Synthesis and biological activity investigation of azole and quinone hybridized phosphonates

Article abstract of DOI:10.1016/j.bmcl.2018.08.002

Phosphonates, azoles and quinones are pharmacophores found in bioactive compounds. A series of phosphonates conjugated to azoles and quinones with variable carbon chain lengths were synthesized in 3–4 steps with good yield. Antifungal assay of these compounds showed that ethyl protected phosphates have excellent inhibitory activity against phytopathogenic fungus Fusarium graminearum, and the free-base phosphates have good activity against human pathogenic fungi Aspergillus flavus and Candida albicans. Structure- activity relationship (SAR) studies showed activity increases with longer carbon chain length between phosphonate and anthraquinone analogs consisting of azole and quinone moieties. These newly synthesized compounds also have mild antibacterial activities to Gram positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytotoxicity analysis of these compounds against HeLa cells reveals that the phosphoric acid analogs are less toxic compared to ethyl protected phosphonates. Three leads compounds have been identified with prominent antifungal activity and low cytotoxicity.

Full text of DOI:10.1016/j.bmcl.2018.08.002

Accepted Manuscript
Synthesis and biological activity investigation of azole and quinone hybridized
phosphonates
Yagya Prasad Subedi, Madher N. Alfindee, Jaya P. Shrestha, Greg Becker,
Michelle Grilley, Jon Y. Takemoto, Cheng-Wei Tom Chang
PII:
S0960-894X(18)30651-6
https://doi.org/10.1016/j.bmcl.2018.08.002
BMCL 25982
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
13 July 2018
28 July 2018
1 August 2018
Please cite this article as: Subedi, Y.P., Alfindee, M.N., Shrestha, J.P., Becker, G., Grilley, M., Takemoto, J.Y.,
Chang, C.T., Synthesis and biological activity investigation of azole and quinone hybridized phosphonates,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.08.002
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Yagya Prasad Subedi, Madher N. Alfindee, Jaya P. Shrestha, Greg Becker, Michelle Grilley, Jon Y. Takemoto,
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Bioorganic & Medicinal Chemistry Letters
Synthesis and biological activity investigation of azole and quinone hybridized
phosphonates
a
a
a
a
b
Yagya Prasad Subedi , Madher N. Alfindee , Jaya P. Shrestha , Greg Becker , Michelle Grilley , Jon Y.
b
a
Takemoto , Cheng-Wei Tom Chang *
a
Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322-0300, USA
Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322-5305, USA
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Phosphonates, azoles and quinones are pharmacophores found in bioactive compounds. A series
of phosphonates conjugated to azoles and quinones with variable carbon chain lengths were
synthesized in 3-4 steps with good yield. Antifungal assay of these compounds showed that
ethyl protected phosphates have excellent inhibitory activity against phytopathogenic fungus
Fusarium graminearum, and the free-base phosphates have good activity against human
pathogenic fungi Aspergillus flavus and Candida albicans. Structure- activity relationship (SAR)
studies showed activity increases with longer carbon chain length between phosphonate and
anthraquinone analogs consisting of azole and quinone moieties. These newly synthesized
compounds also have mild antibacterial activities to Gram positive bacteria, including
methicillin-resistant Staphylococcus aureus (MRSA). Cytotoxicity analysis of these compounds
against HeLa cells reveals that the phosphoric acid analogs are less toxic compared to ethyl
protected phosphonates. Three leads compounds have been identified with prominent antifungal
Keywords:
Phosphonate
Azole antifungal
Anthraquinone analogue
Fusarium graminearum
Aspergilus flavu
Candida albicans
activity and low cytotoxicity.
2009 Elsevier Ltd. All rights reserved.
1
1-15
Compounds containing quinone moieties are in great interest
3).
These compounds can be prepared in 2-3 steps and have a
1
2
because of their wide biological activity . Mitomycin C ,
wide range of biological activities from anticancer to
antibacterial and antifungal activity. For example, when attached
with a linear alkyl chain, the CAAs, 1 are mainly antibacterial.
With the attachment of aryl groups, compound 2 becomes
primarily anticancer and antibacterial. The dimeric CAAs, 3
behave mainly as antifungal and antibacterial agents. These
compounds all have a redox active naphthoquinone core, which
plays the pivotal role in the exerted biological activities.
3
4
streptonigrin , and anthracyclines are the quinone based
anticancer drugs used in the clinical practice (Figure 1). Along
with anticancer properties, compounds containing the quinone
5
-8
motif are also antifungal and anti-bacterial. Azole compounds
have also attracted great attention due to their broad-spectrum
biological activities, especially as antifungal agents like
9
, 10
fluconazole, metconazole and itraconazole (Figure 2).
Figure 2. A caption is positioned left-justified below the figure or scheme.
Figure 1. Structure of quinone-based anticancer drugs.
Organophosphorus compounds are known to exert diverse
1
6
17
18
biological effects including antibacterial , herbicidal , and
anti-inflammatory
Inspired by the success of quinone and azole compounds, we
have pursued the synthesis of cationic anthraquinone analogs
19
activities. To increase the biological
availability and stability, phosphonates are commonly employed
(CAAs), a hybrid of naphthoquinone and 1,2,3-triazole (Figure

as analogs of organophosphorus compounds with a C-P bond.
situ in the presence of excess naphthoquinone, and yielded
12
2
0
Organophosphorus compounds are also used as anticancer , and
compounds 9a-f.
Following the cycloaddition/oxidation
2
1
antiviral prodrugs. Organophosphorus compounds with O-P
process, the ethyl groups of the phosphonate were removed using
trimethylsilyl iodide (TMSI). Deprotection of ethyl groups of 9c
led to the formation of the desired product mixed with
inseparable impurities. Therefore, no corresponding adducts in
the forms of phosphoric acid or potassium phosphonate were
tested. The resulting phosphoric acids, 10a, 10b, and 10d-10f,
were purified and fully characterized. However, to improve
solubility, these phosphoric acids were converted to potassium
salts using IR120 ion exchange resin, and assayed as potassium
salts, 11a, 11b, and 11d-11f. Compounds 9a-9f, ethyl ester of
phosphonates, can be viewed as prodrugs of compounds 11a,
11b, and 11d-11f. (potassium phosphonates), and both were
evaluated for their biological activities. In short, there are two
main structural differences among the synthesized compounds: 1)
chain-length connecting the triazole and phosphonate; and 2) the
forms of phosphonates (ethyl ester vs. potassium phosphonates).
2
2
bonds can be easily hydrolyzed in physiological conditions ,
whereas phosphonates with C-P bonds are stable under these
2
3
conditions.
Similar to organophosphorus compounds,
phosphonates are also used as antifungal, anticancer, and
antiviral prodrugs, such as fosfluconazole, estramustine
phosphate, fludarabine phosphonate, and fosamprenavir (Figure
2
4
4
). Conjugation of these phosphonate groups improves the
solubilities of the parent drugs. Phosphonates conjugated to other
biologically important moieties have shown antifungal and
2
5,26
antibacterial activities (Figure 5).
Motivated by these
applications of phosphonates, we began to systematically
investigate the biological implications of conjugating
phosphonates and anthraquinone analogs.
Scheme 1.
Figure 3. Structure and activity of CAAs.
Figure 4. Structure of bioactive organophosphorus compounds.
1
N/A : Not applicable, compound was purchased from commercial source.
Figure 5. Phosphonate conjugated biologically compound with antibacterial
and antifungal activities.
The synthesized compounds were tested against both plant
and human fungal pathogens, including Aspergillus flavus,
Fusarium graminearum B4-5A, Candida albicans ATCC 64214
(
azole resistance), Candida albicans MYA2876, Cryptococcus
The synthetic design in this work was to combine the redox
active naphthoquinone core, predecessor of the CAA core, and
phosphonate with various linkers. The synthesis began with an
Arbuzov reaction using dibromoalkanes and triethylphosphite
neoformans H99, and Rhotorula pilimanae (Table 1) as
previously described.
aminoglycoside K20 were used as positive controls. F.
graminearum is a well-known plant pathogen causing head blight
in wheat and other grains and destruction of food crops. C.
2
7
Voriconazole and amphiphilic
2
8
(
Scheme 1). Reacting the resulting bromoalkyl phosphonates
with sodium azide provided compound 8a-8f ready for a [2+3]
cycloaddition with 1,4-naphthoquinone. During this
cycloaddition, the cyclization was followed by an oxidation in
2
9
3
0,31
albicans and A. flavus are opportunistic pathogenic fungi.
albicans is the most common Candida species responsible for
C.

candidiasis humans. A. flavus produces toxic mycotoxins in
analogs, 11a-11f. In addition, there was no significant effect
when the linker chain length. Interestingly, among the potassium
phosphonate analogs, 11e is active against A. flavus and C.
albicans with MICs ranging from 8-32 µg/mL. In short, there is
no clear SAR against tested fungi except for F. graminearum. It
is speculated that the variation in antifungal susceptibility is due
to differences in the membrane lipid composition among various
fungal species. Similar results have been observed for the
3
2
mammals and is responsible for aspergillosis in animals and
3
1
humans. Minimum inhibitory concentrations (MICs) from
antifungal assays showed that the ethyl protected analogs (9a-9f)
have excellent inhibitory activity against F. graminearum, and
activity increases with lengthening of the connector chain in
between the phosphonate group and anthraquinone moiety.
Compounds 9a-9d were also active towards C. neoformans but
the activity decreased with an increase in connector chain length.
On the other hand, there was a significant drop in antifungal
activity against F. graminearum for potassium phosphonate
2
8
recently described amphiphilic antifungals.
a
Table 1. Minimum inhibitory concentration of phosphonate compounds against fungi .
Aspergillus
flavus
64 - 128
64 - 128
>128
Fusarium
graminearum B4-5A albicans 64124
Candida
Candida albicans
MYA2876
>128
Cryptococcus
neoformans H99
Rhodotorula
pilimanae
64 - 128
64 - 128
128
64 - 128
>128
>128
Compound
9
9
a
b
16
16
8
>128
>128
>128
>128
>128
>128
>128
32
32 - 64
16 - 32
128
256
>256
16
16
32
32
>128
>128
64
32
64
32
>128
>128
>128
>128
>128
>128
32
32 - 64
8 - 16
128
9
c
9
d
>128
4
9
e
>128
1-2
0.5
32
32
64
32
32
32
32
b
9
f
ND
11a
11b
11d
>128
16 - 32
64
8 - 16
ND
128
32
128
>128
64
1
1
1e
1f
32
4
0.125
K20
Voriconazole
> 256
1
64
0.125
4
8
a
b
Unit: µg/mL, ND: Not determined
For the evaluation of antibacterial activity, the synthesized
compounds were examined against Gram negative (G-) bacteria,
E. coli (25922), and Gram-positive (G+) bacteria, S. aureus
To investigate the cytotoxicity, the synthesized compounds
9a-9f and 11a, 11b, and 11d-11f were tested against human
HeLa cells and cervical cancer cells. Among the tested
compounds, potassium phosphonate analogs 11a, 11b, and 11d-
11f did not show any toxicity up to 10 µg/mL. These compounds
exert toxicities with cell viabilities around 50% at 100 µg/mL.
Similarly, ethyl-protected analogs with the longest connector
chain 9e and 9f did not show toxicity up to 10 µg/mL and some
toxicity at 100 µg/mL. The calculated IC values showed that the
toxicity of the ethyl protected compound 9a-9f decreases with
increase in the connector chain length increase (Table 3). In
general, potassium phosphonates were less toxic relative to the
ethyl-protected analogs particularly with compounds 11a and
(ATCC 25923), S. aureus (ATCC 33591, MRSA), S. aureus
(ATCC 43300, MRSA) (Table 2). Among the tested compounds,
the MICs against E. coli were more than 128 µg/mL for ethyl
esters and 256 µg/mL for potassium phosphonate analogs. Most
of the tested compounds have moderate activity towards G+
bacterial with MIC ranges from 16 – 128 µg/mL. Among these
compounds, 9e is the most active against S. aureus (ATCC
5
0
4
3300) (MRSA) with MIC value 16-32 µg/mL. However, these
newly synthesized compounds show relatively lower antibacterial
activity than their antifungal activity.
1
1b as compared to their ethyl protected counterparts.
Conceivably, the negative charge on the compound, which make
the compound difficult to go inside the cells may be the reason
for reduced toxicity towards mammalian cells.
a
Table 2. MICs of phosphonate compounds against bacteria .
2
1
E. coli
(ATCC
S. aureus
(ATCC
25923)
S. aureus
(ATCC
33591)
S. aureus
(ATCC
43300)
Compound
2
5922)
a
>
128
128
128
128
128
128
256
256
256
256
256
64
64
64
Table 3. IC50 values of the compounds against HeLa cells .
9
9
a
Compound
IC50 value
Compound
IC50 value
>
>
>
>
>
>
>
>
>
>
64
64
64-128
64-128
32-64
16-32
16- 64
128
b
9
9
a
13.83± 0.52
11a
11b
11d
11e
11f
91.80±5.08
87.98±8.37
128
32-64
64
128
64
9
c
6
.97±0.66
b
9
d
9
c
9.11±0.65
73.64±0.94
95.31±4.63
>100
32
9
e
46.99±5.55
9
d
>128
32
64
9e
9
52.00±4.49
65.66±1.30
9
f
f
128
128
64
11a
11b
11d
a
Unit: µg/mL
32
64-128
64
Since the newly synthesized compounds were most active
against F. graminearum, the trend of their antifungal activity and
cytotoxicity was further analyzed. To provide a clear SAR, the
cytotoxicity and MICs of the tested compounds against F.
graminearum was plotted in the same chart (Figure 6). Based on
the comparison of MICs and IC , it is obvious that the antifungal
32-64
64
256
>256
>256
>256
1
1e
1f
32-64
1
a
Unit: µg/mL
5
0

activity of 9a-9f increases with increases in the chain-length of
linkers between anthraquinone moiety and phosphonate group
while the cytotoxicity towards mammalian cells decreases.
Specifically, there is no significant difference in the cytotoxicity
of the compounds with linkers ranging from C2 to C4 (compound
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9
a-9c) and significantly decreases for the compound with linkers
ranging from C6 to C10 (compounds 9c-9f). In contrast, there is
no significant difference in the antifungal activity of the
potassium phosphonate analogs (11a, 11b, and 11d-11f) although
all of these compounds display lower cytotoxicity. Combining
these results, compound 9f stands out as the best lead.
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274-1285.
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fungus, F. graminearum. Potassium phosphonates (ethyl group-
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towards A. flavus and C. albicans. All compounds were less
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We acknowledge the support from Department of Chemistry
and Biochemistry, Utah State University. This work was
supported in part by NSF Award CHE-1429195 for a 500 MHz
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Highlights



Novel bioactive compounds consist of quinone, azole and phosphonate groups.
Leads show excellent activity against Fusarium graminearum and low cytotoxicity.
Optimal structures revealed via SAR study.