Antiprotozoal activities of symmetrical bishydroxamic acids

Article abstract of DOI:10.1016/S0968-0896(03)00522-4

Symmetrical bishydroxamic acids along with their sodium salts containing an alkyl spacer between two aromatic rings were synthesized, and their antiparasitic activities were evaluated. Bishydroxamic acids were conveniently prepared from the alkylation of methyl 4-hydroxybenzoate with various dihalo-alkane, -alkene, and -ether followed by reaction with hydroxylamine. Surprisingly, the bishydroxamic acids and their sodium salts possess strong inhibitory activities against Plasmodium falciparum parasites with IC ₅₀ values in the range of 0.26-3.2 μM. Bishydroxamic acid 3 and its sodium salt 12 also inhibit the growth of Leishmania donovani, albeit at higher concentrations. The corresponding biscarboxylic acids and bismethyl esters are inactive. Presumably, the ability of bishydroxamic acids to complex with metallic iron in hemoglobin may be responsible for antimalarial activity of these compounds.

Full text of DOI:10.1016/S0968-0896(03)00522-4

Bioorganic & Medicinal Chemistry 11 (2003) 4357–4361
Antiprotozoal Activities of Symmetrical Bishydroxamic Acids
Duy H. Hua,a,* Masafumi Tamura,a Masahiro Egi,a Karl Werbovetz,b Dawn Delfın,b
Manar Salemb and Peter K. Chiangc
aDepartment of Chemistry, Kansas State University, Manhattan, KS 66506, USA
bDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue,
Columbus, OH 43210, USA
cDepartment of Applied Biochemistry, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA
Received 7 April 2003; accepted 17 July 2003
Abstract—Symmetrical bishydroxamic acids along with their sodium salts containing an alkyl spacer between two aromatic rings
were synthesized, and their antiparasitic activities were evaluated. Bishydroxamic acids were conveniently prepared from the
alkylation of methyl 4-hydroxybenzoate with various dihalo-alkane, -alkene, and -ether followed by reaction with hydroxylamine.
Surprisingly, the bishydroxamic acids and their sodium salts possess strong inhibitory activities against Plasmodium falciparum
parasites with IC50 values in the range of 0.26–3.2 mM. Bishydroxamic acid 3 and its sodium salt 12 also inhibit the growth of
Leishmania donovani, albeit at higher concentrations. The corresponding biscarboxylic acids and bismethyl esters are inactive.
Presumably, the ability of bishydroxamic acids to complex with metallic iron in hemoglobin may be responsible for antimalarial
activity of these compounds.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
proteinase,6 it is not surprising that bishydroxamic acids
could inhibit malarial parasite growth by affecting
hemoglobin through chelation with iron or inhibiting
proteases such as methionine aminopeptidase 2,1 which
requires Zn, Co, and Mg. Leishmania parasites possess a
transferrin receptor7 and iron chelators reduced the rate
of Leishmania promastigote growth,8 so bishydroxamic
acids are also worthy of investigation against this para-
site.
Malaria remains the world’s most devastating parasitic
disease. According to the World Health Organization,
at least one million deaths and over 300 million acute
illnesses can be attributed to malaria (http://
www.who.int/inf-fs/en/InformationSheet01.pdf). Leish-
maniasis, which is estimated to cause 59,000 deaths each
year (http://www.who.int/tdr/diseases/leish/diseaseinfo.
htm), is triggered by another unrelated protozoan para-
site. In the search for orally active drug candidates
inhibiting the growth of Plasmodium falciparum1 and
Leishmania donovani parasites,2 the causative agents of
severe malaria and visceral leishmaniasis, we decided to
investigate the inhibition potencies of various symme-
trical bishydroxamic acids. Although it has been repor-
ted that histone deacetylation inhibitors such as suberic
acid bisdimethylamide show cytostatic effect against the
acute murine malaria Plasmodium berghei,3 only limited
biologically active bishydroxamic acids have been
reported.4 Since hydroxamic acids are known5 to com-
plex with metallic iron and copper in matrix metallo-
Results and Discussion
Chemistry
Due to the chelation ability of bishydroxamic acids with
metals, three symmetrical bishydroxamic acids 1–3,
along with their corresponding sodium salts, methyl
esters, and carboxylic acids were synthesized, and their
inhibitory activities against Plasmodium and Leishmania
parasites were investigated. As shown in Scheme 1, the
syntheses of the methyl esters, carboxylic acids, and
hydroxamic acids and their sodium salts were carried
out via a typical alkylation of the hydroxy function of
methyl 4-hydroxybenzoate (4) followed by either reac-
tion with hydroxylamine9 to provide bishydroxamic
*Corresponding author. Tel.: +1-785-532-6699; fax: +1-785-532-
6666; e-mail: duy@ksu.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00522-4

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D. H. Hua et al. / Bioorg. Med. Chem. 11 (2003) 4357–4361
Scheme 1.
acids or with base to give biscarboxylic acids. Hence,
treatment of 4 with potassium carbonate and 1,5-dibro-
mopentane, trans-1,4-dichloro-2-butene, and bis-2-bro-
moethyl ether separately in refluxing acetone provided
methyl esters 5 (57% yield), 7 (57% yield), and 9 (40%
yield), respectively. Heating of esters 5,10 7, and 911 with
hydroxylamine and sodium hydroxide in refluxing eth-
anol–water produced bishydroxamic acids 1,4d 2, and 3,
respectively, in good yields. Compounds 2 and 3 have
not been reported previously. Carboxylic acids 6, 8, and
10 were readily obtained from the basic hydrolysis of
esters 5, 7, and 9, respectively, with sodium hydroxide in
refluxing ethanol and water, followed by acidification,
in 79, 82, and 85% yield, respectively. Sodium salts 11
and 12 were obtained by treating bishydroxamic acids 1
and 3, respectively, with sodium hydroxide. Carboxylic
acids 6,12 8,13 and 1012a,14 are known compounds, and
usages in materials have been reported.
Antiparasitic activity
The analogous methyl esters, 5, 7, and 9, and carboxylic
acids, 6, 8, and 10, were used to test whether the bishy-
droxamic acid function is important for the bioactiv-
ities. The sodium salts of 1 and 3, compounds 11 and
12, are soluble in water and were also evaluated for their
biological activities. Table 1 summarizes data of the
inhibition of P. falciparum D6 and W2 intraerythrocytic
forms and L. donovani axenic amastigote-like parasites.
As expected, while bisesters 5, 7, and 9, and biscar-
boxylic acids 6, 8, and 10 do not show inhibitory activ-
ities, hydroxamic acids 1–3 show significant inhibitory
activities. Hence, IC50 values of hydroxamic acids 1, 2,
and 3 are 2.2, 0.69, and 0.24 mM, respectively, for D6,
and 3.2, 1.05, and 0.35 mM, respectively, for W2. Bishy-
droxamic acid sodium salt 12 exhibits the strongest
activities, with IC50 values of 0.26 mM for D6 and

D. H. Hua et al. / Bioorg. Med. Chem. 11 (2003) 4357–4361
4359
Table 1. In vitro inhibitory activities against P. falciparum D6 and W2 and L. donovani parasites
Compounds
D6, IC50 (mM)
D6, IC90 (mM)
W2, IC50 (mM)
W2, IC90 (mM)
L. donovani parasites (mM)
Bisester 5
Bisester 7
Bisester 9
>60
>60
>60
>60
>60
>38
2.2
0.69
0.24
1.0
0.26
0.013
0.013
NT
NT
NT
NT
NT
NT
NT
6.2
1.8
0.47
2.0
>60
>60
>60
>60
>60
>38
3.2
1.05
0.35
1.09
0.36
0.45
8 nM
NT
NT
NT
NT
NT
NT
NT
NT
2.57
0.60
NT
0.60
0.65
0.015
NT
NT
NT
>100
NT
NT
>100
NT
NT
Biscarboxylic acid 6
Biscarboxylic acid 8
Biscarboxylic acid 10
Bishydroxamic acid 1
Bishydroxamic acid 2
Bishydroxamic acid 3
Bishydroxamic acid sodium salt 11
Bishydroxamic acid sodium salt 12
Chloroquine
18.5ꢀ6.0
NT
0.52
0.014
0.024
NT
37.4ꢀ14.0
NT
NT
1.19ꢀ0.12
Mefloquine
Pentamidine
NT, not tested.
0.36 mM for W2. Chloroquine and mefloquine were used
for comparison, their IC50 values being 0.013 mM (both)
for D6, and 0.45 mM and 8 nM, respectively, for W2.
Moreover, bishydroxamic acid 3 and its sodium salt 12
inhibit L. donovani parasites with IC50 values of
18.5ꢀ6.0 and 37.4ꢀ14.0 mM, respectively, with penta-
midine used for comparison. It appears that the inhibi-
tory activities against P. falciparum D6 and W2 and L.
donovani parasites of bishydroxamic acid 3 are similar
to its sodium salt 12. The compounds are more active in
vitro against Plasmodium, perhaps due to the vigorous
metabolism of hemoglobin by the parasite. Compound
12 is soluble in water, while compound 3 is soluble in
DMSO but only slightly soluble in water. The results
demonstrate that bishydroxamic acid functionality is
essential for the inhibition of malaria parasites. It is
possible that the bishydroxamic acids inhibit hypox-
anthine-guanine phosphoribosyltransferases,15 which
lead to antiparasitic activities. The simplicity of the
molecular structures of bishydroxamic acids described
above may provide a new entry in providing potentially
active antimalarial agents that may be used on chlor-
oquine and mefloquine resistant parasites.
Experimental
General methods
Nuclear magnetic resonance spectra were obtained at
either 400 or 200 MHz for 1H and either 100 or
50 MHz for 13C, and reported in ppm. Mass spectra
were taken from an IonSpec HiResMALDI mass
spectrometer using 2,5-dihydroxybenzoic acid as a
matrix.
1,5-Bis-(4-methoxycarbonylphenoxy)pentane (5). A mix-
ture of 11.7 g (0.077 mol) of methyl 4-hydroxybenzoate
(4), 48.0 g (0.347 mol) of K2CO3, and 8.0 g (0.035 mol)
of 1,5-dibromopentane in 100 mL of acetone was stirred
under reflux for 31 h under argon. After the mixture was
cooled to room temperature, the inorganic salts were
removed by filtration, and the filtrate was concentrated
on a rotary evaporator to remove most of the acetone.
The residue was dissolved in 100 mL of dichloro-
methane, washed with aqueous NaOH (30 mL; 0.5 M
solution), dried (MgSO4), and concentrated to dryness
to give 7.37 g (57% yield of 510 as a white solid; mp 93–
95 ꢁC. Ester 5 was crystallized from ethyl acetate to give
1
white crystals. H NMR (CDCl3) d 7.98 (d, J=8.4 Hz,
4H, Ar), 6.90 (d, J=8.4 Hz, 4H, Ar), 4.05 (t, J=6.4 Hz,
4H, OCH2), 3.88 (s, 6H, OCH3), 1.98–1.60 (m, 6H,
CH2); 13C NMR (CDCl3) d 166.8, 162.7, 131.5, 122.4,
114.0, 67.8, 51.8, 28.8, 22.6.
Conclusion
Symmetrical bishydroxamic acids and their sodium salts
were synthesized from the alkylation of methyl 4-
hydroxybenzoate with various dihalogenated alkane,
alkene, and ether, followed by reaction with hydroxyl-
amine, and their antiparasitic activities were evaluated.
These bishydroxamic acids and their sodium salts inhi-
bit P. falciparum with IC50 values in the range of 0.26–
3.2 mM. Compounds 3 and 12 also inhibit the growth of
L. donovani, albeit at higher concentrations. Despite
their simplicity in structure, the above mentioned bish-
ydroxamic acids show significant inhibitory activities
against various parasites. These bishydroxamic acids
can easily be modified to provide different spacers by
changing the middle alkyl chain to other heteroatom
containing chains, and ring systems by changing the
aromatic rings to other heterocyclic rings.
trans-1,4-Bis-(4-methoxycarbonylphenoxy)-2-butene (7).
By
a
procedure similar to that above, 3.30 g
(26.4 mmol) of trans-1,4-dichloro-2-butene, 8.85 g
(58.2 mmol) of 4, 36.5 g (0.264 mol) of K2CO3, and
40 mL of acetone gave 3.75 g (40% yield) of 7 as a white
solid after crystallization from ethyl acetate. The mother
liquor from crystallization was concentrated and col-
umn chromatographed on silica gel to give 1.65 g (17%
yield; a total of 57% yield) of 7. Mp 153–155 ꢁC; H
1
NMR (CDCl3) d 7.98 (d, J=7 Hz, 4H, Ar), 6.92 (d,
J=7 Hz, 4H, Ar), 6.10 (t, J=1.5 Hz, 2H, ¼CH), 4.64 (d,
J=1.5 Hz, 4H, CH2), 3.88 (s, 6H, OCH3); 13C NMR
(CDCl3) d 166.7, 162.1, 131.6, 128.0, 122.9, 114.2, 67.6,
51.8. HRMScalcd for C 20H20O6Na: 379.1158, found
379.1154.

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D. H. Hua et al. / Bioorg. Med. Chem. 11 (2003) 4357–4361
2,20-Bis-(4-methoxycarbonylphenoxy)ethyl ether (9). By
a procedure similar to that above, 2.00 g (7.8 mmol) of
2,20-di(bromoethyl) ether, 2.68 g (17.6 mmol) of 4, 11.0 g
(79.6 mmol) of K2CO3, and 15 mL of acetone gave
2.47 g (85% yield) of 911 as a white solidꢁ after crystal-
water, dried under vacuum, and crystallized from etha-
nol to give 0.22 g (79% yield) of 6:12 mp 283–286 ꢁC; 1H
NMR (DMSO-d6) d 7.86 (d, J=8.4 Hz, 4H, Ar), 6.99
(d, J=8.4 Hz, 4H, Ar), 4.06 (t, J=7 Hz, 4H, OCH2),
1.79 (pent, J=7 Hz, 4H, CH2), 1.56 (pent, J=7 Hz, 2H,
CH2); 13C NMR (DMSO-d6) d 164.0, 160.8, 124.7,
122.8, 114.0, 67.5, 28.2, 22.0.
1
lization from ethyl acetate. Mp 120–123 C; H NMR
(CDCl3) d 7.97 (d, J=8.5 Hz, 4H, Ar), 6.92 (d,
J=8.5 Hz, 4H, Ar), 4.21 (t, J=5 Hz, 4H, CH2), 3.95 (t,
J=5 Hz, 4H, CH2), 3.88 (s, 6H, OCH3); 13C NMR
(CDCl3) d 166.7, 162.4, 131.5, 122.8, 114.1, 69.7, 67.5,
51.8.
trans-1,4-Bis-[4-(hydroxycarbonyl)phenoxy]-2-butene (8).
A mixture of 0.10 g (0.28 mmol) of ester 7, 3.0 mL of
ethanol, and 2.0 mL (12 mmol) of 6 N NaOH was trea-
ted similarly to that described above for the preparation
ꢁ
1
1,5-Bis-[4-(hydroxyaminocarbonyl)phenoxy]pentane (1).
To a mixture of 0.9 g (2.4 mmol) of ester 5 in 24 mL of
.
0.5 M solution of NH2OH HCl (12 mmol) was added
of 6 to give 67 mg (74% yield) of 8:13 mp >290 C; H
NMR (DMSO-d6) d 7.88 (d, J=9 Hz, 4H, Ar), 7.03 (d,
J=9 Hz, 4H, Ar), 6.09 (bs, 2H, ¼CH), 4.69 (bs, 4H,
CH2O); 13C NMR (DMSO-d6) d 166.9, 161.7, 131.3,
128.2, 123.2, 114.5, 67.4.
4.8 mL (28.8 mmol) of 6 M aqueous NaOH solution.
The mixture was refluxed for 5 min, then cooled to
room temperature, and 28.8 mL (28.8 mmol) of 1 N HCl
was added. The precipitated solids, was collected by fil-
tration and dried thoroughly under vacuum, to weight
2,20-Bis-[(4-hydroxycarbonyl)phenoxy]ethyl ether (10). A
mixture of 0.15 g (0.40 mmol) of 9, 5.0 mL of ethanol,
and 2.7 mL (16.2 mmol) of 6 N NaOH was treated
similarly to that described above for the preparation of
6 to give 0.115 g (83% yield) of 10:12a,14 mp 308–310 ꢁC;
1H NMR (DMSO-d6) d 7.86 (d, J=7.5 Hz, 4H, Ar),
7.00 (d, J=7.5 Hz, 4H, Ar), 4.18 (t, J=3.5 Hz, 4H,
OCH2), 3.83 (t, J=3.5 Hz, 4H, OCH2); 13C NMR
(DMSO-d6) d 166.9, 162.0, 131.3, 123.0, 114.3, 68.9,
67.4.
1
0.74 g (82% crude yield). H NMR spectrum indicated
the presence of 1 and a small amount of biscarboxylic
acid 6. Recrystallization from DMSO–water (10:1) three
times gave 1,4d 0.51 g (57% yield): mp 200–202 ꢁC (lit.4d
202–203 ꢁC); 1H NMR (DMSO-d6) d 7.69 (d, J=8.8 Hz,
4H, Ar), 6.96 (d, J=8.8 Hz, 4H, Ar), 4.03 (t, J=6.4 Hz,
4H, OCH2), 1.78 (m, 4H, CH2), 1.55 (m, 2H, CH2); 13C
NMR (DMSO-d6) d 165.6, 156.7, 131.2, 128.5, 114.1,
67.6, 28.1, 22.0.
1,5-Bis-[4-(hydroxy-sodio-aminocarbonyl)phenoxy]pentane
(11). To a solution of 0.10 g (0.27 mmol) of 1 in 1 mL of
water was added 21.4 mg (0.53 mmol) of NaOH. The
solution was concentrated to dryness to give 0.112 g
(100% yield) of sodium salt 11. Compound 11 is soluble
trans-1,4-Bis[4-(hydroxyaminocarbonyl)phenoxy]-2-butene
(2). A mixture of 0.45 g (1.26 mmol) of ester 7, 12 mL of
.
0.5 M solution of NH2OH HCl (6 mmol), and 1.0 mL
(6 mmol) of 6 M aqueous NaOH was treated similarly to
that described above for the preparation of 1. Three
crystallizations of the crude product from DMSO–water
1
in water and H NMR is similar to that of 1.
(10:1) gave 2, 0.18 g (40% yield): mp 205–210 ꢁC; H
2,20-Bis-[(4-hydroxy-sodio-aminocarbonyl)phenoxy]ethyl
ether (12). By a procedure similar to that above descri-
bed for the preparation of 11, 0.10 g of 3 gave 0.112 g
(100% yield) of 12. Compound 12 is soluble in water
1
NMR (DMSO-d6) d 7.70 (d, J=8.8 Hz, 4H, Ar), 6.99
(d, J=8.8 Hz, 4H, Ar), 6.07 (bs, 2H, ¼CH), 4.62 (bs,
4H, OCH2); 13C NMR (DMSO-d6) d 166.9, 160.4,
131.3, 128.6, 125.2, 114.3, 67.3. HRMScalcd for
C18H18N2O6Na: 381.1063, found 381.1064.
1
and the H NMR spectrum is similar to that of 3.
Inhibition of the growth of P. falciparum. A reported
method16 for the inhibition of the growth of P. falci-
parum was followed. The incubation period of the
parasites was 66 h and the starting parasitemia was
0.2% with a 1% hematocrit. The medium used was
RPMI-1640 culture medium with no folate or p-amino-
benzoic acid and 10% normal heat-inactivated human
plasma, and P. falciparum D6 and W2 clones were used.
D6 is a clone from the Sierra I/UNC isolates and is
susceptible to chloroquine and pyrimethamine but has
reduced susceptibilities to mefloquine and halofantrine.
W2 is a clone of the Indochina I isolate and is resistant
to chloroquine and pyrimethamine but susceptible to
mefloquine. Compounds were dissolved in DMSO,
diluted 400-fold with complete culture media, and then
diluted 2-fold, 11 times, to give a concentration range of
1048-fold by a Biomek 1000 or 2000 liquid handling
system into 96-well microtiter plates. The diluted com-
pounds were transferred (25 mL) to test plates, 200 mL of
parasitized erythrocytes (0.2% parasitemia and 1%
hematocrit) was added, and was incubated at 37 ꢁC in a
2,20-Bis-[(4-hydroxyaminocarbonyl)phenoxy]ethyl ether
(3). A mixture of 1.0 g (2.67 mmol) of ester 9, 27 mL of
.
0.5 M solution of NH2OH HCl (13.5 mmol), and 5.3 mL
(32 mmol) of 6 M aqueous NaOH was treated similarly
to that described above for the preparation of 1. Two
crystallizations of the crude product from DMSO–water
(10:1) gave 2, 0.804 g (80% yield): mp 220–226 ꢁC; H
1
NMR (DMSO-d6) d 7.68 (d, J=8 Hz, 4H, Ar), 6.97 (d,
J=8 Hz, 4H, Ar), 4.17 (t, J=4 Hz, 4H, OCH2), 3.80 (t,
J=4 Hz, 4H, OCH2); 13C NMR (DMSO-d6) d 164.2,
160.8, 128.8, 125.1, 114.2, 69.0, 67.5. HRMScalcd for
C18H20N2O7Na: 399.1168, found 399.1155.
1,5-Bis-[4-(hydroxycarbonyl)phenoxy]pentane (6). To a
solution of 0.30 g (0.81 mmol) of ester 5 in 20 mL of
ethanol, 1.6 mL of 6 M aqueous NaOH was added and
the solution was stirred under reflux for 10 min. After
being cooled to room temperature, the solution was
acidified with 1 N HCl to pH 1, and the precipitated
white solids were collected by filtration, washed with

D. H. Hua et al. / Bioorg. Med. Chem. 11 (2003) 4357–4361
4361
controlled environment of 5% CO2, 5% O2, and 90%
N2. After 42 h, 25 mL of [3H]-hypoxanthine was added
and the plates were incubated for an additional 24 h.
The plates were then frozen at ꢂ70 ꢁC to lyse the red
cells and later thawed and harvested onto glass fiber fil-
ter mats by using a 96-well cell harvester. The filter mats
were counted in a scintillation counter and the data
were downloaded with the custom, automated analysis
software developed by WRAIR. For each compound,
the concentration–response profile was determined and
50% inhibitory concentrations (IC50) were determined
by using a nonlinear, logistic dose–response analysis
program.
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Inhibition of the growth of L. donovani parasites. A
reported method17 for the inhibition of the growth of L.
donovani parasites was followed. L. donovani axenic
amastigotes were maintained in modified RPMI med-
ium as previously described.17 Inhibition of growth by
the compounds was measured in a 3-day assay using the
tetrazolium dye-based CellTiter 96 Aqueous Non-
Radioactive Cell Proliferation Assay Kit (Promega,
Madison, WI, USA) as previously described.17 The
known anti-leishmaniasis drug, pentamidine, was used
as a control.
Acknowledgements
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D.H.H. gratefully acknowledges financial support from
the Walter Reed Army Institute of Research, National
Science Foundation (CHE-0078921), National Insti-
tutes of Health (National Cancer Institute, CA86842),
and American Heart Association, Heartland Affiliate
(0051658Z, 9951177Z).
References and Notes
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