Synthesis and antifungal activity of substituted salicylaldehyde hydrazones, hydrazides and sulfohydrazides

Article abstract of DOI:10.1016/j.bmc.2014.07.022

Efficient synthetic procedures for the preparation of acid hydrazines and hydrazides were developed by converting the corresponding carboxylic acid into the methyl ester catalyzed by Amberlyst-15, followed by a reaction with hydrazine monohydrate. Sulfohydrazides were prepared from the corresponding sulfonyl chlorides and hydrazine monohydrate. Both of these group of compounds were condensed with substituted salicylaldehydes using gradient concentration methods that generated a large library of hydrazone, hydrazide and sulfohydrazide analogs. Antifungal activity of the prepared analogs showed that salicylaldehyde hydrazones and hydrazides are potent inhibitors of fungal growth with little to no mammalian cell toxicity, making these analogs promising new targets for future therapeutic development.

Full text of DOI:10.1016/j.bmc.2014.07.022

Bioorganic & Medicinal Chemistry 22 (2014) 4629–4636
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antifungal activity of substituted salicylaldehyde
hydrazones, hydrazides and sulfohydrazides
Gregory L. Backes a, Donna M. Neumann a,b, Branko S. Jursic c,d,
⇑
a Louisiana State University Health Sciences Center, Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido
St., New Orleans, LA 70112, United States
b Department of Ophthalmology, LSUHSC, New Orleans, United States
c Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United States
d STEPHARM, LLC., PO Box 24220, New Orleans, LA 70184, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Efficient synthetic procedures for the preparation of acid hydrazines and hydrazides were developed by
converting the corresponding carboxylic acid into the methyl ester catalyzed by Amberlyst-15, followed
by a reaction with hydrazine monohydrate. Sulfohydrazides were prepared from the corresponding sul-
fonyl chlorides and hydrazine monohydrate. Both of these group of compounds were condensed with
substituted salicylaldehydes using gradient concentration methods that generated a large library of
hydrazone, hydrazide and sulfohydrazide analogs. Antifungal activity of the prepared analogs showed
that salicylaldehyde hydrazones and hydrazides are potent inhibitors of fungal growth with little to no
mammalian cell toxicity, making these analogs promising new targets for future therapeutic
development.
Received 4 June 2014
Revised 7 July 2014
Accepted 15 July 2014
Available online 23 July 2014
Keywords:
Salicylaldehyde
Hydrazones
Antifungals
Hydrazides
Ó 2014 Elsevier Ltd. All rights reserved.
Opportunistic fungal infections are common and potentially
life-threatening infections.1 For example, invasive fungal infections
from Candida spp. are a significant cause of morbidity and mortal-
ity in at-risk populations, particularly transplant recipients, cancer
patients and those infected with HIV and AIDS.2,3 Further, invasive
Candida spp. infections rapidly develop resistance to currently
marketed antifungal agents,4 making vigilant treatment difficult
at best. Clinically, candidiasis incidence is commonly associated
with Candida albicans, Candida glabrata and Candida krusei microor-
ganisms most frequently.5,6 Currently, the first-line treatment for
these infections are azoles, yet the Candida spp. have developed
multiple resistance mechanisms to azoles, including overexpres-
sion of efflux pumps1 and point mutations in the ERG11 gene7 in
Candida albicans. In addition, Candida glabrata develops resistance
during prolonged treatment with azole antifungals.8 For these rea-
sons, there is a continuous demand for the discovery of novel ther-
apeutics to treat fungal infections, particularly Candida spp.
infections.
albicans and Candida glabrata.9 A number of those derivatives,
including phenylhydrazones of 5-acylpyrimidinetrione exhibited
potent growth inhibition at or below 10 lM with minimal mam-
malian cell toxicity, making this class of compounds an interesting
alternative for exploration and development to treat fungal infec-
tions. In addition, it has also been well documented that some
substituted salicylaldehyde hydrazones also possess antimicrobial
activity while salicylaldehydes by themselves do not possess anti-
fungal activity.10–17 Nonetheless, there are significant drawbacks
that have limited development of salicylaldehyde hydrazones as
a broad class of antifungal agents. These drawbacks include the
lack of appropriately developed synthetic procedures that can pro-
duce a range of structural variations within the class of salicylalde-
hyde hydrazones for antifungal and SAR studies. In this work, we
present the efficient synthetic methodology used to generate three
structurally diverse groups of salicylaldehyde derivatives; salicyl-
aldehyde phenylhydrazones (group I), benzohydrazides (group
II), and sulfohydrazides (group III; Scheme 1). The antifungal activ-
ity for Candida albicans and Candida glabrata was assessed for each
class of salicylaldehyde derivatives. Finally, mammalian cell toxic-
ity screens were performed against all compounds with MICs of
Hydrazine and hydrazone derivatives are emerging in the liter-
ature as novel classes of potential antifungal agents with activity
against numerous Candida spp. Recently, we reported an extensive
collection of substituted pyrimidinetrione derivatives with potent
antifungal activity against two human pathogenic species, Candida
8 lg/mL or less. In summary, we found that nitro salicylaldehyde
hydrazones, in addition to a broad range of unsubstituted, substi-
tuted and halogenated salicylaldehyde hydrazides were potent
inhibitors of fungal growth with minimal cytotoxicity to liver or
kidney cells.
⇑
Corresponding author. Tel.: +1 504 858 0515.
E-mail address: bjursic1@uno.edu (B.S. Jursic).
http://dx.doi.org/10.1016/j.bmc.2014.07.022
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

4630
G. L. Backes et al. / Bioorg. Med. Chem. 22 (2014) 4629–4636
Salicylaldehyde Hydrazones
R1
R1
R1
H
N
H
N
H
N
H
N
H
H
N
R2
R2
R4
N
R5
R2
OH
OH
III
S
O
OH
R3
O
O
II
I
Scheme 1. Three groups of salicylaldehyde hydrazone libraries selected for antifungal studies.
H2NNH2xH2O
1. Chemistry
R
SO2Cl
R SO2NHNH2
THF/H2O/between 0ºC and 8ºC
To our surprise, we could find no simple and high yielding gen-
eral synthetic procedures applicable to the preparation of large
quantities of structurally diverse acid hydrazides18–22 even though
their application in medicine and medicinal chemistry has been
well documented.23–27 Several approaches were documented for
the preparation of acid hydrazides,18 including starting with car-
boxylic acids,28 acid esters,29 acid chlorides,20 amides30 and even
N-acylbenzotriazoles.31 In general, acid chlorides and acid anhy-
drides double acylate hydrazines32 and subsequently are not viable
intermediates for the preparation of acid hydrazides. On the other
hand, single hydrazine acylation with esters seemed to be easier to
control. For this reason, we have developed two parallel methods
(Methods I and II, Scheme 2) for the preparation of acid hydrazides.
The first approach uses thionyl chloride catalyzed acid esterifica-
tion with ethanol, which is commonly used for the preparation of
amino acid hydrazides.33 Thus, the prepared esters were isolated,
purified and used in the next reaction with hydrazine monohy-
drate to yield the desirable acid hydrazide (Method 1, Scheme 2).
The second preparation method utilized Amberlyst-15,34 a strong
acid exchange resin catalyst for esterification with methanol. After
the reaction was complete, the catalyst was removed by filtration
and the same clear methanol solution that contained an ester
was mixed with an excess of hydrazine monohydrate to give the
targeted acid hydrazide in high isolated yields (Method II,
Scheme 2). Additionally, the catalyst is recyclable35 and the reac-
tion could also tolerate the presence of acid sensitive moieties such
as phenol, OH, t-butyl, or even the pyridine moiety. In this way
Method II was developed as the method of choice for the prepara-
tion of a structurally diverse library of acid hydrazides.
SO2NHNH2
87%
CH3
SO2NHNH2
92%
CH3O
SO2NHNH2
88%
SO2NHNH2
93%
NO2
SO2NHNH2
89%
Br
SO2NHNH2
89%
Scheme 3. Preparation of sulfonohydrazides.
increasingly important synthetic intermediates37 for preparation
of valuable biologically active compounds.38–42
In general, hydrazones were prepared from the corresponding
carbonyl (aldehyde or ketone) compound and the mono and asym-
metric double substituted hydrazines43–46 in refluxing methanol or
ethanol without acid or base catalyst (Scheme 4). This reaction was
generally completed after three hours. Isolated yields and purifica-
tion of the final products were simplified when the reaction began
as a very diluted reaction mixture (100 ml of methanol per 1 mmol
of aldehyde and 1 mmol of substituted hydrazine) and then the
reagent concentration was gradually increased by slowly distilling
the solvent from the refluxing reaction mixture. Distillation of the
solvent was stopped at the point when a solid started to form in
the reaction mixture or if the reaction mixture volume was
reduced to ꢀ5 ml. At room temperature or at ꢁ5 °C, all of the pre-
pared compounds crystallized readily. They were further purified
by washing with ice cold methanol, ether, or hexane as solvents
for which the corresponding products are only slightly soluble. It
is important to emphasize that the formation of the hydrazone
product was enhanced by gradually increasing the reactant con-
centration through slow solvent distillation. In this way, the iso-
lated yields of structurally diverse hydrazones was >85%
(Scheme 4).
Sulfonohydrazides (Scheme 3) were all prepared from the
corresponding sulfonyl chloride by modifying procedures outlined
by Friedman and co-workers.36 The reaction involved the slow
addition of a tetrahydrofuran solution of the corresponding sulfo-
nyl chloride into ice-water cooled aqueous hydrazine. It was
important to maintain the reaction mixture temperature close to
0 °C to avoid the formation of the double sulfonation byproduct.
It was also important to evaporate tetrahydrofuran at a reduced
pressure without heating. Furthermore, we found that this
procedure was an appropriate general method for the preparation
of sulfonohydrazides, a class of compounds that are becoming
2. Antifungal activity
The antifungal activity of the salicylaldehyde hydrazones,
hydrazides and sulfohydrazides described in Schemes 2–4 were
evaluated in vitro using Candida albicans (ATCC No. 10231) and
Candida glabrata (ATCC No. 48435). All assays were done in
Method I
Method II
H2NNH2xH2O/CH3OH
reflux 3 hrs
H2NNH2xH2O/CH3OH
reflux 3 hrs
Amberlyst-15/CH3OH
reflux 3 days
SO2Cl2/C2H5OH
overnight reflux
RCO2H
RCO2H
RCONHNH2
80-90%
HO
CONHNH2
93%
CONHNH2
87%
OH
CONHNH2
91%
O2N
CONHNH2
82%
CH3O
CONHNH2
92%
HO
CH3O
CONHNH2
89%
(CH3)3C
CONHNH2
86%
HO
HO
CONHNH2
(CH3)3C
CONHNH2
91%
93%
Scheme 2. Two different pathways for preparation of hydrazides.

G. L. Backes et al. / Bioorg. Med. Chem. 22 (2014) 4629–4636
4631
R1
(cat.
#
G4000). Representative compounds are presented in
R1
H
N
H2NNHX
Figure 1. Briefly, all cytotoxicity studies were done using a negative
control (wells of either kidney or liver cells in the absence of the
analog), a positive control (wells of either kidney or liver cells trea-
ted with a known cytotoxic agent known as saponin) and the serial
dilutions of the novel analogs. All control (negative) wells were
normalized to 100% viable cells to accurately show the percentage
of either liver or kidney cells that remain alive and viable following
overnight incubation with either saponin or the analogs tested. Of
the analogs assayed, SA1, SA6, SA10 and SA55 had minimal reduc-
tions in viable kidney and liver cells, where the only marked cell
death was observed at concentrations 100ꢂ greater than the estab-
lished MIC (Fig. 1). In contrast, the two most potent analogs, SA48
and SA49 exhibited toxicity toward kidney cells, with an ꢀ20%
reduction in viable cells even at the MIC relative to the negative
control (Fig. 1). While this was a somewhat discouraging finding,
more studies are currently underway to determine the mechanism
of toxicity and possible ways to reduce kidney toxicity associated
with these two analogs. Nonetheless, the majority of analogs tested
showed minimal cytotoxicity in our in vitro assays. All results
from the toxicity screenings outside of Figure 1 are provided in
Supplemental information.
H
X
CHO
methanol reflux 3 hrs
R2
N
R2
slow methanol distillation
OH
SA
OH
substituted
salycilaldehyde
Yield 85-97%
R1 = H, Cl, Br, CH3, CH3O, NO2, OH, R2 = H, Cl, Br, CH3, CH3O, NO2, OH
R3 = H, OH, NO2
X = H, C6H5, C6H4-4-OCH3, C6H3-2,4-(CH3)2, C6H4-4-NO2, C6H3-2,4-(NO2)2, COC6H5,
COC6H4-4-NO2, COC6H4-2-OH, COC6H4-4-C(CH3)3, COC6H4-4-OCH3, SO2C6H5,
SO2C6H4-4-CH3, SO2C6H4-4-OCH3, SO2C6H4-4-Br, SO2C6H4-NO2, SO2-2-naphthyl
Scheme 4. General route for preparation our hydrazone library.
accordance with NCCLS reference documents.47 The results of
these screenings are summarized in Tables 1–8 as the minimal
inhibitory concentrations that inhibited more than 80% fungal
growth as compared to the positive controls in 1% DMSO and
HEPES buffered RPMI media. All MIC screens were done using a
visual scoring method as opposed to spectroscopic methods of
analysis, due to the physical properties of many of the compounds
altering their absorbance spectra relative to the positive, negative
and drug controls.
Of the analogs tested, none of the salicylaldehyde sulfohydraz-
3. Chemistry experimental
ides showed antifungal activity through 125 lg/mL, regardless of
the nature of the aromatic ring attached (Table 8). Similarly, lim-
Thin-layer chromatographic analysis (TLC) was performed
using silica gel on aluminum foil glass plates and was detected
under ultraviolet (UV) light. The 1H and 13C NMR spectra were
run on Varian 400 MHz Unity instruments in CDCl3 or in DMSO-
d6. Solvent signals were use as internal NMR chemical shift refer-
ences. If necessary, products were purified by short silica gel
(40–70 mm) filtration. Silica gel was purchased from Sorbent Tech-
nologies. Substituted phenylhydrazines were prepared from corre-
sponding anilines by following the preparation procedure outlined
in Vogel.48 All reagents and solvents were purchased from Sigma–
Aldrich and were analytical grade.
ited fungal growth inhibition was observed with 2-(p-nitro-
phenylhydrazonomethyl)phenols presented in Scheme
4
(Table 3). In contrast, however, virtually every salicylaldehyde
hydrazone and hydrazide synthesized, regardless of whether the
aromatic ring substitutions included halogens, electron donating
or electron withdrawing moieties, showed antifungal activity
through fungal growth inhibition (Tables 1, 2 and 4–7). The most
potent inhibitors of fungal growth included the nitrobenzohydraz-
ides SA6 and SA49, Table 5 and the halogenated benzohydrazide
SA10, Table 4, each of which had antifungal activity at a concentra-
tion below 2 lM for both fungal species tested.
To further test the mammalian cell toxicity of the salicylalde-
hyde analogs presented, we screened selected compounds against
liver and kidney cells to establish toxicity parameters. All analogs
presented in this manuscript with antifungal activity of less than
3.1. Typical procedure for preparation of aryl hydrazides.
Preparation Method I (see Scheme 2)
Preparation of 3,4,5-trihydroxybenzohydrazide (H1-Supple-
mental material). A methanol (300 ml) suspension of 3,4,5-trihy-
droxybenzoic acid and (17 g; 0.1 mol) and the strongly acidic
ion-exchange resin Amberlyst-15 (5 g) was stirred with refluxing
for three days. The insoluble catalyst was separated by filtration,
and washed with methanol (3 ꢂ 10 ml). The combined methanol
filtrates were mixed with hydrazine hydrate (20 ml; 20.5 g;
0.4 mol) and refluxed for 3 h. The volume of the reaction mixture
or equal to 8 lg/mL were further subjected to in vitro mammalian
cell toxicity studies using mammalian kidney cells and human liver
cells (Vero (kidney) cells-ATCC No. CRL-1651 and Hep G2 (liver)
ATCC No. HB-8065). Multiple concentrations of the analog were
used, including the concentration at which fungal inhibition was
observed as well as at 5ꢂ, 10ꢂ and 100ꢂ the minimum inhibition
concentration. Cytotoxicity studies were performed in accordance
with Promega CellTiter 96 Non-RadioactivCell Proliferation Assay
Table 1
Isolated yield and antifungal activity of 2-(hydrazonomethyl)phenols
Entry
R1
R2
Compound
Yield (%)
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
1
2
3
4
5
6
H
H
Cl
Cl
Br
NO2
H
SA1
92
93
94
96
91
85
16
125
62
16
16
4
62
31
62
8
C(CH3)3
H
Cl
Br
H
SA50
SA19
SA28
SA23
SA33
NA
31

4632
G. L. Backes et al. / Bioorg. Med. Chem. 22 (2014) 4629–4636
Table 2
Antifungal activity of 2-(phenylhydrazonomethyl)phenols
Entry
R1
R2
Compound
Yield (%)
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
1
2
3
4
5
6
7
8
9
H
CH3
H
CH3O
Cl
Cl
Br
H
Br
NO2
NO2
H
H
SA2
89
92
97
92
93
92
91
87
88
91
83
16
62
NA
31
31
125
31
8
16
8
SA46
SA38
SA42
SA20
SA29
SA15
SA51
SA24
SA34
SA55
(CH3)3C
H
H
Cl
H
Br
Br
H
NA
31
31
125
31
16
62
16
4
NA
31
2
10
11
NO2
Table 3
Antifungal activity of 2-(p-nitrophenylhydrazonomethyl)phenols
Entry
R1
R2
Compound
Yield (%)
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
1
2
3
4
5
6
7
8
9
H
CH3
H
Cl
Cl
Br
H
Br
NO2
NO2
H
H
SA4
94
93
89
92
93
91
85
88
85
82
125
NA
NA
NA
NA
NA
NA
NA
125
125
4
SA47
SA39
SA21
SA30
SA16
SA52
SA25
SA35
SA56
NA
NA
62
62
NA
NA
62
62
125
(CH3)3C
H
Cl
H
Br
Br
H
10
NO2
was reduced to ꢀ30 ml and left at room temperature for 1 h and
then at ꢁ5 °C. The formed white precipitate was separated by fil-
tration, washed with ice cold methanol (3 ꢂ 5 ml), followed by
ice cold ether (3 ꢂ 10 ml) and dried on air to give pure product
(17.1 g; 93%). 1H NMR (DMSO-d6) d 9.34 (1H, br s, NH), 6.79 (2H,
s, 2-H), and 4.33 (2H, br s, NH2) ppm. 13C NMR (DMSO-d6) d
167.1, 146.1, 136.9, 124.3, and 107.2 ppm.
(3 ꢂ 15 ml) and dried on air overnight to give pure product
(4.86 g; 89%). 1H NMR (DMSO-d6) d 8.67 (1H, br s, NH), 8.39 (2H,
d, J = 7.6 Hz, 3-H), 8.04 (2H, d, J = 7.6 Hz, 2-H), and 4.30 (2H, br s,
NH2) ppm. 13C NMR (DMSO-d6)
d 150.4, 144.8, 129.9, and
124.9 ppm.
3.3. Typical procedure for preparation of
hydrazinomethylphenols. Preparation Method III (see
Scheme 4)
3.2. Typical procedure for preparation of aryl
sulfonohydrazides. Preparation Method II (see Scheme 3)
Preparation of 2-(hydrazonomethyl)-4-nitrophenol (SA33). A
mixture of methanol (300 ml), 2-hydroxy-5-nitrobenzaldehyde
(250 mg; 1.5 mmol), and hydrazine monohydrate (130 mg;
4.5 mmol) was refluxed for three hours. The solvent was evapo-
rated to a solid residue. This residue was mixed with hot methanol
(10 ml) and sonicated at 60 °C for a few minutes. The resulting
mixture was mixed with water (ꢀ100 ml) and then cooled in ice
bath. The resulting insoluble product was separated by filtration,
washed with cold water (3 ꢂ 5 ml) and dried under reduced pres-
sure to give pure product (230 mg; 85%). 1H NMR (DMSO-d6) d 12.0
Preparation of 4-nitrobenzenesulfonohydrazide (H5). A water
(300 ml) solution of hydrazine hydrate (12.5 g; 0.25 mol) was
cooled in ice-water bath to ꢀ5 °C. The temperature was then main-
tained below 8 °C, while the tetrahydrofuran (50 ml) solution of 4-
nitrobenzenesulfonyl chloride (5.54 g, 0.025 mol) was slowly
added with stirring. After the addition was completed the reaction
mixture was stirred at room temperature for thirty minutes and
tetrahydrofuran was evaporated at reduced pressure. The white
solid product was separated by filtration, washed with water

G. L. Backes et al. / Bioorg. Med. Chem. 22 (2014) 4629–4636
4633
Table 4
Antifungal activity of N0-(2-hydroxybenzylidene)benzohydrazides
Entry
R1
R2
Compound
Yield (%)
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
1
2
3
4
5
6
7
8
9
H
CH3
H
CH3O
Cl
Cl
Br
H
Br
NO2
NO2
H
H
SA5
87
92
89
91
95
91
93
94
86
87
81
2
1
NA
4
0.5
8
2
4
8
16
NA
1
1
SA48
SA40
SA44
SA10
SA31
SA17
SA53
SA26
SA36
SA57
C(CH3)3
H
H
Cl
H
Br
Br
H
NA
0.5
0.5
16
2
2
31
16
NA
10
11
NO2
Table 5
Antifungal activity of N0-(2-hydroxybenzylidene)-4-nitrobenzohydrazide
Entry
R1
R2
Compound
Yield (%)
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
1
2
3
4
5
6
7
8
9
H
CH3
H
CH3O
Cl
Cl
Br
H
Br
NO2
NO2
H
H
SA6
92
95
93
89
94
89
91
93
87
87
83
0.5
0.5
31
125
16
31
1
31
1
NA
NA
0.5
0.125
125
2
62
125
2
31
125
16
SA49
SA41
SA45
SA22
SA32
SA18
SA54
SA27
SA37
SA58
(CH3)3C
H
H
Cl
H
Br
Br
H
10
11
NO2
NA
(1H, br s, OH), 8.22 (1H, d, J = 2.8 Hz, 6-H), 7.98 (1H, s, CH@N), 7.96
(1H, d of d, J1 = 8.8 Hz, J2 = 2.4 Hz, 4-H), 7.25 (2H, br s, NH2), and
6.95 (1H, d, J = 9.2 Hz, 3-H) ppm. 13C NMR (DMSO-d6) d 162.6,
140.4, 138.1, 124.7, 123.5, 121.5, and 117.1 ppm.
(2H, d, J = 8.4 Hz, phenyl 2-H), and 6.82 (1H, t, J = 7.3 Hz, phenyl
4-H) ppm. 13C NMR (DMSO-d6) d 151.0, 144.5, 136.8, 130.1, 128.6,
126.7, 124.0, 123.7, 121.9, 120.6, and 112.6 ppm.
3.5. Typical procedure for preparation of benzohydrazones.
Preparation Method V (see Scheme 4)
3.4. Typical procedure for preparation phenylhydrazones.
Preparation Method IV (see Scheme 4)
Preparation of N0-(2-hydroxy-5-methoxybenzylidene)-4-nitro-
benzohydrazide (SA45). A methanol (100 ml) mixture of 4-nitro-
benzohydrazide (181 mg; 1 mmol) and 5-methoxysalicylaldehyde
(152 mg; 1 mmol) was refluxed for 4 h. The solvent volume was
reduced to ꢀ10 ml by atmospheric evaporation followed by evapo-
ration at reduced pressure to yield a white solid residue. The solid
was mixed with ether (20 ml) and sonicated with refluxing for
10 min. The suspension was left at room temperature for 30 min.
An insoluble white precipitate was separated by filtration, washed
with ether (3 ꢂ 5 ml) and dried on air to give pure product
(280 mg; 89%). 1H NMR (DMSO-d6) d 12.31 (1H, br s, OH)
10.52 (1H, s, NH), 8.64 (1H, s, CH@N), 8.34 (2H, d, J = 8.4 Hz,
Preparation of 3,5-dichloro-6-((2-phenylhydrazono)methyl)-
phenol (SA29). A methanol (50 ml) solution of 3,5-dichloro-2-
hydroxybenzaldehyde (191 mg; 1 mmol) and phenylhydrazine
(1.08 mg; 1 mmol) was refluxed for 4 h. Solvent was evaporated to
an oily residue that crystalized upon cooling. This residue was dis-
solved in hot hexane (ꢀ100 ml) and left at room temperature. The
formed crystalline product was separated by filtration, washed with
hexane (3 ꢂ 10 ml) and dried on air to give pure product (260 mg;
92%). 1H NMR (DMSO-d6) d 11.50 (1H, br s, OH), 10.81 (1H, s, NH),
8.07 (1H, s, CH@N), 7.54 (1H, d, J = 2.0 Hz, salicylic 6-H), 7.42 (1H,
d, J = 2.0 Hz, salicylic 4-H), 7.26 (2H, J = 7.6 Hz, phenyl 3-H), 6.96

4634
G. L. Backes et al. / Bioorg. Med. Chem. 22 (2014) 4629–4636
Table 6
Antifungal activity of N0-(2-hydroxybenzylidene)hydroxybenzohydrazide
Entry
R
OH
Compound
Yield (%)
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (
l
g/mL)
1
2
3
4
5
6
7
8
9
H
2
2
2
2
2
4
4
4
4
4
SA83
94
92
89
95
91
92
91
87
89
91
16
16
16
8
8
CH3
CH3O
Cl
Br
H
CH3
CH3O
Cl
SA143
SA123
SA96
SA109
SA84
SA144
SA127
SA97
NA
16
NA
62
4
1
4
62
62
8
NA
NA
NA
NA
125
10
Br
SA110
Table 7
Antifungal activity of N0-substituted 4-(hydrazonomethyl)benzene-1,3-diol
Entry
R
Compound
Yield (%)
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
1
2
3
4
5
6
H
C6H5
4-(O2N)C6H4
2,4-(O2N)2C6H3
C6H5CO
SA8
SA9
SA11
SA68
SA12
SA13
85
95
87
93
91
88
62
31
N/A
NA
31
31
2
1
NA
1
0.5
4-(O2N)C6H4CO
NA
Table 8
Lack of antifungal activity for N0-(2-hydroxybenzylidene)sulfonohydrazides
Entry
R1
R2
Compound
Yield (%)
MIC80 C. albicans (
l
g/mL)
MIC80 C. glabrata (lg/mL)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
C6H5
SA75
SA74
SA73
SA76
SA77
SA78
SA135
SA134
SA133
SA136
SA137
SA138
SA132
SA131
SA130
SA128
91
93
88
94
85
77
88
89
92
93
88
75
91
93
89
87
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
125
NA
125
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
4-(CH3)C6H4
4-(CH3O)C6H4
4-BrC6H4
4-(O2N)C6H4
2-Naphthyl
C6H5
4-(CH3)C6H4
4-(CH3O)C6H4
4-BrC6H4
4-(O2N)C6H4
2-Naphthyl
C6H5
4-(CH3)C6H4
4-BrC6H4r
4-(O2N)C6H4

G. L. Backes et al. / Bioorg. Med. Chem. 22 (2014) 4629–4636
4635
Table 8 (continued)
Entry
R1
R2
Compound
Yield (%)
MIC80 C. albicans (
l
g/mL)
MIC80 C. glabrata (lg/mL)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
OCH3
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
NO2
NO2
NO2
NO2
NO2
NO2
2-Naphthyl
C6H5
4-(CH3)C6H4
4-(CH3O)C6H4
4-BrC6H4
4-(O2N)C6H4
2-Naphthyl
C6H5
4-(CH3)C6H4
4-(CH3O)C6H4
4-BrC6H4
4-(O2N)C6H4
2-Naphthyl
C6H5
4-(CH3)C6H4
4-(CH3O)C6H4
4-BrC6H4
SA129
SA88
SA86
SA87
SA89
SA90
SA91
SA101
SA100
SA99
SA102
SA103
SA104
SA119
SA120
SA121
SA118
SA117
SA116
74
93
95
93
91
88
83
93
91
92
90
88
78
88
89
83
86
79
72
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
4-(O2N)C6H4
2-Naphthyl
Figure 1. Evaluation of mammalian cell toxicity of select active compounds. Cytotoxicity was determined using non-cancerous Vero cells (kidney-african green monkey) and
liver cells (Hep-2G) in accordance with Promega CellTiter 96 Non-RadioactivCell Proliferation Assay (cat. # G4000). Compounds were diluted in media and all assays were
done in triplicate. Average values are presented, with SD. Cells were incubated in the presence of the compounds for 24 h at 37 °C and 5% CO2. Tetrazolium dye solution was
added to each well and allowed to incubate for 1–4 h. Stabilization/stop solution was added and allowed to sit at room temperature for 1 h. Formazan product was scored
spectrophotometrically with an automatic plate reader set at 570 nm. 0.1% saponin was used as a control to measure cell death. Wells containing 1% DMSO and no drug were
used as a positive control and the average absorbance at 570 nm were set to 100%. The percent of cells viable were calculated as a ratio between the average absorption at
570 nm of the drug containing wells/average absorption at 570 nm of the control (no drug). Ratios were multiplied by 100 to give the values as a percentage.
benzohydrazide 3-H), 8.15 (2H, d, J = 8.4 Hz, benzohydrazide 2-H),
7.14 (1H, d, J = 2.4 Hz, salicylic 6-H), 6.90 (1H, d of d, J1 = 8.8 Hz,
J2 = 2.4 Hz, salicylic 4-H), 6.84 (1H, d, J = 8.8 Hz, salicylic 2-H) and
3.71 (3H, s, OCH3) ppm. 13C NMR (DMSO-d6) d 152.8, 152.2,
150.0, 148.9, 139.3, 129.9, 124.3, 119.6, 119.3, 118.0, 112.5, and
65.1 ppm.
3.6. Typical procedure for preparation sulfonohydrazones.
Preparation Method VI (see Scheme 4)
Preparation of N0-(2-hydroxybenzylidene)naphthalene-2-sul-
fonohydrazide (SA78). A methanol (20 ml) solution of salicylalde-
hyde (122 mg; 1 mmol) and naphthalene-2-sulfonohydrazide

4636
G. L. Backes et al. / Bioorg. Med. Chem. 22 (2014) 4629–4636
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(222 mg; 1 mmol) was refluxed for 2 h. Solvent was evaporated at
reduced pressure to an oily residue. This residue was mixed with
ether (ꢀ10 ml) and sonicated at 5 °C for 5 min. Ether solution
was decanted and mixed with hexane (ꢀ50 ml). The resulting
slightly milky mixture was left at room temperature for 1 h. The
formed white powdery product was separated by filtration,
washed with hexane (3 ꢂ 5 ml) and dried on air to give pure prod-
uct (250 mg; 77%). 1H NMR (DMSO-d6) d 11.61 (1H, s, OH), 10.15
(1H, s, NH), 8.55 (1H, s, naphthalene 1-H), 8.19 (1H+1H, d+s, naph-
thalene 4-H+CH@N), 8.12 (1H, d, J = 8.8 Hz, naphthalene 3-H), 8.00
(1H, d, J = 8.00 Hz, naphthalene 8-H), 7.85 (1H, d of d, J1 = 8.8 Hz,
J2 = 1.6 Hz, naphthalene 5-H), 7.66 (2H, m, naphthalene 6,7-H),
7.44 (1H, d of d, J1 = 8.0 Hz, J2 = 1.6 Hz salicylic 6-H), 7.17 (1H, d
of t, J1 = 8.0 Hz, J2 = 1.6 Hz, salicylic 4-H), 6.80 (1H, d, J = 7.6 Hz,
salicylic 3-H), and 6.77 (1H, t, J = 7.2 Hz, salicylic 5-H) ppm.
13C NMR (DMSO-d6) d 157.2, 146.8, 136.5, 135.1, 132.4, 132.2,
130.1, 130.0, 129.8, 129.1, 128.6, 128.4, 127.9, 123.1, 120.1,
119.7, and 116.8 ppm.
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Acknowledgments
This work was supported in part by funding from the Louisiana
State Health Sciences Center, School of Medicine, Department of
Pharmacology, Louisiana Lions Eye Foundation and by an unre-
stricted grant from Research to Prevent Blindness, New York, NY
(LSU Department of Ophthalmology-LSUHSC).
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Supplementary data
Supplementary data (spectroscopic characterization, including
electronic images of NMR data, literature data and CAS numbers
and additional cytotoxicity screens are provided for all new com-
pounds as well as compounds with biological activity) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2014.07.022.
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