzed in the same manner as reported.¹⁵

General experimental procedures

To confirm the structures determined by NMR spectroscopy, high-resolution mass

spectrometry (HR/MS) was carried out on a ultraperformance liquid chromatography

(UPLC)-hybrid quadrupole-time-of-flight mass spectrometer with a Waters Acquity UPLC

system (Waters Corp., Milford, MA) with the help of Prof. Choong Hwan Lee at Konkuk

University, Korea.¹⁶All HR/MS data were collected in M⁺mode, except for the data for

derivatives 1, 14, and 16 that were collected in [M+H]⁺mode.

Results and discussion

Eighteen 5-(naphthalene-1-yl)-3-(2-hydroxyphenyl)pyrazoline derivatives (1-18)

were analyzed; the procedure used to assign derivative 16, 5-(2,3-dimethoxynaphthalene-1-

yl)-3-(2-hydroxy-4,5-dimethoxyphenyl)pyrazoline, is described as a representative. The most

deshielded ¹³C peak at 153.4 ppm was long-range coupled to three protons at 3.37, 3.68, and

6.81 ppm. In the HMQC spectrum, two protons with signals at 3.37 and 3.68 ppm were

attached to the carbon with a peak at 40.4 ppm, which was determined to be a methylene

carbon based on DEPT. Because derivative 16 contains only one methylene carbon, C-py4,

the protons were assigned as H-py4. The carbon peak at 153.4 ppm mentioned above was

assigned as C-py3. Because the proton with the peak at 6.81 ppm was attached to the carbon

with the peak at 111.5 ppm, this proton was assigned as H-6’. Two methylene protons of H-

py4 were correlated with the proton at 5.74 ppm in the COSY spectrum, and were thus

determined to be H-py5. Five protons at 7.30, 7.38, 7.39, 7.82, and 8.08 ppm were correlated

with each other in the COSY spectrum. These protons were assigned as H-7, H-6, H-4, H-5,

and H-8 of the naphthalenyl group, respectively, based on comparison with previously

reported chemical shifts.¹⁴The two carbons with peaks at 126.5 and 131.7 ppm were assigned

as C-9 and C-10, respectively, because they were long-range coupled to H-4 and H-8,

respectively, in the HMBC analysis. H-8 also showed long-range coupling with the carbon

peak at 128.4 ppm; thus the carbon was assigned as C-1. The carbon peak at 147.9 ppm

showed long-range coupling with H-4 and was assigned as C-2. Because this carbon was

long-range coupled to the methoxy proton at 3.81 ppm, it was assigned as 2-OCH₃. The two

carbons with signals at 150.6 and 152.3 ppm were long-range coupled to H-6’ in the HMBC

profile. They could be assigned as C-4’ or/and C-2’. Because the former showed long-range

coupling with the methoxy proton at 3.79 ppm, it was assigned as C-4’. Consequently, the

latter was C-2’ and this methoxy proton was 4’-OCH₃. The two carbon peaks that were

deshielded below 100 ppm were not immediately assignable, that is, the signals at 108.0 and

151.3 ppm. Because C-1’ of the phenyl ring and C-3 of the naphthalenyl group were not

assigned, these peaks could be assigned to C-1’ and C-3, respectively. H-3’ was assigned

based on the long-range coupling with C-1’, with a signal at 6.64 ppm. The carbon peak at

141.8 ppm showed long-range coupling with H-3’ and was assigned as C-5’. The methoxy

proton at 3.68 ppm showed long-range coupling with C-5’ and thus should be 5’-OCH₃. The

remaining methoxy group could be assigned as 3-OCH₃. The two undetermined proton peaks

at 7.73 and 11.17 ppm were assigned to NH and OH, respectively, based on comparison with

the chemical shifts reported previously.¹⁷The important correlations obtained from the COSY

and HMBC spectra are shown in Fig. 2A. The complete assignments of the ¹H and ¹³C NMR

data

for

derivative

16,

5-(2,3-dimethoxynaphthalene-1-yl)-3-(1-hydroxy-4,5-

dimethoxyphenyl)pyrazoline, are listed in Tables 2 and 3, respectively. The NMR data for the

other naphthalenylphenylpyrazolines containing the naphthalene-1-yl group were assigned in

1

13

a similar manner, and the complete assignments of the H and C NMR data are listed in

Tables 2 and 3, respectively.

Of the six derivatives 19-24 with the naphthalen-2-yl group, the procedure used to

assign derivative 21, 5-(naphthalen-2-yl)-3-(2-hydroxy-6-methoxyphenyl)pyrazoline is

described as a representative. As in the case of derivative 16, the methylene carbon and

proton could be assigned because this compound contains only one methylene group. Seven

protons at 7.48, 7.51, 7.57, 7.89, 7.898, 7.904, and 7.92 ppm showing correlations in the

COSY spectrum were determined based on comparison of the chemical shifts with those

reported previously.¹⁴The three carbons signals at 132.4, 132.9, 140.1 ppm were assigned to

C-10, C-9, and C-2, respectively, based on the long-range coupling with H-1, H-7, and H-4,

respectively. Of the two carbons at 106.4 and 140.1 ppm that showed long-range coupling

with H-py4, because the latter was assigned as C-2, the former was determined to be C-1’.

Given that there is one non-aromatic methine group in this compound, the methine carbon at

62.1 ppm was determined to be C-py5. The carbon with a signal at 151.4 ppm showing long-

range coupling with H-py5 (4.96 ppm) was assigned as C-py3. Because this compound

contains one methoxy group, the methoxy proton (3.73 ppm) and carbon (55.56 ppm) were

determined. The carbon peak at 158.0 ppm showing long-range coupling with the methoxy

proton was assigned to C-6’. The three protons with signals at 6.51, 6.54, and 7.16 ppm were

correlated with each other in the COSY spectrum and were determined to be H-5’, H-3’, and

H-4’, respectively, based on comparison with previously reported chemical shifts.¹⁶The

remaining carbon peak at 158.1 ppm should be C-2’. As with derivative 16, the two protons

at 7.83 and 12.19 ppm were assigned as NH and OH, respectively. The important correlations

obtained from the COSY and HMBC spectra are shown in Fig. 2B. The complete

1

assignments of the H and ¹³C NMR data for derivative 21, 5-(naphthalen-2-yl)-3-(2-

hydroxy-6-methoxyphenyl)pyrazoline, are listed in Tables 2 and 3, respectively. The NMR

data for the other naphthalenylphenylpyrazolines containing the naphthalene-2-yl group were

1

13

assigned, and the complete assignments of the H and C NMR data are listed in Tables 2

and 3, respectively. To confirm the structures of the twenty-four derivatives determined by

NMR, HR/MS data were obtained and compared with the calculated mass data, which are

listed in Table 1. In this study, the NMR data of the eighteen derivatives with the naphthalen-

1-yl group (1-18) and the six derivatives with the naphthalen-2-yl group (19-24) were

assigned. As might be expected, a remarkable difference between the chemical shifts of the

two derivatives was observed for H-py5 and C-py5. The proton chemical shifts of H-py5 for

the derivatives with the naphthalen-1-yl group were more deshielded than those of the

derivatives with the naphthalen-2-yl group. On the contrary, the carbon chemical shifts of the

C-py5 of the derivatives with the naphthalen-1-yl group were more shielded than those of the

derivatives with the naphthalen-2-yl group. Derivatives 1-18 contain the naphthalen-1-yl

group. While R₁of the naphthalen-1-yl group in derivatives 1-6 and 13-18 is the methoxy

group, R₁in derivatives 7-12 is proton. While in the derivatives containing the 2-

methoxynaphthalen-1-yl group (1-6 and 13-18) the difference of the chemical shifts of two

py-4 methylene protons is less than 0.4 ppm, in the derivatives without the 2-methoxy group

in the naphthalen-1-yl group (7-12) the difference of two py-4 methylene protons is more

than 0.8 ppm. Among eighteen derivatives with the naphthalen-1-yl group, derivatives 3, 5, 9,

11, 15 contain the 6’-methoxy group and the others do not have substituents. The chemical

shifts of two py-4 methylene protons of 6’-methoxylated derivatives are deshielded more than

those of derivatives without the 6’-methoxy group. Therefore, the methoxy groups at 6’-

position and 2-position influence the chemical shifts of py-4 methylene protons in derivatives

1

1-18 containing the naphthalen-1-yl group. The H and ¹³C NMR data compiled herein

should aid in the identification of naphthalenylphenylpyrazolines. The spectra obtained from

the NMR and HR/MS experiments are provided as Supplementary Material.

Acknowledgments

This work was supported by the Priority Research Centers Program (NRF, 2009-0093824),

Basic Science Research Program (NRF-2016R1D1A1B03931623), and the Agri-Bio Industry

Technology Development Program (316028-3, Korea Institute of Planning and Evaluation for

Technology in Food, Agriculture, Forestry, and Fisheries). SY Shin was supported by the KU

Research Professor Program of Konkuk University.

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Table 1. Structures and names of naphthopyrazolines 1-24, and their mass data and melting points. All HR/MS data were collected in M⁺mode, except for those with an *, which

denotes HR/MS data collected in [M+H]⁺mode

py1

py2

N

R₁

OH

NH

2

2'

py5

R₂

R₃

1

1'

3'

py3

3

py4

6'

9

4

4'

8

7

R₄

R₆

10

5

5'

R₅

6

deriv

ative

mass

(calcd./found)

melting

point/℃

R₁

R₂

R₃

R₄

R₅

R₆

name

5-(2-methoxynaphthalene-1-yl)-3-(2-

hydroxyphenyl)pyrazoline

OCH₃

H

OCH₃

H

1

2

3

4

5

160-161

114

319.1447/319.1446*

348.1474/348.1473

348.1474/348.1471

378.1580/378.1578

5-(2-methoxynaphthalene-1-yl)-3-(2-hydroxy-5-

methoxyphenyl)pyrazoline

5-(2-methoxynaphthalene-1-yl)-3-(2-hydroxy-6-

methoxyphenyl)pyrazoline

H

OCH₃

H

183-186

110-113

180-189

5-(2-methoxynaphthalene-1-yl)-3-(2-hydroxy-

4,5-dimethoxyphenyl)pyrazoline

OCH₃

H

5-(2-methoxynaphthalene-1-yl)-3-(2-hydroxy-

4,6-dimethoxyphenyl)pyrazoline

OCH₃

5-(2-methoxynaphthalene-1-yl)-3-(2-hydroxy-4-

methoxyphenyl)pyrazoline

OCH₃

H

OCH₃

H

OCH₃

H

6

120

378.1580/378.1579

348.1474/348.1474

318.1368/318.1369

348.1474/348.1471

348.1474/348.1472

378.1580/378.1584

378.1580/378.1578

348.1474/ 348.1476

379.1658/ 379.1662*

378.1580/ 378.1581

409.1763/409.1753*

408.1685/ 408.1681

378.1580/ 378.1581

5-(4-methoxynaphthalene-1-yl)-3-(2-

hydroxyphenyl)pyrazoline

7

149

5-(4-methoxynaphthalene-1-yl)-3-(2-hydroxy-5-

methoxyphenyl)pyrazoline

H

OCH₃

H

8

146-147

163-164

184-189

184-192

157

5-(4-methoxynaphthalene-1-yl)-3-(2-hydroxy-6-

methoxyphenyl)pyrazoline

H

OCH₃

H

9

5-(4-methoxynaphthalene-1-yl)-3-(2-hydroxy-

4,5-dimethoxyphenyl)pyrazoline

H

OCH₃

H

OCH₃

H

10

11

12

13

14

15

16

17

18

5-(4-methoxynaphthalene-1-yl)-3-(2-hydroxy-

4,6-dimethoxyphenyl)pyrazoline

H

OCH₃

H

5-(4-methoxynaphthalene-1-yl)-3-(2-hydroxy-4-

methoxyphenyl)pyrazoline

H

5-(2,3-dimethoxynaphthalene-1-yl)-3-(2-

hydroxyphenyl)pyrazoline

OCH₃

H

158-159

100-105

168-172

130-136

150-152

126-127