Synthesis and in vitro studies of novel pyrimidinyl peptidomimetics as potential antimalarial therapeutic agents

Article abstract of DOI:10.1021/jm020104f

A class of new pyrimidinyl peptidomimetic agents (compounds 1-6) were synthesized, and their in vitro antimalarial activities against Plasmodium falciparum were evaluated. The core structure of the new agents consists of a substituted 5-aminopyrimidone ri

Full text of DOI:10.1021/jm020104f

J . Med. Chem. 2002, 45, 3491-3496
3491
Syn th esis a n d In Vitr o Stu d ies of Novel P yr im id in yl P ep tid om im etics a s
P oten tia l An tim a la r ia l Th er a p eu tic Agen ts
Shuren Zhu, Thomas H. Hudson, Dennis E. Kyle, and Ai J . Lin*
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue,
Silver Spring, Maryland 20910
Received March 5, 2002
A class of new pyrimidinyl peptidomimetic agents (compounds 1-6) were synthesized, and
their in vitro antimalarial activities against Plasmodium falciparum were evaluated. The core
structure of the new agents consists of a substituted 5-aminopyrimidone ring and a Michael
acceptor side chain methyl 2-hydroxymethyl-but-2-enoate. The synthesis of 1-6 featured a
Baylis-Hillman reaction of various aldehydes with methyl acrylate catalyzed by 1,4-
diazabicyclo[2.2.2]octane (DABCO) and a SN2′ Mitsunobu reaction under the conditions of
diethyl azadicarboxylate (DEAD), triphenylphosphine (Ph3P), and various acids. The new
compounds exhibited potent in vitro growth inhibitory activity (IC ) 10-30 ng/mL) against
50
both chloroquine sensitive (D-6) and chloroquine resistant (W-2) Plasmodium falciparum clones.
Compound 6 (IC50 ) 6-8 ng/mL) is the most active compound of the class, the antimalarial
efficacy of which is comparable to that of chloroquine. In general, this class of compound
exhibited weak to moderate in vitro cytotoxicity against neuronal and macrophage cells with
IC
in the range of 1-16 µg/mL and showed less toxicity in a colon cell line. Preliminary
50
results indicated that compounds 3 and 6 are active against P. berghei, prolonged the life span
of parasite-bearing mice from 6 days for untreated control to 16-24 days for drug-treated
animals.
In tr od u ction
of aspartic protease and/or cysteine protease as potential
malaria therapeutic agents are of significant interest
to medicinal chemists, and the approach has attracted
broad attention.6-8
Malaria is one of the most common infectious diseases
in over 100 countries in Africa, Southeast Asia, and
South America. Every year, there are an estimated 300
million new cases of malaria throughout the world, and
the mortality associated with malaria is estimated at
1.1 million deaths per year.1 The increasing prevalence
of multiple-drug-resistant (MDR) strains of Plasmodium
falciparum in most malaria endemic areas has signifi-
cantly reduced the efficacy of current antimalarial drugs
for prophylaxis and treatment of this disease.2 For
instance, resistance to the inexpensive antimalarial
mainstays, such as chloroquine, is worldwide. Similarly,
resistance to mefloquine, which was proposed as the
drug of choice for chloroquine-resistant malaria, has
been reported from Africa and Southeast Asia.3,4 Al-
though drug resistance is a common problem in the
treatment of most microbial infections, malaria, and
many neoplasm, the impact is more acute for malaria
chemotherapy because of the limited number of clini-
cally useful antimalarial drugs. Thus, identification of
new targets for antimalarial therapy and synthesis of
new agents specifically against these targets are im-
portant tasks for scientists involved in malaria research.
Parasitic proteases, cysteine protease (falcipain), and
aspartic protease (plasmepsin) are two of the many
potential targets for malaria chemotherapy.5 Both en-
zymes are involved in degradation of human hemoglobin
as a source of amino acids for protein synthesis of the
schizontes, resulting in destruction of the erythrocytes.
Therefore, design and synthesis of selective inhibitors
Low molecular weight synthetic protease inhibitors
are well documented, including peptidyl(acyloxy)meth-
yl,9 halomethyl,10 and diazomethyl ketones11 and vari-
ous epoxysuccinyl peptide derivatives12 as irreversible
affinity labels, peptidyl aldehyde13 and peptidyl nitrile14
as reversible potent cysteine protease inhibitors, pep-
tidyl Michael acceptors as substrate analogues,15 vinyl
sulfones as mechanism-based inhibitors,16 and others.17
However, the stability of these synthetic inhibitors in
living organisms is poor, presumably because of the fact
that peptide moieties can be readily cleaved by various
enzymes in vivo. In addition, some of the compounds
are so reactive that nonspecific side reactions often
occur, resulting in host toxicity.
As part of an ongoing malaria chemotherapy project
in our laboratory, we undertook the synthesis and
antimalarial activity evaluation of a novel class of
pyrimidinyl peptidomimetics (compounds 1-6, Chart 1).
The core structure of these new agents comprises a
peptidomimetic segment18,19 for recognition and binding
to the target enzyme, and an active group, which can
be considered a Michael acceptor for enzyme alkylation.
The latter differs from ordinary Michael acceptor or
vinyl sulfones because the new agent features a unique
“addition-elimination” mechanism when it reacts with
the cysteine residue of the target enzyme (Scheme 1),
resulting in irreversible inhibition of the target enzyme.
The use of a peptidomimetic segment in place of the
natural peptides renders the compounds more stable to
in vivo enzymatic degradation. The activity of the
* To whom correspondence should be addressed. Phone: 301-319-
9084. Fax: 301-319-9449. E-mail: ai.lin@na.amedd.army.mil.
10.1021/jm020104f This article not subject to U.S. Copyright. Published 2002 by the American Chemical Society
Published on Web 07/09/2002

3492 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Zhu et al.
Ch a r t 1
Sch em e 1
products, (E)-2-(hydroxymethyl) 3-substituted 2-alke-
noate derivatives, in >70% yield with SN2′:SN2 ratio of
22:1 to >50:1.21a Since SN2 and SN2′ reactions take place
in a competitive manner and are sensitive to steric
hindrance, the bulky 2-phenylpyrimidinyl group in
compounds 8a and 8b may have prevented the SN2 and
favored the SN2′ displacement reaction. Identification
of the Mitsunobu reaction products was achieved by
NMR studies. Distinctive differences in NMR spectra
of SN2 and SN2′ products involve the number of olefinic
proton(s) (2H vs 1H), the split patterns (two doublets
2
with a small J HH in the range of 0-3 Hz vs a triplet),
and the chemical shift of the olefinic proton(s). The
olefinic proton cis to the ester function showed a very
downfield chemical shift (∼7.0 ppm) on 1H NMR,
apparently caused by conjugative and anisotropic
deshielding effects of the ester group. The exclusive
formation of the (E) isomer suggested that this reaction
proceeds through a stepwise mechanism involving car-
bonium ion formation, as suggested by Farina.21b Reac-
tion by a concerted SN2′ mode would have furnished (E)
and (Z) isomers in equal amounts, since the starting
alcohols 8a and 8b are each a racemic mixture.
compounds as a Michael acceptor can be finely tuned
by systematic modification of the Michael acceptor
leaving group to balance the chemical reactivity versus
cell growth inhibitory potency and host toxicity.
Ch em istr y
Scheme 2 illustrated the synthesis of compounds 1-5.
Aldehydes 7a and 7b were synthesized by the methods
of Bernstein.18a Treatment of these aldehydes with
methyl acrylate in the presence of 1,4-diazabicyclo[2.2.2]-
octane (DABCO)20 gave allylic alcohols 8a or 8b in good
yield. Both 8a and 8b are mixtures of R and S enanti-
omers. Compound 8a was subjected to the SN2′ Mit-
sunobu reaction21a in the presence of diethyl azadicar-
boxylate (DEAD), triphenylphosphine (Ph3P), and
p-nitrobenzoic acid or benzoic acid to yield compound 1
or 2, respectively. Likewise, treatment of 8a or 8b with
DEAD, Ph3P, and acetic acid produced compound 3 or
4. The products 1-4 were solely trans alkenes, a result
of SN2′ displacement reactions. In all reactions, no SN2
displacement products such as 1′-4′ were detected in
the crude products by NMR. This observation fully
agreed with the literature report that the Mitsunobu
reaction of acyclic allylic alcohols bearing an ester group
at the â position gave high regio- and stereoselective
The synthesis of compound 6 is shown in Scheme 3.
Chiral amino acid aldehyde 9, reported by Fehrentz and
Castro,22 was treated with methyl acrylate in the
presence of DABCO to yield methyl 4-tert-butoxycarbo-
nylamino-3-hydroxyl-2-methylenepentanoate 10 in 78%
yield. The SN2′ Mitsunobu reaction of compound 10
furnished methyl 4-tert-butoxycarbonylamino-2-acetoxy-
trans-2-pentenoate 11. Again, the trans alkene was the
only isomer isolated and displacement occurred exclu-
sively through the SN2′ mode. Removal of the BOC
protecting group with dry HCl in diethyl ether gave the
corresponding amine hydrogen chloride salt 12. The
intermediate pyrimidinyl acid 1318a was prepared in
good yield by oxidation of aldehyde 7a with NaClO2.
Compound 6 can be readily anticipated from the coup-
ling of acid 13 and the amine HCl salt of 12. However,

Pyrimidinyl Peptidomimetics
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3493
Sch em e 2a
a
(a) DABCO, methyl acrylate, 72-91%; (b) DEAD, Ph3P, AcOH (or benzoic acid or p-nitrobenzoic acid), THF, 59-75%; (c) MeOH,
K2CO3, 71%.
Sch em e 3a
Ta ble 1. In Vitro Activity against Malarial Parasite (IC50
)
IC50 (ng/mL)
compound
D6 clone
W2 clone
1
2
3
4
5
6
24
27
17
17
16
9
24
28
17
18
17
10
chloroquine (control)
6
114
3-(3-dimethylamino)propylcarbodiimide (EDC) alone also
gave poor yield. Nevertheless, a combination of DCC and
HOBT makes an excellent coupling reagent for this final
reaction.
Resu lts a n d Discu ssion
These compounds (1-6) exhibited potent in vitro
growth inhibitory activity (IC50 ) 10-30 ng/mL) against
Plasmodium falciparum clones, D-6 and W-2 (Table 1).
The former (D-6) is a chloroquine sensitive clone, and
the latter (W-2) is a chloroquine-resistant cell line.
These new compounds not only showed potent activity
against chloroquine sensitive malarial strain (D-6) but
are equally active against chloroquine-resistant malarial
strain (W-2). Compound 6 is the most active compound
of the class. The antimalarial efficacy of compound 6 is
comparable to that of chloroquine with IC50 ) 6-8
ng/mL against D-6. Compound 6 differs from compound
1-5 by having an extra unit of peptide, which may have
contributed to the enhancement of its efficacy. However,
no significant inhibitory activity against plasmepsin was
observed, suggesting that plasmepsin may not be the
primary target enzyme of the new peptidomimetic
agents synthesized in this study.
a
(a) DABCO, methyl acrylate, 88%; (b) DEAD, Ph3P, AcOH,
THF, 77%; (c) dry HCl, Et2O, 99%; (d) NaClO2, H2O, tert-butyl
alcohol, 62%; (e) 12, DCC, HOBT, DMAP, CHCl3, 79%.
several reaction conditions were attempted before con-
ditions with satisfactory yield were found. We first
investigated the mixed anhydride method for the coup-
ling reaction, using isobutyl chloroformate and triethyl-
amine. It turned out that the quickly formed mixed
anhydride from compound 13 and isobutyl chlorofor-
mate was too stable to react with the free amine
generated in situ from the amine salt 12. The mixed
anhydride can be purified over silica gel column chro-
matography. Dicyclohexylcarbodiimide (DCC) or 1-ethyl-
This study has uncovered the core structure of a new
class of peptidomimetic antimalarial agents, which

3494 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Ta ble 2. In Vitro Cytotoxicity Study (IC50
Zhu et al.
)
Infrared spectra were recorded on
a
Bio-Rad FTS 3000
spectrophotometer and are reported in reciprocal centimeters
IC50 (ng/mL)
macrophage
(cm-1). 1H NMR and 13C NMR spectra were recorded in
compound
neuronal
colon
7600
13500
>50000
>50000
>50000
1600
deuteriochloroform, unless otherwise noted, on
a Bruker
Avance 600 spectrometer at frequency of 600.1 MHz.
a
1
2
3
4
5
6
1750
2600
920
13800
950
3600
1600
1080
16000
780
Chemical shifts are reported in parts per million on the δ scale
from an internal standard of tetramethylsilane. Combustion
analyses were performed by Atlantic Microlab, Inc. (Norcross,
Georgia). When necessary, solvents and reagents were dried
as follows. Ether, tetrahydrofuran, benzene, and toluene were
stored and distilled from sodium benzophenone ketyl; dichlo-
romethane, triethylamine, pyridine, and hexane were distilled
over calcium hydride. Unless otherwise stated, the reagents
were purchased from Fisher Scientific, Aldrich Chemical Co.,
Lancaster, and Fluka and used as received.
1100
5400
consists of a substituted 5-aminopyrimidone ring and a
Michael acceptor side chain methyl 2-hydroxymethyl-
but-2-enoate. Acetylation of the hydroxymethyl function
of the Michael acceptor of compound 5 to give compound
3 did not affect their antimalarial efficacy in the test
system. Since acetyloxy is a better leaving group than
the hydroxy function, one would expect that acetylation
of the hydroxy group will enhance the efficacy substan-
tially if addition-elimination is a mode of action. How-
ever, the results suggest that the addition-elimination
mechanism may play a small or no role in antimalarial
activity of this class of compounds. This contention is
further substantiated by the observation that benzoyl
esters (1 and 2), which are an even better leaving group
than the acetyloxy group, are less active than acetyl
esters 3 and 4. Furthermore, the comparable activity
of 3 and 4 indicated that the substituent on the amino
group at the 5-position of the pyrimidone ring has
minimum effect on the activity. However, elongation of
the Michael acceptor side chain by one additional
peptide unit, such as compound 6, enhanced the anti-
malarial activity 1- to 2-fold.
The in vivo antimalarial activity of compounds 3 and
6 were assessed against P. berghei under the reported
Thompson test protocol.23a The preliminary results
indicated that both compounds exhibited antimalarial
activity at 40-160 mg/kg dosages administered subcu-
taneously and prolonged the life span of parasite-
bearing mice from 6 days for untreated control to
16-24 days for those treated with test compounds.
To evaluate the potential human toxicity of the new
peptidomimetic agents, the cell growth inhibitory activ-
ity in three human cell lines (neuronal, macrophage, and
colon) were assessed. The results are shown in Table 2.
In general, this class of compound exhibited weak to
moderate cytotoxicity against neuronal and macrophage
cells with IC50 of ∼1 µg/mL for 5 to 16 µg/mL for 4. In
most cases the toxicity to colon cell line were weak (IC50
) 1.6 µg/mL for 6) to insignificant (>50 µg/mL for
compound 3-5). Compound 4, the most polar among the
compounds tested, was the least toxic compound.
2-(2-ter t-Bu toxycar bon ylam in o-1-h ydr oxy-pr opyl)acr yl-
ic Acid Meth yl Ester (10). DABCO (150 mg) was added to a
solution of aldehyde 9 (1.19 g) in methyl acrylate (5 mL), and
the resulting mixture was stirred at ambient temperature for
6 days. Excess methyl acrylate was evaporated. The resulting
oil was dissolved in 30 mL of EtOAc and washed successively
with 0.5 N HCl (20 mL), saturated aqueous NaHCO3 (20 mL),
and brine (20 mL). The ethyl acetate solution was then dried
over anhydrous Na2SO4 and evaporated in a rotary evaporator
1
to give 1.57 g of compound 10 as a viscous oil. Yield: 88%. H
NMR: δ 6.32 (s, 1H), 5.91 (s, 1H), 4.78 (br, 1H), 4.42 (m,1H),
3.92(m, 1H), 3.79(s, 3H), 3.42(br, 1H), 1.41 (s, 9H), 1.24 (d, J
) 6.9 Hz, 3H). 13C NMR: δ 166.7, 156.3, 140.5, 126.2, 79.5,
74.2, 51.9, 49.8, 28.4, 15.4. Anal. (C12H21NO5) C, H, N.
2-Acetoxym eth yl-4-ter t-bu toxyca r bon yla m in op en t-2-
en oic Acid Meth yl Ester (11). Triphenylphosphine (3.8 g)
was added to a solution of 1.25 g of 10 in 15 mL of THF. The
resulting solution was cooled to -78 °C, and acetic acid (0.83
mL) was added, followed by slow addition of DEAD (2.3 mL)
via a syringe. The mixture was slowly warmed to -30 °C and
stirred for 1 h. It was then further warmed to 0 °C and stirred
for another hour. The mixture was then diluted with 40 mL
of EtOAc and washed successively with saturated aqueous
NaHCO3 (20 mL) and brine (20 mL). The EtOAc solution was
dried over anhydrous Na2SO4 and evaporated in a rotary
evaporator to give an oil, which was loaded onto a silica gel
column and eluted with 25% EtOAc/hexanes to give 1.12 g of
compound 11 as a viscous oil. Yield: 77%. 1H NMR: δ 6.75
(d, J ) 9.2 Hz, 1H), 5.00 (d, J ) 12.1 Hz, 1H, AB of A), 4.90
(d, J ) 12.1 Hz, 1H, AB of B), 4.60 (br, 1H), 4.58 (m, 1H), 3.78-
(s, 3H), 2.05 (s, 3H), 1.42 (s, 9H), 1.26 (d, J ) 6.4 Hz, 3H). 13C
NMR: δ 170.6, 166.8, 154.8, 149.0, 126.9, 79.8, 58.3, 52.2, 44.7,
28.4, 20.9, 20.7. Anal. (C14H23NO6) C, H, N.
2-(2-{5-[(4-Acetylp ip er a zin e-1-ca r bon yl)a m in o]-6-oxo-
2-ph en yl-6H-pyr im idin -1-yl}-1-h ydr oxyeth yl)acr ylic Acid
Meth yl Ester (8b). The same procedure as for the synthesis
of 10 was used to synthesize compound 8b, starting from
aldehyde 7b, except the purification was achieved by elution
from the column with 15% MeOH/EtOAc to give 8b as a white
solid, mp 211.7 °C. Yield: 72%. 1H NMR: δ 8.30 (s, 1H), 7.52-
7.47 (m, 5H), 6.25 (s, 1H), 5.80 (s, 1H), 4.62 (m, 1H), 4.47 (dd,
J ) 13.9, 6.8 Hz, 1H), 4.38 (dd, J ) 13.9, 4.5 Hz, 1H), 3.78 (m,
4H), 3.59 (s, 3H), 3.46 (m, 4H), 2.16 (s, 3H). 13C NMR: δ 167.9,
166.1, 157.8, 154.7, 152.0, 138.8, 136.5, 134.9, 131.8, 130.1,
128.5, 127.4, 125.7, 74.6, 56.9, 49.5, 45.9, 43.3, 21.8. Anal.
(C23H27N5O6) C, H, N.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Mettler
FP62 melting point apparatus and are uncorrected. Unless
otherwise noted, all nonaqueous reactions were performed
under an oxygen-free atmosphere of nitrogen with rigid
exclusion of moisture from reagents and glassware. Analytical
thin-layer chromatography (TLC) was performed using EM
Reagents 0.25 mm silica gel 60-F plates. Visualization of the
developed chromatogram was performed with UV absorbance,
aqueous potassium permanganate, or ethanolic anisaldehyde.
Liquid chromatography was performed using a force flow (flash
chromatography) of the indicated solvent system on EM
Reagents silica gel 60 (70-230 mesh). Preparative TLC was
performed using Whatman silica gel C8 TLC plates (PLK5F).
2-[2-(5-Ben zyloxyca r b on yla m in o-6-oxo-2-p h en yl-6H -
p yr im id in -1-yl)-1-h yd r oxyeth yl]a cr ylic Acid Meth yl Es-
ter (8a ). The same procedure as for the synthesis of 10 was
used to synthesize compound 8a , starting from aldehyde 7a ,
except the purification was achieved by elution from the
column with 25% EtOAc/hexanes to give 8a as a white solid,
mp 185.6 °C. Yield: 91%. 1H NMR: δ 8.78 (br, 1H), 7.56 (s,
1H), 7.50-7.39 (m, 10H), 6.24 (s, 1H), 5.76 (s, 1H), 5.24 (s,
2H), 4.63 (m, 1H), 4.46 (dd, J ) 13.8, 7.5 Hz, 1H), 4.39 (dd, J
) 13.8, 4.4 Hz, 1H), 3.69 (d, J ) 7.6 Hz, 1H), 3.57 (s, 3H). 13C
NMR: δ 167.5, 158.1, 152.7, 152.4, 139.6, 135.7, 134.9, 133.6,
131.0, 129.1, 128.8, 128.5, 128.2, 127.3, 125.4, 74.5, 68.1, 56.4,
49.7. Anal. (C24H23N3O6) C, H, N.

Pyrimidinyl Peptidomimetics
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3495
2-Acet oxym et h yl-4-{5-[(4-a cet ylp ip er a zin e-1-ca r b o-
n yl)a m in o]-6-oxo-2-p h en yl-6H-p yr im id in -1-yl}bu t-2-en o-
ic Acid Meth yl Ester (4). Compound 4 was obtained in 75%
yield from 8b according to the same procedure as for the
synthesis of compound 11. The purification was achieved using
a silica gel column and elution with 15% MeOH/EtOAc, mp
stirring at room temperature to a mixture that consisted of
acid 13 (260 mg), DCC (142 mg), HOBT (93 mg), and DMAP
(910 mg) in 5 mL of chloroform. The resulting mixture was
stirred overnight, then diluted with 40 mL of EtOAc, and
washed successively with saturated aqueous NaHCO3 solution
(20 mL) and brine (20 mL). The ethyl acetate solution was then
dried over anhydrous Na2SO4 and evaporated in vacuo to
dryness to give a crude product as an oil. The crude product
was purified by the use of a silica gel column and elution with
50% EtOAc/hexanes to give 306 mg of compound 6 as a white
solid, mp 214.6 °C. Yield: 79%. 1H NMR: δ 8.79 (br, 1H), 7.53-
7.39 (m, 11H), 6.69 (d, J ) 9.3 Hz, 1H), 6.10 (d, J ) 7.0 Hz,
1H), 5.23 (s, 2H), 4.99 (d, J ) 12.3 Hz, 1H), 4.93 (d, J ) 12.3
Hz, 1H), 4.91 (m, 1H), 4.54 (d, J ) 15.3 Hz, 1H), 4.45 (d, J )
15.3 Hz, 1H), 3.79 (s, 3H), 2.03 (s, 3H), 1.30 (d, J ) 6.8 Hz,
3H). 13C NMR: δ 170.8, 166.6, 165.5, 158.1, 153.0, 146.7, 136.0,
135.5, 134.3, 130.8, 129.23, 129.18, 129.09, 129.00, 128.91,
128.7, 128.4, 125.1, 73.6, 58.3, 52.3, 50.0, 44.3, 20.9, 20.4. IR
(film), cm-1: 3310, 3061, 2953, 1719, 1655, 1605, 1515, 1491,
1443, 1366, 1216, 1191, 1090, 1029, 980, 772, 735, 700. Anal.
(C29H30N4O8) C, H, N.
1
231.3 °C. H NMR: δ 8.24 (s, 1H), 7.55-7.41 (m, 5H), 6.24 (t,
J ) 5.3 Hz, 1H), 5.12 (d, J ) 5.3 Hz, 2H), 4.72 (s, 2H), 3.75
(m, 4H), 3.65 (s, 3H), 3.42 (m, 4H), 2.14 (s, 3H), 2.10 (s, 3H).
13C NMR: δ 169.2, 162.7, 153.2, 141.2, 133.7, 130.9, 129.5,
129.2, 128.9, 128.2, 127.9, 127.8, 124.6, 120.6, 63.4, 51.9, 47.1,
45.9, 42.3, 21.4, 21.0. IR (film), cm-1: 2999, 2927, 1718, 1668,
1624, 1523, 1492, 1424, 1369, 1242, 1161, 1022, 996, 924, 778,
729, 704. Anal. (C25H29N5O7) C, H, N.
2-Acetoxym eth yl-4-(5-ben zyloxyca r bon yla m in o-6-oxo-
2-p h en yl-6H-p yr im id in -1-yl)bu t-2-en oic Acid Meth yl Es-
ter (3). Compound 3 was obtained in 65% yield from 8a
according to the same procedure as for the synthesis of
compound 11. The purification was achieved by using a silica
gel column and elution with 25% EtOAc/hexanes, mp 184.1
°C. 1H NMR: δ 8.76 (br, 1H), 7.51-7.40 (m, 11H), 6.82 (t, J )
5.9 Hz, 1H), 5.24 (s, 2H), 4.88 (d, J ) 5.9 Hz, 2H), 4.56 (s,
2H), 3.77 (s, 3H). 13C NMR: δ 170.8, 166.4, 157.9, 152.8, 152.3,
138.5, 136.2, 129.2, 128.8, 128.4, 128.1, 125.5, 68.1, 62.3, 52.4,
44.8, 20.6. Anal. (C26H25N3O7) C, H, N.
In Vitr o An tim a la r ia l Stu d ies. The in vitro assays were
conducted by using a modification of the semiautomated
microdilution technique of Desjardins et al.23b and Chulay et
al.24 Two P. falciparum malaria parasite clones, from CDC
Indochina III (W-2) and CDC Sierra Leone I (D-6), were
utilized in susceptibility testing. They were derived by direct
visualization and micromanipulation from patient isolates.25
The W-2 clone is susceptible to mefloquine but resistant to
chloroquine, sulfadoxine, pyrimethamine, and quinine, whereas
the D-6 clone is naturally resistant to mefloquine but suscep-
tible to chloroquine, sulfadoxine, pyrimethamine, and quinine.
Test compounds were initially dissolved in DMSO and diluted
400-fold in RPMI 1640 culture medium supplemented with 25
mM Hepes, 32 mM NaHCO3, and 10% Albumax I (Gibco BRL,
Grand Island, NY). These solutions were subsequently serially
diluted 2-fold with a Biomek 1000 (Beckman, Fullerton, CA)
to give over 11 different concentrations. The parasites were
exposed to serial dilutions of each compound for 48 h and
incubated at 37 °C with 5% O2, 5% CO2, and 90% N2 prior to
the addition of [3H]hypoxanthine. After a further incubation
of 18 h, parasite DNA was harvested from each microtiter well
using Packard Filtermate 196 harvester (Meriden, CT) onto
glass filters. Uptake of [3H]hypoxanthine was measured with
a Packard Topcount scintillation instrument. Concentration-
response data were analyzed by a nonlinear regression logistic
dose response model, and the IC50 values (50% inhibitory
concentrations) for each compound were calculated (Table 1).
Ben zoic Acid 4-(5-Ben zyloxyca r b on yla m in o-6-oxo-2-
p h e n yl-6H -p yr im id in -1-yl)-2-m e t h oxyca r b on ylb u t -2-
en yl Ester (2). Compound 2 was prepared using the same
method as for the synthesis of compound 3 to give white solid,
1
mp 219.4 °C. Yield: 62%. H NMR: δ 8.77 (br, 1H), 7.88 (d, J
) 7.4 Hz, 2H), 7.55 (s, 1H), 7.54 (d, J ) 7.1 Hz, 1H), 7.44-
7.35 (m, 12H), 6.89 (t, J ) 6.2 Hz, 1H), 5.24 (s, 2H), 4.96 (d, J
) 6.2 Hz, 2H), 4.79 (s, 2H), 3.79 (s, 3H). 13C NMR: δ 165.9,
165.7, 157.7, 153.0, 152.5, 141.2, 135.6, 134.8, 133.9, 133.2,
130.4, 129.64, 129.62, 129.55, 129.0, 128.7, 128.5, 128.4,
128.24, 128.21, 125.3, 67.6, 58.3, 52.4, 45.4. Anal. (C31H27N3O7)
C, H, N.
4-Nitr oben zoic Acid 4-(5-Ben zyloxyca r bon yla m in o-6-
oxo-2-p h en yl-6H-p yr im id in -1-yl)-2-m eth oxyca r bon ylbu t-
2-en yl Ester (1). Compound 1 was prepared using the same
method as for the synthesis of compound 3 to give a light-
yellow solid, mp 234.9 °C. Yield: 59%. 1H NMR: δ 8.77 (br,
1H), 8.25 (d, J ) 8.9 Hz, 2H), 8.08 (d, J ) 8.9 Hz, 2H), 7.54 (s,
1H), 7.47-7.30 (m, 10H), 6.92 (t, J ) 6.3 Hz, 1H), 5.24 (s, 2H),
4.96 (d, J ) 6.3 Hz, 2H), 4.86 (s, 2H), 3.80 (s, 3H). 13C NMR:
δ 165.5, 164.1, 157.7, 152.9, 152.3, 150.7, 141.7, 134.9, 134.8,
133.9, 130.8, 130.5, 129.1, 129.0, 128.9, 128.8, 128.7, 128.6,
128.3, 125.4, 123.6, 67.7, 59.0, 52.5, 45.2. IR (film), cm-1: 3378,
3274, 3059, 2953, 1720, 1653, 1605, 1517, 1491, 1348, 1267,
1213, 1190, 1096, 1064, 967, 770, 736, 719, 699. Anal.
Toxicity Assa y. Selected compounds were tested for toxic-
ity in vitro against three mammalian cell lines. A subclone
(G8) of the murine monocyte-like macrophage line J 774 was
obtained from Dr. J ose Alunda, Departmento de Sanidad
Animal, Facultad de Veterinaria, Universidad Complutense,
Madrid, Spain. Murine cells were maintained in 75 cm2 tissue
culture flasks in Dulbecco’s modified Eagle medium (GIBCO)
supplemented with 10% fetal calf serum, 2 mM l-glutamine,
and 50 mg/mL gentamicin under humidified 5% CO2/95% air
at 37 °C. A subclone (NG-108-15) of a hybrid rat neruoblas-
toma/mouse glioma cell line was the gift of Dr. Marshall
Nirenberg, National Heart Lung and Blood Institute, National
Institutes of Health, Bethesda, MD. Neuronal cells were
maintained in 75 cm2 tissue culture flasks in Dulbecco’s
modified essential medium, including a hypoxanthine-ami-
nopterin-thymidine (HAT) supplement, 10% fetal calf serum,
and 50 mg/mL gentamicin under humidified 5% CO2/95% air
at 37 °C. The human colon cell line SW620 was provided by
Robert Robey, National Cancer Institute, National Institutes
of Health, Bethesda, MD. Colon cells were maintained and
tested in RPMI-1640 supplemented with 10% fetal bovine
serum. Toxicity tests were performed in 96-well tissue culture
plates using the protein-binding dye sulforhodamine B (SRB)
as described.26 Test compounds were serially diluted and added
to empty wells of the 96-well plate. Appropriate cells in their
respective culture medium were immediately seeded into the
C
31H26N4O9) C, H, N.
4-(5-Ben zyloxyca r b on yla m in o-6-oxo-2-p h en yl-6H-p y-
r im id in -1-yl)-2-h yd r oxym eth ylbu t-2-en oic Acid Meth yl
Ester (5). Granular potassium carbonate (20 mg) was added
into a stirred solution of compound 3 (100 mg) in 2 mL of
methanol at ambient temperature. After being stirred for 30
min, the mixture was partitioned between EtOAc (20 mL) and
H2O (15 mL). The separated organic layer was washed with
brine (10 mL), dried over anhydrous Na2SO4, and evaporated
in a rotary evaporator to give 65 mg of compound 5 as a white
solid, mp 188.5 °C. Yield: 71%. 1H NMR: δ 8.79 (br, 1H), 8.54-
7.38 (m, 11H), 6.63 (t, J ) 6.9 Hz, 1H), 5.23 (s, 2H), 4.84 (d, J
) 6.9 Hz, 2H), 4.21 (s, 2H), 3.77 (s, 3H), 3.00 (br, 1H). 13C
NMR: δ 166.7, 157.8, 152.9, 152.4, 138.6, 136.1, 135.6, 135.0,
134.6, 133.9, 130.5, 129.1, 128.7, 128.5, 128.2, 125.4, 67.6, 56.9,
52.3, 44.9. Anal. (C24H23N3O6) C, H, N.
2-Acet oxym et h yl-4-[2-(5-b en zyloxyca r b on yla m in o-6-
oxo-2-p h en yl-6H-p yr im id in -1-yl)a cetyla m in o]p en t-2-en o-
ic Acid Meth yl Ester (6). Compound 11 (207 mg) was
dissolved in 3.0 mL of Et2O. To the solution was added 0.5
mL of 2.0 N HCl in ether, and the mixture was stirred for 15
min at ambient temperature. The solvent was removed in
vacuo to give the amine salt 12, which was dissolved in 2 mL
of chloroform. The amine solution was added dropwise with

3496 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Zhu et al.
Inhibitors. J . Med. Chem. 1995, 38, 3193-3196. (c) Simpkins,
N. S. The Chemistry of Vinyl Sulfones. Tetrahedron 1990, 46,
6951-6984. (d) Fuchs, P. L.; Braish, T. F. Multiply Convergent
Syntheses Via Conjugate-Addition Reactions to Cycloalkenyl
Sulfones. Chem. Rev. 1986, 86, 903-918.
wells. Appropriate solvent blanks (no compound) were run in
each test. After 72 h under culture conditions, cells were fixed
to the plate by layering 50% TCA (4 °C) over the growth media
in each well to produce a final TCA concentration of 10%. After
incubating for 1 h at 4 °C, cultures were washed five times
with tap water and air-dried. Wells were stained for 30 min
with 0.4% (w/v) SRB in 1% acetic acid and washed four times
with 1% acetic acid. Cultures were air-dried, and bound dye
was solubilized with 10 mM Tris base (pH 10.5) for 15 min on
a gyratory shaker at room temperature. A Spectra MAX Plus
microtiter plate reader (Molecular Devices) was used to
measure the optical density (OD) at wavelengths of 490-530
nm. The 50% cell growth inhibitory concentration (IC50) of each
compound is shown in Table 2.
(17) (a) Angelastro, M. R.; Mehdi, S.; Burkhart, J . P.; Peet, N. P.;
Bey, P. R-Diketone and R-Keto Ester Derivatives of N-Protected
Amino Acids and Peptides as Novel Inhibitors of Cysteine and
Serine Proteinases. J . Med. Chem. 1990, 33, 11-13. (b) Hu, L.
Y.; Abeles, R. H. Inhibition of Cathepsin B and Papain by
Peptidyl alpha-Keto Esters, alpha-Keto Amides, alpha-Dike-
tones, and alpha-Keto Acids. Biochim. Biophys. 1990, 281, 271-
274. (c) Li, Z.; Patel, G. S.; Golubski, Z. E.; Hori, H.; Tehrani,
K.; Foreman, J . E.; Eveleth, D. D.; Bartus, R. T.; Powers, J . C.
Peptide R-Keto Ester, R-Keto Amide, and R-Keto Acid Inhibitors
of Calpains and Other Cysteine Proteases. J . Med. Chem. 1993,
36, 3472-3480. (d) Harbeson, S. L.; Abelleira, S. M.; Akiyama,
A.; Barrett, R., III; Carroll, R. M.; Straub, J . A.; Tkacz, J . N.;
Wu, C.; Musso, G. F. Stereospecific Synthesis of Peptidyl R-Keto
Amides as Inhibitors of Calpain. J . Med. Chem. 1994, 37, 2918-
2929. (e) Shaw, E. Peptidyl Sulfonium Salts. A New Class of
Protease Inhibitors. J . Biol. Chem. 1988, 263, 2768-2772.
(18) This ring system has been used as a mimetic for the P3-P2 (Val-
Pro) in the human leukocyte elastase inhibitor 4-((4-Cl-Ph)SO2-
NHCO)PhCO-Val-Pro-Val-COCF3. (a) Bernstein, P. R.; Ed-
ward, P. D.; Shaw, A.; Thomas, A. M.; Veale, C. A.; Warner, P.;
Wolanin, D. J . Pyrimidinyl Acetamides as Elastase Inhibitors.
European Patent Application, No. WO 9321210, October, 1993.
(b) Bernstein, P. R.; Edward, P. D.; Shaw, A.; Shenvi, A. B.;
Thomas, A. M.; Veale, C. A.; Warner, P.; Wolanin, D. J . Alpha-
Aminoboronic Acid Peptides and Their Use as Elastase Inhib-
otors. PTC Publication No. WO 9321214, October, 1993. (c)
Veale, C. A.; Steelman, G. B.; Chow, M. M. Efficient Method for
the Synthesis of 1,4-Disubstituted 5-Carbomethoxypyrimidin-
6-ones. J . Org. Chem. 1993, 58, 4490-4493. (d) Brown, F. J .;
Andisik, D. W.; Bernstein, P. R.; Bryant, C. B.; Ceccarelli, C.;
Damewood, J . R., J r.; Edwards, P. D.; Earley, R. A.; Feeney, S.;
Green, R. C.; Gomes, B.; Kosmider, B. J .; Krell, R. D.; Shaw, A.;
Steelman, G. B.; Thomas, R. M.; Vacek, E. P.; Veale, C. A.;
Tuthill, P. A.; Warner, P.; Williams, J . C.; Wolanin, D. J .;
Woolson, S. A. Design of Orally Active, Non-Peptidic Inhibitors
of Human Leukocyte Elastase. J . Med. Chem. 1994, 37, 1259-
1261.
Ack n ow led gm en t. Dr. Shuren Zhu held a National
Research Council Research Associateship while he
conducted the reported research.
Refer en ces
(1) The World Health Report; World Health Organization: Geveva,
1999.
(2) Winstanley, P. A. Chemotherapy for Falciparum Malaria: The
Armoury, the Problems and the Prospects. Parasitol. Today
2000, 16, 146-115.
(3) Nosten, F.; Ter Kuile, F.; Chongsuphajaisiddhi, T.; Luxemburger,
C.; Webster, H. K.; Edstein, M.; Phaipun, L.; Thew, K. L.; White,
N. J . Mefloquine-Resistant Falciparum Malaria on the Thai-
Burmese Border. Lancet 1982, 337, 1140-1143.
(4) Oduola, A. M.; Milhous, W. K.; Salako, L, A.; Walker, O.;
Desjardins, R. E. Reduced in Vitro Susceptibility to Mefloquine
in West African Isolates of Plasmodium falciparum. Lancet 1987,
2, 1304-1305.
(5) Semenov, A.; Olson, J . S.; Rosenthal, P. J . Antimalarial Synergy
of Cysteine and Aspartic Protease Inhibitors. Antimicrob. Agents
Chemother. 1998, 42, 2254-2258.
(6) Rothenthal, P. J . Plasmodium falciparum: Effects of Proteinase
Inhibitors on Globin Hydrolysis by Cultured Malaria Parasites.
Exp. Parasitol. 1995, 80, 272-281.
(7) Rothenthal, P. J . Protease of Malaria Parasites: New Targets
for Chemotherapy. Emerging Infect. Dis. 1998, 4, 49-57.
(8) Otto, H. H.; Schirmeister, T. Cysteine Proteases and Their
Inhibitors. Chem. Rev. 1997, 97, 133-171 and references therein.
(9) Smith, R. A.; Copp, L. J .; Coles, P. J .; Pauls, H. W.; Robinson,
V. J .; Spencer, R. W.; Heard, S. B.; Krantz, A. New Inhibitors of
Cysteine Proteinases. Peptidyl Acyloxymethyl Ketones and the
Quiescent Nucleofuge Strategy. J . Am. Chem. Soc. 1988, 110,
4429-4431.
(19) This ring system has also been used as a mimetic for the P3-P2
(Val-Ala) in the design of interleukin-1â converting enzyme
inhibitor. Dolle, R. E.; Prouty, C. P.; Prasad, C. V. C.; Cook, E.;
Saha, A.; Ross, T. M.; Salvino, J . M.; Helaszek, C. T.; Ator, M.
A. First Examples of Peptidomimetic Inhibitors of Interleukin-1
â Converting Enzyme. J . Med. Chem. 1996, 39, 2438-2440.
(20) Basavaiah, D.; Hyma, R. S. Synthetic Applications of the Baylis-
Hillman Reaction: Simple Synthesis of [2E]-2-Butyloct-2-enal
and [2E]-2-Tridecylheptadec-2-enal. Tetrahedron 1996, 52, 1253-
1258.
(21) (a) Charette, A.; Cote, B.; Monroc, S.; Prescott, S. Synthesis of
Monoprotected 2-Alkylidene-1,3-propanediols by an Unusual
SN2′ Mitsunobu Reaction. J . Org. Chem. 1995, 60, 6888-6894.
(b) Farina, V. Complex Stereochemical Course of the Mitsunobu
Inversion of Allylic Alcohols. Tetrahedron Lett. 1989, 30, 6645-
6648.
(10) Rasnick, D. Synthesis of Peptide Fluoromethyl Ketones and the
Inhibition of Human Cathepsin B. Anal. Biochem. 1985, 149,
461-465.
(11) Crawford, C.; Mason, R. W.; Wikstrom, P.; Shaw, E. The Design
of Peptidyldiazomethane Inhibitors To Distinguish between the
Cysteine Proteinases Calpain II, Cathepsin L and Cathepsin B.
Biochem. J . 1988, 253, 751-758.
(12) Gour-Salin, B. J .; Lachance, P.; Magny, M. C.; Plouffe, C.;
Menard, R.; Storer, A. C. E64 [trans-Epoxysuccinyl-l-leucyl-
amido-(4-guanidino)butane] Analogues as Inhibitors of Cysteine
Proteinases: Investigation of S2 Subsite Interactions. Biochem.
J . 1994, 389-392.
(13) Hanzlik, R. P.; J acober, S. P.; Zygmunt, J . Reversible Binding
of Peptide Aldehydes to Papain. Structure-Activity Relation-
ships. Biochim. Biophys. Acta 1991, 1073, 33-42.
(14) Hanzlik, R. P.; Zygmunt, J .; Moon, J . B. Reversible Covalent
Binding of Peptide Nitriles to Papain. Biochim. Biophys. Acta
1990, 1035, 62-70.
(15) (a) Hanzlik, R. P.; Thompson, S. A. Vinylogous Amino Acid
Esters: A New Class of Inactivators for Thiol Proteases. J . Med.
Chem. 1984, 27, 711-712. (b) Thompson, S. A.; Andrews, P. R.;
Hanzlik, R. P. Carboxyl-Modified Amino Acids and Peptides as
Protease Inhibitors. J . Med. Chem. 1986, 29, 104-111. (c) Liu,
S.; Hanzlik, R. P. Structure-Activity Relationships for Inhibition
of Papain by Peptide Michael Acceptors. J . Med. Chem. 1992,
35, 1067-1975.
(16) (a) Rosenthal, P. J .; Olson, J . E.; Lee, G. K.; Palmer, J . T.; Klaus,
J . L.; Rasnick, D. Antimalarial Effects of Vinyl Sulfone Cysteine
Proteinase Inhibitors. Antimicrob. Agents Chemother. 1996, 40,
1600-1603. (b) Palmer, J . T.; Rasnick, D.; Klaus, J . L.; Bro¨mme,
D. Vinyl Sulfones as Mechanism-Based Cysteine Protease
(22) Fehrentz, J . A.; Castro, B. An Efficient Synthesis of Optically
Active R-(t-Butoxycarbonylamino)-aldehydes from R-Amino
Acids. Synthesis 1983, 676-678.
(23) (a) Lin, A. J .; Zikry, A. B.; Kyle, D. E. Antimalarial activity of
new dihydroartemisinin derivatives. 7. 4-(p-substituted phenyl)-
4-(R or S)[10-(R or â)-dihydroartemisininoxy]butyric acids. J .
Med. Chem. 1997, 40, 1396-1400. (b) Desjardins, R. E.; Canfield,
C. J .; Haynes, D. E.; Chulay, J . D. Quantitative Assessment of
Antimalarial Activity in Vitro by a Semiautomated Microdilution
Technique. Antimicrob. Agents Chemother. 1979, 16, 710-718.
(24) Chulay, J . D.; Haynes, J . D.; Diggs, C. L. Plasmodium falci-
parum: Assessment of in Vitro Growth by [3H]hypoxanthine
Incorporation. Exp. Parasitol. 1983, 55, 138-146.
(25) Oduola, A. M.; Weatherly, N. F.; Bowdre, J . H.; Desjardins, R.
E. Plasmodium falciparum: Cloning by Single-Erythrocyte
Micromanipulation and Heterogeneity in Vitro. Exp. Parasitol.
1988, 66, 86-95.
(26) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer-drug screen-
ing. J . Natl. Cancer Inst. 1990, 82, 1107-1112.
J M020104F