Potent antimicrobial agents against azole-resistant fungi based on pyridinohydrazide and hydrazomethylpyridine structural motifs

Article abstract of DOI:10.1016/j.bmc.2015.04.040

Abstract Schiff base derivatives have recently been shown to possess antimicrobial activity, and these derivatives include a limited number of salicylaldehyde hydrazones. To further explore this structure-activity relationship between salicylaldehyde hydrazones and antifungal activity, we previously synthesized and analyzed a large series of salicylaldehyde and formylpyridinetrione hydrazones for their ability to inhibit fungal growth of both azole-susceptible and azole-resistant species of Candida. While many of these analogs showed excellent growth inhibition with low mammalian cell toxicity, their activity did not extend to azole-resistant species of Candida. To further dissect the structural features necessary to inhibit azole-resistant fungal species, we synthesized a new class of modified salicylaldehyde derivatives and subsequently identified a series of modified pyridine-based hydrazones that had potent fungicidal antifungal activity against multiple Candida spp. Here we would like to present our synthetic procedures as well as the results from fungal growth inhibition assays, mammalian cell toxicity assays, time-kill assays and synergy studies of these novel pyridine-based hydrazones on both azole-susceptible and azole-resistant fungal species.

Full text of DOI:10.1016/j.bmc.2015.04.040

Accepted Manuscript
Potent Antimicrobial Agents Against Azole-Resistant Fungi Based on Pyridi-
nohydrazide and Hydrazomethylpyridine Structural Motifs
Gregory L. Backes, Branko S. Jursic, Donna M. Neumann
PII:
S0968-0896(15)00348-X
http://dx.doi.org/10.1016/j.bmc.2015.04.040
BMC 12252
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 February 2015
2 April 2015
13 April 2015
Please cite this article as: Backes, G.L., Jursic, B.S., Neumann, D.M., Potent Antimicrobial Agents Against Azole-
Resistant Fungi Based on Pyridinohydrazide and Hydrazomethylpyridine Structural Motifs, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.040
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Potent Antimicrobial Agents Against Azole-Resistant Fungi Based on Pyridinohydrazide and
Hydrazomethylpyridine Structural Motifs
Authors: Gregory L. Backes a, Branko S. Jursicb,c, and Donna M. Neumanna,d
Affiliations: aLouisiana State University Health Sciences Center, Department of Pharmacology
and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901
Perdido St. New Orleans, LA. 70112; bDepartment of Chemistry, University of New Orleans,
New Orleans, LA 70148; cSTEPHARM, LLC., P.O. Box 24220, New Orleans, LA. 70184;
dDepartment of Ophthalmology, LSUHSC, New Orleans
*Corresponding Author: Donna M. Neumann, PhD. LSUHSC Department of Pharmacology
and Experimental Therapeutics, New Orleans LA 70112
Email: dneum1@lsuhsc.edu
Phone: 504-568-3179

Abstract
Schiff base derivatives have recently been shown to possess antimicrobial activity, and these
derivatives include a limited number of salicylaldehyde hydrazones. To further explore this
structure-activity relationship between salicylaldehyde hydrazones and antifungal activity, we
previously synthesized and analyzed a large series of salicylaldehyde and formylpyridinetrione
hydrazones for their ability to inhibit fungal growth of both azole-susceptible and azole-resistant
species of Candida. While many of these analogs showed excellent growth inhibition with low
mammalian cell toxicity, their activity did not extend to azole-resistant species of Candida. To
further dissect the structural features necessary to inhibit azole-resistant fungal species, we
synthesized a new class of modified salicylaldehyde derivatives and subsequently identified a
series of modified pyridine-based hydrazones that had potent fungicidal antifungal activity
against multiple Candida spp. Here we would like to present our synthetic procedures as well as
the results from fungal growth inhibition assays, mammalian cell toxicity assays, time-kill assays
and synergy studies of these novel pyridine-based hydrazones on both azole-susceptible and
azole-resistant fungal species.

Introduction
The incidence rate for fungal infections has increased dramatically in the last few decades.
For instance, recent analyses of hospital data in France revealed that the annual rate of invasive
fungal infections increased by 16% and the annual rate of deaths from invasive fungal infections
increased 33% in the last decade (1), and it is reasonable to assume that world trends are similar.
The most common culprits for the onset of invasive infections arise from the Candida spp.
Candidiasis is most commonly associated with Candida albicans, Candida glabrata and
Candida krusei microorganisms (2;3). The immune-compromised -- specifically transplant
recipients, cancer patients and those infected with AIDS – are particularly susceptible to
candidiasis infections (4;5). While classes of antifungal therapies, such as the polyenes, azoles
and the echinocandins, are available for the treatment of invasive Candida spp. infections, many
have adverse effects or other drawbacks associated with their use, including nephrotoxicity,
hepatotoxicity and the development of resistance in the target microorganism (6;7). In addition,
oral candidiasis is one of the most frequent opportunistic infections associated with HIV and
AIDS, and these infections have largely been controlled with the use of topical polyenes or
systemic azoles (8). However, the overuse of azoles in the treatment of oral candidiasis has
ultimately led to the development of drug resistance. For example, it has been reported that up to
33% of AIDS patients have azole-resistant oral candidiasis infections (8). This resistance to the
azoles stems not only from the drugs’ fungistatic effects, but also from overexpression of efflux
pumps (9) and point mutations in the ERG11 gene (10) in Candida albicans. In addition,
Candida glabrata develops resistance during prolonged treatment with azole antifungals (11).
For these reasons, there is a continuous demand for the discovery of novel therapeutics to treat
Candida spp. infections, particularly if they have a different method of action than the azoles.

Some literature data suggest that simple Schiff bases possess antimicrobial activity and
might have different biological targets than currently available antifungal agents (12; 13). Others
have previously studied the antimicrobial activity of a limited number of salicylaldehyde
hydrazones, however the mechanism of action for these derivatives has not been clearly defined
(14). It was suggested that the activity of these molecules may be based on the formation of
Schiff bases with important amino groups of the microbial cells. To further explore this
structure-activity relationship and to identify possible pharmacophores, we previously
synthesized and analyzed a large series of salicylaldehyde (15) and formylpyridinetrione
hydrazones (16) for their ability to inhibit fungal growth of both azole-susceptible and azole-
resistant species of Candida. Additionally, we explored potential mechanisms of action for these
compounds. Many of these analogs showed excellent growth inhibition with low mammalian cell
toxicity. However, this activity did not extend to azole-resistant species of Candida.
Furthermore, our preliminary mechanistic studies indicated that these compounds were likely
fungistatic, as with azoles. Therefore, a new structure-activity relationship study was designed
using modified salicylaldehyde derivatives not previously studied, with the goal of determining
the pharmacophore required to produce antifungal activity against azole-resistant fungi. We
subsequently identified a series of modified pyridine-based hydrazones that had potent fungicidal
antifungal activity against multiple Candida spp. Here we would like to present our synthetic
procedures as well as the results from fungal growth inhibition assays, mammalian cell toxicity
assays, time-kill assays and synergy studies of these novel pyridine-based hydrazones on both
azole-susceptible and azole-resistant fungal species (8;17). In summary, we have identified the
structural components that illicit potent fungal growth inhibition of both azole-susceptible and
azole-resistant fungal species and have synthesized a broad class of structural analogs.

Furthermore, these analogs show limited toxicity against mammalian cells in vitro and several
exhibit fungicidal activity. Considering these data, the analogs presented here may be a
promising alternative route for the development of novel antifungal agents.
Results
1.A Chemistry
All hydrazone derivatives were prepared from the corresponding aromatic aldehydes and
hydrazine derivatives (phenylhydrazines, hydrazides, or sulfonohydrazides). All aromatic
aldehydes, phenylhydrazine, 4-nitrophenylhydrazine, and 2,4-dinitrophenylhydrazine were
purchased from either Sigma-Aldrich or Ark Pharm. All hydrazines were prepared from the
corresponding carboxylic acid through Amberlyst-15 catalyzed ethanol esterification, as
previously reported (18). Following esterification, the catalyst was removed by filtration. The
filtrate was then mixed with hydrazine hydrate and refluxed for several hours to yield the
desirable acid hydrazide (Scheme 1). Because the catalyst is recyclable and isolation of the
intermediate ester is not necessary, this synthetic procedure is a one-pot reaction. This synthetic
method also tolerates the presence of the phenol group and pyridine moiety. The
sulfonohydrazides were synthesized using the large scale preparation method that we previously
published (Scheme 2) (15). Hydroxybenzylidenepyridinohydrazides were prepared from 5-
substituted salicylaldehyde and pyridinohydrazides in refluxing ethanol without an acid catalyst
(Scheme 3) (19;20). This reaction was driven by increasing the reactant concentration,
allowing the product to selectively precipitate from the reaction mixture. To efficiently
synthesize these derivatives in greater quantities, we developed a large scale synthetic method (3
L round bottom flask using 1.5 L of ethanol to make 100g of product) using a modified

Hickman still head with an attached condenser and distillation receiving flask (Scheme 3).
Following this scheme, individual reactions were prepared in 50 ml round bottom flasks to
produce about 300 mg of each member of the diverse pyridine-based hydrazone library. This
was accomplished by slow distillation of the mixture to remove the ethanol. The rate of
distillation was controlled by both the size of pre-column and the temperature of the reaction
mixture. Distillation continued until precipitate started to form in the reaction flask. The
distilled ethanol was recycled and reused in the preparation of the more than fifty members of
our hydrazone library. The same procedure was used for the preparation of individual members
of the hydrazonomethylpyridine library (Scheme 4).
1.B Antifungal activity
1.b.1 Minimum Inhibitory Concentration (MIC) studies
The antifungal activity of the pyridinohydrazide and hydrazomethylpyridine derivatives
described in the above schemes were evaluated in vitro using Candida albicans (ATCC no.
10231) and Candida glabrata (ATCC no. 48435). In addition, the compounds were evaluated
for activity against a previously-described clinical isolate of C. albicans known to have
developed azole-resistance (a kind gift from Dr. Glen Palmer, LSUHSC). These isolates were
obtained over a two year period from a single AIDS patient and the development of the azole-
resistance mechanisms have been extensively characterized (8). MIC assays were performed on
the clinical isolates 1 and 17 (annotated TW1 and TW17, respectively), where isolate 1 was used
as an azole-susceptible control and isolate 17 was considered azole-resistant. All assays were
done in accordance with NCCLS reference documents (21). The results of these screenings are
summarized in Tables 1-9 as the minimum concentrations that inhibited more than 80% of

fungal growth (MIC80) as compared to positive controls containing 1% DMSO. Assays were
performed in HEPES-buffered RPMI-1640 media. All MIC screens used a visual scoring method
as opposed to spectroscopic analysis, due to the altered absorbance spectra of many of the
compounds.
Table 1 lists the MICs of the picolinohydrazides (AR1-AR6). These derivatives are all
hydrazones of salicylaldehyde and five-substituted salicylaldehyde. All of the synthesized
derivatives in this group consistently inhibited fungal growth of the azole-susceptible fungal
species at very low concentrations (~3-15 uM - Table 1). In addition, 4 of the 6 derivatives
(namely AR2, AR4, AR5 and AR6- Table 1) inhibited fungal growth in the azole-resistant
fungi TW17 and C. glabrata at a concentration range of 3-30 uM (Table 1). Furthermore, the
position of the nitrogen in the pyridine ring proved to have little effect on the ability of the
resulting hydrazone analog to act as a fungal growth inhibitor. For example, 5-substituted
salicylaldehyde hydrazones of nicotinohydrazide (Table 2) also consistently inhibited fungal
growth, even though several derivatives were less active against azole-resistant isolates (AR8
and AR12). Additionally, substituted salicylaldehyde hydrazones of isonicotinohydrazide all
possessed antifungal activity with the most potent again being the 5-nitrosalicylaldehyde
hydrazone (Table 3; AR18).
To determine the effects of an OH-group or oxidizing pyridine moiety on fungal growth
inhibition, these two functionalities were individually incorporated on the nicotinohydrazide
derivatives. Unlike the previous nicotinohydrazide tested where all derivatives were active (see
Table 2) the incorporation of the OH-functionality in the two position of the pyridine ring led to
inconsistent results. Introducing the hydroxyl group diminished or even eliminated the
antifungal activity previously observed (Table 4). However, we did observe growth inhibition at

very low concentrations across all Candida albicans isolates treated with AR19. In addition,
AR22 was one of our most potent compounds against Candida glabrata. In the case of the
pyridine oxides presented in Table 5, activity of the analogs was consistent, with all derivatives
exhibiting inhibition of fungal growth, regardless of the azole-sensitivity or resistance. However,
in general the MICs of these analogs were higher than other classes of compounds (Table 5).
The most potent analog in this class against azole-resistant TW17 was AR28 (activity at 2 µM).
Our data suggested that the pyridine structural motif might be essential for antifungal
activity, particularly for azole-resistant isolates. Various hydrazones of 2-pyridinecarbaldehyde
were prepared for further analysis of this motif (Table 6 and Table 7). Each of these analogs
lacks the corresponding salicylaldehyde moiety present on the active derivatives presented in
Tables 1-5. The phenylhydrazone AR29, and the picolinohydrazide AR34 (Table 6) showed
moderate activity, likely due to structural similarities to compounds shown in Table 1, but in
general, the loss of the salicyl moiety resulted in a significant loss of antifungal activity.
Furthermore, sulfohydrazide derivatives showed no antifungal activity (Table 7) which is
consistent with our previously published data showing that compounds bearing a sulfonyl group
are essentially inactive (15).
Because phenylhydrazone AR29 showed moderate activity, two new groups of
phenylhydrazones were prepared and tested to determine if we could increase the antifungal
activity. One set of analogs was synthesized from 3-pyridinecarbaldehyde (Table 8) and the
other from 4-pyridinecarbaldehyde (Table 9). As in the case of 2-pyridinecarbaldehyde,
moderate antifungal activity was observed with phenylhydrazones AR44 and AR48, but not with
any other derivatives. Based on these data, our most consistent results with respect to growth

inhibition of both azole-susceptible and azole-resistant fungal isolates were obtained with
hydrazones bearing both salicylaldehyde and pyridine structural motifs.
1.b.2 Mammalian Cell Toxicity studies
Following the fungal growth inhibition assays, compounds displaying antifungal activity at
concentrations below or equal to 8 g/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). Active analogs meeting this criterion
were screened at multiple concentrations, including the lowest MIC among the Candida isolates
as well as at multiples of 5, 10 and 100 times the MIC. Cytotoxicity studies were performed in
accordance with Promega CellTiter 96 Non-RadioactivCell Proliferation Assay (cat # G4000).
All cytotoxicity studies used a negative control (wells of either kidney or liver cells containing
1% DMSO but lacking the analog), a positive control (wells of either kidney or liver cells treated
with the known cytotoxic agent, 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 remained alive and viable following either overnight
incubation with the analogs or 5-minute treatment with 0.1% saponin. We tested representative
active compounds from each of the structural classes synthesized. We found that the
picolinohydrazide AR2 (Table 1) showed only minimal toxicity, even at the highest
concentration of 100 times greater than the MIC (Fig 1, Panel A). Similar trends in toxicity
were observed with other active compounds in Table 1, as shown in the Supplemental Materials.
However, the nicotinohydrazides, represented by AR12 and shown in Table 2, had elevated
toxicity in the kidney cells, even at the lower concentrations of 5 times the MIC (Fig 1 Panel B

and Supplemental Material). Isonicotinohydrazide AR18 (Table 3) was not toxic through 10X
and showed minimal cytotoxicity at the 100X concentration. Similarly, the 2-hydroxy
nicotinohydrazide AR19 showed minimal toxicity at concentrations through 10X (Fig1 panels C
and D). Unfortunately, one of the more promising pyridine oxide analogs (Table 5) was
cytotoxic to liver and kidney cells, even at the 5X concentrations (AR27 Fig 1 Panel E). Finally,
the aryl hydrazone pyridines of AR44 and AR48 (Tables 8 and 9) showed no cell toxicity
through 10X concentrations of the MIC, with the 100X concentrations being toxic to both kidney
and liver cells (Fig 1). In summary, while some toxicity was observed with a few of the
analogs tested, the majority of compounds analyzed had limited cytotoxicity in both mammalian
cell lines. All results from the toxicity screenings outside of Fig 1 are provided in the
Supplemental Information.
1.b.3 Time-Kill Assay
One of the objectives of this study was to determine the cidal or static nature of the analogs
that were active against TW17, the azole-resistant clinical isolate. Azoles affect Candida spp. by
inhibiting ergosterol biosynthesis, which ultimately prevents their reproduction. This effect has
led to the classification of azoles as fungistats. Determining the fungistatic or fungicidal nature
of our synthesized compounds allows a comparison to the azoles, in addition to improving our
understanding of how the structural motifs might lend toward an alternate mechanism of action.
Therefore, time-kill assays were performed using the representative analogs that were active
against the azole-resistant TW17 at concentrations at or below 8 ug/mL. Solid drugs were
dissolved in DMSO to a concentration of 10 mg/ml. This stock solution was then further diluted
in water to the desired experimental concentration, where the experimental concentration was

determined to be four times that of the MIC for TW17 (as reported in the Tables). Individual
colonies of TW17 were suspended in 5 ml of sterile water and adjusted to achieve a McFarland
reading between 0.08 and 0.12. The inoculum was then further diluted in sterile water at a ratio
of 1:100. The experimental “Kill Curve” tubes were prepared by combining 2.5 ml of above
diluted inoculum, 21.7 ml of HEPES buffered RPMI-1640 media, and 800 μl of experimental
drug dilution in a 50-ml tissue culture tube. A “Growth Control” tube was prepared by
combining 2.5 ml of inoculum, 21.7 HEPES buffered RPMI-1640 media, and 800 μl of water-
DMSO solution equal to the DMSO concentration of the experimental drug dilution. A “Sterile
Control” tube was prepared by combining 24.2 ml of HEPES-buffered RPMI-1640 media and
800 μ of water-DMSO solution as in the “Growth Control”. All tubes were incubated for 48
hours at 35°C, 50 RPM. 500 μl samples were taken from the tubes at timepoints of
predetermined intervals during this incubation.
Timepoints for the “Kill-Curve” and “Growth Control” tubes were taken at 0, 1, 2, 4, 6, 8,
24, and 48 hours after the start of incubation. Timepoints for “Sterile Control” were taken at 0
and 48 hours. Each timepoint sample was serially diluted 1:10 in sterile 0.9% NaCl solution up
to 5 times. 100 μl of each sample was spread onto YM agar plates with a sterile bent spreading
rod. Plates were incubated at 35°C overnight and colonies were then counted on each plate.
Colonies on plates from diluted timepoints were used to estimate uncountable undiluted samples.
The number of colonies in undiluted samples was estimated to be 10n times greater than the
diluted sample, where n was the number of dilutions made.
From our time-kill assays, we identified several compounds that were fungistatic. In
general, analogs with the picolinohydrazide structure (Figure 2), the nicotinohydrazide structure
(supplemental material) and the isonicotinohydrazide structure were fungistatic and no increase

in fungal growth could be observed after 48 hours in the presence of these analogs. An
exception to this generalization regards the presence of a chloro- or bromo-substituent in the 4-
position of the salicylaldehyde moiety; these analogs showed fungicidal activity (see Figure 2,
Panel B AR5). With respect to the active pyridine oxide compound AR27 (Table 5), the
presence of the bromo-substituent did not render the analog fungicidal; this compound instead
showed fungistatic activity. Finally, the aryl substituted pyridines lacking a salicylaldehyde
moiety, AR44 and AR48 (Tables 8 and 9) were fungicidal, with no observed colonies after 24
hours, making these derivatives promising analogs for future exploration. Considering their
high cell toxicity and their fungistatic nature, the pyridine oxide derivatives show the least
promise for future antifungal use.
1.b.4 Synergy studies: Checkerboard Assay
Considering that two different modes of activity (cidal and static) were observed
depending on the structural motifs present in our analogs, we performed a series of checkerboard
assays to determine if representative active compounds could act in synergy with the azole,
fluconazole. Each of these assays tested multiple concentrations of the selected analog in the
presence of multiple concentrations of fluconazole. All checkerboard assays were prepared in
96-well plates. 200 μl of previously prepared drug solutions were added to each well in column
2. Using a multichannel pipette, 100 μl from each well in column 2 was serially diluted (1:2)
horizontally across the plate, stopping at column 10. Contents of each well were mixed
thoroughly. Column 11 was reserved for fluconazole MIC control and contained no other drug.
100 μl of media was added to each well in columns 2-10 except row B. Each well was mixed
thoroughly with a multichannel pipette. 100 μl was then removed and discarded from these

wells. 100 μl of previously prepared fluconazole dilution was added to each well in row B. Using
a multichannel pipette, 100 ul from each well was serially diluted (1:2) vertically down the plate,
stopping at row F. Contents of each well were mixed thoroughly. Row G was reserved for tested
drug MIC control and contained no fluconazole. Well G11 contained neither drug and served as
a drug-free growth control.
The fungal samples were prepared from glycerol stocks of Candida albicans ATCC. No
10231. The inoculum’s concentration was adjusted to a McFarland standard between 0.08 and
0.12. Inocolum was then diluted at a ratio of 1:37.5 in buffered media. 100 μl of inoculum-media
solution was added to each well. Following inoculation, the 96-well plates were wrapped in
parafilm to prevent evaporation and incubated for overnight at 35°C. After 18 hours, the wells
showing growth were recorded.
Synergistic activity was determined based on the MIC read from these plates. The
fractional inhibitory concentration (FIC) for a given well is the ratio of the minimum inhibitory
concentration of one drug A in the presence another drug B to the minimum inhibitory
concentration with only drug A. The FIC was calculated for the test drug in each of the wells
along the growth/no-growth interface at varying concentrations of fluconazole, then these values
were averaged to determine the average FIC for the test drug. The same process was used to
calculate the average FIC for fluconazole at varying concentrations of the test drug. The sum of
these two FICs gives the FIC index which can be used to determine the synergistic effect
between the two drugs (22). An FIC index value less than 1 indicates synergistic interactions
between the two drugs while an FIC index value greater than or equal to 1 indicates antagonistic
interactions. Table 10 shows the results from our checkerboard assays, indicating no synergy
exists between fluconazole and our new analogs.

Chemistry Experimental
Thin-layer chromatographic analysis (TLC) was performed on each compound using silica gel
on aluminum foil-backed glass plates and was detected under ultraviolet (UV) light. The 1H and
13C NMR spectra were run on Varian 400 MHz Unity instruments in DMSO-d6. The solvent
signals were used as internal NMR chemical shift references. All products were purified by
crystallization from ethanol. When necessary during the preparation of the hydrazides (HD1-
HD8, Scheme 1), some intermediate esters were purified by short silica gel (40-70 mm) filtration
with various mixtures of ethyl acetate and dichloromethane as eluents. Silica gel was purchased
from Sorbent Technologies. Substituted phenylhydrazines were prepared from corresponding
anilines by following an existing preparation procedure (23). All solvents were purchased from
Fisher Scientific. All reagents were purchased from Sigma-Aldrich and were analytical grade.
Preparation 5-nitrosalycilaldehyde. Acetic acid (20 ml) was added to a large (190x100 mm)
crystallization dish equipped with a magnetic stirrer and placed under a fume hood with aqueous
sodium carbonate and a nitrogen oxide trap. This acetic acid salicylaldehyde (12.2 g; 0.1 mol)
was added to concentrated nitric acid (80 ml). The resulting dark red reaction mixture was
stirred at room temperature for approximately 10 minutes, when the reaction started to
spontaneously heat up forming a large quantity of nitric oxide (WARNING: Do not use a
reaction scale four times or greater than this under these conditions, as the reaction mixture will
explode due to the its exothermicity). The mixture was stirred at room temperature for an
additional hour; no cooling of the reaction mixture was necessary. During this period, the color
of the reaction mixture changed from dark red to orange. This reaction mixture was then poured
on crushed ice (400 g) and left at room temperature for one hour. The resulting yellow

suspension was filtered and the solid precipitate was collected. The precipitate was washed with
ice cold water (3x20 ml) and -5°C methanol (3x15 ml), then dried at room temperature to give
pure product (14.6 g; 82%).
Typical procedure for the preparation of hydrazides. Preparation of 2-
Hydroxybenzohydrazide (HD2). An ethanol (300 ml) suspension of salicylic acid (13.8 g; 0.1
mol) and strongly acidic ion-exchange resin, Amberlyst-15 (5g) were stirred with refluxing for
three days. Insoluble catalyst was separated by filtration, and washed with ethanol (3x20 ml).
Combined ethanol filtrates were mixed with hydrazine hydrate (20 ml; 20.5 g; 0.4 mol) and
refluxed with slow solvent distillation using the modified Hickman still apparatus (Scheme 3).
After three hours of refluxing, the volume of the reaction mixture was reduced to about 50 ml
and white precipitate started to form. The white suspension was cooled to room temperature and
then left at -5°C for one hour. Insoluble product was separated by filtration, washed with ice
cold ethanol, and dried on air to give pure product (13.2 g; 87%). 1H-NMR (DMSO-d6) ) δ 12.3
(1H, broad s, OH), 10.0 (1H, broad s, NH), 7.0 (1H, d of d, J1 = 8.4 Hz, J2 = 1.6 Hz, 6-H), 7.33
(1H, t, J =8. 4 Hz. 4-H), 6.89 (1H, d, J =8.4 Hz, 3-H), 6.86 (1H, t, J = 8.4 Hz, 5-H), and 4.70
(2H, brad s, NH2) ppm. 13C-NMR (DMSO-d6) δ 168.7, 160.3, 134.1, 127.8, 119.3, 118.0, and
115.1 ppm.
1
Picolinohydrazide (HD4). Isolated yield 81% (11.1 g). H-NMR (DMSO-d6) δ 9.23 (1H, broad
s, NH), 8.56 (1H, d, J = 4.8 Hz, 6-H), 7.98 (1H, d, J = Hz, 3-C), 7.93 (1H, t = 7.6 Hz, 5-H), 7.51
(1H, t, J = 4.8 Hz, 4-H), and 4.64 (2H, broad s, NH2) ppm. 13C-NMR (DMSO-d6) δ 163.4,
150.5, 149.2, 138.3, 126.9, and 122.5 ppm.

1
Nicotinohydrazide (HD5). Isolated yield 92% (12.6 g). H-NMR (DMSO-d6) δ 9.99 (1H, s,
NH), 8.95 (1H, s, 2-H), 8.64 (1H, m, 4-H), 8.13 (1H, m, 6-H), 7.44 (1H, m, H-5), and 4.61 (2H,
NH2) ppm. 13C-NMR (DMSO-d6) δ 165.2, 152.4, 148.8, 135.4, 129.5, and 124.2 ppm.
1
Isonicotinohydrazide (HD6). Isolated yield 94% (12.9 g). H-NMR (DMSO-d6) δ 10.13, 8.66
(2H, d, J = 6.0 Hz, 3-H), 7.71 (2H, d, J = 6.0 Hz, 2-H), and 4.69 (2H, NH2) ppm. 13C-NMR
(DMSO-d6) δ 164.8, 150.8, 140.9, and 121.7 ppm.
2-Hydroxynicotinohydrazide (HD7). Isolated yield 84% (12.9 g). 1H-NMR (DMSO-d6) δ 7.93
(1H, d, J = 6.0 Hz), 7.85-7.80 (2H, m), and 8.32 (1H, d, J = 9.6 Hz) ppm. 13C-NMR (DMSO-d6)
δ 164.3, 163.1, 142.0, 139.4, 137.7, 119.8, and 111.9 ppm.
1
3-(Hydrazinecarbonyl)pyridine 1-oxide (HD8). Isolated yield (12.1 g; 79%). H-NMR
(DMSO-d6) δ 10.06 (1H, broad s, NH), 8.50 (1H, s, 2-H), 8.32 (1H, d, J = 6.4 Hz, 4-H), 7.69
(1H, d, J = 8.0 Hz, 6-H), 7.49 (1H, t, J = 7.2 Hz, 5-H), and 4.60 (2H, broad s, NH2) ppm. 13C-
NMR (DMSO-d6) δ 162.6, 141.3, 137.9, 133.2, 127.2, and 124.4 ppm.
Typical Procedure for preparation of aryl sulfonohydrazides. Preparation of 4-
methoxybenzenesulfonohydrazide (SH3). A water (300 ml) solution of hydrazine hydrate
(12.5 g; 0.25 mol) was cooled in an ice-water bath to 5°C. A tetrahydrofuran (50ml) solution
containing 4-methoxybenzenesulfonyl chloride (5.16 g, 0.025 mol) was slowly added while
stirring slowly. Following this addition, the reaction mixture was stirred at 5°C for an additional
thirty minutes, and then the tetrahydrofuran was evaporated at reduced pressure and room
temperature. The white solid product was separated by filtration, washed with cold water (3x15
1
ml) and dried on air overnight to give pure product (4.4 g; 88%). H-NMR (DMSO-d6) δ 8.20

(1H, s, NH), 7.72 (2H, d, J = 8.8 Hz, 2-H), 7.09 (2H, d, J = 8.8 Hz), 3.99 (2H, s, NH2), and 3.81
(3H, s OCH3) ppm. 13C-NMR (DMSO-d6) δ 163.1, 130.5, 130.0, 114.9, and 56.3 ppm.
4-bromobenzenesulfonohydrazide (SH4). Isolated yield 5.6 g (89%). 1H-NMR (DMSO-d6) δ
8.47 (1H, s, NH), 7.78 (2H, d, J = 8.0 Hz, 2-H), 7.70 (2H, d, J = 8.0 Hz, 3-H), and 4.16 (2H, s,
NH2) ppm. 13C-NMR (DMSO-d6) δ 138.1, 132.8, 130.4, and 127.3 ppm.
4-Nitrobenzenesulfonohydrazide (SH5). Isolated yield 4.86 g (89%). 1H-NMR (DMSO-d6) δ
8.67 (1H, broad 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, broad s, NH2) ppm. 13C-NMR (DMSO-d6) δ 150.4, 144.8, 129.9, and 124.9 ppm.
2-Naphthalenesulfonohydrazide (SH6). Isolated yield 5.2 g (93%). 1H-NMR (DMSO-d6) δ
8.53 (1H, s, NH), 8.47 (1H, s), 8.12 (2H, m), 8.01 (1H, d, J = 7.6 Hz), 7.84 (1H, m), 7.67 (2H,
m), and 4.00 (2H, s, NH2) ppm. 13C-NMR (DMSO-d6) δ 135.9, 135.1, 132.4, 129.9, 129.8,
129.7, 129.4, 128.5, 128.2, and 123.8 pmm.
Typical procedure for preparation of individual members of pyridinohydrazide (Scheme 3) and
hydrazonomethylpyridine (Scheme 4) libraries (Only NMR characterization is presented for
AR1-AR6, Table 1). Preparation of (E)-N'-(2-hydroxybenzylidene)picolinohydrazide (AR1).
An ethanol (15 ml) mixture of salycilaldehyde (122 mg; 1 mmol) and picolinohydrazide (137
mg; 1 mmol) was refluxed with slow solvent distillation using the modified Hickman still
(Sheme 3). After the volume of the reaction mixture was reduced to about 3-5 ml white
precipitate started to form. This suspension was left at room temperature for one hour followed
by an additional hour at 0ºC. Insoluble product was removed by filtration, washed with ice cold
1
ethanol (3x2 ml) and dried on air to give pure product (220 mg) with a yield of 91%. H-NMR
(DMSO-d6) δ 12.51 (1H, s, NH), 11.42 (1H, s, OH), 8.83 (1H, s, CH=N), 8.71 (1H, d, J = 4.4

Hz, picolino 3-H), 8.12 (1H, d, J = 8.0 Hz, picolino 6-H), 8.04 (1H, t, J = 8.0 Hz, picolino 5-H),
7.66 (1H, m, picolino 4-H), 7.45 (1H, d, J = 7.6 Hz, salicylic 6-H), 7.29 (1H, t, J = 8.0 Hz,
salicylic 4-H) and 6.92 (2H, m, salicylic 3-H and 5-H) ppm. 13C-NMR (DMSO-d6) δ 161.1,
158.4, 150.8, 149.8, 149.3, 138.8, 132.2, 130.6, 127.9, 123.5, 120.1, 119.3, and 117.2 ppm.
(E)-N'-(2-Hydroxy-5-methylbenzylidene)picolinohydrazide (AR2). Isolated yield 240 mg
(94%). 1H-NMR (DMSO-d6) δ 12.47 (1H, s, NH), 11.14 (1H, s, OH), 8.77 (1H, s, CH=N), 8.71
(1H, d, J = 4.8 Hz, picolino 3-H), 8.12 (1H, d, J = 7.6 Hz, picolino 6-H), 8.05 (1H, t, J = 8.0 Hz,
picolino 5-H), 7.67 (1H, t, J = 6.0 Hz, picolino 4-H), 7.26 (1H, s, salicylic 6-H), 7.10 (1H, d, J =
8.4 Hz, salicylic 4-H), 6.82 (1H, d, J = 8.4 Hz, salicylic 3-H), and 2.24 (3H, s, CH3) ppm. 13C-
NMR (DMSO-d6) δ 156.2, 150.7, 149.9, 149.3, 138.8, 132.9, 130.5, 128.6, 127.9, 123.5, 118.9,
117.0, 115.6, and 20.6 ppm.
(E)-N'-(2-Hydroxy-5-methoxybenzylidene)picolinohydrazide (AR3). Isolated yied 250 mg
1
(92%). H-NMR (DMSO-d6) δ 12.47 (1H, s, NH), 10.80 (1H, s, OH), 8.79 (1H, s, CH=N), 8.71
(1H, d, J = 4.8 Hz, picolino 3-H), 8.11 (1H, d, J = 7.6 Hz, picolino 6-H), 8.07 (1H, t, J = 7.6 Hz,
picolino 5-H), 7.66 (1H, m, picolino 4-H), 7.26 (1H, s, salicylic 6-H), 7.03 (1H, d, J = 2.8 Hz,
salicylic 6-H), 6.91 (1H, d of d, J1 = 8.8 Hz, J2 = 3.2Hz, salicylic 4-H), 6.85 (1H, d, J = 8.8 Hz),
and 3.72 (3H, s, CH3) ppm. 13C-NMR (DMSO-d6) δ 161.2, 152.8, 152.4, 150.0, 149.3, 138.8,
135.5, 127.8, 123.5, 119.5, 119.1, 118.0, 113.3, and 56.2 ppm. EMS m/z 272.2 (M+H+) and
294.2 (M+Na+). Anal. Calcd for C14H13N3O3: C, 61.99; H, 4.83; N, 15.49. Found: C, 62.08; H,
4.98; N, 15.36.
(E)-N'-(5-Chloro-2-hydroxybenzylidene)picolinohydrazide (AR4). Isolated yield 255 mg
(92%). 1H-NMR (DMSO-d6) δ 12.56 (1H, s, NH), 11.33 (1H, s, OH), 8.80 (1H, s, CH=N), 8.70
(1H, d, J = 4.8 Hz, picolino 3-H), 8.12 (1H, d, J = 7.6 Hz, picolino 6-H), 8.04 (1H, t of d, J1= 7.6

Hz, J2 = 1.6 Hz, picolino 5-H), 7.65 (1H, m, picolino 4-H), 7.57 (1H, d, J = 2.8 Hz, salicylic 6-H,
7.30 (1H, d of d, J1 = 8.8 Hz, J2 = 2.8 Hz, salicylic 4-H), and 6.93 (1H, d, J = 8.8 Hz) ppm. 13C-
NMR (DMSO-d6) δ 161.3, 156.9, 149.8, 149.3, 148.3, 138.7, 131.6, 128.6, 127.9, 123.6, 123.5,
121.3, and 118.9 ppm. EMS m/z 276.2 (M+H+) and 298.1 (M+Na+). Anal. Calcd for
C13H10ClN3O2xH2O: C, 53.49; H, 4.07; N, 14.40. Found: C, 53.40; H, 4.05; N, 14.49.
(E)-N'-(5-bromo-2-hydroxybenzylidene)picolinohydrazide (AR5). Isolated yield 300 mg
(94%). 1H-NMR (DMSO-d6) δ 12.55 (1H, s, NH), 11.34 (1H, s, OH), 8.79 (1H, s, CH=N), 8.69
(1H, d, J = 4 Hz, picolino 3-H), 8.11 (1H, d, J = 7.6 Hz, picolino 6-H), 8.03 (1H, t, J = 7.6 Hz,
picolino 5-H), 7.69 (1H, d, J = 2.4 Hz, salicylic 6-H), 7.65 (1H, m, picolino 4-H), 7.40 (1H, d od
d, J1 = 8.8 Hz, J2 = 2.4 Hz, salicylic 4-H), and 6.88 (1H, d, J = 8.8 Hz, salicylic 3-H) ppm. 13C-
NMR (DMSO-d6) δ 161.3, 157.3, 149.8, 149.3, 148.1, 138.7, 134.4, 131.5, 127.9, 123.6, 121.9,
119.4, and 111.1 ppm. EMS m/z 320.2 (79Br-M+H+), 322.1 (81Br-M+H+), 342.1 (79Br-M+Na+),
and 344.0 (81Br-M+H+) and 298.1 (M+Na+). Anal. Calcd for C13H10BrN3O2x0.2H2O: C, 48.23;
H, 3.17; N, 12.98. Found: C, 48.22; H, 3.19; N, 13.01
(E)-N'-(2-hydroxy-5-nitrobenzylidene)picolinohydrazide (AR6). Isolated yield 260 mg
(90%). 1H-NMR (DMSO-d6) δ 12.62 (1H, s, NH), 12.30 (1H, s, OH), 8.88 (1H, s, CH=N), 8.68
(1H, d, J = 4 Hz, picolino 3-H), 8.45 (1H, d, J = 2.8 Hz, salicylic 6-H), 8.11 (2H, m), 8.03 (1H,
m), 7.64 (1H, m) and 7.40 (1H, d, J = 9.2 Hz, salicylic 3-H) ppm. 13C-NMR (DMSO-d6) δ 163.5,
161.4, 149.7, 149.3, 146.8, 140.5, 138.7, 127.3, 127.3, 124.8, 123.6, 120.5, and 117.8 ppm.
Acknowledgements: 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 unrestricted grant from Research to Prevent Blindness, New York, NY

(LSU Department of Ophthalmology-LSUHSC). Isolates TW1 and TW17 were a kind gift from
Dr. Glen Palmer, LSUHSC.
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Table 1. Antifungal Activity of N'-(2-hydroxybenzylidene)picolinohydrazides
3MIC80
C. albicans C. glabrata
(g/ml)
ATCC no.
10231
4MIC80
1MIC80
TW1 (g/ml)
2MIC80
TW17
(g/ml)
Entry
R
Compound Yield
(g/ml)
ATCC no.
48435
1
2
3
4
5
6
H
CH3
CH3O
Cl
91%
94%
92%
92%
94%
90%
62
4
NA
8
16
2
8
2
1
1
4
8
AR1
AR2
AR3
AR4
AR5
AR6
8
31
8
4
4
1
Br
2
4
1
NO2
8
8
4
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant

Table 2. Antifungal Activity of N'-(2-hydroxybenzylidene)nicotinohydrazides
3MIC80
C. albicans
(g/ml)
ATCC no.
10231
4MIC80
C. glabrata
(g/ml)
ATCC no.
48435
1MIC80
TW1
(g/ml) (g/ml)
2MIC80
TW17
Entry
R
Compound Yield
1
2
3
4
5
6
H
91%
93%
86%
91%
92%
86%
4
16
16
8
31
NA
4
4
4
16
8
AR7
AR8
CH3
CH3O
Cl
62
4
4
AR9
62
16
8
8
AR10
AR11
AR12
Br
8
4
16
NA
NO2
8
4
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant

Table 3. Antifungal Activity of N'-(2-hydroxybenzylidene)isonicotinohydrazides
3MIC80
C. albicans
(g/ml)
ATCC no.
10231
4MIC80
C. glabrata
(g/ml)
ATCC no.
48435
1MIC80
TW1
(g/ml) (g/ml)
2MIC80
TW17
Entry
R
Compound Yield
1
2
3
4
5
6
H
90%
92%
91%
94%
97%
86%
4
31
31
8
4
16
31
4
AR13
AR14
AR15
AR16
AR17
AR18
CH3
CH3O
Cl
16
16
16
16
8
16
4
125
125
8
31
16
4
16
64
31
Br
NO2
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant

Table 4. Antifungal Activity of 2-hydroxy-N'-(2-hydroxybenzylidene)nicotinohydrazides
3MIC80
C. albicans
(g/ml)
ATCC no.
10231
4MIC80
C. glabrata
(g/ml)
ATCC no.
48435
1MIC80
TW1
(g/ml)
2MIC80
TW17
(g/ml)
Entry
R
Compound Yield
1
2
3
4
H
89%
87%
91%
93%
4
4
NA
NA
NA
NA
1
2
AR19
AR20
AR21
AR22
CH3
Cl
NA
NA
NA
NA
NA
NA
NA
0.5
Br
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant

Table 5. Antifungal Activity of 3-(2-(2-hydroxybenzylidene)hydrazinecarbonyl)pyridine 1-
oxides
3MIC80
C. albicans
(g/ml)
ATCC no.
10231
4MIC80
C. glabrata
(g/ml)
ATCC no.
48435
1MIC80
TW1
(g/ml) (g/ml)
2MIC80
TW17
Entry
R
Compound
Yield
1
2
3
4
5
6
H
87%
89%
88%
91%
93%
85%
31
16
62
31
8
31
16
31
31
8
31
16
16
16
16
8
16
31
4
AR23
AR24
AR25
AR26
AR27
AR28
CH3
CH3O
Cl
4
Br
4
NO2
4
2
4
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant

Table 6. Antifungal activity of phenyl and aroyl N’-substituted 2-(hydrazonomethyl)pyridines
3MIC80
C. albicans
(g/ml)
ATCC no.
10231
4MIC80
C. glabrata
(g/ml)
ATCC no.
48435
1MIC80
TW1
(g/ml) (g/ml)
2MIC80
TW17
Entry
R
Compound Yield
1
2
Phenyl
93%
91%
90%
88%
86%
92%
93%
91%
88%
90%
16
NA
31
16
NA
62
16
NA
62
31
NA
62
AR29
AR30
AR31
AR32
AR33
AR34
AR35
AR36
AR37
2,4-dinitrophenyl
benzoyl
3
4
2-hydroxybenzoyl
4-nitrobenzoyl
31
62
31
4
5
NA
16
NA
16
NA
31
NA
16
6
Pyridine-2-carbonyl
Pyridine-3-carbonyl
Pyridine-4-carbonyl
2-hydroxynicotinoyl
1-oxidopyridine-3-
carbonyl
7
62
62
125
125
NA
125
125
NA
8
62
125
NA
9
NA
10
62
125
125
125
AR38
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant

Table 7. Antifungal activity of aryl substituted N'-(2-pyridinylmethylene)sulfonic hydrazides
3MIC80
C. albicans C. glabrata
(g/ml)
ATCC no.
10231
4MIC80
1MIC80 2MIC80
TW1 TW17
(g/ml) (g/ml)
Entry
R
Compound Yield
(g/ml)
ATCC no.
48435
1
2
3
4
5
2-naphthyl
4-methylphenyl
4-methoxyphenyl
4-bromophenyl
4-nitrophenyl
91%
88%
93%
97%
91%
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
AR39
AR40
AR41
AR42
AR43
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant

Table 8. Antifungal activity of aryl substituted 3-(hydrazonomethyl)pyridines
3MIC80
C. albicans
(g/ml)
ATCC no.
10231
4MIC80
C. glabrata
(g/ml)
ATCC no.
48435
1MIC80 2MIC80
TW1 TW17
(g/ml) (g/ml)
Entry
R
Compound Yield
1
2
3
4
Phenyl
2,4-dinitrophenyl
Benzoyl
93%
96%
91%
86%
16
8
16
16
AR44
AR45
AR46
AR47
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
4-nitrobenzoyl
1Azole-susceptible; 2Azole-resistant; 3Azole-susceptible; 4Azole-resistant