Synthesis and antifungal activity of substituted 2,4,6-pyrimidinetrione carbaldehyde hydrazones

Article abstract of DOI:10.1016/j.bmc.2013.12.010

Opportunistic fungal infections caused by the Candida spp. are the most common human fungal infections, often resulting in severe systemic infections - a significant cause of morbidity and mortality in at-risk populations. Azole antifungals remain the mainstay of antifungal treatment for candidiasis, however development of clinical resistance to azoles by Candida spp. limits the drugs' efficacy and highlights the need for discovery of novel therapeutics. Recently, it has been reported that simple hydrazone derivatives have the capability to potentiate antifungal activities in vitro. Similarly, pyrimidinetrione analogs have long been explored by medicinal chemists as potential therapeutics, with more recent focus being on the potential for pyrimidinetrione antimicrobial activity. In this work, we present the synthesis of a class of novel hydrazone-pyrimidinetrione analogs using novel synthetic procedures. In addition, structure-activity relationship studies focusing on fungal growth inhibition were also performed against two clinically significant fungal pathogens. A number of derivatives, including phenylhydrazones of 5-acylpyrimidinetrione exhibited potent growth inhibition at or below 10 μM with minimal mammalian cell toxicity. In addition, in vitro studies aimed at defining the mechanism of action of the most active analogs provide preliminary evidence that these compound decrease energy production and fungal cell respiration, making this class of analogs promising novel therapies, as they target pathways not targeted by currently available antifungals.

Full text of DOI:10.1016/j.bmc.2013.12.010

Bioorganic & Medicinal Chemistry 22 (2014) 813–826
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antifungal activity of substituted 2,4,6-pyrimidinetrione
carbaldehyde hydrazones
a
a
c
d,e
Donna M. Neumann a,b, , Amy Cammarata , Gregory Backes , Glen E. Palmer , Branko S. Jursic
⇑
a 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 Microbiology, Immunology and Parasitology, LSUHSC-New Orleans, United States
d Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United States
e STEPHARM, LLC., P.O. 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:
Opportunistic fungal infections caused by the Candida spp. are the most common human fungal infec-
tions, often resulting in severe systemic infections—a significant cause of morbidity and mortality in
at-risk populations. Azole antifungals remain the mainstay of antifungal treatment for candidiasis, how-
ever development of clinical resistance to azoles by Candida spp. limits the drugs’ efficacy and highlights
the need for discovery of novel therapeutics. Recently, it has been reported that simple hydrazone deriv-
atives have the capability to potentiate antifungal activities in vitro. Similarly, pyrimidinetrione analogs
have long been explored by medicinal chemists as potential therapeutics, with more recent focus being
on the potential for pyrimidinetrione antimicrobial activity. In this work, we present the synthesis of a
class of novel hydrazone-pyrimidinetrione analogs using novel synthetic procedures. In addition, struc-
ture–activity relationship studies focusing on fungal growth inhibition were also performed against
two clinically significant fungal pathogens. A number of derivatives, including phenylhydrazones of
Received 21 October 2013
Revised 25 November 2013
Accepted 4 December 2013
Available online 12 December 2013
Keywords:
Antifungal
Hydrazine
Carbaldehyde
Pyrimidinetrione
Hydrazones
5-acylpyrimidinetrione exhibited potent growth inhibition at or below 10 lM with minimal mammalian
cell toxicity. In addition, in vitro studies aimed at defining the mechanism of action of the most active
analogs provide preliminary evidence that these compound decrease energy production and fungal cell
respiration, making this class of analogs promising novel therapies, as they target pathways not targeted
by currently available antifungals.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
include the first-line treatments of azoles. However, the develop-
ment of clinical resistance to azoles by the Candida spp. occurs
Opportunistic fungal infections caused by the Candida spp. repre-
sent the most common fungal infections of humans.1 Candida spp.
infections can result in a broad spectrum of clinical manifestations,
ranging from superficial mucocutaneous infections to the more se-
vere invasive systemic fungal infections, and these invasive fungal
infections (IFI’s) are a significant cause of morbidity and mortality
in at-risk populations, particularly transplant recipients, cancer pa-
tients and those infected with HIV and AIDS.2,3 IFI’s present further
diagnostic and therapeutic challenges due to the fact that they are
difficult to diagnose early and are associated with high resistance
rates to currently marketed antifungal agents.4 Finally, the incidence
of candidiasis caused by non-albicans Candida spp. is increasing,
with Candida glabrata and Candida krusei most frequently isolated
in clinical settings, in addition to Candida albicans.5,6 Currently avail-
able clinical therapies for both cutaneous and systemic candidiasis
through multiple mechanisms and limits azole efficacy. For exam-
ple, resistance to azoles by C. albicans has been shown to be largely
due to both the overexpression of efflux pumps1 and point muta-
tions in ERG11 gene.7 The opportunistic yeast pathogen C. glabrata
is also recognized for its ability to acquire resistance during pro-
longed treatment with azole antifungals.8 For these reasons, there
is a continuous demand for the discovery of novel therapeutics to
treat fungal infections, particularly Candida spp. infections.
Hydrazine derivatives have recently begun to emerge in the
literature as novel classes of antifungal agents with therapeutic
potential against numerous Candida spp., including species com-
monly resistant to azole antifungals. For example, it was shown
that (4-aryl-thiazol-2-yl)hydrazines possessed potent antifungal
activities against a number of clinically relevant Candida spp.9 Ana-
logs from derivatives of the C2 and C4 positions of this hydrazine
skeleton also yielded a number of promising antifungal agents that
had synergistic effects when used in combination with an azole,
while maintaining low mammalian cell toxicity. Finally, the
hydrazine pharmacophore with substitutions of N1, together with
⇑
Corresponding author. Tel.: +1 504 568 3179.
E-mail address: dneum1@lsuhsc.edu (D.M. Neumann).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.010

814
D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
4-substituted phenyls at the C4 of a thiazole nucleus produced a
number of potent and selective hydrazine derivatives that pos-
a number of derivatives, including 5-formyl-1,3-dimethylpyrimi-
dinetrione using a modified Reimer–Tiemann reaction.15,16 How-
ever, the isolated yields using this method were <70% and
furthermore, this method could not be used in the preparation of
base sensitive 5-formylpyrimidinetriones. For these reasons, we
developed a new and more efficient method for preparation of
5-formyl and 5-acetylpyrimidinetriones by using trimethyl
orthoformate or triethyl orthoacetate as acylation reagents, respec-
tively.17 This method involved refluxing the corresponding pyrim-
idinetrione in trimethyl orthoformate with a catalytic amount of
an acid catalyst (preferably with PTSA) for several hours
(Scheme 2). For the preparation of acetyl derivatives, certain reac-
tion precautions were taken. Namely, if the reaction was carried out
with a temperature above 100 °C, a substantial amount of black tar
was formed and isolation of the product was difficult. However, if
the reaction was carried out at or below 80 °C overnight, the forma-
tion of black tar was minimal and the isolated yield was almost
quantitative. New methods were also developed for the preparation
of 5-aroylpyrimidinetriones (Scheme 2). The freshly prepared potas-
sium salt of the corresponding pyrimidinetrione was condensed with
aroyl chloride in THF–water as a reaction media. Although this meth-
od was excellent for pyrimidinetrione acylation with aromatic acid
chlorides, dismal yields were obtained with aliphatic acid chlorides
due to their fast hydrolysis in the reaction media. For this reason,
other acyl pyrimidinetriones were prepared in pyridine as a reaction
media by following our previously published procedures.18
sessed antifungal activity in the l
M range.9
Other literature reports have shown that hydrazone derivatives
have also emerged as compounds with the ability to potentiate anti-
fungal activities in vitro. For example, the ability of hydrazone deriva-
tives to inhibit the growth of Candida spp. was recently explored by
Altintop et al.10 Hydrazone derivatives bearing 5-thio-1-methyl 1H
tetrazole moiety were synthesized and evaluated for potential antifungal
activity and mammalian cell toxicity, with a number of compounds
showing potential for further development as antifungal agents.10
Finally, pyrimidinetrione analogs have long been explored by
medicinal chemists as not only psychotropic compounds, but as
anti-seizure, anticancer and antimicrobial compounds as well.
For example, pyrazole and isoxazole derivatives have gained
importance as potential chemotherapeutics that have applications
as antimicrobials and are active against a number of different fun-
gal species,11 while other pyrimidinetrione derivatives, including
bisoxadiazolyl and bisthiadiazolyl pyrimidinetriones have use as
antibiotic and antifungal therapies.12 In this manuscript, we pres-
ent the synthesis of an extensive collection of substituted pyrimi-
dinetrione derivatives using novel synthetic procedures. In
addition, structure–activity relationship studies focusing on fungal
growth inhibition were also performed against two clinically sig-
nificant fungal pathogens, namely C. albicans and C. glabrata A
number of derivatives, including phenylhydrazones of 5-acylpyr-
imidinetrione exhibited potent growth inhibition at or below
10
lM
with minimal mammalian cell toxicity. In addition,
2.1.3. Synthesis of 5-arylidene-2,4,6-pyrimidinetriones
in vitro studies aimed at defining the mechanism of action of the
most active analogs provide preliminary evidence that these com-
pound decrease energy production and fungal cell respiration,
making this class of analogs promising novel therapies, as they tar-
get pathways not targeted by currently available antifungals.
The majority of condensation products between pyrimidinetri-
ones and aromatic aldehydes (Knoevenagel condensation) were
prepared by following our previously reported procedures.19
However, when the condensation reaction was performed with
electron-rich aromatic aldehydes, such as salicylaldehyde,
4-dimethylaminobenzaldehyde, or pyridinecarbaldehyde, special
precautions were taken, due to fact that two rather than one pyr-
imidinetrione molecule can add easily to these aldehydes.20 This
2. Results
is because the formed
a,b-conjugates (Knoevenagel condensates)
2.1. Synthesis
are very reactive Michael acceptors.21 To control the second pyrim-
idinetrione addition, we eliminated the Knoevenagel condensate
from the reaction mixture in the course of the reaction, in a
manner similar to work presented by Deb and Bhuyan, where
Knoevenagel condensation was done in aqueous media.22 We
applied the same approach to the electron rich aromatic aldehydes
and pyrimidinetrione derivatives in water. Both pyrimidinetriones
and the electron rich aromatic aldehydes were partially soluble in
water, while the Knoevenagel condensation product was less solu-
ble. Precipitation of the Knoevenagel product in aqueous media
prevented the second pyrimidinetrione addition, and using this
procedure, we were able to prepare a structurally diverse library
of Knoevenagel condensates (Scheme 3).
2.1.1. Preparation of 1,3-substituted 2,4,6-pyrimidinetriones
The pyrimidinetrione building blocks used for all subsequent
syntheses reported here were first prepared by the condensation
of diethyl malonate with substituted urea in the presence of so-
dium ethoxide and ethanol following the classic Dickey–Gray pro-
cedure.13 If the appropriate substituted urea was not commercially
available, then the desired substituted urea was prepared from the
corresponding amines and phenyl chloroformate by following the
procedure outlined in Scheme 1.14 Using this method, a small
library of 1,3-di and mono-substituted pyrimidinetrione deriva-
tives were generated, and then used to further synthesize all
substituted pyrimidinetrione analogs presented in this work.
2.1.4. Synthesis of 5-mono and 5,5-dialkylated-2,4,6-
pyrimidinetriones
2.1.2. Synthesis of 5-acyl-2,4,6-pyrimidinetriones
Selecting the optimal preparation method for 5-acyl pyrimidin-
etriones depends on both the nature of the substituted pyrimidin-
etrione moiety as well as the acyl moiety. Previously, we prepared
Previously, we prepared a several mono and di-alkylated pyrim-
idinetrione analogs using a different method of synthesis.23 Here,
we prepared both mono and di-alkylated pyrimidinetrione analogs
R1
N
O
CH2(CO2C2H5)2
H2NR2
C6H5OCOCl
THF/H2O/0oC
10-30 minutes
R1NHCONHR2
O
R1NH2
R1NHOCOC6H5
NaOC2H5/HOC2H5
reflux overnight
N
-methylmorpholine/acetonintrile
50oC/sonication/30 minutes
N
R2
O
Scheme 1. Preparation of 1,3-substituted-2,4,6-pyrimidinetriones. Reagents and conditions: R1 = C6H5, 2-HOC6H4, 3-HOC6H4, 4-HOC6H4, 4-CH3OC6H4, 4-(CH3)2NC6H4,
4-O2NC6H4, 4-HO2CC6H4, 4-NCC6H4, 2-naphthyl, 2-phenanthrenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, (CH2)3CH3, (CH2)2CO2H, (CH2)5CO2H; R2 = H, CH3, C6H5, 4-HOC6H4,
2-pyridinyl, 3-pyridinyl, 4-pyridinyl.

D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
815
R1
O
O
R1
O
O
R1
N
O
O
N
N
O
O
H
(CH3CH2O)3OCCH3
(CH3O)3CH
catalytic amount of
H2SO4 or p-CH3C6H4SO3H
O
O
O
catalytic amount of
H2SO4 or p-CH3C6H4SO3H
N
N
CH3
N
R2
R2
R2
Method B (R3
alkyl)
=
O
R 3
H2
-
C
Method A (R3 = Aryl)
F
F
O
f
H
C
T
H
rs
/
T
o
h
l
/
P
/
O 3
3
R
3
l
r
.
t
y
r
C
.
.
C
t
i
O
C
d
o
K 2
i
r.
r
n
e
)
o
t
a
n
e
(
s
r
s
y
)
b
(
h
s
p
e
n
s
h
2
o
u
o
C
r
0
i
o
n
R1
N
O
O
R1
O
O
O
N
O
O
O
N
R3
N
R4
R2
R2
Scheme 2. Preparation of 5-acyl-2,4,6-pyrimidinetriones. Reagents and conditions: R1 = H, CH3, (CH2)3CH3, (CH2)2CO2H, (CH2)5CO2H, C6H5, 4-O2NC6H4, 3-O2NC6H4,
4-HOC6H4, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R2 = H, CH3, C6H5, 4-HOC6H4, CH2CO2H, (CH2)5CO2H; R3 = C5H5, 4-O2NC6H4, 4-CH3OC6H4, 2-pyridinyl, 3-pyridinyl,
4-pyridinyl; R4 = (CH2)4CH3, (CH2)11CH3, (CH2)2CO2H.
CHO
R1
N
O
O
R1
N
O
O
R1
O
O
CHO
N
O
Z
Y
O
O
N
water, methanol,
water, methanol,
N
R2
Y
N
acetic acid, or DMSO
acetic acid, or DMSO
R2
R2
Z
Scheme 3. Preparation of 5-arylidene-2,4,6-pyrimidinetriones. Reagents and conditions: R1 = C6H5, 4-O2NC6H4, 2-HOC6H4, 3-HOC6H4, 4-HOC6H4, 2-pyridinyl, 3-pyridinyl,
4-pyridinyl, n-butyl, methyl; R2 = H, C6H5, 4-HOC6H4, CH2CO2H, (CH2)4CO2H; Y = H, 2-OH, 3-OH, 4-OH, 2-NH2, 3-NH2, 4-NH2, 3-(CH3)2N, 4-(CH3)2N, 2-O2N, 3-O2N, 4-O2N,
2-HO2C, 3-HO2C, 4-HO2C; Z = H, 2-HO, 4-HO, 2-CH3O, 4-CH3O, 4-(CH3)2N.
R3
R1
N
O
R1
N
O
R1
N
O
R1
N
O
O
R3
R4
R5
R5
R3
R4
R4
H2/Pd-C
CH3OH or
CH3CO2H
R5CHO
H2/Pd-C in CH3OH
or CH3COOH
O
O
O
O
CH3OH or
CH3COOH
Sonication at 0ºC
N
N
N
N
R2
O
R2
O
R2
O
R2
O
Scheme 4. Preparation of 5-mono and 5,5-disubstituted-2,4,6-pyrimidinetriones. Reagents and conditions: R1 = H, CH3, C4H9, C6H5, 2-HOC6H4, 3-HOC6H4, 4-HOC6H4,
2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R2 = H, CH3, C4H9, C6H5, CH2CO2H, (CH2)4CO2H; R3 = H, CH3, n-C7H15, C6H5, 4-HOC6H5, 4-(CH3)2NC6H4; R4 = CH3, n-C7H15, C6H5,
4-HOC6H5; R5 = C6H5, 4-(CH3)2NC6H4.
utilizing a modified and simplistic synthetic procedure that gave
better yields. For the mono-alkylation reactions, a mixture of
equivalent amounts of the corresponding benzaldehyde and
2,4,6-pyridinetrione, was sonicated at 0 °C preferably in methanol,
for 30 min, followed by hydrogenation with wet 10% PdC under
hydrogen pressure of 30 psi. In the first step, the 5-arylidene-
2,4,6-pyrimidine was prepared and without isolation, and the new-
ly formed carbon–carbon double bond was hydrogenated in situ.
By making these modifications, we avoided the double pyrimidin-
etrione addition to the benzaldehyde carbonyl. In the case of the
dialkylated product, a mixture of the corresponding pyrimidinetri-
one and two equivalents of the corresponding benzaldehyde were
used in a similar manner and hydrogenated (Scheme 4).
the presence of starting materials was not detected in the reaction
mixture by thin layer chromatography (between three and eight
hours). If one or both of the reactants were not soluble in hot meth-
anol, then the reaction was performed in hot (ꢀ60 °C) acetic acid.
The transformations were typically quantitative and isolation pro-
cedures involved simply reducing the reaction mixture by evapora-
tion of the solvent and crystallizing the product. Isolated yields of
most of the prepared derivatives were typically >90% (Scheme 5).
2.1.6. Synthesis of substituted hydrazines, hydrazides, and
benzosulfonohydrazides
The majority of hydrazides and semicarbazides used in the
preparation of the Schiff bases were not available commercially
and had to be prepared. The preparation of these hydrazides
started with readily available acid esters. A methanol solution of
an ester with hydrazine hydrate (10 equiv) was refluxed for one
hour, followed by the distillation of methanol at atmospheric pres-
sure. In this way, the amount of hydrazine was gradually increased
to facilitate the reaction. After all the methanol was distilled from
the reaction mixture, the remaining residue was mixed with water
and extracted in ethyl acetate.24 In the case of semicarbazides,
there are many different methods for the preparation of substi-
tuted semicarbazides. However, the two most commonly used
2.1.5. Synthesis of Schiff bases and hydrazones of 5-acyl-2,4,6-
pyrimidinetriones
Similar reaction conditions were used for the preparation of all
three classes of pyrimidinetrione derivatives: Schiff bases, hydra-
zones, and semicarbazones. They were all prepared from the corre-
sponding 5-acyl pyrimidinetriones and substituted amines,
hydrazines, hydrazides, or semicarbazides respectively, by follow-
ing our previously published procedures.15 Preferably, the metha-
nol solution or suspension was refluxed for several hours until

816
D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
R1
O
O
R5
H
R1
N
O
O
N
N
N
N R4
R3
O
O
N
R3
N
5
R2
H2
R
R2
-
NH
N
m
c
r
R
4
2N
o
i
e
l
R1
O
O
t
h
H
o
a
Hydrazones and
a
a
n
ic
a
n
c
d
a
o
l
Schiff Bases
e
h
t
e
c
t
PhenylHydrazones
o
i
c
r
N
O
m
t
e
i
d
c
a
O
r
N
R3
6
R
H
2N
N
O
R2
C
H
H
o
S
N
l
O
o
me
a
c
e
O
o
7
N
2R
r
n
c
d
t
h
a
O
H 2
ci
O
N N
R3
a
n
a
h
c
R1
N
O
O
S R7
a
o
c
t
l
e
a
t
i
c
i
t
R1
N
O
O
R6
m
e
i
d
N N
O
H
O
H
N
R3
N
R2
R2
Benzenesulfonohydrazones
Acylhydrazones
Scheme 5. Preparation of Schiff bases an hydrazones of 5-acyl-2,4,6-pyrimidinetriones. Reagents and conditions: R1 = H, CH3, (CH2)3CH3, C6H5, 4-O2NC6H4, 2-HOC6H4,
3-HOC6H4, 4-HOC6H4, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R2 = H, CH3, (CH2)3CH3, C6H5, p-HOC6H4, CH2CO2H, (CH2)4CO2H; R3 = H, CH3, CH3(CH2)4, C6H5, 2-HOC6H4,
3-HOC6H4, 4-HOC6H4, 4-(CH3)2NC6H4, 4-CH3OC6H4, 4-CH3C6H4, 4-HO2CC6H4, 2-O2NC6H4, 3-O2NC6H4, 4-O2NC6H4, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R4 = (CH2)5CH3,
(CH2)2CO2H, (CH2)5CO2H, C6H5, 2-HOC6H4, 3-HOC6H4, 4-HOC6H4, 4-CH3OC6H4, 4-O2NC6H4, 4-HO2CC6H4, 4-NCC6H4; R5 = CH3, (CH2)5CH3, CH2C6H5, C6H5, 4-CH3C6H4,
4-CH3OC6H4, 4-O2NC6H4, 2,4-(O2N)2C6H3, 4-HO2CC6H4; R6 = CH3, (CH2)5CH3, C6H5, 2-HOC6H5, 3-HOC6H4, 4-HOC6H4, 4-(CH3)2NC6H4, 4-CH3OC6H4, 3-O2NC6H4, 4-O2NC6H4,
2-pyridinyl, 3-pyridinyl, 4-pyridinyl; R7 = C6H5, 4-CH3C6H4.
HCl/H2O/NaNO2/0ºC
Na2SO3/H2O r.t.
NH2
NHNH2
X
X
LiAlH4
Ether, r.t.
H2NNH2
C2H5OH, 70ºC
R1CH2NHNH2
R1CO2C2H5
R1CONHNH2
H2NNH2
THF/H2O/0ºC
SO2NHNH2
SO2Cl
Y
Y
Scheme 6. Preparation of substituted hydrazines, hydrazides, and benzosulfonohydrazides. Reagents and conditions: X = H, 4-CH3, 4-CH3O; Y = H, 4-CH3O, 4-O2N; R3 = C5H9,
C6H5, 4-O2NC6H4.
are (a) from substituted urea and hydrazine hydrate25 and (b) from
substituted isocyanate.26 We have further developed a very effi-
cient synthetic procedure for the preparation of semicarbazides
that starts with the corresponding amine, phenyl chloroformate
and hydrazine14 (Scheme 6).
BA53 (shown in Table 2) modestly inhibited fungal growth of
either C. albicans or C. glabrata, indicating that extended conjuga-
tion of the pyrimidinetrione ring may be required for growth
inhibition.
The possibility that extended conjugation of the 2,4,6-pyrimi-
dinetrione ring is a prerequisite for antifungal activity was an
intriguing prospect. To explore this possibility more thoroughly,
we synthesized and analyzed the antifungal activity (by growth
inhibition) of a number of Schiff base derivatives of 5-acylpyrimi-
dinetriones, including several analogs with substituted and unsub-
stituted phenyl groups, all of which would extend conjugation
through the aromatic ring system. Making these modifications to
the pyrimidinetrione ring yielded a number compounds that had
inhibitory activity of C. albicans and/or C. glabrata (compounds
BA47 and BA48, Table 2 and BA93 and BA94, Table 5). Interestingly,
all of these compounds had a hydroxyl group in the 2-position of
the aromatic ring. Substitution of the aromatic system with an
ortho hydroxyl group would position the non-bonded electron pair
of the hydroxyl close to the carbon–nitrogen double bond of BA93
and BA94, further extending and stabilizing the conjugation of
the system. In contrast, compounds with a hydroxyl in the 3-
position (BA95 and BA96) or in the 4-position (BA79–81, Table 5;
BA44–46, Table 2) of the aromatic ring lack that extended conjuga-
tion and stabilization, and were subsequently found to have no
inhibition of fungal growth. Collectively, these data indicate that
the contribution from extended conjugation of molecule through
the presence of the non-bonding electron pair on the OH in the 2
position might be integral for antifungal activity.
2.2. Antifungal activity
The antifungal activity of the 2,4,6-pyrimidinetrione analogs
shown in Schemes 2–6 were evaluated in vitro using C. albicans
(ATCC no. 10231) and C. glabrata (ATCC no. 48435). All assays were
done in accordance with NCCLS reference documents.27 The results
of these screenings are summarized in Tables 1–9 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 5-acyl-2,4,6-pyrimidinetri-
ones, 5-alkylated pyrimidinetriones or the 5,5-dialkylated pyrim-
idinetriones showed antifungal activity through 125 lg/mL,
regardless of the nature of the acyl or alkyl group attached at
the 5-position of the pyrimidinetrione ring (Tables 1, 3 and 4).
In contrast, however, the addition of an arylidene moiety to the
pyrimidinetrione led to increased growth inhibition. Specifically,
the 5-arylidene derivatives, such as BA40, BA41, BA42, BA43 and

D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
817
Table 1
5-Acyl-2,4,6-pyrimidinetriones synthesized following procedures in Scheme 2*18,15
R1
N
O
O
O
O
N
R3
R2
Compound
R1
R2
R3
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
BA1
BA2
BA3
BA4
BA5
BA6
BA7
BA8
H
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
H
H
CH3
H
CH3
H
H
H
C6H5
C6H5
C6H5
3-O2NC6H4
3-O2NC6H4
4-O2NC6H4
4-O2NC6H4
3,5-(O2N)2C6H3
3,5-(O2N)2C6H3
4-HOC6H4
4-HOC6H4
4-HOC6H4
4-HOC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
3-Pyridinyl
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
CH3
CH3
H
CH3
H
CH3
H
CH3
H
CH3
C4H9
C6H5
H
CH3
C4H9
CH3
H
BA9
BA10
BA11
BA12
BA13
BA14
BA15
BA16
BA17
BA18
BA19
BA20
BA21
CH3
H
C6H5
H
CH3
CH3
H
H
*
Compounds previously synthesized.
Table 2
5-Arylidene-2,4,6-pyrimidinetriones synthesized following procedures in Scheme 3
R1
N
O
O
O
N
R3
R2
Compound
R1
R2
R3
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
BA39
BA40
BA41
BA42
BA43
BA44
BA45
BA46
BA47
BA48
BA50
BA51
BA52
BA53
BA54
BA55
BA56
H
H
H
H
CH3
H
CH3
H
H
H
H
H
C6H5
—
—
—
—
2-Naphthyl (C10H7)
1-Naphthyl (C10H7)
CH@CHAC6H5
CH@CHAC6H5
4-HOC6H4
4-HOC6H4
4-HOC6H4
2-HOC6H4
2,4-(HO)2C6H3
4-CH3OC6H4
2,3,4-(CH3O)3C6H4
2,4,6-(CH3O)3C6H4
4-(CH3)2NC6H4
4-(CH3)2NC6H4
3-Furanyl (C4H3O)
2-Furanyl (C4H3O)
125
125
62
62
—
—
—
—
—
—
—
—
62
—
CH3
H
CH3
C6H5
H
CH3
CH3
CH3
CH3
H
125
—
—
—
8
H
CH3
CH3
CH3
CH3
H
CH3
H
H
2
—
—
—
—
—
—
—
CH3
H
H
—
—
Functionally and structurally, derivatives of 2,4,6-pyrimidine-
triones with hydrazone moieties share similar characteristics to
compounds BA93 and BA94, namely extended conjugation through
the hydrazone moiety. To test whether this structural component
was important in antifungal function, hydrazones of 5-acylpyrim-
idinetriones and phenylhydrazones of 5-acylpyrimidinetriones
synthesized in Schemes 5 and 6 were evaluated for antifungal
activity. We found minimal inhibition resulting from treatment
with 5-acylpyrimidinetrione hydrazones, even though extended
conjugation existed through the C–N and N–N double bonds
(Table 6). Similar results were observed in the case of N-acylhydra-
zones of 5-acylpyrimidinetriones and sulfonohydrazones (Tables 8
and 9). However, in the cases where the substitution to 5-acylpyr-
imidinetriones was a phenylhydrazone, growth inhibition signifi-
cantly increased, and over 80% inhibition could be observed
through lower dilutions, typically in the range of 1–4
lg/mL
(<10 M) (Table 7, BA22, BA23, BA25, BA73, BA74, BA78 and
l
BA85). The addition of one NO2 group in the 4-position of the
phenyl ring did not decrease or increase antifungal activity of
1,3-dimethyl or 1,3-unsubstituted molecules, but the addition of
a phenyl group to the 3-position of the pyrimidinetrione ring
greatly reduced inhibition of fungal growth (Table 7, BA28). Fungal
growth inhibition from treatment with N-phenylhydrazones of
5-acylpyrimidine triones (Table 8, compounds 127–161) yielded

818
D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
Table 3
5-Alkylated pyrimidinetriones synthesized following procedures in Scheme 4
R1
N
O
O
O
R3
N
R2
Compound
R1
R2
R3
MIC80 C. albicans (
l
g/mL)
MIC80 C. glabrata (lg/mL)
BA60
BA61
BA62
BA63
BA64
BA65
BA66
BA67
BA68
BA69
H
H
H
H
H
H
H
CH3
H
H
C6H5
H
H
H
C6H5
H
CH3
H
n-C7H15
CH(CH3)2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Cyclohexyl (C6H11
CH2CH2CH2C6H11
CH2C6H10-4-OH
CH2C6H10-4-OH
CH2CH2CH2C6H5
CH2CH2CH2C6H5
CH2C6H3-4-OH
CH2-2-C10H7
)
H
H
Table 4
5,5-Dialkylated pyrimidinetriones synthesized following procedures in Scheme 4
R1
N
O
O
R3
R4
O
N
R2
Compound
R1
R2
R3
R4
CH2C6H5
CH2C6H4-4-N(CH3)2
CH2C6H4-4-N(CH3)2
MIC80 C. albicans (
lg/mL)
MIC80 C. glabrata (lg/mL)
BA70
BA71
BA72
H
H
H
H
H
H
CH2C6H5
CH2C6H4-4-N(CH3)2
CH2CH2CH2C6H5
—
—
—
—
—
—
variable results, however, with no significant fungal growth inhibi-
tion, indicating that the carbonyl functionality did not contribute
to activity.
One significant drawback to the use of antifungals clinically is
the fact that many exhibit either hepatotoxicity or nephrotoxicity.
Therefore any new class of analogs that have the potential to be
used as antifungals must be screened diligently to establish
toxicity parameters in kidney and liver cells. Therefore, all analogs
presented in this manuscript with antifungal activity of less than
remained healthy and viable, indicating that analogs with
structural and functional similarities may not be ideal candidates
for further therapeutic development. Similar results were observed
with respect to the cell viability assays using hepatocytes (Fig. 1).
Considering the significant growth inhibition and lack of overall
lack of mammalian cell toxicity of many of the phenylhydrazones,
we further explored potential mechanisms of action using the
compound BA22 as a representative drug. We initially observed
that on agar plates enriched with a range of BA22 concentrations,
C. albicans colony formation was significantly delayed in a dose
dependent manner, eventually forming small colonies (data not
shown). This phenotype somewhat resembles that of respiration
deficient ‘petite’ mutants, which are unable to utilize non-ferment-
able carbon sources such as glycerol and ethanol.28 We therefore
determined if BA22 affected the ability of C. albicans or C. glabrata
to use glycerol or ethanol as carbon sources. The concentrations of
BA22 required to inhibit the growth of either fungi were much
lower when glycerol or ethanol is provided as a carbon source ver-
sus glucose (Fig. 2). This suggests that BA22 may preferentially
interfere with the utilization of non-fermentable carbon sources,
and thus may cause a defect in respiration. Growth inhibition oc-
curred at higher BA22 concentrations in glucose medium, possibly
indicating an additional secondary mechanism.
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 5ꢁ, 10ꢁ and 100ꢁ the fungal growth inhibition concentra-
tion. Cytotoxicity studies were performed in accordance with
Promega CellTiter 96 Non-RadioactivCell Proliferation Assay
(cat
# G4000). Representative compounds are presented in
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, BA22, BA23 and BA73 had minimal reductions
in viable kidney cells, where over 80% cells remain viable even at a
concentration 10 times higher than the fungal growth inhibition
concentration (Table 7). In contrast, when we tested the cytotoxic-
ity of compounds BA25, BA78 and BA94 we found that each caused
a significant reduction (ꢀ35%) in the number of kidney cells that
3. Discussion and conclusions
In this manuscript, we have outlined new and optimized
synthetic preparations of a wide range of 2,4,6-pyrimidinetrione
analogs, and their potential for inhibition of fungal growth. A
small number of the compounds presented in this manuscript
were previously prepared in our laboratory. However, new and
optimized synthetic procedures presented in Schemes 1–6

D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
819
Table 5
Schiff bases of 5-acylpyrimidinetriones synthesized following procedures in Scheme 5
R1
N
O
O
N
R4
O
N
R3
R2
Compound
R1
R2
R3
R4
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
BA2915
BA35
BA79
BA80
BA81
BA82
BA83
BA84
BA87
BA88
BA89
BA90
BA91
BA92
BA93
BA94
BA95
H
H
H
H
CH3
4-CH3OC6H4
H
CH3
H
H
H
CH3
CH3
CH3
H
(CH2)5CO2H
(CH2)5CO2H
4-HOC6H4
4-HOC6H4
4-HOC6H4
4-Pyridinyl (C5H4N)
4-Pyridinyl (C5H4N)
4-Pyridinyl (C5H4N)
4-HOC6H4
4-Pyridinyl (C5H4N)
C6H5
C6H5
C6H5
C6H5
2-HOC6H4
2-HOC6H4
3-HOC6H4
3-HOC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
4-O2NC6H4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
8*
8*
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
CH3
CH3
H
CH3
H
CH3
H
H
CH3
CH3
H
CH3
H
CH3
H
H
H
H
CH3
CH3
H
CH3
CH3
H
H
CH3
CH3
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
CH3
CH3
H
CH3
CH3
H
BA96
BA97
BA98
BA99
H
H
CH3
CH3
H
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
CH3
H
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
BA100
BA101
BA102
BA103
BA104
BA105
BA106
BA107
BA108
BA109
BA110
BA111
BA112
4-O2NC6H4
2-Pyridinyl (C5H4N)
2-Pyridinyl (C5H4N)
4-C6H4-C6H5
4-C6H4-C6H5
1,2,4-Triazol-3-yl (C2H2N3)
1,2,4-Triazol-3-yl (C2H2N3)
1,2,4-Triazol-4-yl (C2H2N3)
1,2,4-Triazol-4-yl (C2H2N3)
5-Tetrazolyl (CN4H)
H
H
H
H
CH3
CH3
H
5-Tetrazolyl (CN4H)
*
Compound displayed MIC with <50% inhibition.
Table 6
Hydrazones of 5-acylpyrimidinetriones synthesized following procedures in Scheme 5
R1
O
R4
H
N
N
N
O
N
R3
R2
O
Compound
R1
R2
R3
R4
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
BA116
BA117
BA118
BA119
BA120
BA121
BA122
BA123
H
CH3
H
CH3
H
H
H
CH3
H
CH3
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
125
125
—
—
—
16
16
—
CH3
CH3
H
CH3
H
—
—
—
31*
125
—
CH3
CH3
CH3
CH3
62*
—
CH3
*
Compound displayed MIC with <50% inhibition.
subsequently allowed for the preparation of a large library of
these derivatives, regardless of the physical properties of the sub-
stituents, and all are therefore included in this manuscript. Of the
compounds synthesized and analyzed, we found that pyrimidin-
etriones containing either a 2-OH phenyl moiety or a phenylhyd-
razone moiety possess potent growth inhibition for C. albicans
and C. glabrata. In contrast, we found that either phenyl
hydrazines or unsubstituted 2,4,6-pyrimidinetriones were unable
to inhibit fungal growth alone (data not shown). Based on these
results, we hypothesize that the pyrimidinetrione moiety to-
gether with the hydrazone moiety in one stable molecule elicit
antifungal activity through the extension of the 2,4,6-pyrimidin-

820
D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
Table 7
Phenylhydrazones of 5-acylpyrimidinetriones synthesized following procedures in Scheme 5
R1
N
O
O
Y
N
N
H
O
N
R3
R2
Compound
R1
R2
R3
Y
MIC80 C. albicans (
lg/mL)
MIC80 C. glabrata (lg/mL)
BA2234
BA2334
BA24
H
CH3
H
H
H
CH3
H
H
H
H
CH3
CH3
H
CH3
H
H
CH3
H
H
H
CH3
C6H5
H
C4H9
C6H5
CH3
CH3
H
H
H
H
4-Nitro (NO2)
4-Nitro (NO2)
2,4-Dinitro (NO2)2
4-Nitro (NO2)
2,4-Dinitro (NO2)2
2,4-Dinitro (NO2)2
4-Nitro (NO2)
2,4-Dinitro (NO2)2
2,4-Dinitro (NO2)2
2,4-Dinitro (NO2)2
4-Carboxy (COOH)
2,4-Dinitro (NO2)2
4-Carboxy (COOH)
H
2
2
—
2
1
2
125
2
BA2534
BA2634
BA2734
BA2815
BA3034
BA3118
BA3218
BA3318
BA3418
BA38
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
C6H5
4-HOC6H4
—
—
31.5
—
—
—
—
—
—
4
—
—
16
—
—
—
—
—
125
2
5-Pyrimidinetrione
H
H
BA73
BA74
CH3
H
H
4
2
BA78
BA85
CH3
H
CH3
H
CH3
CH3
H
H
2
2
2
2
Table 8
N-Acylhydrazones of 5-acylpyrimidinetriones synthesized following procedures in Scheme 5
O
N
R1
N
O
O
R4
N
O
H
N
R3
R2
Compound
R1
R2
R3
R4
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
BA36
BA37
BA75
BA76
BA77
BA86
BA124
BA125
BA126
BA127
BA128
BA129
BA130
BA131
BA132
BA133
BA134
BA135
BA160
BA161
CH3
H
CH3
CH3
H
H
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
CH3
H
H
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
4-HOC6H4
4-O2NC6H4
CH3
H
H
CH3
H
H
CH3
CH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
C6H5
C6H5
C6H5
C6H5
4-O2NC6H4
4-O2NC6H4
4-O2NC6H4
4-O2NC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
4-CH3OC6H4
(CH2)4CH3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
62*
62*
—
16*
125
125
—
H
CH3
CH3
H
—
—
62*
31
—
H
62*
—
CH3
CH3
H
—
NT
NT
—
NT
NT
CH3
CH3
CH3
(CH2)4CH3
NT = not screened.
*
Compound displayed MIC with <50% inhibition.
etrione ring conjugation, either from the non-bonded electron
pair of the 2-OH group or the phenyl hydrazone moiety. Our re-
sults strongly support this hypothesis.
We initially observed that in the presence of BA22, C. albicans
formed small colonies similar to respiration deficient ‘petite’ mu-
tants previously described for Saccharomyces. Thus the severe
growth inhibition caused by BA22 could indicate a defect in energy
production. Consistent with this argument, BA22 inhibits fungal
growth at significantly lower concentrations when non-ferment-
able carbon sources such as ethanol and glycerol are provided as
compared to glucose, suggesting that phenylhydrazones of pyrim-
idinetriones may cause defects in respiration. Alternatively, it is
possible that when energy production is dependent upon respira-
tion, both C. albicans and C. glabrata are somehow sensitized to
the effects of BA22. Either way, at higher concentrations growth
in the presence of glucose was also inhibited, indicating a potential

D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
821
Table 9
Benzenesulfonohydrazones of 5-acylpyrimidinetrione synthesized following procedures in Scheme 5
O
O
R1
O
O
S
N
N NH
Y
O
N
R3
R2
Compound
R1
R2
R3
Y
MIC80 C. albicans (lg/mL)
MIC80 C. glabrata (lg/mL)
BA113
BA114
BA115
BA136
BA137
BA138
BA139
H
H
CH3
H
CH3
H
H
H
CH3
H
CH3
H
H
CH3
H
H
H
4-CH3
4-CH3
4-CH3
H
H
H
16*
—
—
—
—
—
—
—
—
—
—
—
CH3
CH3
—
—
CH3
CH3
H
*
Compound displayed MIC with <50% inhibition.
secondary mechanism of growth inhibition. More comprehensive
mechanistic studies are currently underway that will precisely de-
fine the effects of BA22 on fungal energy production, however,
based on these preliminary data, we expect that energy production
and respiration are deficient following treatment with the pyrimi-
dinetrione carbaldehyde analogs. This is particularly exciting, con-
sidering that targeting energy production and respiration of fungal
species would provide an alternative mechanism of action to that
of more commonly used antifungals, such as the azoles.
ogy below), giving final concentrations tested in the range of 128–
0.5 g/mL.
l
4.2. MIC determination
Antifungal susceptibility studies and minimal inhibitory con-
centrations (MIC) values for C. albicans (ATCC no. 10231) and C.
glabrata (ATCC no. 48435) were determined by the broth microdi-
lution technique in accordance with NCCLS reference documents.
Finally, it should be pointed out that hydroxyl phenyl hydra-
zones and 5-alkylidene barbituric acids have both been identified
as Pan-Assay Interference Compound (PAINS) substructures.29 It
has been reported that PAINS substructures are often associated
with false-positives in high throughput screenings, due to their
ability to form aggregates that interfere in binding, are protein
reactive or directly interfere in assay signaling.29 These findings
are largely based on photometric assays, and the fact that PAINS
substructures may ultimately interfere with the absorption of light
or interact/react with proteins. In this study, we performed MIC
assays on over 120 analogs. However, these MIC assays were all
done manually according to the NCCLS guidelines, and each series
of drug dilutions was visually scored (as opposed to spectroscopic
analysis), to eliminate possible false positives based on light
interference. Further, PAINS cannot be categorically ruled out for
potential drug development, as marketed drugs contain PAINS sub-
structures.29 Finally, reactive, photosensitive and/or redox-active
compounds have been characterized as being particularly suited
therapeutics for areas of microbiology, regardless of their PAINS
classification.29 Considering this, our findings identify several com-
pounds that could be further developed as potential novel anti-
fungal compounds with the ability to inhibit fungal growth in
Microdilution panels ranged from 0.5 to 128 lg/mL. All organisms
were subcultured on Sabouraud agar and passaged to ensure purity
and viability. The initiating inocula were prepared by picking 5–7
isolated colonies ꢀ1 mm in diameter from freshly prepared YM
agar plates. Colonies were resuspended in sterile water, vortexed
and the cell density was adjusted spectrophotometrically to the
transmittance of a 0.5 McFarland standard at a wavelength of
530 nm, to yield a stock suspension of 1 ꢁ 106–5 ꢁ 106 cells/mL.
The stock was allowed to hydrate for 30 min at room temperature.
A working suspension was then made by diluting the stock 1:50
with RPMI 1640 media containing HEPES buffer. The working sus-
pension was used for all assays. The plates were incubated at 35 °C
for 48–72 h in a humid atmosphere. Growth was scored visually.
MIC values are defined as the lowest concentration of agent that
prevents any discernible growth, ꢀ80% reduction of growth, as
compared with drug-free control wells. All assays were run in par-
allel using either fluconazole or amphotericin B at the NCCLS rec-
ommended concentrations27 as a drug control (Sigma–Aldrich).
4.3. In vitro mammalian cell toxicity assays
Cytotoxicity was determined using non-cancerous Vero cells
(African green monkey kidney cells-ATCC no. CRL-1651) and Hep
G2 (liver) (ATCC no. HB-8065) cells in accordance with Promega
CellTiter 96 Non-RadioactivCell Proliferation Assay (cat # G4000).
Cells were grown for 24 h at 37 °C and 5% CO2 in a 96-well culture
the lM range.
4. Experimental section
plate. All compounds with MIC 68 lg/mL were further screened in
4.1. Preparation of diluted compounds
the MTT assays using both cell types. Compounds that fit these cri-
teria were diluted in media to the testing dilution amounts: the
MIC concentration (shown in respective tables; 5ꢁ MIC; 10ꢁ MIC
and 100ꢁ MIC). The media used to grow cells was DMEM contain-
ing 10% FBS and 2%. 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 spectrophoto-
metrically with an automatic plate reader set at 570 nm. 0.1%
None of the compounds tested over the course of these studies
were soluble in water and DMSO was used to dilute each com-
pound tested. Master stock concentrations of all tested compounds
were prepared to ensure that the maximum final concentration
of DMSO was 1% or less. Subsequent twofold serial dilutions were
made using sterile water to a final concentration of 1280–5.00 lg/
mL. A final 10-fold dilution of each drug was made by aliquotting
0.1 mL of each drug dilution into 0.9 mL of fungal suspension in
RPMI 1640 media with 10 mM HEPES buffer added (see methodol-

822
D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
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.

D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
823
Figure 2. BA22 activity is enhanced when Candida growth depends upon non-fermentable carbon sources. C. albicans strain SC5314 (A) or C. glabrata strain CS117.93 (B) were
grown in medium with 2% glucose (YPD), 3% glycerol (YPG) or 3% ethanol (YPE) at pH 6.5, in dose response experiments with BA22. After 24 h incubation at 30 °C, growth was
measured by OD600nm. Growth at each concentration of BA22 is shown as% growth relative to the minus drug (DMSO) control. Data shown is representative of two repeat
experiments.
Saponin was used as a positive control. 1% DMSO controls were
also used since the compounds were reconstituted in DMSO.
at room temperature for two hours. The dark red solid was sepa-
rated by filtration, washed with water (5 ꢁ 100 mL) and dried at
110 °C for two hours to give pure product (4.1 g; 89%). 1H NMR
(DMSO-d6) d 11.47 (2H, NH), 7.55 (2H, d, J = 7.6 Hz, o-H), 5.55
(1H, t, J = 7.2 Hz, p-H), and 7.41 (2H, t, J = 7.6 Hz) ppm. 13C NMR
(DMSO-d6) d 190.8, 149.9, 135.8, 132.2, 129.2, 128.2, and 95.7 ppm.
4.4. Testing for respiratory deficiencies
Respiratory deficiencies were examined using YPD (2% glucose),
YPE (3% ethanol), or YPG (3% glycerol).30 Each medium was buf-
fered with 0.3 M MOPS, and pH adjusted to 6.5 with KOH. Serial
1:1 dilutions of BA22 were performed in DMSO from a 20 mM
stock. Each drug stock or DMSO alone was then diluted further
1:99 with each of the above media. Medium + drug suspensions
4.5.2. Typical procedure for preparation of N-substituted 5-
aroyl-2,4,6-pyrimidinetriones (Table 1)
Preparation of 5-benzoyl-1,3-dimethylpyrimidine-2,4,6-trione
(BA3). A mixture of 3,3-dimethyl-2,4,6-pyrimidinetrione (1,56 g;
0.01 mol) and sodium bicarbonate (1.26 g; 1.5.mol) was dissolved
in water. Into this solution with stirring at room temperature, a
tetrahydrofuran (60 mL) solution of benzoyl chloride (1.4 g;
0.01 mol) was added. The resulting clear reaction mixture was stir-
red at room temperature for one hour. The colorless reaction mix-
ture changed from yellow to brown and finally to red, indicating
the progress of product formation. The reaction volume was re-
duced to approximately 10 mL by evaporation of solvent. The ice
water-cooled reaction mixture was acidified with 10% HCl to pH
ꢀ2. Water was decanted from the white gummy precipitate. This
precipitate was washed with cold water (3 ꢁ 10 mL) and crystal-
lized from ethanol to give 2.2 g (85% yield) pure product. 1H
NMR (DMSO-d6) d 7.51 (3H, d+t), 7.42 (2H, d, J = 8.0 Hz), and 3.17
(6H, s) ppm. 13C NMR (DMSO-d6) d 185, 163, 146, 131, 127, 124,
123, 92, and 24 ppm.
(100 ll) were then transferred to a round bottomed 96 well plate.
C. albicans strain SC531431 and C. glabrata strain CS 177.93,32
grown overnight in YPD broth at 30 °C (180 rpm) were washed in
distilled water, and resuspended at 1 ꢁ 104 cells mLꢂ1 in each of
the above three media. Then 100 ll of each cell suspension
(approx. 103 cells) was added to wells of the 96 well plate contain-
ing the corresponding medium + drug combinations. Final DMSO
concentration was 0.5% in all wells, with final drug concentrations
of 0 and 0.195–100 lM. After 24 and 48 h incubation at 30 °C,
growth was quantified by measuring OD600nm using a BioTek plate
reader. Growth at each BA concentration was then expressed as%
growth versus the DMSO control of the same medium.
4.5. Chemistry experimental
Thin-layer chromatographic analysis (TLC) was performed
using silica gel on aluminum foil glass plates and products were
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 with solvent signals as internal standards. When neces-
sary, products were purified by flash chromatography on silica
gel (40–70 mm) from Sorbent Technologies. Hydroxyphenylhydra-
zines were prepared from corresponding aminophenol by follow-
ing preparation procedure33 and/or following the procedure for
preparation of 3-hydroxyphenylhydrazine. All reagents and sol-
vents were purchased from Sigma–Aldrich and were analytical
grade.
4.5.3. Typical procedure for 5-formylation of 2,4,6-pyrimidine
triones with trimethyl orthoformate (Table 1)
Preparation of 1,3-dimethyl-2,4,6-trioxohexahydropyrimidine-
5-carbaldehyde (BA19). A white suspension of trimethyl orthofor-
mate (300 mL), 1,3-dimethyl-2,4,6-pyrimidinetrione (50 g;
0.32 mol), and sulfuric acid (two drops) was stirred with refluxing
for five hours. After approximately 10 min, the reaction mixture
transformed into a clear yellow solution that shortly turns again
into yellow suspension. The reaction suspension was left at room
temperature for several hours. Solid product was separated by fil-
tration, washed with ether (3 ꢁ 30 mL), and dried on air to give
pure product (51 g; 86%). 1H NMR (CDCl3) d 9.25 (1H, s, CH), 8.64
(1H, s, 5-H), 3.35 (3H, s, NCH3), and 3.33 (3H, s, NCH3) ppm. 13C
NMR (CDCl3) d 178.2, 156.2, 154.4, 101.0, 28.2 and 27.6 ppm;
MS-EI, m/z 184 (M+, 20%), 169 ((MꢂCH3)+, 28%), 156 (MꢂCO)+,
39%).
4.5.1. Typical procedure for the preparation of N-unsubstituted
5-aroyl-2,4,6-pyrimidinetriones (Table 1)
Preparation of 5-benzoyl-pyrimidine-2,4,6-trione (BA1). A pyri-
dine (100 mL) suspension of pyrimidinetrione (2.56 g; 0.02 mol)
was heated at 70 °C until dissolved. Into this dark yellow solution,
benzoyl chloride was slowly added (2.8 g; 0.02 mol). Immediately
the color changed to red and then to a dark red solution. Stirring
at 70 °C continued for an additional two hours, and then at room
temperature overnight. The solvent was evaporated under reduced
pressure. The dark solid was dissolved in hot water (400 mL) and
acidified with concentrated hydrochloric acid to pH ꢀ2 and left
4.5.4. Typical procedure for 5-acetylation of 2,4,6-pyrimidine
triones with triethyl orthoacetate (Table 1)
Preparation of 5-acetyl-1,3-dimethylpyrimidine-2,4,6-trione
(BA20). A triethyl orthoacetate (20 mL) suspension of 2,4,6-pyrim-
idinetrione (2.56 g; 0.02 mol), and p-toluenesulfonic acid monohy-
drate (200 mg) was heated at 80 °C overnight. The reaction

824
D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
mixture was a suspension the entire time. The reaction mixture
was concentrated to about 1/3 of the original volume and left at
room temperature overnight. Solid material was separated by filtra-
tion from the dark solution, washed with a small portion of ice–
water chilled acetone, ether (3 ꢁ 10 mL) and dried on air to give pure
product (2.9 g; 85%). 1H NMR (DMSO-d6) d 11.77 (1H, s, NH), 11.04
(1H, s, NH), and 2.56 (3H, s, CH3) ppm. 13C NMR (DMSO-d6) d
195.67, 149.8, 96.1, and 24.5 ppm. Elemental Analysis Calculated:
C 42.36, H 3.55, N 16.47. Found: C 42.25, H. 3.68, N 16.35.
4.5.9. Typical procedure for preparation of Schiff bases with
amino acids (Table 5)
Preparation of 6-{(E)-[1-(2,4,6-trioxohexahydropyrimidin-5-
yl)ethylidene]amino}hexanoic acid (BA29). A methanol (500 mL)
suspension of 5-acetyl-2,4,6-pyrimidinetrione (1.7 g; 0.01 mol)
and 5-aminohexanoic acid (1.3 g; 0.01 mol) with kaolin clay (1 g)
was refluxed with stirring overnight. Insoluble material was sepa-
rated by hot filtration. The filtrate volume was reduced to ꢀ30 mL
and cooled at ice water for 30 min. The formed white powder was
separated by filtration, washed with ether (3 ꢁ 10 mL) and dried
on air to give pure product (2.1 g; 74%). 1H NMR (DMSO-d6) d
10.0 (2H, broad s, NH), 2.74 (2H, t, J = 7.6 Hz, NCH2), 2.25 (3H, s,
CH3), 2.16 (2H, t, J = 7.6 Hz), 1.51 (2H, m), 1.45 (2H, m), and 1.26
(2H, m) ppm. 13C NMR (DMSO-d6) d 194.7, 175.2, 166.4, 151.7,
95.6, 39.3, 34.1, 32.7, 27.3, 26.0, and 24.6 ppm.
4.5.5. Typical procedure for preparation of 5-rylidene-2,4,6-pyri
midinetrione (Table 2)
Preparation of 5-(2-hydroxybenzylidene)pyrimidine-2,4,6-tri-
one (BA47). Salicylic aldehyde (1.22 g; 0.01 mol) was added into
a
stirring water (75 mL) solution of 2,4,6-pyrimidinetrione
(1.28 g; 0.01 mol). Immediately after mixing, an orange precipitate
started to form. The resulting orange suspension was stirred at
room temperature for additional 10 min. The solid product was
separated by filtration, washed with water (3 ꢁ 10 mL) and dried
on air to give 2.2 g (91%) of pure product. 1H NMR (DMSO-d6) d
11.29 (1H, s), 11.12 (1H, s), 10.58 (1H, s), 8.60 (1H, s), 8.14 (1H,
d, J = 8.0 Hz), 7.35 (1H, t, J = 8.0 Hz), 6.91 (1H, d, J = 8.4 Hz), 6.81
(1H, t, J = 8.0 Hz) ppm. 13C NMR (DMSO-d6) d 164.4, 162.5, 159.7,
151.0, 135.4, 133.5, 120.6, 118.9, 117.8, and 116.1 ppm.
4.5.10. Typical procedure for preparation of Schiff bases with
aromatic and aromatic heterocyclic amines (Table 5)
5-{(E)-[(4-Hydroxyphenyl)imino]methyl}-1,3-dimethylpyrimidine-
2,4,6-trione (BA79). Methanol (150 mL) solution of 5-formyl-1,
3-dimethyl-2,4,6-pyridinetrione (1.84 g; 0.01 mol), and 4-amino-
phenol (1.2 g; 0.011 mol) was refluxed for two hours. The reaction
mixture became a suspension after refluxing for a few minutes. The
pale yellow solid was separated by filtration, washed with cold
methanol (3 ꢁ 20 mL) and dried on air to give 2.6 g (95%) of pure
product. 1H NMR (DMSO-d6) d 11.78 (1H, d, J = 1.32 Hz, 5-H),
9.66 (1H, s, OH), 8.32 (d, J = 1.28, CH), 7.23 (2H, d, J = 8.8 Hz,
m-H), 6.75 (2H, d, J 8.8 Hz), and 3.09 (6H, s, NCH3) ppm. 13C NMR
(DMSO-d6) d 164.5, 152.5, 156.7, 152.1, 151.9, 130.9, 120.7,
116.7, 92.0, 28.1, and 27.4 ppm. Elemental Analysis Calculated: C
56.72; H 4.76; N 15.27. Found: C 56.65, H 4.87, N 15.16.
4.5.6. Typical procedure for preparation of 5-alkyl-2,4,6-pyrimid
one (Table 3)
Preparation
of
5-(3-phenylpropyl)pyrimidine-2,4,6-trione
(BA66). A methanol (30 mL) suspension of 5[3-phenylprop-2-en-
1-ylidene]pyrimidine-2,4,6-trione (BA42) (500 mg; 2 mmol) and
5% Pt/C (50 mg) was shaken under hydrogen pressure (32 psi) at
room temperature for 5 h. The catalyst was separated by filtration
and filtrate was evaporated to white powdery material (480 mg;
96%). 1H NMR (DMSO-d6) d 11.19 (2H, s NH), 7.24 (2H, t,
J = 7.2 Hz), 7.15 (3H, m), 3.53 (1H, m, 5-H), 2.53 (2H, t, J = 7.
6 Hz), 1.89 (2H, m), and 1.55 (2H, m) ppm. 13C NMR (DMSO-d6) d
171.2, 151.6, 142.2, 128.95, 128.92, 126.4, 48.5, 35.6, 28.6, and
28.4 ppm.
4.5.11. Preparation of Schiff bases with amino pyridines
(Table 5)
Preparation of (E)-1,3-dimethyl-5-((pyridin-4-ylimino)methyl)
pyrimidine-2,4,6-trione (BA82). A methanol (150 mL) solution of
5-formyl-1,3-dimethylpyrimidine-2,4,6-trione (1.84 g; 0.01 mol)
and 4-aminopyridine (0.94 g; 0.01 mol) was refluxed for three
hours. The volume of the reaction mixture was reduced to 1/3
(ꢀ50 mL) by distilling off methanol at atmospheric pressure. A
deep red colored reaction mixture was left at room temperature
overnight. The formed yellow crystals were separated by filtration,
and washed with cold methanol to give 2.1 g (81%) pure product.
1H NMR (CDCl3) d 11.95 (1H, d, J = 13.6 Hz, 5-H), 8.75 (1H, d, J
=13.6 Hz, CH), 8.63 (2H, d, J = 6.2 Hz, 2-H pyridine), 7.16 (2H, d,
J = 6.0 Hz, 3-H pyridine), and 3.38 (6H, s, NCH3) ppm. 13C NMR
(CDCl3) d 165, 152, 151, 145, 115, 112, 96, 28, and 27 ppm.
4.5.7. Typical procedure for preparation of 5,5-dialkylated pyri
midinetrione (Table 4)
Preparation of 5,5-di[4-(dimethylamino)benzyl]pyrimidine-
2,4,6-trione (BA71). A methanol (50 mL) suspension of 2,4,6-pyri-
dinetrione (256 mg; 2 mmol), 4-dimethylaminobenzaldehyde
(600 mg; 4 mmol) and 5% Pt–C (50 mg) was first shaken under
nitrogen for two hours and then under hydrogen pressure (32
psi) for 10 h. Catalyst was separated by filtration and the filtrate
evaporated to a yellow powdery product (750 mg; 94%). 1H NMR
(DMSO-d6) d 11.10 (2H, s, NH), 6.84 (4H, d, J = 8.4 Hz), 6.56 (4H,
d, J = 8. 4 Hz), 3.11 (4H, s, CH2), and 2.80 (12H, s, NCH3) ppm. 13C
NMR (DMSO-d6) d 173.3, 150.1, 149.9, 130.5, 123.0, 112.8, 71.1,
60.3, and 43.9 ppm.
4.5.12. Typical procedure for preparation of unsubstituted 2,4,6-
pyrimidinetrione hydrazones (Table 6)
Preparation of 5-[(E)-hydrazinylidenemethyl]pyrimidine-2,4,6-
trione (BA116). A methanol (200 mL) suspension of 5-formylpyrim-
idine-2,4,6-trione (1.56 g; 0.01 mol) and hydrazine (2 g; 0.04 mol)
was refluxed for one hour, sonicated for 30 min and refluxed for an
additional hour. The still hot, yellow insoluble material was sepa-
rated by filtration, washed with hot methanol (3 ꢁ 20 mL) and dried
at 110 °C for 10 min to give pure product (1.6 g; 94%). 1H NMR
(DMSO-d6) d 11.01 (1H, br s, 5-H), 10.55 (1H, s, NH), 10.49 (1H, s,
NH), 7.97 (1H, s, CH), and 5.64 (2H, s, NH2) ppm. 13C NMR (DMSO-
d6) d 161.9, 155.5, 151.6, and 87.5 ppm. Elemental Analysis
Calculated: C 35.30, H 3.55, N 32.93; Found: C 35.21, H 3.61, N 32.85.
4.5.8. Preparation of (E)-1,3-dimethyl-5-((2-phenylhydrazono)
methyl)pyrimidine-2,4,6-trione (BA73)
A
methanol solution (100 ml) of carbaldehyde (1.82 g;
0.01 mol) and phenylhydrazine (1,3 g; 0.012 mol) was refluxed
for two hours. The reaction mixture was left at room temperature
overnight and cooled in ice water for 10 min before being filtered.
The solid product was washed with ice–water cooled methanol
(3 ꢁ 10 ml) and dried on air to give pure product in 95% (2.6 g) iso-
lated yield. 1H NMR (DMSO-d6) d 11.15 (1H, d, J = 11 Hz, NH), 8.79
(1H, s), 8.14 (1H, d, J = 11 Hz), 7.19 (2H, t, J = 8 Hz), 6.83 (1H, t,
J = 7 Hz), 6.73 (2H, d, J = 8 Hz) 3.10 (3H, s), and 3.09 ppm (3H, s);
13C NMR (DMSO-d6) d 163.9, 162,6, 160.8, 158.3, 152.1, 148.4,
129.7, 121.3, 113,7, 90.3, 28.1, and 27.5 ppm.
4.5.13. Typical procedure for preparation of hydrazones of 2,4,6-
pyrimidinetriones (Table 7)
Preparation
of
5-{(E)-[2-(4-nitrophenyl)hydrazinylidene]
methyl}pyrimidine-2,4,6-trione (BA22). A hot methanol (200 mL)

D. M. Neumann et al. / Bioorg. Med. Chem. 22 (2014) 813–826
825
solution
of
2,4,6-trioxohexahydropyrimidine-5-carbaldehyde
two hours and concentrated to volume of about 100 mL. Still
hot methanol suspension was filtered and white solid product was
washed with methanol (3 ꢁ 10 mL) and dried at 110 °C for 10 min
to give 1.2 g (90%) white crystalline product. 1H NMR (DMSO-d6) d
11.09 (1H, s, NH), 10.78 (1H, s, NH), 10.67 (1H, s, NH), 7.90 (1H, s,
CH), 2.13 (2H, t, J = 7.2 Hz, COCH2), 1.50 (2H, m, COCH2CH2), 1.22
(4H, m, CH2CH2), and 0.82 (3H, t, J = 6.4 Hz, CH3) ppm. 13C NMR
(DMSO-d6) d 171.4, 166.0, 164.2, 156.4, 151.5, 89.9, 33.7, 31.4,
24.9, 22.5, and 14.4 ppm.
(1.56 g, 0.01 mol) and a methanol solution (200 mL) of 4-nitro-
phenylhydrazine were mixed together and refluxed with stirring
for two hours. The still hot reaction mixture was filtered. A dark
brown solid was discarded and the filtrate was reduced
(ꢀ75 mL). The formed yellow precipitate was separated by filtra-
tion, washed with cold methanol (3 ꢁ 15 mL) and dried on air to
give pure product (2.2 g; 76%). 1H NMR (DMSO-d6) d 11.25 (1H,
br s), 10.85 (1H, s), 10.75 (1H, s), 9.86 (1H, s), 8.11 (2H, d,
J = 9.2 Hz), 6.80 (2H, d, J = 9.2 Hz) ppm. 13C NMR (DMSO-d6) d
165.8, 164.2, 159.8, 154.3, 151.5, 140.3, 126.5, 112.5, and
91.3 ppm. Elemental Analysis Calculated: C 45.37, H 3.11, N
24.05; Found: C 45.33, H 3.20, N 23.94.
4.5.18. (E)-N0-(1-(1,3-Dimethyl-2,4,6-trioxohexahydropyri
midin-5-yl)ethylidene)hexanehydrazide (BA161)
Methanol (200 mL) solution of 5-acetyl-1,3-dimethylbarbitu-
ric acid (990 mg; 5 mmol) and hexanehydrazide (650 mg;
5 mmol) was refluxed for 2 h. Reaction mixture was concentrated
to 10 mL and diluted with ether (100 mL). Resulting solution was
left at room temperature for two hours. Formed white crystalline
product was separated by filtration, washed with ether
(3 ꢁ 10 mL) and dried on air to give 1.45 g (94%) of pure product.
1H NMR (CDCl3), d 13.61 (1H, s, CH), 9.10 (1H, s, NH), 3.23 (3H,s,
NCH3), 3.21 (3H, s, NCH3), 2.59 (3H, s, CH3), 2.30 (2H, t, J = 7.6 Hz,
COCH2), 1.65 (2H, q, J = 7.6 Hz, COCCH2), 1.29 (4H, m), and 0.87
(3H, t, J = 6.8 Hz, CH3) ppm. 13C NMR (CDCl3) d 174.3, 172.4,
166.1, 162.6, 151.4, 90.3, 34.2, 31.5, 28.2, 27.9, 25.2, 22.5, 16.7,
and 14.1 ppm.
4.5.14. Typical procedure for preparation of methyl hydrazono
carboxylates (Table 8)
Preparation of (Z)-methyl 2-((1,3-dimethyl-2,4,6-trioxohexahy-
dropyrimidin-5-yl)(4-hydroxyphenyl)methylene)hydrazinecarboxy-
late (BA36). A methanol (150 mL) suspension of (4-hydroxybenzoyl)-
1,3-dimethylpyrimidine-2,4,6-trione (2.7 g; 0.01 mol) and methyl
hydrazinocarboxylate (0.9 g; 0.01 mol) was refluxed with stirring
for three hours. The dark reaction mixture was evaporated to dry-
ness. The solid residue was mixed with ether (150 mL) and soni-
cated with stirring at room temperature for two hours. Insoluble
product was separated by filtration, washed with ether
(3 ꢁ 30 mL) and dried on air to give 3.05 g (88%) pure product.
1H NMR (DMSO-d6) d 12.7 (1H, s, 5-H), 9.8 (2H, br s, NH and
OH), 6.97 (2H, d, J = 8.4 Hz, m-H), 6.73 (2H, d, J = 8.4 Hz, o-H), 3.5
(3H, s, OCH3), and 3.06 (6H, s, NCH3) ppm. 13C NMR (DMSO-d6) d
159.0, 151.6, 129.4, 123.4, 115.2, 90.7, 53.1, and 28.2 ppm. Elemen-
tal Analysis Calculated: C 51.72, H 4.63, N 16.09. Found: C 51.64, H
4.75, N 15.98.
4.5.19. Typical procedure for preparation of
arylsulfohydrazones
(E)-4-Methyl-N0-((2,4,6-trioxohexahydropyrimidin-5-yl)meth-
ylene)benzenesulfonohydrazide (BA113). Methanol (400 mL) sus-
pension of 5-formylpyrimidinetrione (1.56 g; 0.01 mol) and 4-
methylbenzenesulfonohydrazide (1.86 g; 0.01 mol) was refluxed
with stirring and sonication for four hours and left at room tem-
perature for additional four hours. White solid product was sepa-
rated by filtration, washed with methanol (3 ꢁ 20 mL), ether
(3 ꢁ 10 mL) and dried on air to give 2.95 g (91%) of pure product.
1H NMR (DMSO-d6) d 11.10 (1H, s, 5-H), 10.84 (1H, s, NH, 10.78
(1H, s, NH), 10.65 (1H, s, NH), 7.81 (1H, s, CH), 7.64 (2H, d,
J = 7.6 Hz, o-H), 7.41 (2H, d, J = 8 Hz, m-H), and 2.37 (3H, s, CH3)
ppm. 13C NMR (DMSO-d6) d 165.9, 163.8, 159.2, 151.3, 145.5,
134.1, 130.6, 128.6, 92.1, and 21.7 ppm. Elemental Analysis Calcu-
lated: C 44.44; H 3.73; N 17.28. Found: C 44.32; H 3.84; N 17.13
4.5.15. Typical procedure for preparation aromatic hydrazide–
hydrazones (Table 8)
Preparation
(E)-N0-((2,4,6-trioxohexahydropyrimidin-5-
yl)methylene)benzohydrazide (BA124). Methanol (200 mL) suspen-
sion of 5-formyl-2,4,6-pyrimidinetrione (1.56 g; 0.01 mol) and
benzohydrazide (1.36 g; 0.01 mol) was sonicated with stirring at
60 °C for six hours and refluxed for one hour. The still hot, white sus-
pension was filtered and solid washed with hot methanol
(3 ꢁ 20 mL), ether (3 ꢁ 20 mL) and dried on air to give 2.3 g (84%)
white powdery product. 1H NMR (DMSO-d6) d 11.86 (1H, s, 5-H),
10.87 (1H, s, NH), 10.74 (1H, s, CH), 8.15 (1H, s, NH), 7.86 (2H, d,
J = 8.4 Hz, o-H), 6.10 (1H, t, J = 8.4 Hz, p-H), and 7.53 (2H, t, J = 8 Hz,
m-H) ppm. 13C NMR (DMSO-d6) d 166.2, 165.1, 164.1, 155.5, 151.5,
133.1, 132.0, 129.4, 128.2, and 90.1 ppm. Elemental Analysis Calcu-
lated: C 52.56, H 3.68, N 20.43. Found: C 52.51, H 3.75, N 20.05.
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 unre-
stricted Grant from Research to Prevent Blindness, New York, NY
(LSU Department of Ophthalmology-LSUHSC). Strain CS 177.93
was a kind gift from Dr. Paul Fidel (LSUHSC).
4.5.16. Typical procedure for preparation of alkanehydrazides
Preparation of hexanehydrazide. A methanol solution (50 mL) of
hydrazine hydrate (5 g; 0.1 mol) and methyl hexanoate (1.3 g;
0.01 mol)wasrefluxed for90 min. Methanolwasdistilled off at atmo-
spheric pressure and the resulting oily residue was mixed with ethyl
acetate (200 mL) and water (50 mL). The ethyl acetate layer was sep-
arated, washed with water (3 ꢁ 10 mL), and dried over anhydrous so-
dium sulfate. Evaporation of ethyl acetate yields a white solid product
in 93% isolated yield. 1H NMR (CDCl3) d 6.98 (1H, s, NH), 3.91 (2H, s,
NH2),2.13(2H, t,J = 7.6 Hz, COCH2),1.62(2H, q,J = 7.6 Hz, COCH2CH2),
1.29 (4H, m, CH2CH2), and 0.88 (3H, t, J = 7.6 Hz, CH3) ppm. 13C NMR
(CDCl3) d 174.4, 34.7, 31.6, 25.4, 22.6, and 14.1 ppm.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.010.
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