Hydrazide-hydrazones of 3-methoxybenzoic acid and 4-tert-butylbenzoic acid with promising antibacterial activity against Bacillus spp

Article abstract of DOI:10.3109/14756366.2016.1170012

A series of 28 hydrazide-hydrazones of 3-methoxybenzoic and 4-tert-butylbenzoic acid were synthesized and screened in vitro against the panel of reference strains of bacteria and fungi with the use of the broth microdilution method according to EUCAST and CLSI guidelines. Five of the synthesized compounds were found to exhibit high bacteriostatic or bactericidal activity against Gram-positive bacteria. The antimicrobial activity of compounds 13, 14, and 16 against Bacillus spp. was higher than that of commonly used antibiotics, like cefuroxime or ampicillin.

Full text of DOI:10.3109/14756366.2016.1170012

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Hydrazide-hydrazones of 3-methoxybenzoic
acid and 4-tert-butylbenzoic acid with promising
antibacterial activity against Bacillus spp
Łukasz Popiołek & Anna Biernasiuk
To cite this article: Łukasz Popiołek & Anna Biernasiuk (2016): Hydrazide-hydrazones
of 3-methoxybenzoic acid and 4-tert-butylbenzoic acid with promising antibacterial
activity against Bacillus spp, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.3109/14756366.2016.1170012
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Published online: 13 Apr 2016.
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J Enzyme Inhib Med Chem, Early Online: 1–8
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1170012
!
RESEARCH ARTICLE
Hydrazide-hydrazones of 3-methoxybenzoic acid and 4-tert-butylben-
zoic acid with promising antibacterial activity against Bacillus spp
Łukasz Popiołek1 and Anna Biernasiuk2
2
1Department of Organic Chemistry and Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Lublin, Lublin,
Poland
Abstract
Keywords
A series of 28 hydrazide-hydrazones of 3-methoxybenzoic and 4-tert-butylbenzoic acid
were synthesized and screened in vitro against the panel of reference strains of bacteria and
fungi with the use of the broth microdilution method according to EUCAST and CLSI guidelines.
Five of the synthesized compounds were found to exhibit high bacteriostatic or bactericidal
activity against Gram-positive bacteria. The antimicrobial activity of compounds 13, 14, and 16
against Bacillus spp. was higher than that of commonly used antibiotics, like cefuroxime or
ampicillin.
Antimicrobial activity, Bacillus spp., hydrazide-
hydrazones, MBC, MIC
History
Received 22 January 2016
Revised 2 March 2016
Accepted 4 March 2016
Published online 12 April 2016
Introduction
hydrazide-hydrazones which exhibit significant antibacterial activity
towards Gram-positive bacteria, especially against Bacillus spp.
One of the main reasons why the infectious microbial diseases
still remain pressing problems worldwide is the widespread
excessive use of antibacterial agents, which leads to the devel-
opment of bacteria more resistant to commonly used antibiotics1. Chemistry
Materials and methods
Consequently, there is still need to explore safe and potent
All required chemicals and solvents were purchased from Sigma-
chemotherapeutic agents. It is desirable to discover drugs with
improved potency, wide activity spectrum and low toxicity2.
In many reports, besides being useful building blocks for the
synthesis of a variety of heterocyclic rings and in drug develop-
ment3,4, hydrazide-hydrazones are considered to be good scaf-
folds for different pharmaceutical applications5–11 and have a
significant role to play as antimicrobial agents12–16. The bio-
logical activity associated with these compounds is attributed to
the presence of the highly reactive azomethine group (–CO–NH–
N¼CH–). Moreover, some widely used chemotherapeutic drugs
such as nitrofurazone and furazolidone are known to contain this
Aldrich (Munich, Germany) and Merck Co. (Darmstadt,
Germany) and used without further purification. Melting points
were determined with Fisher-Johns blocks (Fisher Scientific,
1
Schwerte, Germany) and are uncorrected. The H NMR and 13C
NMR spectra were recorded on the Bruker Avance 300 apparatus
(Bruker BioSpin GmbH, Munich, Germany) in DMSO-d6 with
TMS as the internal standard. The chemical shifts are expressed
on the ꢀ (ppm) scale with the use of TMS as the standard
reference. The coupling constants (J) are given in Hertz. Spin
multiples are given as s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), q (quartet), m (multiplet), and b (broad). The
progress of the reaction and purity of obtained compounds were
monitored by TLC using precoated aluminum sheet 60 F254
plates (Merck Co., Kenilworth, NJ), in a CHCl3/C2H5OH (10:1,
v/v) solvent system. The spots were detected by exposure to the
UV lamp at 254 nm. The elemental analysis of obtained
compounds was carried out with the AMZ 851 CHX analyzer
group (Figure 1)17–19
.
Additionally, as we have reported earlier, we synthesized some
hydrazide–hydrazone derivatives which were shown to exhibit
antimicrobial activity20–22. Some of these compounds had inter-
esting in vitro activity against Gram-positive bacteria. Especially
N-[2–(3-bromo-4-methoxyphenyl)ethenyl]furan-2-carboxamide
and N-[2–(3-bromo-4-methoxyphenyl)ethenyl]thiophene-2-car-
boxamide which showed the significant inhibitory activity
towards Bacillus cereus ATCC 1087620.
´
(PG, Gdansk, Poland). The results of elemental analysis (C, H,
and N) were within 0.4% of the calculated values. C log P
values of synthesized compounds were calculated using
HypeChemTM 8.0.823.
Considering the above-mentioned facts and in continuation of our
research of new chemotherapeutic agents, in this paper, we would
like to present synthesis and in vitro antimicrobial evaluation of new
Preparation of 3-methoxybenzoic acid hydrazide (1)
The compound was prepared according to a procedure from the
literature24.
CAS registry number: 5785-06-8. Analytical and spectral data
is consistent with those reported in the literature24. Yield: 85%;
Address for correspondence: Łukasz Popiołek, Department of Organic
Chemistry, Faculty of Pharmacy, Medical University of Lublin,
´
4A
lukasz.popiolek@umlub.pl
Chodzki
Street,
Lublin
20-093,
Poland.
E-mail:

2
Ł. Popiołek & A. Biernasiuk
J Enzyme Inhib Med Chem, Early Online: 1–8
(b)
(a)
Figure 1. Chemotherapeutic drugs containing
hydrazide–hydrazone moiety: nitrofurazone
(a) and furazolidone (b).
O
O2N
O2N
O
N
N
O
O
NH
N
NH2
O
m.p.: 93–95 ꢀC. 1H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.80 (s, 3H, 56.8 (CH3), 100.3, 107.1, 113.5, 117.5, 118.9, 123.6, 128.4,
CH3), 4.26 (s, 2H, NH2), 7.10–7.12 (m, 1H, ArH), 7.41–7.43 (m, 129.7, 133.5 (9Car), 150.7 (¼CH), 160.2, 160.9, 162.8 (3Car),
1H, ArH), 7.49–7.53 (m, 2H, ArH), 8.81 (s, 1H, NH); 13C NMR 163.7 (C¼O). Analysis for C17H18N2O4 (314.33) Calculated: C:
(DMSO) ꢀ (ppm) ¼ 56.1 (CH3), 113.3, 118.7, 122.1, 128.7, 134.2, 64.96%, H: 5.77%, N: 8.91%; Found: C: 64.91%, H: 5.78%, N:
160.5 (6Car), 166.9 (C¼O). Analysis for C8H10N2O2 (166.18) 8.79%.
Calculated: C: 57.82%, H: 6.07%, N: 16.86%; Found: C: 57.93%,
H: 6.03%, N: 16.89%.
N-[(3,4-Dimethoxyphenyl)methylidene]-3-
methoxybenzhydrazide (5)
Preparation of 4-tert-butylbenzoic acid hydrazide (2)
CAS registry number: 1396397-12-8. Yield: 66%; m.p.: 138–
1
The compound was prepared according to a procedure from the 140 ꢀC. C log P ¼ 2.67. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.81 (s,
literature25.
3H, CH3), 3.83 (s, 3H, CH3), 3.84 (s, 3H, CH3), 7.02–7.05 (d, 1H,
CAS Registry Number: 43100-38-5. Analytical and spectral ArH, J ¼ 9 Hz), 7.15–7.20 (m, 2H, ArH), 7.34 (s, 1H, ArH), 7.42–
data is consistent with those reported in the literature25. Yield: 7.50 (m, 3H, ArH), 8.39 (s, 1H, ¼CH), 11.71 (s, 1H, NH); 13C
1
78%; m.p.: 121–123 ꢀC. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.36 (s, NMR (DMSO) ꢀ (ppm) ¼ 56.1 (CH3), 56.7 (CH3), 56.8 (CH3),
9H, 3 ꢁ CH3), 4.16 (s, 2H, NH2), 7.45–7.47 (d, 2H, ArH, 110.1, 113.5, 114.1, 118.9, 121.1, 122.6, 128.4, 129.4, 133.5
J ¼ 6 Hz), 7.80–7.82 (d, 2H, ArH, J ¼ 6 Hz), 8.28 (s, 1H, NH); 13C (9Car), 148.7 (¼CH), 149.8, 150.5, 160.2 (3Car), 163.7 (C¼O).
NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3), 34.3 (Ct-butyl), 126.1, Analysis for C17H18N2O4 (314.33) Calculated: C: 64.96%, H:
131.4, 131.7, 153.6 (6Car), 167.4 (C¼O). Analysis for C11H16N2O 5.77%, N: 8.91%; Found: C: 64.92%, H: 5.74%, N: 8.94%.
(192.26) Calculated: C: 68.72%, H: 8.39%, N: 14.57%; Found: C:
68.77%, H: 8.43%, N: 14.63%.
N-[(3-Ethoxy-4-hydroxyphenyl)methylidene]-3-
methoxybenzhydrazide (6)
Preparation of hydrazide-hydrazones of 3-methoxyben-
zoic and 4-tert-butylbenzoic acids (3–30)
CAS registry number: 362711-54-4. Yield: 52%; m.p.: 172–
1
174 ꢀC. C log P ¼ 2.98. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.35–
General procedure
1.39 (t, 3H, CH3, J ¼ 6 Hz), 3.83 (s, 3H, CH3), 4.04–4.11 (q, 2H,
CH2, J ¼ 6 Hz), 6.84–6.87(d, 2H, ArH, J ¼ 9 Hz), 7.07–7.17 (m,
2H, ArH), 7.29–7.30 (m, 1H, ArH), 7.41–7.49 (m, 3H, ArH), 8.33
(s, 1H, ¼CH), 9.50 (s, 1H, OH), 11.63 (s, 1H, NH); 13C NMR
(DMSO) ꢀ (ppm) ¼ 15.2 (CH3), 55.8 (CH3), 64.3 (CH3), 111.2,
113.2, 115.9, 118.5, 120.2, 123.2, 126.1, 130.1, 135.5, 147.7,
148.8 (11Car), 149.7 (¼CH), 159.6 (Car), 163.1 (C¼O). Analysis
for C17H18N2O4 (314.33) Calculated: C: 64.96%, H: 5.77%, N:
8.91%; Found: C: 64.90%, H: 5.79%, N: 8.87%.
To the solution of 0.01 mole of adequate carboxylic acid
hydrazide: 3-methoxybenzhydrazide (1) or 4-tert-butylbenzhy-
drazide (2) in 10 mL of ethanol, an appropriate substituted
aromatic aldehyde (0.011 mole) was added. The mixture was
heated under reflux for 3 h. Then it was allowed to cool and the
formed precipitate was filtered off and recrystallized form
methanol (3–16) or ethanol (17–30).
N-[(2,3-dimethoxyphenyl)methylidene]-3-
methoxybenzhydrazide (3)
3-Methoxy-N-[(4-methoxyphenyl)methylidene]benzhydrazide (7)
CAS registry number: 341945-25-3. Yield: 62%; m.p.: 152–
1
154 ꢀC. C log P ¼ 2.92. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.79 (s,
CAS registry number: 351365-58-7. Yield: 79%; m.p.: 169–
1
171 ꢀC. C log P¼ 2.67. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.80 (s,
3H, CH3), 3.80 (s, 3H, CH3), 6.92–6.94 (d, 2H, ArH, J ¼ 6 Hz),
7.11–7.13 (m, 1H, ArH), 7.41–7.44 (t, 1H, ArH, J ¼ 3 Hz), 7.49
(s, 1H, ArH), 7.52–7.54 (d, 2H, ArH, J ¼ 6 Hz), 7.57–7.59 (m, 1H,
ArH), 8.39 (s, 1H, ¼CH), 11.06 (s, 1H, NH); 13C NMR (DMSO) ꢀ
(ppm) ¼ 56.0 (CH 3), 56.1 (CH3), 113.5, 114.3, 118.9, 122.6,
127.1, 128.4, 129.1 133.5 (10Car), 149.3 (¼CH), 160.1, 160.2
(2Car), 163.7 (C¼O). Analysis for C16H16N2O3 (284.31)
Calculated: C: 67.59%, H: 5.67%, N: 9.85%; Found: C: 67.64%,
H: 5.65%, N: 9.83%.
3H, CH3), 3.84 (s, 6H, 2 ꢁ CH3), 7.11–7.18 (m, 3H, ArH), 7.42–
7.53 (m, 4H, ArH), 8.75 (s, 1H, ¼CH), 11.86 (s, 1H, NH); 13C
NMR (DMSO) ꢀ (ppm) ¼ 55.8 (CH3), 56.2 (CH3), 61.7 (CH3),
111.3, 114.7, 118.0, 120.3, 124.9, 128.2, 130.1, 135.2, 143.8
(10Car), 148.4 (¼CH), 153.1, 159.6 (2Car), 169.2 (C¼O).
Analysis for C17H18N2O4 (314.33) Calculated: C: 64.96%, H:
5.77%, N: 8.91%; Found: C: 64.90%, H: 5.80%, N: 8.95%.
N-[(2,4-Dimethoxyphenyl)methylidene]-3-
methoxybenzhydrazide (4)
N-[(3-Ethoxy-4-methoxyphenyl)methylidene]-3-methoxybenzhy-
drazide (8)
CAS Registry Number: 329720-10-7. Yield: 54%; m.p.: 137–
1
139 ꢀC. C log P ¼ 2.67. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.83 (s,
CAS registry number: 396655-61-1. Yield: 53%; m.p.: 157–
1
159 ꢀC. C log P ¼ 3.01. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.34–
6H, 2 ꢁ CH3), 3.87 (s, 3H, CH3), 6.63–6.66 (m, 2H, ArH), 7.13–
7.16 (m, 1H, ArH), 7.40–7.52 (m, 3H, ArH), 7.80–7.82 (m, 1H,
ArH), 8.72 (s, 1H ¼ CH), 11.68 (s, 1H, NH); 13C NMR (DMSO) ꢀ
(ppm) ¼ 56.1 (CH3), 56.5 (CH3), 56.8 (CH3), 100.3, 107.1, 113.5,
117.5, 118.9, 123.6, 128.4, 129.7, 133.5 (9Car), 150.7 (¼CH),
160.2, 160.9, 162.8 (3Car), 163.7 (C¼O) 56.1 (CH3), 56.5 (CH3),
1.39 (t, 3H, CH3, J ¼ 6 Hz, J ¼ 9 Hz), 3.82 (s, 3H, CH3), 3.83 (s,
3H, CH3), 4.03–4.10 (q, 2H, CH2, J ¼ 6 Hz), 7.02–7.05 (d, 1H,
ArH, J ¼ 9 Hz), 7.15–7.22 (m, 2H, ArH), 7.33–7.34 (m, 1H, ArH),
7.41–7.50 (m, 3H, ArH), 8.38 (s, 1H, ¼CH), 11.70 (s, 1H, NH);
13C NMR (DMSO) ꢀ (ppm) ¼ 15.2 (CH3), 55.8 (CH3), 55.9

DOI: 10.3109/14756366.2016.1170012
Hydrazide-hydrazones of 3-methoxybenzoic and 4-tert-butylbenzoic acids
3
(CH3), 64.2 (CH2), 109.7, 112.0, 113.3, 117.8, 120.2, 122.3, J ¼ 3 Hz), 8.38 (s, 1H, ¼CH), 11.84 (s, 1H, NH); 13C NMR
127.4, 130.1, 135.4, 148.6 (10Car), 148.7 (¼CH), 151.3, 159.7 (DMSO) ꢀ (ppm) ¼ 56.0 (CH3), 56.8 (CH3), 113.5, 115.5, 118.9,
(2Car), 163.2 (C¼O). Analysis for C18H20N2O4 (328.36) 122.6, 123.3, 125.5, 127.4, 128.4, 130.7, 133.5 (10Car), 148.7
Calculated: C: 65.84%, H: 6.14%, N: 8.53%; Found: C: 65.88%, (¼CH), 156.2, 160.2 (2Car), 163.3 (C¼O). Analysis for
H: 6.16%, N: 8.47%.
C16H15ClN2O3 (318.75) Calculated: C: 60.29%, H: 4.74%, N:
8.79%; Found: C: 60.35%, H: 4.78%, N: 8.83%.
N-[(4-Hydroxy-3,5-dimethoxyphenyl)methylidene]-3-methoxy-
benzhydrazide (9)
N-[(3-Bromo-4-methoxyphenyl)methylidene]-3-methoxybenzhy-
drazide (14)
CAS registry number: 544661-09-8. Yield: 67%; m.p.: 149–
1
151 ꢀC. C log P ¼ 2.38. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.83 (s,
CAS registry number: 904253-53-8. Yield: 71%; m.p.: 149–
1
151 ꢀC. C log P ¼ 3.71. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.84 (s,
6H, 2 ꢁ CH3), 3.84 (s, 3H, CH3), 6.99 (s, 2H, ArH), 7.14–7.18
(m, 1H, ArH), 7.42–7.49 (m, 3H, ArH), 8.34 (s, 1H, ¼CH), 8.93
(s, 1H, OH), 11.69 (s, 1H, NH); 13C NMR (DMSO) ꢀ
(ppm) ¼ 56.2 (CH3), 56.5 (2 ꢁ CH3), 105.1, 113.3, 120.2,
124.9, 130.0, 135.1, 137.9, 139.4 (10Car), 148.6 (¼CH), 149.1,
155.3 (2Car), 159.6 (C¼O). Analysis for C17H18N2O5 (330.33)
Calculated: C: 61.81%, H: 5.49%, N: 8.48%; Found: C: 61.87%,
H: 5.46%, N: 8.52%.
3H, CH3), 3.91 (s, 3H, CH 3), 7.15–7.23 (m, 2H, ArH), 7.41–7.51
(m, 3H, ArH), 7.70–7.73 (d, 1H, ArH, J ¼ 9 Hz), 7.95–7.97 (t, 1H,
ArH, J ¼ 6 Hz), 8.37 (s, 1H, ¼CH), 11.84 (s, 1H, NH); 13C NMR
(DMSO) ꢀ (ppm) ¼ 55.8 (CH3), 56.9 (CH3), 111.7, 113.3, 118.3,
120.1, 128.8, 130.1, 131.3, 136.1 (10Car), 146.2 (¼CH), 157.7,
159.7 (2Car), 163.9 (C¼O). Analysis for C16H15BrN2O3 (363.20)
Calculated: C: 52.91%, H: 4.16%, N: 7.71%; Found: C: 52.96%,
H: 4.18%, N: 7.67%.
N-[(4-Propoxyphenyl)methylidene]-3-methoxybenzhydrazide (10)
CAS registry number: 351365-16-7. Yield: 68%; m.p.: 96–98 ꢀC. C
N-[(2-Bromo-3-hydroxy-4-methoxyphenyl)methylidene]-3-meth-
oxybenzhydrazide (15)
1
log P ¼ 4.13. H NMR (DMSO-d6) ꢀ (ppm) ¼ 0.96–1.01 (t, 3H,
CAS registry number: 1798917-09-5. Yield: 97%; m.p.: 212–
1
CH3, J ¼ 9 Hz, J ¼ 6 Hz), 1.69–1.81 (m, 2H, CH2), 3.83 (s, 3H,
CH3), 3.96–4.01 (t, 2H, CH2, J ¼ 6 Hz), 7.00–7.03 (d, 2H, ArH,
J ¼ 9 Hz), 7.14–7.17 (m, 1H, ArH), 7.41–7.50 (m, 3H, ArH), 7.65–
7.68 (d, 2H, ArH, J ¼ 9 Hz), 8.40 (s, 1H, ¼CH), 11.69 (s, 1H, NH);
13C NMR (DMSO) ꢀ (ppm) ¼ 10.8 (CH3), 22.5 (CH2), 30.5 (CH2),
55.8 (CH3), 69.5 (CH2), 113.2, 115.2, 117.3, 120.2, 127.2, 129.2,
130.2, 135.4 (10Car), 148.2 (¼CH), 159.6, 160.8 (2Car), 163.1
(C¼O). Analysis for C18H20N2O3 (312.36) Calculated: C: 69.21%,
H: 6.45%, N: 8.97%; Found: C: 69.26%, H: 6.42%, N: 8.94%.
214 ꢀC. C log P ¼ 2.64. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.84 (s,
3H, CH3), 3.89 (s, 3H, CH3), 7.09–7.18 (m, 2H, ArH), 7.42–7.53
(m, 4H, ArH), 8.78 (s, 1H, ¼CH), 9.65 (s, 1H, OH), 11.94 (s, 1H,
NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 56.0 (CH3), 56.8 (CH3),
108.1, 113.5, 115.0, 118.9, 119.9, 122.6, 126.2, 128.4, 133.5
(9Car), 144.8 (¼CH), 145.0, 148.9, 160.2 (3Car), 163.6 (C¼O).
Analysis for C16H15BrN2O4 (379.20) Calculated: C: 50.68%, H:
3.99%, N: 7.39%; Found: C: 50.71%, H: 4.01%, N: 7.43%.
N-[((2-Hydroxy-3,5-diiodophenyl)methylidene]-3-methoxybenz-
hydrazide (16)
3-Methoxy-N-{[4-(trifluoromethyl)phenyl]methylidene}benzhy-
drazide (11)
CAS registry number: 339011-17-5. Yield: 94%; m.p.: 205–
1
CAS registry number: 710291-60-4. Yield: 65%; m.p.: 122–
1
207 ꢀC. C log P ¼ 5.58. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.84 (s,
124 ꢀC. C log P ¼ 4.06. H NMR (DMSO-d6) ꢀ (ppm) ¼ 3.84 (s,
3H, CH3), 7.19–7.22 (m, 1H, ArH), 7.45–7.55 (m, 3H, ArH),
7.88–7.89 (d, 1H, ArH, J ¼ 3 Hz), 8.05–8.06 (d, 1H, ArH,
J ¼ 3 Hz), 8.44 (s, 1H, ¼CH), 12.51 (s, 1H, OH), 12.94 (s, 1H,
NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 55.8 (CH3), 82.6, 88.3, 113.5,
118.5, 120.4, 120.8, 130.3, 133.9, 139.1, 146.9 (10Car), 147.7
(¼CH), 157.1, 159.7 (2Car), 163.2 (C¼O). Analysis for
C15H12I2N2O3 (522.08) Calculated: C: 34.51%, H: 2.32%, N:
5.37%; Found: C: 34.56%, H: 2.30%, N: 5.39%.
3H, CH3), 7.17–7.20 (m, 1H, ArH), 7.33–7.39 (m, 1H, ArH),
7.41–7.53 (m, 2H, ArH), 7.81–7.84 (d, 2H, ArH, J ¼ 9 Hz), 7.94–
7.97 (d, 2H, ArH, J ¼ 9 Hz), 8.54 (s, 1H, ¼CH), 12.03 (s, 1H,
NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 55.8 (CH3), 111.9, 113.4,
118.1, 119.3, 120.4, 126.2, 126.3, 128.1, 130.2, 135.0, 138.8
(11Car), 146.5 (¼CH), 159.7 (Car), 163.5 (C¼O). Analysis for
C16H13F3N2O2 (322.28) Calculated: C: 59.63%, H: 4.07%, N:
8.69%; Found: C: 59.68%, H: 4.11%, N: 8.73%.
4-Tert-butyl-N-[(2,3-dimethoxyphenyl)methylidene]benzhydrazide
(17)
N-[(2-Chloro-3-methoxyphenyl)methylidene]-3-methoxybenzhy-
drazide (12)
Yield: 97%; m.p.: 204–206 ꢀC. C log P ¼ 3.44. 1H NMR (DMSO-
d6) ꢀ (ppm) ¼ 3.84 (s, 3H, CH3), 3.89 (s, 3H, CH3), 7.17–7.23 (m,
2H, ArH), 7.37–7.54 (m, 4H, ArH), 7.61–7.63 (d, 2H, ArH,
J ¼ 9 Hz), 8.90 (s, 1H, ¼CH), 12.06 (s, 1H, NH); 13C NMR
(DMSO) ꢀ (ppm) ¼ 55.8 (CH3), 56.8 (CH3), 113.4, 113.5, 118.1,
118.7, 120.7, 122.1, 128.3, 130.2, 133.2, 134.9 (10Car), 144.5
(¼CH), 155.4, 159.7 (2Car), 163.3 (C¼O). Analysis for
C16H15ClN2O3 (318.75) Calculated: C: 60.29%, H: 4.74%, N:
8.79%; Found: C: 60.33%, H: 4.71%, N: 8.84%.
CAS registry number: 325798-99-0. Yield: 87%; m.p.: 216–
1
218 ꢀC. C log P ¼ 4.55. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.32 (s,
9H, 3 ꢁ CH3), 3.80 (s, 3H, CH3), 3.84 (s, 3H, CH3), 7.11–7.15 (m,
2H, ArH), 7.47–7.49 (m, 1H, ArH), 7.53–7.56 (d, 1H, ArH,
J ¼ 6 Hz), 7.86–7.89 (d, 1H, ArH, J ¼ 9 Hz), 11.83 (s, 1H, NH); 13C
NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3), 35.2 (Ct-butyl), 56.2
(CH3), 61.7 (CH3), 115.1, 117.4, 124.9, 125.7, 127.9, 122.3, 131.0,
132.8, 143.5 (10Car), 148.4 (¼CH), 153.1, 154.9 (2Car), 163.9
(C¼O). Analysis for C20H24N2O3 (340.42) Calculated: C: 70.56%,
H: 7.11%, N: 8.23%; Found: C: 70.63%, H: 7.08%, N: 8.25%.
N-[(3-Chloro-4-methoxyphenyl)methylidene]-3-methoxybenzhy-
drazide (13)
4-Tert-butyl-N-[(2,4-dimethoxyphenyl)methylidene]benzhydrazide
(18)
Yield: 81%; m.p.: 148–150 ꢀC. C log P ¼ 3.44. 1H NMR (DMSO-
d6) ꢀ (ppm) ¼ 3.84 (s, 3H, CH3), 3.92 (s, 3H, CH3), 7.15–7.18 (m,
1H, ArH, J ¼ 9 Hz), 7.23–7.26 (d, 1H, ArH, J ¼ 9 Hz), 7.42–7.51
(m, 3H, ArH), 7.66–7.70 (m, 1H, ArH), 7.81–7.82 (d, 1H, ArH,
CAS registry number: 328921-38-6. Yield: 65%; m.p.: 198–
1
200 ꢀC. C log P ¼ 4.55. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.31 (s,
9H, 3 ꢁ CH3), 3.82 (s, 3H, CH3), 3.86 (s, 3H, CH3), 6.63–6.65

4
Ł. Popiołek & A. Biernasiuk
J Enzyme Inhib Med Chem, Early Online: 1–8
(m, 2H, ArH), 7.51–7.54 (d, 2H, ArH, J ¼ 9 Hz), 7.80–7.83 (m, Analysis for C21H26N2O3 (354.44) Calculated: C: 71.16%, H:
1H, ArH), 7.85–7.86 (d, 2H, ArH, J ¼ 9 Hz), 8.72 (s, 1H, ¼CH), 7.39%, N: 7.90%; Found: C: 71.21%, H: 7.37%, N: 7.94%.
11.66 (s, 1H, NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3),
34.3 (Ct-butyl), 56.0 (CH3), 56.8 (CH3), 100.3, 107.1, 117.5, 125.8, 4-Tert-butyl-N-[(4-hydroxy-3,5-dimethoxyphenyl)methylidene]-
129.7, 130.3, 132.9 (9Car), 150.7 (¼CH), 152.96, 160.9, 162.8 benzhydrazide (23)
CAS registry number: 524730-78-7. Yield: 69%; m.p.: 236–
(3Car), 164.3 (C¼O). Analysis for C20H24N2O3 (340.42)
Calculated: C: 70.56%, H: 7.11%, N: 8.23%; Found: C: 70.61%,
1
238 ꢀC. C log P ¼ 4.26. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.32 (s,
H: 7.09%, N: 8.26%.
9H, 3 ꢁ CH3), 3.82 (s, 6H, 2 ꢁ CH3), 6.98 (s, 2H, ArH), 7.53–
7.55 (d, 2H, ArH, J ¼ 6 Hz), 7.82–7.85 (d, 2H, ArH, J ¼ 9 Hz),
8.33 (s, 1H, ¼CH), 8.92 (s, 1H, OH), 11.66 (s, 1H, NH); 13C
4-Tert-butyl-N-[(3,4-dimethoxyphenyl)methylidene]benzhydrazide
(19)
NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3), 35.1 (Ct-butyl), 56.5 (2
ꢁ CH3), 105.0, 125.1, 125.7, 127.9, 131.3, 138.3 (9Car), 147.8
CAS registry number: 328921-37-5. Yield: 79%; m.p.: 214–
1
216 ꢀC. C log P ¼ 4.55. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.31 (s,
9H, 3 ꢁ CH3), 3.81 (s, 3H, CH3), 3.82 (s, 3H, CH3), 7.02–7.04 (d,
1H, ArH, J ¼ 6 Hz), 7.19–7.22 (m, 1H, ArH), 7.35 (s, 1H, ArH),
7.51–7.55 (d, 2H, ArH, J ¼ 9 Hz), 7.84–7.86 (d, 2H, ArH,
J ¼ 6 Hz), 8.39 (s, 1H, ¼CH), 11.69 (s, 1H, NH); 13C NMR
(DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3), 34.30 (Ct-butyl), 56.8 (CH3),
56.9 (CH3), 110.1, 114.1, 122.1, 125.8, 129.4, 130.3, 132.8 (9Car),
148.7 (¼CH), 149.7, 150.5, 152.9 (3Car), 164.3 (C¼O). Analysis
for C20H24N2O3 (340.42) Calculated: C: 70.56%, H: 7.11%, N:
8.23%; Found: C: 70.62%, H: 7.14%, N: 8.20%.
(¼CH), 148.8, 152.9 (3Car), 164.3 (C¼O). Analysis for
C20H24N2O4 (356.41) Calculated: C: 67.40%, H: 6.79%, N:
7.86%; Found: C: 67.47%, H: 6.76%, N: 7.89%.
4-Tert-butyl-N-[(4-propoxyphenyl)methylidene]
benzhydrazide (24)
CAS registry number: 544689-51-2. Yield: 73%; m.p.: 196–
198 ꢀC. C log P ¼ 6.01. 1H NMR (DMSO-d6) ꢀ (ppm) ¼ 0.96–1.01
(t, 3H, CH3, J ¼ 9 Hz, J ¼ 6 Hz), 1.32 (s, 9H, 3 ꢁ CH3), 1.69–1.80
(m, 2H, CH2), 3.96–4.00 (t, 2H, CH2, J ¼ 6 Hz), 7.00–7.03 (d, 2H,
ArH, J ¼ 9 Hz), 7.52–7.55 (d, 2H, ArH, J ¼ 9 Hz), 7.64–7.67 (d,
2H, ArH, J ¼ 9 Hz), 7.83–7.86 (d, 2H, ArH, J ¼ 9 Hz), 8.39 (s,
1H, ¼CH), 11.66 (s, 1H, NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 10.85
(CH3), 21.46 (CH2), 30.5 (CH2), 31.4 (3 ꢁ CH3), 35.2 (Ct-butyl),
69.5 (CH2), 115.2, 125.7, 127.2, 127.9, 129.1, 131.3 (10Car), 147.8
(¼CH), 154.9, 160.7 (2Car), 163.3 (C¼O). Analysis for
C21H26N2O2 (338.44) Calculated: C: 74.52%, H: 7.74%, N:
8.28%; Found: C: 74.58%, H: 7.72%, N: 8.31%.
4-Tert-butyl-N-[(3-ethoxy-4-hydroxyphenyl)methylidene]benzhy-
drazide (20)
CAS registry number: 525562-83-8. Yield: 76%; m.p.: 216–
1
217 ꢀC. C log P ¼ 4.86. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.31 (s,
9H, 3 ꢁ CH3), 1.34–1.39 (t, 3H, CH3, J ¼ 9 Hz, J ¼ 6 Hz), 4.04–
4.11 (q, 2H, CH2, J ¼ 6 Hz, J ¼ 9 Hz), 6.84–6.87 (d, 1H, ArH,
J ¼ 9 Hz), 7.06–7.10 (m, 1H, ArH), 7.30 (s, 1H, ArH), 7.52–7.55
(d, 2H, ArH, J ¼ 9 Hz), 7.82–7.85 (d, 2H, ArH, J ¼ 9 Hz), 8.32 (s,
1H, ¼CH), 9.50 (s. 1H, OH), 11.61 (s, 1H, NH); 13C NMR
(DMSO) ꢀ (ppm) ¼ 15.2 (CH3), 31.4 (3 ꢁ CH3), 35.1 (Ct-butyl),
64.3 (CH2), 110.7, 115.9, 122.5, 125.7, 126.2, 127.9, 131.3, 147.6
(10Car), 148.6 (¼CH), 149.6, 154.9 (2Car), 163.3 (C¼O).
Analysis for C20H24N2O3 (340.42) Calculated: C: 70.56%, H:
7.11%, N: 8.23%; Found: C: 70.63%, H: 7.09%, N: 8.26%.
4-Tert-butyl-N-{[4-(trifluoromethyl)phenyl]methylidene}benzhy-
drazide (25)
Yield: 74%; m.p.: 198–200 ꢀC. C log P ¼ 5.94. 1H NMR (DMSO-
d6) ꢀ (ppm) ¼ 1.32 (s, 9H, 3 ꢁ CH3), 7.55–7.57 (d, 2H, ArH,
J ¼ 6 Hz), 7.81–7.89 (m, 4H, ArH), 7.94–7.97 (d, 2H, ArH,
J ¼ 9 Hz), 8.52 (s, 1H, ¼CH), 12.00 (s, 1H, NH); 13C NMR
(DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH ), 35.2 (Ct-butyl), 124.6 (CF3),
3
4-Tert-butyl-N-[(4-methoxyphenyl)methylidene]benzhydrazide
(21)
125.8, 126.2, 128.1, 129.5, 130.2, 132.9, 135.9 (11Car), 149.3
(¼CH), 152.9 (Car), 164.3 (C¼O). Analysis for C19H19F3N2O
(348.36) Calculated: C: 65.51%, H: 5.50%, N: 8.04%; Found: C:
65.57%, H: 5.53%, N: 8.01%.
CAS registry number: 328921–35-3. Yield: 98%; m.p.: 196–
1
198 ꢀC. C log P ¼ 4.80. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.32 (s,
9H, 3 ꢁ CH3), 3.81 (s, 3H, CH3), 7.01–7.04 (d, 2H, ArH, J ¼ 9 Hz),
7.52–7.55 (d, 2H, ArH, J ¼ 9 Hz), 7.66–7.69 (d, 2H, ArH,
J ¼ 9 Hz), 7.83–7.86 (d, 2H, ArH, J ¼ 9 Hz), 8.39 (s, 1H, ¼CH),
11.67 (s, 1H, NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3),
35.2 (Ct-butyl), 55.8 (CH3), 114.8, 125.7, 127.4, 127.9, 129.1, 131.3
(10Car), 147.9 (¼CH), 154.9, 161.2 (2Car), 163.3 (C¼O). Analysis
for C19H22N2O2 (310.39) Calculated: C: 73.52%, H: 7.14%, N:
9.03%; Found: C: 73.58%, H: 7.11%, N: 9.06%.
4-Tert-butyl-N-[(2-chloro-3-methoxyphenyl)methylidene]benzohy-
drazide (26)
Yield: 92%; m.p.: 259–261 ꢀC. C log P ¼ 5.32. 1H NMR (DMSO-
d6) ꢀ (ppm) ¼ 1.32 (s, 9H, 3 ꢁ CH3), 3.89 (s, 3H, CH3), 7.20–7.23
(d, 1H, ArH, J ¼ 9 Hz), 7.37–7.42 (t, 1H, ArH, J ¼ 6 Hz,
J ¼ 9 Hz), 7.54–7.57 (d, 2H, ArH, J ¼ 9 Hz), 7.61–7.63 (d, 1H,
ArH, J ¼ 6 Hz), 7.87–7.90 (d, 2H, ArH, J ¼ 9 Hz), 8.89 (s,
1H, ¼CH), 12.03 (s, 1H, NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 31.4
(3 ꢁ CH3), 35.2 (Ct-butyl), 36.8 (CH3), 114.6, 119.7, 122.8, 125.8,
128.0, 128.3, 132.3, 142.3 (10Car), 149.9 (¼CH), 155.4, 157.6
(2Car), 164.0 (C¼O). Analysis for C19H21ClN2O2 (344.83)
Calculated: C: 66.18%, H: 6.14%, N: 8.12%; Found: C: 66.23%,
H: 6.11%, N: 8.16%.
4-Tert-butyl-N-[(3-ethoxy-4-methoxyphenyl)methylidene]benzhy-
drazide (22)
CAS registry number: 394685-88-2. Yield: 95%; m.p.: 206–
1
208 ꢀC. C log P ¼ 4.89. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.31 (s,
9H, 3 ꢁ CH3), 1.34–1.39 (t, 3H, CH3, J ¼ 6 Hz, J ¼ 9 Hz), 3.81 (s,
3H, CH3), 4.03–4.10 (q, 2H, CH2, J ¼ 6 Hz, J ¼ 9 Hz), 7.02–7.04
(d, 1H, ArH, J ¼ 6 Hz), 7.18–7.21 (m, 1H, ArH), 7.33 (s, 1H, ArH),
7.52–7.55 (d, 2H, ArH, J ¼ 9 Hz), 7.83–7.86 (d, 2H, ArH,
J ¼ 9 Hz), 8.37 (s, 1H, ¼CH), 11.67 (s, 1H, NH); 13C NMR
(DMSO) ꢀ (ppm) ¼ 15.2 (CH3), 31.4 (3 ꢁ CH3), 35.2 (Ct-butyl), 55.9
(CH3), 64.2 (CH2), 109.6, 112.0, 122.2, 125.7, 127.5, 127.9, 131.3,
148.2 (10Car), 148.7 (¼CH), 151.2, 154.9 (2Car), 163.4 (C¼O).
4-Tert-butyl-N-[((3-chloro-4-methoxyphenyl)methylidene]benzo-
hydrazide (27)
CAS registry number: 328921-99-9. Yield: 75%; m.p.: 227–
1
229 ꢀC. C log P ¼ 5.32. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.32 (s,
9H, 3 ꢁ CH 3), 3.92 (s, 3H, CH3), 7.23–7.26 (d, 1H, ArH,

DOI: 10.3109/14756366.2016.1170012
Hydrazide-hydrazones of 3-methoxybenzoic and 4-tert-butylbenzoic acids
5
J ¼ 9 Hz), 7.53–7.56 (d, 2H, ArH, J ¼ 9 Hz), 7.66–7.69 (d, 1H, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC
ArH, J ¼ 9 Hz), 7.81–7.86 (m, 3H, ArH), 8.37 (s, 1H, ¼CH), 13883, Proteus mirabilis ATCC 12453, Salmonella typhimurium
11.80 (s, 1H, NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3), ATCC 14028, and Pseudomonas aeruginosa ATCC 9027) and
35.2 (Ct-butyl), 56.8 (CH3), 113.5, 122.12, 125.7, 127.8, 128.3, against the fungi belonging to yeasts (Candida albicans ATCC
128.4, 130.1, 133.4 (10Car), 147.9 (¼CH), 155.9, 156.2 (2Car), 2091, Candida albicans ATCC 10231, and Candida parapsilosis
164.2 (C¼O). Analysis for C19H21ClN2O2 (344.83) Calculated: ATCC 22019). These micro-organisms came from American
C: 66.18%, H: 6.14%, N: 8.12%; Found: C: 66.23%, H: 6.12%, N: Type Culture Collection (ATCC), routinely used for the evalu-
8.15%.
ation of antimicrobials. All the used microbial cultures were first
subcultured on a suitable medium at 30–35 ꢀC for 18–48 h.
The surfaces of Mueller–Hinton agar (for bacteria) and RPMI
1640 with MOPS (for fungi) were inoculated with the suspensions
of bacterial or fungal species. The microbial suspensions were
prepared in sterile saline with an optical density of McFarland
standard scale 0.5 – approximately 1.5 ꢁ 108 CFU (Colony
Forming Units)/ml for bacteria, and 5 ꢁ 105 CFU/ml) for fungi.
The samples containing the examined compounds were
dissolved in 1 ml dimethyl sulfoxide (DMSO). Furthermore, the
bacterial and fungal suspensions were put onto Petri dishes with
the solid media containing 2 mg/ml of the tested compounds and
incubated under appropriate conditions. The inhibition of micro-
bial growth was evaluated by the comparison with a control
culture prepared without any sample tested. Ciprofloxacin,
cefuroxime, ampicillin (Sigma, St. Louis, MO) were used as a
reference antibacterial compounds and fluconazole (Sigma) as
reference antifungal compound.
N-[(3-Bromo-4-methoxyphenyl)methylidene]-4-tert-butylbenzohy-
drazide (28)
CAS registry number: 328921-88-6. Yield: 79%; m.p.: 234–
1
236 ꢀC. C log P ¼ 5.59. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.31 (s,
9H, 3 ꢁ CH3), 3.91 (s, 3H, CH3), 7.19–7.22 (d, 1H, ArH,
J ¼ 9 Hz), 7.53–7.55 (d, 2H, ArH, J ¼ 6 Hz), 7.63–7.65 (d, 1H,
ArH, J ¼ 6 Hz), 7.83–7.86 (d, 2H, ArH, J ¼ 9 Hz), 7.96–7.97 (d,
1H, ArH, J ¼ 9 Hz), 8.36 (s, 1H, ¼CH), 11.80 (s, 1H, NH); 13C
NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3), 35.2 (Ct-butyl), 56.9
(CH3), 111.7, 113.3, 125.7, 126.9, 127.9, 128.9, 129.1, 131.3
(10Car), 147.2 (¼CH), 152.9, 157.2 (2Car), 164.3 (C¼O).
Analysis for C19H21BrN2O2 (389.29) Calculated: C: 58.62%, H:
5.44%, N: 7.20%; Found: C: 58.68%, H: 5.40%, N: 7.25%.
N-[((2-Bromo-3-hydroxy-4-methoxyphenyl)methylidene]-4-tert-
butylbenzhydrazide (29)
Subsequently minimal inhibitory concentration (MIC) of the
compounds was examined by the microdilution broth method,
Yield: 95%; m.p.: 212–214 ꢀC. C log P ¼ 4.52. 1H NMR (DMSO- with the use of their two-fold dilutions in Mueller–Hinton broth
d6) ꢀ (ppm) ¼ 1.32 (s, 9H, 3 ꢁ CH3), 3.89 (s, 3H, CH3), 7.09–7.12 (for bacteria) and RPMI 1640 broth with MOPS (for fungi),
(d, 1H, ArH, J ¼ 9 Hz), 7.50–7.56 (t, 3H, ArH, J ¼ 9 Hz), 7.86– prepared in 96-well polystyrene plates. The final concentrations
7.89 (d, 2H, ArH, J ¼ 9 Hz), 8.78 (s, 1H, ¼CH), 9.65 (s, 1H, OH), of the compounds ranged from 2000 to 0.98 mg/ml. The microbial
11.92 (s, 1H, NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 31.4 (3 ꢁ CH3), suspensions were prepared in sterile 0.85% NaCl with an optical
35.2 (Ct-butyl), 56.5 (CH3), 111.4, 112.2, 118.9, 125.7, 126.5, density of 0.5 McFarland standard. Next 2 ml of each bacterial or
127.9, 131.3, 144.2 (10Car), 147.8 (¼CH), 150.1, 154.8 (2Car), fungal suspensions were added to each well containing 200 ml
163.8 (C¼O). Analysis for C19H21BrN2O3 (405.28) Calculated: broth and various concentrations of the examined compounds.
C: 56.31%, H: 5.22%, N: 6.91%; Found: C: 56.35%, H: 5.20%, N: After incubation, the MIC was assessed spectrophotometrically as
6.95%.
the lowest concentration of the samples showing complete
bacterial or fungal growth inhibition. Appropriate DMSO,
growth and sterile controls were carried out. The medium with
no tested substances was used as control28,29
.
4-Tert-butyl-N-[(2-hydroxy-3,5-diiodophenyl)methylidene]benzo-
hydrazide (30)
The minimal bactericidal concentration (MBC) or minimal
fungicidal concentration (MFC) is defined as the lowest concen-
tration of the compounds that is required to kill a particular
bacterial or fungal species. MBC or MFC were determined by
removing 20 ml of the culture using for MIC determinations from
each well and spotting onto appropriate agar medium. The plates
were incubated. The lowest compound concentrations with no
visible growth observed were assessed as bactericidal or fungi-
cidal concentrations. All the experiments were repeated three
CAS registry number: 325990-74-7. Yield: 79%; m.p.: 250–
1
252 ꢀC. C log P ¼ 7.28. H NMR (DMSO-d6) ꢀ (ppm) ¼ 1.32 (s,
9H, 3 ꢁ CH3), 7.57–7.59 (d, 2H, ArH, J ¼ 6 Hz), 7.88 (s, 1H,
ArH), 7.89–7.91 (d, 2H, ArH, J ¼ 6 Hz), 8.05–8.06 (d, 1H, ArH,
J ¼ 3 Hz), 8.44 (s, 1H, ¼CH), 12.46 (s, 1H, OH), 12.99 (s, 1H,
NH); 13C NMR (DMSO) ꢀ (ppm) ¼ 31.3 (3 ꢁ CH3), 35.3 (Ct-
butyl), 82.9. 88.3, 120.8, 125.9, 128.1, 129.8, 139.5 (9Car), 147.9
(¼CH), 148.2, 155.9, 157.1 (3Car), 164.3 (C¼O). Analysis for
C18H18I2N2O2 (548.16) Calculated: C: 39.44%, H: 3.31%, N:
5.11%; Found: C: 39.48%, H: 3.35%, N: 5.08%.
times and representative data were presented28,29
.
In this study, no bioactivity was defined as a MIC41000 mg/
ml, mild bioactivity as a MIC in the range 501–1000 mg/ml,
moderate bioactivity with MIC from 126 to 500 mg/ml, good
bioactivity as a MIC in the range 26–125 mg/ml, strong bioactivity
with MIC between 10 and 25 mg/ml and very strong bioactivity as
a MIC510 mg/ml. The MBC/MIC or MFC/MIC ratios were
calculated in order to determine bactericidal/fungicidal (MBC/
MIC ꢂ 4, MFC/MIC ꢂ 4) or bacteriostatic/fungistatic (MBC/
MIC44, MFC/MIC44) effects of the tested compounds30.
Microbiology
In vitro antimicrobial assay
The examined compounds 3–30 were screened in vitro for
antibacterial and antifungal activities with the use of the broth
microdilution method according to European Committee on
Antimicrobial Susceptibility Testing (EUCAST)26 and Clinical
and Laboratory Standards Institute guidelines27 against a panel of
reference strains of microorganisms, including Gram-positive
bacteria (Staphylococcus aureus ATCC 6538, S. aureus ATCC
25923, S. aureus ATCC 43300, Staphylococcus epidermidis
ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus
ATCC 10876, and Micrococcus luteus ATCC 10240), Gram-
negative bacteria (Bordetella bronchiseptica ATCC 4617,
Results and discussion
Chemistry
Titled compounds were synthesized according to the stages
presented in Scheme 1. First hydrazides of 3-methoxybenzoic
acid (1) and 4-tert-butylbenzoic acid (2) were synthesized with

6
Ł. Popiołek & A. Biernasiuk
J Enzyme Inhib Med Chem, Early Online: 1–8
the use of the methods described earlier24,25 by the reaction of subtilis ATCC 6633 and Bacillus cereus ATCC 10876 reveal the
commercially available methyl esters of above mentioned highest vulnerability to synthesized hydrazide-hydrazone deriva-
carboxylic acids with hydrazine hydrate. Then the synhtesized tives (13, 14, 16, 23, and 30).
hydrazides were subjected to the condensation reactions with
In the case of Bacillus subtilis ATCC 6633, the activity of
corresponding substituted aromatic aldehydes what allowed us to compound 16 (MIC¼1.95 mg/ml) was almost 10 times higher than
obtain appropriate hydrazide-hydrazone derivatives (3–30). The cefuroxime (MIC¼15.63 mg/ml), whereas for compounds 13 and
reactions were carried out by heating substrates in ethanol under 14, their values of MIC 3.91 mg/ml and 7.81 mg/ml, respectively,
reflux. The structures of all synthesized (1–30) compounds were were almost five and two times lower than the MIC of
fully confirmed by means of 1H NMR, 13C NMR data and cefuroxime. Compound 30 showed the same value of MIC
elemental analysis. Appropriate spectral and physico-chemical (15.63 mg/ml) as cefuroxime against B. subtilis ATCC 6633. It is
properties are presented in the Experimental section. To the also worth to add that compounds 14 and 23 (MIC¼31.25 mg/ml)
best of our knowledge among the synthesized derivatives (3–30) had bactericidal effects (MBC/MIC ꢂ 4), other compounds (13,
only compound 524 has any reference in the literature. As for 16, and 30) showed bacteriostatic effects (MBC/MIC44) against
compounds 1–4, 7–11, 14–24, 27, 28 and 30 their structures are this pathogen.
known, but there are no references reporting their use, synthesis
As for the Bacillus cereus ATCC 10876, compounds 13, 14,
and physico-chemical characterization. Moreover, our synthesized and 16 were the most active against this bacterium. Their activity
hydrazide-hydrazones 12, 13, 25, 26, and 29 are new and their (MIC¼7.81 mg/ml) was fourfold higher than that of cefuroxime
structures and synthesis have not been reported in the literature (MIC¼31.25 mg/ml) and eight-fold times higher than for ampi-
so far.
cillin (MIC¼62.5 mg/ml). Compound 23 inhibited the growth of
B. cereus ATCC 10876 with the same strength as ampicillin. In
addition, compound 16 showed bactericidal effect (MBC/
MIC ꢂ 4) against this microorganism.
In vitro antimicrobial activity against a panel of reference
strains
Considering M. luteus ATCC 10240, compounds 16 and 30
with MIC values of 3.91 mg/ml and 15.63 mg/ml, respectively,
were the most active with bacteriostatic effect against this
pathogen. Compounds 13 and 14 didn’t show any activity against
this microorganism.
The results of our in vitro antimicrobial activity study indicated,
that synthesized compounds 3–6, 8–12, 15, and 17–24 had no
inhibitory effect on the growth of the reference strains of Gram-
positive and Gram-negative bacteria and yeasts belonging to
fungi. To the contrary, compounds 13, 14, 16, 23, and 30 had
significant inhibitory effect on the growth of the reference strains
of Gram-positive bacteria.
The activity of synthesized hydrazide-hydrazones (13, 14,
16, 23, and 30) against Staphylococcus spp. can be assessed as
very strong to good (MIC¼7.81 – 125 mg/ml) or in some cases
as moderate (MIC4125 mg/ml). The most active compound
On the basis of the MIC values obtained with the broth
microdilution method (Table 1), we can conclude that Bacillus
CHO
O
O
O
NH2
N
NH2NH2
OCH3
NH
R2
NH
R2
EtOH, reflux, 3h
MeOH, reflux, 3h
R1
R1
R1
(
-
)
3
30
(
,
)
1 2
Compound No
R1
R2
-
Compound No
R1
R2
-
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
2
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
2,3-diOCH3
2,4-diOCH3
3,4-diOCH3
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2,3-diOCH3
2,4-diOCH3
3,4-diOCH3
3-OC2H5-4-OH
4-OCH3
3-OC2H5-4-OH
4-OCH3
3-OC2H5-4-OCH3
3,5-diOCH3-4-OH
4-OC3H7
3-OC2H5-4-OCH3
3,5-diOCH3-4-OH
4-OC3H7
4-CF3
4-CF3
2-Cl-3-OCH3
3-Cl-4-OCH3
3-Br-4-OCH3
2-Br-3-OH-4-OCH3
2-OH-3,5-diI
2-Cl-3-OCH3
3-Cl-4-OCH3
3-Br-4-OCH3
2-Br-3-OH-4-OCH3
2-OH-3,5-diI
Scheme 1. Synthesis scheme of hydrazide-hydrazone derivatives (3–30).

DOI: 10.3109/14756366.2016.1170012
Hydrazide-hydrazones of 3-methoxybenzoic and 4-tert-butylbenzoic acids
Table 1. The activity data expressed as MIC (mg/ml) against the reference strains of Gram-positive bacteria.
MIC (mg/ml) of the tested compounds and antibiotic standards
7
Species
13
14
16
23
30
CPX
CFX
APC
Gram-positive bacteria
Staphylococcus aureus ATCC 25923
Staphylococcus aureus ATCC 6538
Staphylococcus aureus ATCC 43300
Staphylococcus epidermidis ATCC 12228
Micrococcus luteus ATCC 10240
Bacillus subtilis ATCC 6633
7.81
7.81
7.81
15.62
–
15.62
15.62
15.62
15.62
–
7.81
62.5
250
125
500
500
250
31.25
62.5
125
0.488
0.244
0.244
0.122
0.976
0.031
0.061
0.49
0.98
nd
0.24
0.98
15.63
31.25
nd
nd
nd
nd
nd
nd
62.5
125
250
500
15.63
15.63
500
7.81
15.62
3.91
1.95
7.81
3.91
7.81
7.81
7.81
Bacillus cereus ATCC 10876
The standard antibiotic used as positive control for bacteria: ciprofloxacin (CPX); cefuroxime (CFX), ampicillin (APC); ‘‘–’’– no activity; nd – not
determined; MIC – minimal inhibitory concentration; MBC – minimal bactericidal concentration; compounds with bactericidal effect (MBC/
MIC ꢂ 4) are marked in bold.
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The authors report that they have no conflicts of interest. This
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Outstanding Scientific Achievements from the Medical University
of Lublin (Poland).
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