O-alkoxyamidine prodrugs of furamidine: In vitro transport and microsomal metabolism as indicators of in vivo efficacy in a mouse model of Trypanosoma brucei rhodesiense infection

Article abstract of DOI:10.1021/jm030604o

Five O-alkoxyamidine analogues of the prodrug 2,5-bis[4- methoxyamidinophenyl]furan were synthesized and evaluated against Trypanosoma brucei rhodesiense in the STIB900 mouse model by oral administration. The observed in vivo activity of these prodrugs de

Full text of DOI:10.1021/jm030604o

J . Med. Chem. 2004, 47, 4335-4338
4335
Brief Articles
O-Alk oxya m id in e P r od r u gs of F u r a m id in e: In Vitr o Tr a n sp or t a n d Micr osom a l
Meta bolism a s In d ica tor s of in Vivo Effica cy in a Mou se Mod el of Tr ypa n osom a
br u cei r h od esien se In fection
J ohn H. Ansede,*,† Mariappan Anbazhagan,‡ Reto Brun,§ J udy D. Easterbrook,§ J ames Edwin Hall,† and
David W. Boykin‡
Division of Drug Delivery and Disposition, School of Pharmacy, The University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-7360, Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, and
Swiss Tropical Institute, Basel, CH4002, Switzerland
Received December 5, 2003
Five O-alkoxyamidine analogues of the prodrug 2,5-bis[4-methoxyamidinophenyl]furan were
synthesized and evaluated against Trypanosoma brucei rhodesiense in the STIB900 mouse
model by oral administration. The observed in vivo activity of these prodrugs demonstrates
that compounds with an O-methoxyamidine or O-ethoxyamidine group effectively cured all
trypanosome-infected mice, whereas prodrugs with larger side-chains did not completely cure
the mice. Permeability across Caco-2 cell monolayers and microsomal metabolism were used
to identify the underlying mechanisms of prodrug efficacy.
Ta ble 1. Activity of Amidoxime Prodrugs of Furamidine in an
Acute Mouse Model of Trypanosomiasis
In tr od u ction
Aryl diamidines exhibit broad spectrum antimicrobial
activity; however, their potential as chemotherapeutic
agents has not been realized, in part due to their low
oral bioavailability.1 Several reports have appeared
which have demonstrated that prodrug strategies which
in vitroa
in vivob
lower the pK of the functional group can provide orally
effective molecules.2-5 We have reported that a prodrug
of furamidine (1, Table 1), 2,5-bis[4-methoxyamidino-
phenyl]furan (3), was effective in an immunospressed
rat model for Pneumocystis carinii pneumonia (PCP).6
The prodrug, 3, is currently in Phase II clinical trials
against PCP, human African trypanosomiasis and ma-
laria.1 In our earlier studies, we noted that 2,5-bis[4-
ethoxyamidinophenyl]furan (4) was not effective in the
PCP rat model and speculated that the bis-ethoxy-
bearing molecule was either poorly metabolized to
furamidine or was transported across the intestinal
epithelial at an insufficient rate.6 More recently, in a
study of aza-analogues of furamidine we found that a
bis-ethoxyamidino prodrug was quite effective in a
mouse model for Trypanosoma brucei rhodesiense infec-
tion.7 Albeit in two different animal models and with
two different molecules, these contradictory results have
led us to study the effect of variation of the alkyl group
on the prodrug efficacy of O-alkoxyamidines.
IC50
(nM)
dose
survival
code
R
(mg/kg)c curesd (days)e
1, furamidine na
2, DB290
3, DB289
4.5
4 × 10
0/8
0/4
4/4
2/4
1/4
4/4
0/4
1/4
2/4
0/4
0/4
>46
50
H
66.4 × 103 4 × 100
14.6 × 103 4 × 100
4 × 50
Me
>60
>60
>60
>60
32
4 × 25
4, DB377
Et
645
4 × 100
4 × 25
5, DB1005
6, DB1009
7, DB1010
8, DB1011
n-Pr
i-Pr
n-Bu
472
4 × 100
>47.5
48
12.6 × 103 4 × 100
1.9 × 103
4 × 100
4 × 100
9
6
n-Hex 5.7 × 103
Average of duplicate determinations; see ref 7 for details. b See
ref 7 for experimental details of STIB900 mouse model. c Dosage
for furamidine (1) is intraperitoneal, all others dosage by oral
a
d
administration. Number of mice that survive and are parasite
free for 60 days. e Average days of survival. Untreated controls
expire between day 7 and 8 postinfection.
have reported the synthesis of 3 and related compounds
by first preparing the requisite O-alkyl-4-bromobenz-
amidoxime by reaction of 4-bromobenzonitrile with
hydroxylamine to form 4-bromobenzamidoxime and
subsequent O-alkylation.8 Reaction of O-alkyl-4-bromo-
benzamidoximes with 2,5-bis(tri-n-butylstannyl)furan
under Stille coupling conditions readily produced the
desired O-alkoxyamidines.8 We have used this latter
approach to prepare the compounds used in this study
as outlined in Scheme 1.
Ch em istr y
Earlier we synthesized 3 from the corresponding bis-
nitrile using Pinner methodology.6 More recently, we
* Current address and to whom correspondence should be ad-
dressed: Qualyst Inc., 7030 Kit Creek Road, P.O. Box 12199, Research
Triangle Park, NC, 27709. Tel: (919) 313-0162. Fax: (919) 313-6504.
E-mail: J ohnAnsede@Qualyst.com.
† The University of North Carolina at Chapel Hill.
‡ Georgia State University.
§ Swiss Tropical Institute.
10.1021/jm030604o CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/16/2004

4336 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Brief Articles
Sch em e 1a
a
Reagents and conditions: (a) NH2OH‚HCl/KO-t-Bu, DMSO; (b)R2SO4 or RBr/NaOH, dioxane, 0 °C; (c) 2,5-bis(tri-n-butylstannyl)furan,
Pd(P Ph3)4.
Biologica l Resu lts
impose a barrier for the transcellular diffusion of this
compound across the monolayers. A low Papp value and
high sensitivity to extracellular Ca2+ suggests furami-
dine may be transported via a paracellular route;9,10
however, the roles of organic cation and other trans-
porters in furamidine transport have not been deter-
mined.
Table 1 contains the results of both in vitro and in
vivo evaluation of these O-alkoxyamidine compounds,
furamidine (1) and 2,5-bis[4-hydroxyamidinophenyl]-
furan (2), against T. b. rhodesiense. As expected, the
prodrugs do not exhibit significant in vitro activity due
to the absence of the cytochrome P450 and cytochrome
b5 enzymes from trypanosomes needed for bioconversion
of the prodrug to the active furamidine.9-11 The results
from the in vivo studies show that significant absorption
and bioconversion to furamidine occurs on oral admin-
istration of the amidoxime and the O-alkoxyamidines
when the alkyl group contains three carbons or less
(Table 1). It is quite clear that the O-n-butyl and O-n-
hexyl analogues are not effective, as the average sur-
vival time of the treated animals is not significantly
different from untreated controls. The O-n-propyl and
O-i-propyl prodrugs had intermediate activity in vivo,
failing to cure all mice at the highest dose tested, but
greatly prolonging mean survival time compared to
untreated control mice. The O-methyl analogue was
most active, curing all animals treated orally with 4 ×
100 mg/kg/day and greatly prolonging mean survival
time of animals treated with 4 × 50 and 4 × 25 mg/kg/
day. The O-ethyl analogue was nearly as active, curing
all animals at the highest dose. However, no animals
were cured at 4 × 25 mg/kg/day, and the mean survival
time was shortened considerably compared to the O-
methyl analogue.
In an effort to gain a better understanding behind the
in vivo efficacy of these prodrugs, the uptake and
metabolism of the O-alkoxyamidines were studied in
Caco-2 cell monolayers and human liver microsomes,
respectively. The transport of a compound across Caco-2
cell monolayers represents an in vitro model for the
intestinal absorption of orally administered drugs and
thus may predict whether intestinal absorption may be
the cause for poor oral activity. Furthermore, the
metabolism of the prodrug to its active metabolite must
occur at a sufficient rate in order for the prodrug to exert
its therapeutic effect. The metabolism of the O-alkoxy-
amidine prodrugs to furamidine was measured in hu-
man liver microsomes to gain a better understanding
on how the length of the alkyl side-chains may affect
the dealkylation of the compounds and thus the in vivo
efficacy of the prodrugs.
Strategies for increasing the oral bioavailability for
amidines have focused on masking the positive charges.
3 is an O-methoxyamidine prodrug derivative of fur-
amidine which exhibits enhanced oral activity and
reduced acute toxicity in animal models for pneumo-
cystosis and trypanosomiasis.6 The transport in Caco-2
cell monolayers of 3 increased over 100-fold in compari-
son to furamidine [6.04 × 10-5 ((8.68 × 10-7) and 3.75
× 10-5 ((1.53 × 10-6) for AP to BL and BL to AP,
respectively]. These results were consistent with previ-
ously reported values and support the hypothesis that
3 is translocated via transcellular passive diffusion.9,10
P-glycoprotein (P-gp) does not seem to have an effect
on the transport of this compound across the monolayers
as indicated by no significant difference in the rate of
transport for either direction (i.e. AP to BL and BL to
AP). The O-ethoxyamidine prodrug, 4, exhibited a
slightly lower Papp value than did 3 [0.59 × 10-5 (( 1.73
× 10-7) and 0.37 × 10-5 ((1.11 × 10-7), AP to BL and
BL to AP, respectively.] Again, the transport of 4 showed
no significant difference in either the AP to BL or BL
to AP directions. The n-propyl (5), i-propyl (6), n-butyl
(7) and n-hexyl (8) O-alkoxyamidine prodrugs of fur-
amidine were not transported across the cell monolayers
as indicated by no detectable levels of compound in the
receiver compartments. These results may help to
explain why some of the higher alkyl analogues of these
furamidine prodrugs were unable to cure trypanosomal
infections in the animal model.
Meta bolism of O-Alk oxya m id in e P r od r u gs in
Hu m a n Liver Micr osom es. Thus far, 3 is a promising
candidate for treating primary stage trypanosomal
infections most likely due to both its increased intestinal
absorption in comparison to furamidine and its suf-
ficient rate of metabolism to furamidine.1,9,10 The me-
tabolism of the O-alkoxyamidine analogues of 3 was
investigated in human liver microsomes by measuring
the loss of the parent compound over time. Quantifying
the disappearance of the parent compound measures the
first step in the biotransformation of the prodrugs to
furamidine and provides some indication of the meta-
bolic stability of these compounds toward dealkylation
(an essential and presumably rate-limiting step in the
production of furamidine). However, hydroxylation of
the O-alkyl side-chains (i.e. analogues with side-chains
two carbons or more) would also cause a decrease in
prodrug concentration and therefore LC-MS analyses
was used to identify and quantify the major metabolites
formed in the microsomal incubations.
Tr a n p or t a cr oss Ca co-2 Cell Mon ola yer s. The
apparent permeability (Papp) of furamidine and its
prodrugs was determined across Caco-2 cell monolayers
in both the apical (AP) to basolateral (BL) and BL to
AP directions. In agreement with previous results,
furamidine transport in both directions increased with
time and resulted in a low Papp value across the cell
monolayers (3.8 ( 0.2 × 10-7 and 8.5 ( 0.2 × 10-7 for
AP to BL and BL to AP, respectively).9,10 At physiologi-
cal pH, the cationic charges of the diamidine groups

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4337
F igu r e 1. Scheme depicting the metabolism of 3 to furamidine (1). Metabolites were identified by LC-MS. The m/z for each
metabolite is provided.
of ca. 60 min. More than 10 metabolites were detected
by LC-MS analysis after 30, 60 and 90-min of incuba-
tion. The metabolic transformation of 5 was similar to
that of 4 with one additional methylene group of the
O-n-propyl side-chain susceptible to hydroxylation. The
O-i-propyl analogue, 6, was less susceptible to hydroxyl-
ation and dealkylation in comparison to 3, 4 and 5. No
metabolites were detected after 90 min of incubation
with 6, suggesting that the isopropyl side-chain hin-
dered the direct attack on this molecule by oxidizing
F igu r e 2. The structure of the most abundant metabolites
enzymes such as cytochrome P450. The O-n-butyl
analogue, 7, underwent minor metabolism with a greater
than 3 h microsomal half-life. Hydroxylation(s) on the
butyl side-chain produced the only minor metabolites
identified by LC-MS. Metabolic stability of 8 based on
microsomal half-life suggested that this O-alkoxyami-
dine analogue was not metabolized at a detectable rate.
In summary, the size of the O-alkyl side-chain deter-
mined the metabolic stability of the prodrug with the
O-methyl analogue (3) being most susceptible to me-
tabolism and the larger O-n-butyl and O-n-hexyl groups
least susceptible to metabolism. The production of
furamidine requires a cytochrome P450-catalyzed de-
alkylation of the O-alkoxyamidine prodrug with the
subsequent reduction of the amidoxime intermediate by
a cytochrome b5/cytochrome b5 reductase-mediated reac-
tion.12 However, human liver microsomes are not a
favorable environment for reductive reactions, and
therefore quantifying furamidine production would most
likely not be the best indication for prodrug metabolism
to furamidine.
The in vivo studies in the STIB900 mouse model for
T. b. rhodesiense have shown that O-alkoxyamidine
prodrugs, where the alkyl chain is less than three
carbons, can be effectively used as prodrugs for amidines.
As judged from the Caco-2 cell monolayer studies, the
O-methoxy and O-ethoxy analogues are transported at
a high rate in comparison to the higher alkyl analogues.
The human liver microsome studies demonstrate, at
least to a first approximation, that dealkylation of
O-methoxy, and to a lesser extent, O-ethoxy analogues
occurs relatively rapidly and is the likely rate-limiting
step leading to the bioactive amidines. Taken together,
the in vitro transport and metabolism studies are
consistent with the in vivo results from the STIB900
mouse model for T. b. rhodesiense.
formed during the metabolism of 4 in human liver microsomes.
The O-methoxyamidine prodrug, 3, was rapidly me-
tabolized in human liver microsomes (HLM) with a
microsomal half-life of ca. 4.16 min and was completely
metabolized within 40 min. Five metabolites were
formed during the metabolism of 3 (Figure 1). LC-MS
was used to verify the structure of each metabolite. The
methoxyamidine/amidoxime metabolite (M1) reached
the highest concentration of all metabolites and was
subsequently metabolized to either the diamidoxime
(M3) or the methoxyamidine/amidine (M2) metabolites.
The concentration of M3 also increased to significant
levels and reached a steady-state concentration after 60
min. The amidine metabolites were also identified;
however, at much lower concentrations. A detailed
description of the metabolism of 3 in both human and
rat liver microsomes has been previously noted.9,11
The O-ethoxyamidine prodrug, 4, was metabolized
less efficiently than its O-methoxyamidine analogue
with a microsomal half-life of ca. 35 min. More than
twelve metabolites were detected during the metabolism
of 4 to furamidine in HLM (Figure 2). The metabolites
were identified by LC-MS based on their m/z and their
MSn fragmentation patterns. The three reactions that
resulted in the production of these metabolites included
(i) hydroxylation of the â-carbon of the ethoxy group,
(ii) dealkylation of the ethoxy (or ethoxy-OH) group(s)
and (iii) amidoxime reduction to form an amidine group.
The O-ethoxy side-chain of 4 was rapidly hydroxylated
to form M1, reaching a maximum concentration at 20
min and only slightly decreasing thereafter. The dihy-
droxylated metabolite, M3, was also formed in signifi-
cant quantities and steadily increased in concentration
over the 90-min incubation. The nonhydroxylated and
hydroxylated ethoxy side-chains were susceptible to
cleavage as indicated by the production of both mono-
and diamidoxime metabolites. Low concentrations of the
several monoamidine metabolites were also observed;
however, their concentrations were much lower than
some of the O-ethoxyamidine metabolites.
Exp er im en ta l Section
In Vivo a n d in Vitr o Assa ys. Antitrypanosomal assays
were performed as previously described.7 Transport of fur-
amidine prodrugs across Caco-2 cell monolayers were per-
formed as previously described.9,10
P r od r u g Met a b olism in H u m a n Liver Micr osom es.
Furamidine prodrugs were prepared as 5 mM stocks in
The O-n-propyl analogue, 5, was more stable in
comparison to both 3 and 4 with a microsomal half-life

4338 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Brief Articles
methanol prior to each experiment. Reactions consisted of the
following: 100 mM potassium phosphate buffer (pH 7.4), 10
µM substrate, 0.2 mg/mL pooled human liver microsomes
(GenTest Corp., Woburn, MA) and 1 mM NADPH. Reactions
were preincubated for 5 min prior to the addition of NADPH
to initiate the reaction and aliquots (250 µL) removed at 0, 5,
10, 15, 20, 30, 45, 60 and 90 min. The reactions were
terminated by the addition of 125 µL of acetonitrile and placed
on ice. Precipitated protein was removed by centrifugation
(15 000 rpm for 4 min) and the supernatant analyzed by
HPLC-UV or LC-MS as described previously.10
and dried (Na2SO4). The solvent was removed under reduced
pressure. The crude product was purified by flash chromatog-
raphy using 35-40% ethyl acetate in hexane to yield 294 mg
(70%) of the free base. The hydrochloride salt was obtained
by passing hydrochloride gas into an ethanol solution of the
free base.
Ack n ow led gm en t. This work was supported by the
Bill and Melinda Gates Foundation. The technical
assistance of Tanja Wenzler and Guy Riccio for the
biological evaluation is acknowledged.
Ch em istr y. Melting points were recorded using a Thomas-
Hoover (Uni-Melt) capillary melting point apparatus and are
uncorrected. TLC analysis was carried out on silica gel 60 F254
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis,
1
yields, melting point, mass spectral and H NMR data for all
1
precoated aluminum sheets and detected under UV light. H
compounds. Figure 1 metabolism of O-alkoxyamidine prodrugs
in human liver microsomes. Figures 2 and 3 formation of
metabolites from 3 and 4 in human liver microsomes, respec-
tively. This material is available free of charge via the Internet
at http://pubs.acs.org.
and 13C NMR spectra were recorded employing a Varian
GX400 or Varian Unity Plus 300 spectrometer, and chemical
shifts (δ) are in ppm relative to TMS as internal standard.
Mass spectra were recorded on a VG analytical 70-SE spec-
trometer. Elemental analyses were obtained from Atlantic
Microlab Inc. (Norcross, GA) and are within (0.4 of the
theoretical values. The compounds reported as salts frequently
analyzed correctly for fractional moles by water of solvation.
In each case proton NMR showed the presence of water. All
chemicals and solvents were purchased from Aldrich Chemical
Co. or Fisher Scientific. The synthesis of 2 and 4 have been
previously reported.6,8
Refer en ces
(1) Tidwell, R. R.; Boykin, D. W. Minor Groove Binders as Anti-
microbial Agents. In Small Molecule DNA and RNA Binder:
Synthesis to Nucleic Acid Complexes; Wiley-VCH: New York,
2003; pp 416-460.
(2) Weller, T.; Alig, L.; Beresini, M.; Blackburn, B.; Bunting, S.;
Hadvary, P.; Muller, M. H.; Knopp, D.; Levet-Trafit, B.; Lipari,
M. T.; Modi, N. B.; Muller, M.; Refino, C. J .; Schmitt, M.;
Schonholzer, P.; Weiss, S.; Steiner, B. Orally active fibrinogen
receptor antagonists. 2. Amidoximes as prodrugs of amidines.
J . Med. Chem. 1996, 39, 3139-3147.
(3) Rahmathullah, S. M.; Hall, J . E.; Bender, B. C.; McCurdy, D.
R.; Tidwell, R. R.; Boykin, D. W. Prodrugs for amidines:
synthesis and anti-Pneumocystis carinii activity of carbamates
of 2,5-bis(4-amidinophenyl)furan. J . Med. Chem. 1999, 42, 3994-
4000.
(4) Hall, J . E.; Kerrigan, J . E.; Ramachandran, K.; Bender, B. C.;
Stanko, J . P.; J ones, S. K.; Patrick, D. A.; Tidwell, R. R. Anti-
Pneumocystis activities of aromatic diamidoxime prodrugs.
Antimicrob. Agents Chemother. 1998, 42, 666-674.
(5) Shahrokh, Z.; Lee, E.; Olivero, A. G.; Matamoros, R. A.; Robarge,
K. D.; Lee, A.; Weise, K. J .; Blackburn, B. K.; Powell, M. F.
Stability of alkoxycarbonylamidine prodrugs. Pharm. Res. 1998,
15, 434-441.
(6) Boykin, D. W.; Kumar, A.; Bender, B. C.; Hall, J . E.; Tidwell, R.
R. Anti-pneumocystis activity of bis-amidoximes and bis-O-
alkylamidoximes prodrugs. Bioorg. Med. Chem. Lett. 1996, 6,
3017-3020.
(7) Ismail, M. A.; Brun, R.; Tanious, F. A.; Wilson, W. D.; Boykin,
D. W. Synthesis and anti-protozoal activity of aza-analogues of
furamidine. J . Med. Chem. 2003, 46, 4761-4769.
(8) Anbazhagan, M.; Boykin, D. W. Synthesis of the prodrug 2,5-
bis[4-methoxyamidinophenyl] furan and analogues. Heterocycl.
Commun. 2003, 9, 117-118.
(9) Zhou, L.; Lee, K.; Thakker, D. R.; Boykin, D. W.; Tidwell, R. R.;
Hall, J . E. Enhanced permeability of the antimicrobial agent 2,5-
bis(4-amidinophenyl)furan across Caco-2 cell monolayers via its
methylamidoxime prodrug. Pharm. Res. 2002, 19, 1689-1695.
(10) Zhou, L. Mechanisms for absorption and metabolism of 2,5-bis-
(4-amidinophenyl)furan-bis-o-methylamidoxime, an orally active
prodrug of the antimicrobial agent 2,5-bis(4-amidinophenyl)-
furan. Doctoral Dissertation, Division of Medicinal Chemistry
and Natural Products, The University of North CarolinasChapel
Hill, 2001.
(11) Saulter, J .; Hall, J . E. Personal communication, 2003.
(12) Clement, B. Reduction of N-hydroxylated compounds: amid-
oximes (N-hydroxyamidines) as pro-drugs of amidines. Drug
Metab. Rev. 2002, 34, 565-579.
Syn th esis of O-Alk yloxy-4-br om oben za m id oxim es Il-
lu str ated by th e P r epar ation of O-n -P r opyl-4-br om oben z-
a m id oxim e. Hydroxylamine hydrochloride (6.9 g, 100 mmol)
was suspended in anhydrous DMSO (50 mL), and the mixture
was cooled in an ice bath. KO-t-Bu (11.2 g, 100 mmol) was
added portionwise under nitrogen atmosphere, and the solu-
tion was stirred at room temperature for 1 h. 4-Bromobenzo-
nitrile (1.82 g, 10 mmol) was added to the solution, the reaction
mixture was stirred overnight at room temperature and poured
into ice-water, and the product was filtered. The 4-bromo-
benzamidoxime was recrytallized from ethanol. Yield: 1.95 g,
91%. Mp: 144-45 °C; lit. mp 144-145 °C. The 4-bromobenz-
amidoxime (1.07 g, 5 mmol) was dissolved in dioxane (15 mL)
and cooled to 0 °C. A solution of 2 N NaOH (50 mL) was added
slowly, followed by dropwise addition of n-propyl bromide (923
mg, 7.5 mmol) in dioxane (5 mL). After addition, the ice-bath
was removed, and the mixture was stirred at room tempera-
ture of 1 h. TLC showed the disappearance of the amidoxime.
The mixture was extracted with EtOAc (3 × 50 mL), and the
combined organic layers were washed with water, brine and
dried over Na2SO4. The solvent was removed under reduced
pressure. The crude product was purified by passing through
a short silica gel column (eluent 5% EA in Hexanes) to yield a
pale yellow solid which was recrystallized (hexane).
2,5-Bis (4-O-n -p r op yloxya m id in op h en yl)fu r a n (5). An
oven-dried, 25 mL, round-bottomed flask was charged with 678
mg (1.05 mmol) of 2,5-bis(tri-n-butylstannyl)furan and 456 mg
(2 mmol) of O-n-propyl-p-bromobenzamidoxime under nitro-
gen. To that was added 10 mL of anhydrous dioxane and 115
mg of tetrakis(triphenylphosphene)palladium(0), and the mix-
ture was heated at reflux for 16 h. After complete consumption
of the amidoxime (determined by TLC), the reaction mixture
was cooled, and the solvent was removed using a rotary
evaporator. The residue was diluted with EtOAc and filtered
through Celite, and the Celite layer was washed with EtOAc.
The combined EtOAc layers were washed with water and brine
J M030604O