Identification of isoflavone derivatives as effective anticryptosporidial agents in vitro and in vivo

Article abstract of DOI:10.1021/jm050973f

We report the preparation and antiparasitic activity in vitro and in vivo of a series of isoflavone derivatives related to genistein. These analogues retain the 5,7-dihydroxyisoflavone core of genistein: direct genistein analogues (2-H isoflavones), 2-car

Full text of DOI:10.1021/jm050973f

1450
J. Med. Chem. 2006, 49, 1450-1454
Identification of Isoflavone Derivatives as Effective Anticryptosporidial Agents in Vitro
and in Vivo
Andrew V. Stachulski,*,† Neil G. Berry,† A. C. Lilian Low,† Shelley L. Moores,† Eleanor Row,† David C. Warhurst,‡
Ipemida S. Adagu,‡ and Jean-Franc¸ois Rossignol§
Romark Centre for Drug DiscoVery, Robert Robinson Laboratories, Department of Chemistry, UniVersity of LiVerpool,
LiVerpool L69 7ZD, U.K., Romark Research Laboratory, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, U.K.,
and Romark Institute for Medical Research, 6200 Courtney Campbell Causeway, Suite 200, Tampa, Florida 33607
ReceiVed September 29, 2005
We report the preparation and antiparasitic activity in vitro and in vivo of a series of isoflavone derivatives
related to genistein. These analogues retain the 5,7-dihydroxyisoflavone core of genistein: direct genistein
analogues (2-H isoflavones), 2-carboethoxy isoflavones, and the precursor deoxybenzoins were all evaluated.
Excellent in vitro activity against Cryptosporidium parVum was observed for both classes of isoflavones in
cell cultures, and the lead compound 19, RM6427, shows high in vivo efficacy against an experimental
infection.
Introduction
inhibitor of protein Tyr kinases12 found in epidermal growth
factor receptors (EGRFs). Since the EGRF peptides can mediate
host-parasite infections, they appear to be potential targets in
antiparasitic chemotherapy, and genistein therefore became a
logical starting point.
Although a number of new antiparasitic drugs1 have become
available over the past decade, with specifically mefloquine,2
albendazole,3 ivermectin,4 atovaquone-proguanil,5 and nita-
zoxanide6 having been approved in the United States, there is
still a perceived need for effective new agents. Without question,
intestinal parasitic infections remain a leading cause of mortality,
especially in developing countries, and emerging and resistant
intestinal protozoa pose a wider threat. In the case of Cryptospo-
ridium parVum, especially, significant outbreaks occur in the
Western world and there is a lack of effective therapies, as
discussed in a recent article.7
Chemistry
Condensation of phloroglucinol 3 with commercial arylaceto-
nitriles 4-7, catalyzed by ZnCl2 and gaseous HCl (Hoesch
reaction),13 afforded the open-chain ketones (“deoxybenzoins”)
8-11 (Scheme 1). It proved more reliable to use a preformed
solution of ZnCl2 in Et2O. The first products were imines, which
without isolation were hydrolyzed to ketones 8-11 with aq HCl
using a cosolvent (ethanol, THF) for solubility if required. This
step was often slow: careful TLC monitoring was important.
We found later that addition of EDTA often accelerated this
The nitrothiazole derivative nitazoxanide 1,8 now approved
for both human and veterinary use in the United States and
already extensively used in Latin America, has excellent broad-
spectrum activity against a wide range of intestinal protozoa
and helminths.9 It therefore represents a clear benchmark for
any new antiparasitic agent: in addition it possesses highly
significant antibacterial10 and antiviral11 activity. There could
be advantages, nevertheless, in a very active but highly specific
antiparasitic agent. Absence of other activities, notably anti-
bacterial, could avoid the development of further resistant
bacterial strainssa problem familiar from the overuse of
antibacterial agents, especially penicillins.
step, presumably by complexation of remaining traces of Zn2+
.
An alternative was the direct condensation of arylacetic acids
with phloroglucinol 3 catalyzed by BF3‚Et2O,14 affording
ketones akin to 8-11 directly. We found this procedure worked
very well with aryl groups containing electron-donating sub-
stituents, e.g., 4-methoxyphenylacetic acid, but most of the
substituents of interest to us were electron-withdrawing, and
the corresponding acids did not react satisfactorily.
C-formylation of ketones 8-11 followed by closure to 2-H
isoflavones 12-15 could be achieved in one pot using Vilsmeier
conditions,15 by the use of acetic formic anhydride16 or using
DMF dimethyl acetal17 (Scheme 1).The Vilsmeier conditions
employing methanesulfonyl chloride (MeSO2Cl) activation
reported by Bass18 worked very well provided freshly distilled
MeSO2Cl was used. In cases where a 2′-substituent was present
it was sometimes difficult to remove traces of solvent DMF
from the products, but there were no such difficulties with
compounds 12-15.
Alternatively, condensation of ketones 8-11 with ethyl oxalyl
chloride using K2CO3 as base with a phase-transfer catalyst19,20
afforded the 2-carboethoxyisoflavones 16-19 (Scheme 1). We
found later that the use of a phase-transfer catalyst was
unnecessary, and yields, though variable, approached 80% in
favorable cases. Here again it was important to use freshly
distilled ethyl oxalyl chloride for good yields. In some cases a
We now report the synthesis and biological evaluation of a
selection from a series of isoflavone derivatives having excellent
antiparasitic activity in vitro but lacking antibacterial activity.
Our starting point was the naturally occurring isoflavone
genistein 2,12 which has been extensively studied as a potential
therapeutic agent. In particular, genistein is known to be an
* To whom correspondence should be addressed. Phone: 0151 7943542.
Fax: 0151 7943588. E-mail: stachuls@liv.ac.uk.
† University of Liverpool.
‡ London School of Hygiene and Tropical Medicine.
§ Romark Institute for Medical Research.
10.1021/jm050973f CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/25/2006

IsoflaVones as Anticryptosporidial Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1451
Scheme 1a
Table 1. Antiparasitic Activities of Compounds 8-19, 20, and 21
Expressed as IC50 Values (µM), Number of Observations in Bracketsa
compound
Cryptosporidium parVum
8
9
10
11
12
70.8 (2)
464.5 (2)
15.9 (2)
17.2 (2)
1.8 (2)
13
0.3 (2)
14
1.3 (2)
15
0.4 (3)
16
17
18
64.1 (2)
15.8 (2)
0.6 (3)
19
1.6 (3)
20
21
20.1 (3)
11.7 (2)
3.25 ( 0.85
nitazoxanide 1
a The strain of parasite used is given in the Experimental Section data.
cultures is shown in Table 1. A fuller account of the biological
activity of these and related isoflavones has been published
elsewhere.21 The data are presented to show mean IC50 values
(micromolar) for each compound as measured in a specified
number of determinations. Nitazoxanide 1 has been added to
the Table as a reference compound effective against C. parVum.
It will be seen that in general the open-chain ketones 8-11
were significantly less active in this assay. Also 2′-substituted
analogues, not shown, were less active. From the isoflavones,
the 2-H, 3′- and 4′-NO2 derivative 12 and 13 and the 4′-Br and
3′-Br compounds in both 2-H and 2-CO2Et series, viz., 14, 15,
18, and 19, exhibited excellent activity. Furthermore, when
checked for regrowth of the parasite after up to 7 days these
assays still proved negative. We have selected compounds 18
and 19 as promising candidates for further evaluation in
comparison with nitazoxanide.
a Reagents, conditions: (i) anhydrous HCl, ZnCl2-Et2O, then aq HCl,
heat; (ii) MeSO2Cl, Me2N‚CHO, aq workup; (iii) ClCO‚CO2Et, nBu4N+Br-,
acetone, K2CO3, heat.
little overreaction occurred by acylation of the phenols giving
carbonates, separable by chromatography.
Esters 16-19 are not simply prodrugs for the free carboxylic
acids. Thus, we prepared the free acid form 20 of compound
19 by hydrolysis of 19 with aq NaOH followed by acidification
with an acidic ion-exchange resin, then evaporation and recrys-
tallization of the residue. The activity of 20 was significantly
less than that of 19: see below.
The specificity of activity against C. parVum is striking.
Compounds 18 and 19 have also shown useful activity against
the microsporidian species Vittaforma corneae and Encepha-
litozoon intestinalis with IC50 of ca. 10 µg/mL (E. Didier,
personal communication). However, when we assayed com-
pounds 8-19, 20, and 21 against Trypanosoma brucei, Tri-
chomonas Vaginalis, and Giardia lamblia, very little activity
was seen. Compounds 12 and 14 showed slight activity against
T. brucei (IC50s from 2 to 20 µM) and similar figures against
G. lamblia; also 12 showed weak activity against T. Vaginalis,
IC50 ) 8 µM. The remaining compounds were inactive. Against
a chloroquine-sensitive strain of the malaria parasite Plasmodium
falciparum, compound 12 showed weak activity (IC50 ) 20
µM); the other compounds were inactive.
We also evaluated a partially reduced analogue. Thus,
reduction of the 2,3-double bond in 12 without reduction of
the aryl nitro group was achieved using NaBH4 in 2-propanol:
the saturated ketone 21 resulted in good yield as evinced by a
characteristic A2X pattern in the 1H NMR spectrum. Here again,
21 was less active than 12 against Cryptosporidium parVum:
see below.
In Vivo Activity
The activity of the leading compounds 18 and 19 was studied
in vivo against immunosuppressed gerbils, Meriones unguicu-
latus, infected with Cryptosporidium parVum. Immunosuppres-
sion was achieved by injection with dexamethasone over a 10
day period, and C. parVum was administered by oral inoculation
(see the Experimental Section). The drugs were administered
orally twice daily: compound 19 was administered for 8 days
consecutively, 1 and paromomycin for 12 days (see the
Experimental Section). In addition dexamethasone was admin-
istered until the end of the experiment (12 day point). Compound
19, nitazoxanide 1, and paromomycin were used in direct
comparison, and statistical significance was demonstrated (P
< 0.05 considered significant).
Spectroscopic analysis of these derivatives was generally
straightforward. In particular, the open-chain ketones 8-11
showed characteristic two-proton singlets at around δ 3.0 for
the ArCH2CO protons, while the 3- and 5-protons of the
phloroglucinol moiety resonated as a singlet at around δ 6.5.
After cyclization, the 3- and 5-protons resonated as two narrow
one-proton doublets in both the 2-H and 2-CO2Et isoflavone
classes. The 2-H proton in isoflavones 12-15 resonated as a
sharp singlet around δ 8.0.
Biology: In Vitro Activity
The activity of compounds 8-19, 20, and 21 against the
coccidian parasite Cryptosporidium parVum grown in cell
The results (Table 2) show essentially the same effectiveness
for 19 at doses of either 200 or 400 mg/kg/day; 1 was tested at

1452 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Stachulski et al.
Table 2. In Vivo Activity of Compounds 18 and 19 versus Reference
Compounds against C. ParVum Infection
dropwise to a solution of deoxybenzoin 8 (0.100 g, 0.346 mmol, 1
equiv) in anhydrous DMF (1 mL). A solution of methanesulfonyl
chloride (0.085 mL, 0.126 g, 1.104 mmol, 3 equiv) in anhydrous
DMF (0.3 mL) was added to the mixture, which was heated on a
steam bath for 1.5 h, cooled, and poured into water. The crude
product was filtered off and recrystallized by adding as a hot THF
solution to water and cooling. Filtration afforded pure 13 (0.077 g,
mean oocyst
dose
animals
reduction
in stool
mg/kg/day deparasitized
P value
compound
19
19
1
Paromomycin
18
(no. of days)
at day 8
at day 8 (%) (vs control)
200 (8)
400 (8)
200 (12)
100 (12)
400 (12)
controls
6/10 (60%)
6/10 (60%)
2/14 (14%)
2/14 (14%)
2/15 (13%)a
0/10
90.5
92
<0.0001
<0.0001
<0.0008
NS
1
74%); mp 266-268 °C (THF aq). Anal. (C15H9NO6) C, H, N. H
NMR (400 MHz, CD3OD): 6.21 (1 H, s, ArH), 6.34 (1 H, s, ArH),
7.64 (1 H, dd, J ) 8.3 and 5.1 Hz, ArH), 7.93 (1 H, dd, J ) 9.3
and 5.1 Hz, ArH), 8.20 (1 H, dd, J ) 9.3 and 8.3 Hz, ArH), 8.25
(1 H, s, ArH) and 8.45 (1 H, s, 2-H). m/z (CI, NH3): 300 (MH+,
100%) and 270 (50%). High-resolution mass (ES) calculated for
C15H10NO6 (MH+): 300.0508. Found: 300.0516.
55
17.6
53.5b
0
0.006c
a At day 12. b 67% at day 12. c P ) 0.018 at day 12.
200 mg/kg/day and paromomycin at 100 mg/kg/day. Both 19
(at either dose) and nitazoxanide 1 showed statistically signifi-
cant reduction of oocyst shedding with P < 0.0001 and P <
0.0008, respectively. Under these conditions paromomycin did
not show significant inhibition compared to that of controls.
Most significantly, 19 totally inhibited oocyst shedding in 6/10
gerbils compared to 2/14 for 1 and paromomycin. In summary,
after 8 days, 19 was shown to be clearly superior to the other
agents (Table 2), with 60% of animals fully deparasitized and
a mean oocyst reduction in stools of over 90%. In comparison,
compound 18 was less effective than 19 in vivo, despite its
superior MIC value in vitro. Finally we note that these
compounds have been shown not to be cytotoxic at the dose
used.21
Similarly Prepared Were Compounds 12, 14, and 15 (See
the Supporting Information) (Refs 24, 25). Typical Procedure
for 2-Carboethoxyisoflavones: 3-(3-Bromophenyl)-5,7-dihy-
droxy-4-oxo-4H-chromene-2-carboxylic Acid, Ethyl Ester (19).
Freshly distilled ethyl chlorooxoacetate (1.14 mL, 10.2 mmol, 4
equiv) was added dropwise to a stirred mixture of 11 (0.824 g,
2.55 mmol, 1 equiv) and K2CO3 (2.11 g, 15.3 mmol, 6 equiv) in
anhydrous acetone (50 mL) under N2. The reaction mixture was
stirred at room temperature for 2 h and then heated to 50 °C for a
further 16 h. The solvent was removed, then the residue was
extracted into ethyl acetate and the base neutralized with aqueous
citric acid (20%). The organic layer was separated, washed with
water and brine, and dried over MgSO4 before removal of the
solvent under reduced pressure to afford a brown oil, which was
purified by column chromatography eluting with ethyl acetate-
hexane (3:7). Recrystallization from ethyl acetate-hexane yielded
19 as bright yellow crystals (0.538 g, 52%), mp 202-203 °C. Anal.
(C18H13BrO6) C, H. υmax cm-1 1738 and 1710. 1H NMR (400 MHz,
CD3OD): 1.05 (3 H, t, J ) 7.1 Hz, CH3CH2O), 4.16 (2 H, q, J )
7.1 Hz, CH3CH2O), 6.28 (1 H, d, J ) 1.7 Hz) and 6.42 (1 H, d, J
) 1.7 Hz, 6-H and 8-H), 7.26 (1 H, d, J ) 7.6 Hz, 6′-H), 7.35 (1
H, t, J ) 7.6 Hz, 5′-H), 7.48 (1 H, t, J ) 1.6 Hz, 2′-H) and 7.59
(1 H, d, J ) 7.6 Hz, 4′-H).13C NMR (100 MHz, (CD3)2SO): 13.6,
62.6, 94.4, 99.9, 104.6, 121.2, 123.8, 129.4, 130.3, 131.2, 132.8,
133.5, 150.2, 157.1, 160.3, 161.9, 165.8, and 180.7. m/z (CI, NH3)
405 (100%, MH+). High-resolution mass (CI) calculated for
C18H1479BrO6 (MH+): 404.99738. Found: 404.99786.
Experimental Section
Chemistry. Organic extracts were washed finally with saturated
aq NaCl and dried (over anhydrous Na2SO4 unless otherwise stated)
prior to rotary evaporation at <30 °C. Analytical thin-layer
chromatography was performed using Merck Kieselgel 60 F 254
silica plates. Preparative column chromatography was performed
on Merck 938S silica gel. Infra-red spectra were recorded for Nujol
mulls on a Perkin Elmer RX1 FT1R instrument. Unless otherwise
stated, 1H and 13C NMR spectra were recorded on CDCl3 solutions
using either Bruker 250 or 400 MHz instruments with tetrameth-
ylsilane as internal standard. Mass spectra in the chemical ionization
(CI) mode were obtained using a VG7070E mass spectrometer.
Both low- and high-resolution electrospray mode (ES) mass spectra
were obtained using a Micromass LCT mass spectrometer operating
in the +ve or -ve ion mode as indicated.
Typical Procedure for Open-Chain Ketones: 4′-Nitro-2,4,6-
trihydroxydeoxybenzoin (8). Zinc chloride (3.56 g, 26.2 mmol)
was added to an ice-cold solution of phloroglucinol 3 (3.00 g, 23.8
mmol) and 4-nitrophenylacetonitrile 4 (2.62 g, 16.2 mmol) in ether
(100 mL) at 0 °C, then dry HCl gas was passed through the solution
for 4 h with continuous stirring. Passage of the gas was discon-
tinued, and the mixture was stirred for a further 16 h at 20 °C,
then the mixture was filtered and the solid dissolved in 1 M HCl
(2 equiv) and heated at reflux for 3 h. The solution was cooled and
filtered, then the filtrate was warmed with a solution of EDTA,
sodium salt (4.43 g, 11.9 mmol) in water for 3 h. The solid was
filtered off while hot and washed with warm water, then heated at
reflux in 1 M HCl for 5 h. The resulting solid was filtered off,
washed with water, and dried overnight to give the product 822
(2.39 g, 51%); mp 212-214 °C (lit.23 mp 250-252 °C). Anal.
(C14H11NO6) C, H, N. 1H NMR (250 MHz, CDCl3): 4.64 (2 H, s,
Ar CH2), 6.03 (2 H, s, ArH), 7.49 and 8.20 (4 H, dd, ArH). 13C
NMR (100 MHz, (CD3)2SO): 49.2, 95.1(×2), 104.1(×2), 123.4-
(×2), 144.7, 146.6, 164.6(×3), 165.5 and 201.3. m/z (ES -ve
mode): 289 (M+, 10%), 288 (100%), and 244 (10%). High-
resolution mass (ES) calculated for C14H10NO6 [M - H]+:
288.0502. Found: 288.0508.
Similarly Prepared Were Compounds 16, 17, and 18 (See
the Supporting Information) (Refs 19, 25). 3-(3-Bromophenyl)-
5,7-dihydroxy-4-oxo-4H-chromene-2-carboxylic Acid (20). 1 M
NaOH (8.2 mL, 2.6 equiv) was added in one portion to a stirred
solution of ester 19 (1.90 g, 4.7 mmol) in acetone (70 mL) at 20
°C. After 1 h Amberlite IR-120 (H+: 4.3 g, 8.2 mmol) was added,
and after stirring for 15 min, the resin was filtered off and the filtrate
was evaporated to dryness. The residual oil was partitioned between
EtOAc and 5% aq citric acid, then the organic layer was separated,
washed with water and brine, dried (MgSO4), and evaporated to
give a yellow oil which on recrystallization from EtOAc-hexane
afforded the title compound 20 (1.18 g, 68%), mp 223-224 °C.
1
Anal. (C16H9BrO6) C, H. H NMR ((CD3)2SO, 400 MHz): 3.83,
3.92 (2 H, 2 s, D2O exch., 2×OH), 6.56, 6.75 (2 H, 2 d, J ) 2.2
Hz, 6-H and 8-H), 7.22 (1 H, d, J ) 7.7 Hz, 6′-H), 7.34 (1 H, t, J
) 7.8 Hz, 5′-H), 7.43 (1 H, t, J ) 1.7 Hz, 2′-H) and 7.55 (1 H,
approximately dd, J ) 7.8 and 1.9 Hz, 4′-H). 13C NMR (100
MHz, CD3OD): 94.1, 99.6, 104.8, 123.2, 129.0, 129.6, 131.1,
132.9, 133.4, 152.2, 157.6, 162.2, 162.4, 165.9, and 181.2. m/z (CI,
NH3) 377 (100%, MH+). High-resolution mass (CI) calculated for
C16H10BrO6 (MH+): 376.96606. Found: 376.96630.
5,7-Dihydroxy-3-(4-nitrophenyl)-chroman-4-one (21). Sodium
borohydride (0.014 g, 0.36 mmol) was added to an ice-cold solution
of 12 (0.100 g, 0.33 mmol) in THF (6 mL) and 2-propanol (4 mL).
After 1 h the solution was quenched by addition of 20% aq citric
acid (final pH 6-7), then the solution was evaporated and the
residue was dissolved in ethyl acetate, washed with water and brine,
dried (MgSO4), and evaporated to give an orange solid. Chroma-
tography, eluting with EtOAc-hexane, 3:7, afforded on pooling
and evaporation of appropriate fractions the title compound 21
Similarly Prepared Were Compounds 9, 10, and 11 (See the
Supporting Information). Typical Procedure for 2-H Isofla-
vones: 5,7-Dihydroxy-3-(3-Nitrophenyl)-chromen-4-one (13).
BF3‚Et2O (0.175 mL, 0.196 g, 1.38 mmol, 4 equiv) was added

IsoflaVones as Anticryptosporidial Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1453
(4) Chabala, J. C.; Mrozlk, H.; Tolman, R. L.; Eskola, P.; Lusi, A.;
Peterson, L. H.; Woods, M. F.; Fisher, M. H.; Campbell, W.
C.;Egerton, J. R.; Ostlind, D. A. Ivermectin, a new broad-spectrum
antiparasitic agent. J. Med. Chem. 1980, 23, 1134-1136.
(5) Hudson, A. T.; Dickins, M.; Ginger, C. D.; et al. 566C80sA potent
broad-spectrum antiinfective agent with activity against malaria and
opportunistic infections in AIDS patients. Drugs Exp. Clin. Res. 1991,
17, 427-435.
(6) Rossignol, J.-F.; Maisonneuve, H. Nitazoxanide in the treatment of
taenia-saginata and hymenolepis-nana infections. Am. J. Trop. Med.
Hyg. 1984, 33, 511-512.
(7) Dell, H. Scavengers beware! Drug DiscoVery Today 2004, 9, 425.
(8) Rossignol, J.-F.; Cavier, R. 2-Benzamido-5-nitrothiazoles. Chem.
Abstr. 1975, 83, 28216.
(9) Doumbo, O.; Rossignol, J.-F.; Pichard, E.; Traore, H.; Dembele, M.;
Diakite, M.; Traore, F.; Diallo, D. A. Nitazoxanide in the treatment
of cryptosporidial diarrhoea and other intestinal parasitic infections
associated with AIDS in tropical Africa. Am. J. Trop. Med. Hyg.
1997, 56, 637-639.
(10) (a) Guttner, Y.; Windsor, H. M.; Viiala, C. H.; Dusci, L.; Marshall,
B. J. Nitazoxanide in treatment of Helicobacter pylori: a clinical
and in vitro study. Antimicrob. Agents Chemother. 2003, 47, 3780-
3783. (b) Dubrueil, L.; Houcke, I.; Mouton, Y.; Rossignol, J.-F. In
vitro evaluation of activities of nitazoxanide and tizoxanide against
anaerobes and aerobic organisms. Antimicrob. Agents Chemother.
1996, 40, 2266-2270.
(0.097 g, 98%), mp 194-196 °C. Anal. (C15H11NO6) C, H, N. υmax
cm-1 1644 and 1525 cm-1. 1H NMR (400 MHz, CD3OD): 4.20 (1
H, t, 3-H), 4.63 (2 H, d, 2 × 2-H), 5.93 (2 H, d, ArH), 7.57 and
8.21 (4 H, dd, ArH). 13C NMR (100 MHz, CD3OD): 52.4, 72.2,
96.6, 97.9, 103.7, 125.1, 131.4, 145.0, 149.3, 165.0, 166.4, 169.2,
and 196.7. m/z (CI, NH3): 302 (MH+, 12%), 289 (12%), and 272
(100%). High-resolution mass (CI) calculated for C15H12NO6
(MH+): 302.06647. Found: 302.06649.
Biological Activity. In Vitro Antiparasitic Assays. Anti-
cryptosporidial Screen (Professor L. Favennec and Colleagues).
Human epithelial cells HCT-8 were obtained from the ATCC
collection and seeded in 16-well tissue culture chambers. Sporo-
zoites of C. parVum were obtained from oocysts of experimentally
infected calves and used to infect the above HCT-8 cells by addition
of 50 mL of freshly excysted sporozoite medium (inoculum 2.5 ×
105 mL-1) to each well. After incubation for 2 h at 37 °C, medium
containing nonpenetrated sporozoites was removed and replaced
with either 200 µL of medium containing test compound at a defined
concentration or medium only. Developing parasites were detected
by immunofluorescence using a reported procedure.26 Following
addition of each synthetic compound 8-19, 20, or 21, the effect
of treatment was defined by the percent reduction in development
(treated values vs control).
(11) Korba, B.; Rossignol, J.-F. Antiviral effects of thiazoles against
hepatitis B and C. Antimicrob. Agents Chemother., submitted for
publication.
(12) (a) Genistein. The Merck Index, 13th ed.; O’Neil, M. J., Smith, A.,
Heckelman, P. E., Obenchain, J. R., Jr., Gallipeau, J. A. R., D’Arecca,
M. A., Budavari, S., Eds.; Merck and Co. Inc.: Whitehouse Station,
New Jersey, 2001; entry 4402, p 780. (b) Baker, W.; Robinson, R.
Synthetical experiments in the isoflavone group. III. Synthesis of
genistein. J. Chem. Soc. 1928, 3115-3118. (c) Dixon, R. A.; Ferreira,
D. Genistein. Phytochemistry 2002, 60, 205-211.
(13) Smith, M. B.; March, J. AdVanced Organic Chemistry, 5th ed.; Wiley-
Interscience, New York, 2001; p 723.
(14) Wa¨ha¨la¨, K.; Hase, T. A. Expedient synthesis of polyhydroxyisofla-
vones. J. Chem. Soc., Perkin Trans. 1 1991, 3005-3008.
(15) Pelter, A.; Ward, R. S.; Ashdown, D. H. J. The synthesis of mono-,
di- and trihydroxyisoflavones. Synthesis 1978, 843.
(16) Liu, D. F.; Cheng, C. C. A facile and practical preparation of 5,7-
dihydroxy-3-(4-nitrophenyl)-4H-1-benzopyran-4-one. J. Heterocycl.
Chem. 1991, 28, 1641-1642.
(17) Pelter, A.; Foot, S. A new convenient synthesis of isoflavones.
Synthesis 1976, 326-327.
Other Parasite Screens. The incorporation of tritiated thymidine
or hypoxanthine as a measure of parasite viability, reported by
Adagu et al.,27 was employed in the assessment of the biological
activity of the compounds against T. brucei (strain 427), T. Vaginalis
(strain UCH-1), G. lamblia (strain JKH-1), and P. falciparum (clone
T9-96). To convert the anticryptosporidial data from percent growth
inhibition to IC50 values, as in Table 1, the “logit” transformation
was used.28
In Vivo Testing. Male and female gerbils (Meriones unguicu-
latus) weighing 22-27 g were immunosuppressed by injection with
dexamethasone at 0.8 mg/kg every 2 days for 10 days before oocyst
injection. Prior to the start of treatment, each gerbil was inoculated
orally by gavage with 105 C. parVum oocysts. Compound 19,
nitazoxanide 1, and paromomycin were suspended in 5% car-
boxymethylcellulose and administered twice daily orally, under a
constant volume of 20 mL/kg, commencing 4 h after infection: 19
for 8 consecutive days, 1 and paromomycin for 12 days.
Cryptosporidium infection was assessed by measuring oocyst
shedding in feces collected for 24 h from each animal on days 4,
8, and 12 post infection. Feces were suspended in 10% (w/v)
formalin solution and homogenized, and oocysts were counted by
phase contrast microscopy examination of smears prepared by
mixing fecal suspensions with a carbol fuchsine solution. Oocyst
numbers were expressed per microscopic field at a magnification
of 400.
(18) Bass, R. J. Synthesis of chromones by cyclization of 2-hydroxyphenyl
ketones with boron trifluoride-diethyl ether and methanesulphonyl
chloride. J. Chem. Soc., Chem. Commun. 1976, 78-79.
(19) Baker, W.; Chadderton, J.; Harborne, J. B.; Ollis, W. D. A new
synthesis of isoflavones. J. Chem. Soc. 1953, 1852-1860.
(20) Al-Maharik, N.; Mutikainen, I.; Wa¨ha¨la¨, K. An expedient synthesis
of 2-(ω-carboxyalkyl)polyhydroxyisoflavones. Synthesis 2000, 411-
416.
(21) Gargala, G.; Baishanbo, A.; Favennec, L.; Francois, A.; Ballet, J. J.;
Rossignol, J.-F. Inhibitory activities of epidermal growth factor
receptor tyrosine kinase-targeted dihydroxyisoflavone and trihy-
droxybenzoin derivatives on Sarcocystis neurona, Neospora caninum
and Cryptosporidium parVum development. Antimicrob. Agents
Chemother. 2005, 40, 4628-4634.
(22) (a) Diedrich, D. F.; Scahill, T. A.; Smith, S. L. Preparation and
physical properties of some desoxybenzoins and isoflavones. J. Chem.
Eng. Data, 1977, 22, 448-451. (b) Gowan, J. E.; Lynch, M. F.;
O’Connor, N. S.; Philbin, E. M.; Wheeler, T. S. The synthesis of
isoflavones. J. Chem. Soc. 1958, 2495-2499.
(23) Yamashita, M. Steric hindrance in the Hoesch reaction. Sci. Rep.
Tohoku Imp. UniV. 1929, 18, 615-618; Chem. Abstr. 1930, 24, 2443.
(24) Pivovarenko, V. G.; Khilya, V. P. Mixed anhydride of acetic and
formic acids in the synthesis of chromones. 2. Synthesis of 3-aryl
chromones. Chem. Heterocycl. Compd. (Engl. Transl.) 1992, 28,
497-502.
Acknowledgment. We are most grateful to Professor L.
Favennec, G. Gargala, L. Ballet, and A. Baishanbo (Parasitology
Laboratory, University of Rouen, France) for supplying their
in vitro and in vivo biological data (Tables 1 and 2), to
Alexander Mathis (Institute of Parasitology, University of
Zu¨rich) for corroboration of the in vitro data, and to Romark
Laboratories, LC for funding this work.
Supporting Information Available: Spectroscopic data for
compounds 9-12 and 14-18; analytical data for target compounds
8, 13, and 19-21. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(25) Pelter, A.; Ward, R. S.; Bass, R. J. The carbon-13 NMR spectra of
isoflavones. J. Chem. Soc., Perkin Trans. 1 1978, 666-668 and also
ref 26.
(26) Gargala, G.; Delaunay, A.; Li, X.; Brasseur, P.; Favennec, L.; Ballet,
J. J. Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide
against Cryptosporidium parVum development in sporozoite-infected
HCT-8 enterocytic cells. J. Antimicrob. Chemother. 2000, 46, 57-
60.
(1) Rosenblatt, J. E. Antiparasitic agents. Mayo Clin. Proc. 1999, 74,
1161-1175.
(2) Lutz, R. E.; Ohnmacht, C. J.; Patel, A. R. Antimalarials. 7. Bis-
(trifluoromethyl)-R-2-piperidyl)-4-quinolinemethanols. J. Med. Chem.
1971, 14, 926-928.
(3) Theodorides, V. J. Anthelmintic activity of albendazole against liver
flukes, tapeworms, lung and gastrointestinal roundworms. Experientia
1976, 32, 702-703.

1454 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Stachulski et al.
(27) Adagu, I. S.; Nolder, D.; Warhurst, D. C.; Rossignol, J.-F. In vitro
activity of nitazoxanide and related compounds against isolates of
Giardia intestinalis, Entamoeba histolytica and Trichomonas Vagi-
nalis. J. Antimicrob. Chemother. 2002, 49, 103-111.
(28) QSAR and Advanced CoMFA Manual. In Sybyl, version 7.0; 1699
South Hanley Rd., St. Louis, MO 63144-2319.
JM050973F