Synthesis and pharmacological screening of new isatin-3-[N2-(benzimidazol-1-acetyl)]hydrazone

Article abstract of DOI:10.4314/bcse.v33i2.12

Twenty new isatin-3-[N²-(benzimidazol-1-acetyl)]hydrazones (IV) were synthesized from ten different isatin-3-[N²–(chloroacetyl)] hydrazones (III) by reacting with benzimidazole and 2-methyl benzimidazole. The intermediates were obtained from isatin hydrazones (II) on condensation with chloroacetyl chloride. These compounds were characterized by IR, ¹H NMR and mass spectra. All the compounds were screened for antimicrobial, antioxidant and cytotoxic activity. Some of the new compounds showed promising antibacterial and antifungal activity.

Full text of DOI:10.4314/bcse.v33i2.12

Bull. Chem. Soc. Ethiop. 2019, 33(2), 321-329.
 2019 Chemical Society of Ethiopia and The Authors
DOI: https://dx.doi.org/10.4314/bcse.v33i2.12
ISSN 1011-3924
Printed in Ethiopia
SYNTHESIS AND PHARMACOLOGICAL SCREENING OF NEW ISATIN-3-
[N2-(BENZIMIDAZOL-1-ACETYL)]HYDRAZONE
B. Chaithanya1*, I.V. Kasiviswanath1 and D. Prabhakara Chary2
1Department of Chemistry, Koneru Lakshmaiah Education Foundation, Green Fields,
Vaddeswaram, Guntur-522502, India
2Department of Physical Sciences, Kakatiya Institute of Technology & Science - Warangal,
India
(Received May 26, 2018; Revised March 18, 2019; Accepted March 23, 2019)
ABSTRACT. Twenty new isatin-3-[N2-(benzimidazol-1-acetyl)]hydrazones (IV) were synthesized from ten
different isatin-3-[N2–(chloroacetyl)] hydrazones (III) by reacting with benzimidazole and 2-methyl
benzimidazole. The intermediates were obtained from isatin hydrazones (II) on condensation with chloroacetyl
chloride. These compounds were characterized by IR, 1H NMR and mass spectra. All the compounds were
screened for antimicrobial, antioxidant and cytotoxic activity. Some of the new compounds showed promising
antibacterial and antifungal activity.
KEY WORDS: Antibacterial activity, Antifungal activity, Antioxidant activity, Cytotoxic activity,
Benzimidazole, Isatin
INTRODUCTION
In recent years, several benzimidazole derivatives have been synthesized and reported to possess
varied biological and pharmacological properties. Benzimidazole nucleus is one of the bioactive
heterocyclic compounds that exhibit a range of biological activities. The pharmacological
activities of the benzimidazole containing moiety have been well documented. It is reported that
benzimidazole derivatives were active against Staphylococcus aureus, Escherichia coli, and
Candida albicans [1]. Anthelmintic activities of few synthesized benzimidazole derivatives
were also reported [2]. Further, in-vitro antiprotozoal activity of synthesized N-azopridinium
salts and several betaines were reported adding to the existing pool of biological effects. Few
other observations can be made from the available literature, that the benzimidazole molecule
posses different biological activities such as antihistaminic activity [4], antitumor activity [5],
cytotoxic activity [6], antitubercular activity [7], antifungal [8], antiviral [9] and anti-
inflammatory [10] activities.
An interesting observation one could make from a careful survey of the literature is the
absence of any report on isatin derivatives containing benzimidazole system in the side chain at
3rd position. So, it has been felt worthwhile to undertake the present work of synthesizing such
compounds for the first time by appropriate synthetic routes, with a view to evaluate for
antibacterial, antioxidant and anticancer activities. The required isatins (I) and their hydrazones
(II) were prepared by the standard methods available in literature. Isatin hydrazones on
treatment with chloroacetylchloride afforded their respective isatin-3-[N2-(chloro acetyl)]
hydrazones (III). Each of these compounds were reacted with benzimidazole and 2-methyl
benzimidazole in presence of dry acetone with an anhydrous potassium carbonate to get the
respective isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1-acetyl)]hydrazones (IV). The
intermediates and the title compounds were purified by recrystallization and characterized by
analytical and spectral (IR, NMR and mass) data.
__________
*Corresponding author. E-mail: chaitanya.boga28@gmail.com
This work is licensed under the Creative Commons Attribution 4.0 International License

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B. Chaithanya et al.
EXPERIMENTAL
Materials and instruments
All the chemicals were of synthetic grade and commercially procured from Sigma Aldrich,
Mumbai, India. The melting points were determined by open capillary method and were
uncorrected. Infrared spectra were recorded on FTIR (Bruker Alpha-E) by KBr disc method. 1H
NMR spectra were recorded at 400MHz in CDCl3 as solvent and TMS as an internal standard
using Bruker Advance 400 instrument. In addition, mass spectra were recorded on PEP-SCIUX-
APIQ pulsar mass spectrophotometer. Elemental analysis was performed on Perkin-Elmer
EAL240 elemental analyzer.
Synthesis of compounds
Synthesis of isatin hydrazones (II). An appropriate isatin (indole-2,3-dione) (I, 0.01 mol) was
dissolved in alcohol (20 mL) and added hydrazine hydrate (99%, 0.015 mol) while shaking. The
reaction mixture was stirred well, warmed on a water-bath for 10 min and left in the refrigerator
for 3 hours. The resultant yellow crystalline solid was filtered, washed repeatedly with small
portions of cold water and finally with a small quantity of cold alcohol. The product was dried
and purified by recrystallization from chloroform. The compounds thus obtained were
characterized by comparison with their physical constants reported in the literature.
Synthesis of isatin-3-[N2-(chloroacetyl)] hydrazones (III). An appropriate isatin hydrazone (II,
0.01 mol) was heated under reflux with chloroacetyl chloride (0.01 mol) in dry benzene under
anhydrous conditions using calcium chloride guard-tube for 2 hours. The product thus formed
was filtered and washed with small portions of benzene to remove the un-reacted chloroacetyl
chloride. It was purified by recrystallization from suitable solvent(s).
Synthesis of benzimidazole. O-phenylenediamine of weight 27 g (0.25 mol) is placed in a 250
mL round bottomed flask and added 17.5 g (16 mL, 0.34 mol) of formic acid (90%). The
reaction mixture was refluxed on a water bath for 2 hours, cooled, then added 10 percent sodium
hydroxide solution slowly, with constant rotation of the flask, until the mixture was just alkaline
to litmus. Later, filter off the crude benzimidazole at the pump, washed with ice-cold water
thoroughly. It was completely dried and purified by recrystallization from boiling water. The
yield of pure benzimidazole, m.p. 171 oC (lit. 172 oC), is 25 g (85%). Adopting this procedure,
2-methylbenzimidazole was also prepared by the reaction of O-phenylenediamine and acetic
acid. It was recrystallized from 10% aqueous ethanol, the yield is 2.2 g (56%), m.p. 176 oC (lit.
176 oC).
Synthesis of isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1-acetyl)]hydrazone (IV).
A
mixture of an appropriate isatin-3-[N2-(chloroacetyl)]hydrazone (III, 0.01 mol) and a
benzimidazole (0.01 mol) or 2-methyl benzimidazole (0.01 mol) in dry acetone (20 mL) and
anhydrous potassium carbonate, heated under reflux on a water bath for 5 h. The solvent was
evaporated and poured in crushed ice. The product thus formed was filtered, washed with cold
water and dried. The compound was purified by recrystallization from suitable solvent(s).
Biological evaluation
All the compounds were screened for antibacterial, antifungal, antioxidant and cytotoxic activity
by following the standard protocols as available in the literature.
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Pharmacological screening of new isatin-3-[N2-(benzimidazol -1-acetyl)] hydrazone
323
Antibacterial activity. The antibacterial activity of synthesized compounds was conducted
against two gram positive bacteria viz., Bacillus subtilis and Staphylococcus aureus and two
gram negative bacteria viz., Escherichia coli and Proteus vulgaris by using cup plate method.
Ampicillin sodium was employed as standard to compare the results. Both the test and standard
compounds were studied at 10 µg/mL/cup.
Antifungal activity. All those compounds screened for antibacterial activity were also tested for
their antifungal activity by using potato-dextrose-agar medium against clotrimazole as standard.
Both the test and standard compounds were studied at the same dose of 10 µg/mL. The fungi
employed for screening were: Aspergillus niger and Cunninghamella verticulata.
Antioxidant activity. The model of scavenging the stable DPPH (1,1-diphenyl-2-picryl-hydrazil)
radical is a widely used method to evaluate antioxidant activities in a relatively shorter time
compared with other methods. The effect of antioxidants on DPPH radical scavenging was
thought to be owing to their hydrogen donating ability. The reduction in absorbance is
calculated as percentage inhibition as follows:
Absorbance of Blank - Absorbance of Test
% Inhibition 
100
Absorbance of Blank
Cytotoxic activity. Cytotoxic activity was performed on against HBL-100 cell lines and HeLa
cell lines using Microculture tetrazolium assay (MTT) method. It is based on the metabolic
reduction of 3-(4,5-dimethylthiazol-2,5-diphenyl) tetrazolium bromide (MTT) to water insoluble
formazan crystals with mitochondrial dehydrogenase enzyme, which gives direct correlation of
viable cells.
The reaction sequence used in the synthesis of isatin-3-[N2-(benzimidazol/2-methyl
benzimidazol-1-acetyl)]hydrazone (IV) is depicted in the Scheme 1. The required isatin-3-[N2-
(chloroacetyl)] hydrazones (III) have been prepared by a reaction of respective isatin
hydrazones with chloroacetyl chloride. The isatin hydrazones (II) on the other hand, have been
obtained by the reaction of respective isatins (I) with hydrazine hydrate (99%). These
compounds (XVI) have been purified by recrystallization from suitable solvent(s) and
characterized by their analytical and spectral data.
The IR spectrum of the isatin-3-[N2-(chloroacetyl)]hydrazone (III, R1 = H) exhibited the
absorption frequencies (in cm-1) at: 3238 (NH), 1700 (C=O, lactam), 1666 (C=O, acid
hydrazide), 1624 (C=C, aromatic), 1533 (C=N), 951 (C=C, aromatic). Each of the isatin-3-[N2-
(chloroacetyl)]hydrazones (XVI) has been subjected to a nucleophilic substitution reaction with
benzimidazole and 2-methyl benzimidazole in dry acetone and anhydrous potassium carbonate
to get their respective isatin-3[N2-(benzimidazol/2-methylbenzimidazol-1acetyl)]hydrazone
(IV); similarly twenty compounds were prepared. The resultant products have been purified by
recrystallization from suitable solvents and characterized by their physical data (Table 1) and
spectral (IR, NMR and mass) data as follows:
Iva. Yield 85%, mp 262 ᵒC; IR(KBr) cm-1: 3422(-NH str), 1696 (C=Ostr, lactam), 1625 (C=O,
1
acid hydrazine), 1502 (C=Nstr); HNMR (δppm): 12.71 (s, 1H, lactam), 11.35 (s, 1H, NHCO),
8.33 (s, 1H, -N-CH-N), 6.97-8.27 (m, 8H, Ar-H), 5.82 (s, 2H, COCH2); EI-MS: 319(M+).
IVb. Yield 80%, mp 241 ᵒC; IR (KBr) cm-1: 3422 (-NH str), 1696 (C=Ostr, lactam), 1625 (C=O,
1
acid hydrazine), 1502 (C=Nstr); HNMR (δppm): 12.69 (s, 1H, lactam), 11.21 (s, 1H, NHCO),
8.22 (s, 1H, -N-CH-N), 6.80-8.17 (m, 8H, Ar-H), 5.71 (s, 2H, COCH2); EI-MS: 319 (M+).
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O
R 1
O
N
H
I
1
NN H 2
R1
O
N
H
II
2
NNH COC H 2C l
R1
O
N
H
II I
H
N
R 2
3
N
N
R 2
N
NNH C OC H2
R1
O
5. Results and Discussion
N
H
Scheme 1. Synthesis of isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1-cetyl)]hydrazone
(IV), IVa-t R1 = H, 5-CH3, 5-Cl, 5-Br, 5-NO2, 5-SO2NO2, 5-COOH, 5-COOCH3, 6-
Br, 7-CH3; R2 = H, CH3. Reagents and conditions: 1) isatin (I, 0.01 mol), ethanol,
hydrazine hydrate (99%, 0.015 mol), Δ10 min, 2) chloroacetyl chloride (0.01 mol),
dry benzene, reflux 2h and 3) benzimdazole or 2-methyl benzimidazole (0.01mol),
dry acetone, anhydrous potassium carbonate reflux 5 h.
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Pharmacological screening of new isatin-3-[N2-(benzimidazol -1-acetyl)] hydrazone
325
IVc. Yield 78%, mp 212 ᵒC; IR (KBr) cm-1: 3410 (-NH str), 1690 (C=Ostr, lactam), 1610 (C=O,
1
acid hydrazine), 1480 (C=Nstr); HNMR (δppm): 12.69 (s, 1H, lactam), 11.21 (s, 1H, NHCO),
8.22 (s, 1H, -N-CH-N), 6.80-8.17 (m, 8H, Ar-H), 5.71 (s, 2H, COCH2); EI-MS: 353 (M+).
IVd. Yield 64%, mp 279 ᵒC; IR (KBr) cm-1: 3435 (-NH str), 1710 (C=Ostr, lactam), 1640 (C=O,
1
acid hydrazine), 1515 (C=Nstr); HNMR (δppm): 12.85 (s, 1H, lactam), 11.40 (s, 1H, NHCO),
8.50 (s, 1H, -N-CH-N), 6.98-8.30 (m, 8H, Ar-H), 5.90 (s, 2H, COCH2); EI-MS: 398 (M+).
IVe. Yield 88%, mp 219 ᵒC; IR (KBr) cm-1: 3405 (-NH str), 1680 (C=Ostr, lactam), 1605 (C=O,
1
acid hydrazine), 1475 (C=Nstr); HNMR (δppm): 12.65 (s, 1H, lactam), 11.15 (s, 1H, NHCO),
8.10 (s, 1H, -N-CH-N), 6.75-8.05 (m, 8H, Ar-H), 5.62 (s, 2H, COCH2); EI-MS: 364 (M+).
Biological activity
Antibacterial activity. The antibacterial activity data of isatin-3-[N2-(benzimidazol/2-methyl
benzimidazol-1-acetyl)] hydrazones (IV, Table 2) indicates that the compounds have a
noticeable degree of inhibition, specifically against gram-positive strain, i.e. B. subtilis. Most
significant of them has been found to be the compound IVa showed greater inhibitory effect
against the organisms employed, particularly against B. subtilis and S. aureus with the zones of
inhibition of 22 and 20 mm, respectively, which has been comparable to that of the standard
employed at the concentration of 10 g/mL. This has been closely followed by compound IVd
with a 5-bromo substituent in indolinone which showed significant inhibitory effect specifically
against B. subtilis and E. coli with zones of inhibition of 18 mm, each. Some of the compounds
showed moderate antibacterial activity against both the gram-negative organisms E. coli and P.
vulgaris. The compounds with 5-carboxylic acid and 5-carbomethoxy substituents on indolinone
have not exhibited any activity against gram-negative organisms. Recently, Guo et al. [11] has
excellently reviewed antibacterial potential of isatin derivatives. Intriguingly, azo linked
substituted benzimidazole, benzoxazole, and benzothiazole synthesized by Mishra et al. [12]
through diazo coupling also exhibited in vitro antibacterial activity against Staphylococcus
aureus and Escherichia Coli strains. Mannich bases of benzimidazole derivatives have also
shown in vitro antibacterial activity against Bacillus subtilis, Bacillus pumilus, Escherichia coli
and Pseudomonas aeruginosa organisms [13].
Antifungal activity. The antifungal activity data of isatin-3-[N2-(benzimidazol/2-
methylbenzimidazol-1-acetyl)]hydrazones (IV) as depicted in Table 2 shows that the
compounds of this series have shown antifungal action against A. niger and C. verticulata
except compounds IVh, IVq and IVr, of course, with a degree of variation in their action.
Compounds IVc (R1 = 5-Cl, R2 = H) and IVm (R1 = 5-Cl, R2 = CH3) have more activity against
C. verticulata with the zones of inhibition of 17 mm and 16 mm respectively among all the test
compounds. The data shows that this series of compounds have been found to be comparatively
more effective among all the series tested for antifungal activity. In an interesting work reported
by Jarrahpour et al. [14], twelve new bis-Schiff bases of isatin, benzylisatin and 5-fluoroisatin
3a-3l were prepared by condensation of isatin, benzylisatin and 5-fluoroisatin with primary
aromatic amines. Though these compounds exhibited antiviral and anticancer activity, the
candidates failed to show antifungal activity when tested against S. cerevisiae (ATCC 28383) or
C. albicans (CIP 1180-79). However, Kaplancıklı et al. [15] reported few benzimidazole-
thiazole derivatives with proven anticandidal activity. Several bisbenzimidazole compounds
exhibited moderate to excellent antifungal activities against fungal strains, with MIC values
ranging from 15.6 to 0.975 μg/mL and the antifungal activity were found to depend on alkyl
chain length [16].
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Antioxidant activity. The IC50 values of antioxidant activity of isatin-3-[N2-(benzimidazol/2-
methylbenzimidazol-1acetyl)]hydrazone derivatives (IV) are in the range of 10.45 to 17.56 M
as shown in Table 3. The compound IVa (R1 = R2 = H) has shown highest percentage of free
radical scavenging activity at a concentration of 10.45 M among these compounds. This has
been followed by the compounds IVc, IVd and IVn with an IC50 of 10.52, 10.61 and 10.98 M,
respectively. In an attempt to synthesize gastro-sparing antiinflammatory agents, Sharma et al.
[17] has reported antioxidant activity of 5-methanesulphonamido benzimidazole derivatives.
Other benzimidazole derivatives has also shown remarkable in vitro antioxidant activity [18-20].
Cytotoxic activity. IC50 values of cytotoxic activity of isatin-3-[N2-(benzemidazol/2-
methylbenzimidazol-1-acetyl)]hydrazone (IV) are given the Table 3. They are ranging from
345.59 to 473.9 M. The compound IVm (R1 = 5-Br, R2 = CH3) has shown highest cytotoxic
activity among the series at a concentration of 345.59 M against HeLa cell lines. This has been
followed by the compound IVd (R1 = 5-Br, R2= H) with IC50 of 347.69 M against HBL-100
cell lines. The compounds IVe, IVg, IVh, IVp, IVq, IVr and IVt have not shown any activity
against HBL-100 cell lines and HeLa cell lines. Recent literature also revealed potential
cytotoxic activity of several benzimidazole derivatives [21, 22]
Table 1.Physical data of isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1-acetyl)]hydrazone (IV).
S. No.
1
2
3
4
5
6
7
8
Compd.
IV a
IV b
IV c
IV d
IV e
IV f
IV g
IV h
IV i
IV j
IV k
IV l
IV m
IV n
IV o
IV p
IV q
R1
H
5-CH3
5-Cl
R2
H
H
H
H
H
H
H
H
Mol. Formula
C17H13N5O2
C18H15N5O2
C17H12N5O2Cl
C17H12N5O2Br
C17H12N6O4
C17H14N6O4S
C18H13N5O4
C19H15N5O4
C17H12N5O2Br
C18H15N5O2
C18H15N5O2
C19H17N5O2
C18H14N5O2Cl
C18H14N5O2Br
C18H14N6O4
C18H16N0O4S
C19H15N5O4
m.p. (◦C)
262
241
212
279
210
272
222
229
248
238
259
245
240
265
205
276
210
Yield (%)
85
80
78
64
88
70
60
65
84
82
80
82
75
65
75
72
65
5-Br
5-NO2
5-SO2NH2
5-COOH
5-COOCH3
6-Br
7-CH3
H
5-CH3
5-Cl
9
H
H
10
11
12
13
14
15
16
17
CH3
CH3
CH3
CH3
CH3
CH3
CH3
5-Br
5-NO2
5-SO2NH2
5-COOH
18
IV r
5-COOCH3
CH3
C20H17N5O4
232
68
19
20
IV s
IV t
6-Br
7-CH3
CH3
CH3
C18H14N5O2Br
C19H17N5O2
228
252
75
78
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Pharmacological screening of new isatin-3-[N2-(benzimidazol -1-acetyl)] hydrazone
327
Table 2. Antibacterial activity and antifungal of isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1-
acetyl)]hydrazone (IV).
Zone of inhibition (in mm)
S. No. Compd.
R1
R2
Bacterial species
Fungal species
B. subtilis S. aureus E. coli P. vulgaris A. niger C. verticulata
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
IV a
IV b
IV c
IV d
IV e
IV f
IV g
H
5-CH3
5-Cl
H
H
H
H
H
H
H
H
H
22
15
16
18
13
13
11
13
17
16
18
16
16
18
13
15
12
14
17
18
22
--
20
13
15
17
11
12
--
18
12
14
18
--
11
--
--
15
--
16
--
14
17
14
--
17
11
12
16
--
--
--
--
13
--
15
--
13
15
13
--
--
--
14
15
17
--
15
12
13
10
8
10
8
--
14
13
17
14
10
12
6
--
12
8
12
12
16
13
7
5-Br
5-NO2
5-SO2NH2
5-COOH
IV h 5-COOCH3
--
IV i
IV j
IV k
IV l
IV m
IV n
IV o
IV p
IV q
6-Br
7-CH3
H
5-CH3
5-Cl
16
15
17
15
15
16
12
16
--
12
16
15
20
--
9
H
10
14
11
14
12
10
11
--
CH3
CH3
CH3
CH3
CH3
CH3
CH3
5-Br
5-NO2
5-SO2NH2
5-COOH
7
--
--
11
7
--
--
16
16
18
--
IV r 5-COOCH3 CH3
--
IV s
IV t
6-Br
7-CH3
Ampicillin (10 μg/cup)
Clotrimazole (10 μg/cup)
CH3
CH3
10
11
--
--
20
19
Table 3. Antioxidant activity and cytotoxic activity of isatin-3-[N2-(benzimidazol/2-methylbenzimidazol-1-
acetyl)]hydrazone (IV).
Antioxidant activity
Cytotoxic activity
S. No. Compd.
R1
R2
HBL-100 cell lines
HeLa cell lines
IC50 values (µM)
411.84
NA
IC50 value (µM)
10.45
15.07
10.52
10.61
13.39
15.6
IC50 values (µM)
433.13
441.86
365.28
347.69
NA
1
2
3
4
5
IV a
IV b
IV c
IV d
IV e
IV f
IV g
H
H
H
H
H
H
H
H
H
H
5-CH3
5-Cl
5-Br
5-NO2
5-SO2NH2
5-COOH
349.84
325.46
NA
NA
NA
NA
373.99
NA
463.36
NA
345.59
355.17
NA
6
7
NA
NA
NA
16.61
17.56
11.43
14.91
11.63
14.12
11.39
10.98
14.37
15.13
16.92
17.43
13.67
15.05
8
IV h 5-COOCH3
9
IV i
IV j
IV k
IV l
6-Br
7-CH3
H
5-CH3
5-Cl
367.15
473.9
454.48
NA
375.59
357.79
401.58
NA
NA
NA
391.56
NA
10
11
12
13
14
15
16
17
18
19
20
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
IV m
IV n
IV o
IV p 5-SO2NH2
IV q
5-COOH
IV r 5-COOCH3
5-Br
5-NO2
NA
NA
NA
365.49
NA
IV s
IV t
6-Br
7-CH3
21
22
Ascorbic acid
Cisplatin
5.87
--
--
25.00
--
25.00
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CONCLUSION
The synthesized novel isatin-3-[N2-(benzimidazol-1-acetyl)] hydrazones were found to possess
remarkable biological activities. The compounds showed antibacterial activity against B.
subtilis, S. aureus, E. Coli and P. vulgaris and antifungal activity against A. Niger and C.
verticulata. The results were promising for antioxidant activity in free radical scavenging assay.
The compounds possessed cytotoxic activity also against HeLa cell lines.
ACKNOWLEDGEMENTS
The authors are thankful to K L E F, Guntur, Andhra Pradesh, India and Talla Padmavathi
College of Engineering and Pharmacy, Somidi, Kazipet and Urus, Kareemabad, Warangal,
Telangana State for providing research facilities to carry out research work. The authors are also
grateful to Torrent Pharmaceutical, Ahmedabad for providing NMR and mass spectra.
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