Novel biologically active nonpeptidic inhibitors of myristoylcoa:Protein n-myristoyltransferase

Article abstract of DOI:10.1021/jm980001q

A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been synthesized starting from the octapeptide ALYASKLS- NH₂ (2). The synthetic strategy entailed the preparation of novel protected Ser-Lys mimics 9 and 12 f

Full text of DOI:10.1021/jm980001q

996
J . Med. Chem. 1998, 41, 996-1000
Notes
Novel Biologica lly Active Non p ep tid ic In h ibitor s of Myr istoylCoA:P r otein
N-Myr istoyltr a n sfer a se
Balekudru Devadas,* Sandra K. Freeman, Charles A. McWherter, Nandini S. Kishore, J ennifer K. Lodge,†
Emily J ackson-Machelski,† J effrey I. Gordon,† and J ames A. Sikorski
Department of Medicinal and Structural Chemistry, G. D. Searle and Company, 700 Chesterfield Parkway North,
St. Louis, Missouri 63198, and Department of Molecular Biology and Pharmacology, Washington University School of
Medicine, St. Louis, Missouri 63110
Received December 29, 1997
A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been
synthesized starting from the octapeptide ALYASKLS-NH2 (2). The synthetic strategy entailed
the preparation of novel protected Ser-Lys mimics 9 and 12 from (S)- or (R)-3-iodotyrosine
and then grafting key enzyme recognition elements in a stepwise manner. Like 2, compounds
16, 17, and 18 are competitive Candida NMT inhibitors that bind to the peptide recognition
site of the enzyme. Moreover, 16-18 have an affinity comparable to that of 2 even though
they are devoid of peptide bonds. In contrast to 2, these nonpeptidic inhibitors exhibit antifungal
activity.
In tr od u ction
The design and synthesis of biologically active non-
peptidic inhibitors of disease targets have been a
significant challenge in the discovery of novel thera-
peutic agents. Nonetheless, rapid progress has been
made over the last decade as potent nonpeptidic agents
have been identified for increasingly complex systems
including antagonists of the RGD-based fibrinogen
receptor,1 as well as HIV-I protease inhibitors.2 For the
F igu r e 1. Octapeptide substrate (1) and inhibitor (2) of
past few years we have been interested in finding novel
antifungal agents with a unique mode of action. The
enzyme myristoylCoA:protein N-myristoyltransferase
(NMT; EC 2.1.3.97) is an attractive target for the
generation of such agents.3 NMT catalyzes the cotrans-
lational transfer of myristate (C14:0) from myristoylCoA
to the N-terminal glycine residue of several eukaryotic
proteins, and is present in a variety of pathogenic fungi.3
Genetic studies have demonstrated that C. albicans and
C. neoformans require NMT for their viability.4,5 Al-
though the atomic structure of NMT is not yet known,
several potent and selective peptidomimetic inhibitors
have been developed by exploiting the differences in
peptide substrate specificities between fungal and hu-
man NMTs.6,7 This report describes the design and
synthesis of the first biologically active inhibitors of
fungal NMTs where the key enzyme recognition ele-
ments are tethered to novel scaffolds devoid of peptide
bonds.
Candida albicans NMT.
F igu r e 2. Dipeptide inhibitor of Candida albicans NMT.
enzyme recognition elements responsible for imparting
high affinity to 1.8 Substitution of glycine in 1 with
alanine provided the ALYASKLS-NH2 inhibitor 2 (Ki(app)
) 15.3 ( 6.4 µM), which did not exhibit antifungal
activity even at concentrations of >100 µM. Subsequent
replacement of the first four amino acids in 2 with a
p-[(2-methyl-1-imidazol-1-yl)butyl]phenylacetyl group
and substituting the C-terminal Leu-Ser dipeptide
fragment with a 2-cyclohexylethyl amide moiety pro-
The synthetic design emanated from the previously
described high-affinity octapeptide substrate GLYASKLS-
NH2 1 (Figure 1). Earlier studies have revealed that
the primary amino group of glycine, the hydroxyl group
of serine-5, and ꢀ-amino group of lysine are the critical
vided the potent dipeptide NMT inhibitor 3 (Ki(app)
)
0.031 ( 0.003 µM, Figure 2). Despite its excellent
potency, no antifungal activity was detected against
logarithmically growing cultures of C. albicans (EC50
> 100 µM).6
The lack of antifungal activity for 3 may be partially
attributed to the remaining Ser-Lys dipeptide bond
* To whom correspondence should be addressed.
† Washington University School of Medicine.
S0022-2623(98)00001-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/18/1998

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6 997
Sch em e 1. Synthesis of Ser-Lys Dipeptide Scaffold 9
Sch em e 2. Synthesis of Ser-Lys Dipeptide Scaffold 12
which could be cleaved in vitro. Consequently, we
sought to attenuate the peptide character of 3 in order
to improve its metabolic stability as well as its absorp-
tion and intracellular distribution characteristics. This
rationale led to the design and synthesis of the novel
Ser-Lys dipeptide scaffolds 9 and 12, embodying the key
enzyme recognition elements in a core structure derived
from tyrosine.
saponification of the methyl ester group in 11 followed
by diborane reduction of the corresponding carboxylic
acid, and silylation of the resulting primary alcohol
yielded 12.
Amines 13 and 14 generated by the hydrogenation of
9 and 12 (Scheme 3), were individually condensed with
the imidazole-substituted phenylacetic acid 159 using
DCC/HOBt. The crude reaction products were treated
with trifluoroacetic acid, and the final compounds 16
and 17 were isolated as trifluoroacetate salts by reverse-
phase HPLC.9
Ch em istr y
The synthesis of 9 and 12 began from commercially
available L-3-iodotyrosine as shown in Scheme 1. First,
alkylation of the phenol in the Z-protected L-tyrosine
derivative 4 with 2-cyclohexylethyl bromide afforded the
cyclohexylethyl ether 5. Palladium-catalyzed coupling
of 5 with 1-butyn-4-ol, in the presence of triethylamine
and CuI in acetonitrile, gave the corresponding alkyne
product 6. After the reduction of the triple bond by
catalytic hydrogenation, the Z-group was reintroduced
to give 7a , which was then subjected to iodination using
methyltriphenoxyphosphonium iodide to afford 7b. Re-
action of 7b with sodium azide followed by reduction
with triphenylphosphine in THF/H2O, and subsequent
protection of the resulting primary amine with (BOC)2O
yielded 8. The ester group in 8 was subjected to
methanolysis using 1 M LiOH. After acidification, the
resulting acid was reduced to the primary alcohol and
converted to the silylated derivative 9.
Sch em e 3. Syntheses of Nonpeptidic NMT Inhibitors
or
The synthesis of the R enantiomer 18 (Figure 3) was
also achieved following the same methodologies as
outlined in Schemes 2 and 3, commencing from the
corresponding R isomer of 5.
Analogue 12, having the aminopropyl chain, was also
synthesized starting from 5 according to Scheme 2. The
iodotyrosine derivative 5 underwent smooth palladium
catalyzed insertion of BOC-protected propargylamine in
the presence of CuI and triethylamine to provide the
propargyl derivative 10. Catalytic hydrogenation of 10
followed by carbobenzoxylation gave 11. Subsequent
Resu lts a n d Discu ssion
All seven peptide bonds and seven of the eight chiral
center have been removed from the starting octapeptide

998 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Notes
Ta ble 1. Enzyme Potency and Antifungal Activity of
Nonpeptidic NMT Inhibitors
IC50 (µM)a
EC50 (µM)b
24 h, C. albicans
compd C. albicans NMT human NMT
16
17
18
29.3 ( 0.7
20.0 ( 0.03
17.5
62.3 ( 5.7
56 ( 17
F igu r e 3. Nonpeptidic inhibitor of Candida albicans NMT.
inhibitor, 2. The first four residues (ALYA) were
replaced by a p-[(2-methylimidazol-1-yl)butylphenyl]-
acetyl group in which the imidazole moiety represented
the key N-terminal amino recognition element at ala-
nine. The C-terminal tetrapeptide SKLS was replaced
by a new chiral tyrosinol scaffold that retains the serine
alcohol recognition element and presents the lysine
amine moiety in the form of an attached 3-aminobutyl
side chain.8 The hydrophobic interaction3 fulfilled by
leucine-7 in 2 was represented by the 4-cyclohexylethyl
ether. This design assumed that these nonpeptidic
analogues would have a similar binding mode and
orientation to that of 2.
64
49 (MFC ) 150 µM)
77 (MFC ) 250 µM)
6.5
a
Potency against the indicated NMT as assessed by IC50 using
the peptide substrate GNAASARR-NH2 at its apparent Km and
myristoylCoA at 1 µM. EC50s were determined as described in
ref 6.
b
activity has been discovered. Further optimization of
the spatial orientation of the critical recognition ele-
ments should lead to even more potent and selective
NMT inhibitors with improved antifungal activity.
Exp er im en ta l Section
Unless otherwise stated, starting materials were obtained
from commercial suppliers and were used without further
purification. All reactions were performed under anhydrous
conditions in an atmosphere of argon. Nuclear magnetic
proton were recorded on a Varian XL-300 spectrometer, and
chemical shifts (δ) are reported in ppm relative to tetrameth-
ylsilane. Splitting patterns are designated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad
peak. Low-resolution mass spectra were recorded on a VG40-
250T instrument, and high-resolution mass spectra were
recorded on a Finnigan MAT 90 mass spectrometer operating
in the FAB mode. The purity of all the compounds was shown
to be >95% by 1H NMR and chromatographic techniques.
Analytical reverse-phase high-performance liquid chromatog-
raphy (HPLC) was carried out using a Waters Delta-Pak
cartridge (C-18, 8 × 100 mm), using a linear gradient of (A)
water containing 0.05% trifluoroacetic acid and (B) acetonitrile
containing 0.05% TFA, at a flow rate of 1 mL/min. The elution
was carried out with a linear gradient from 10 to 90% of B in
30 min, and the separation was monitored by UV absorbance
at 215 nm.
Final compounds were purified by reverse-phase HPLC
using a Waters Delta-Pak cartridge (C-18, 40 × 100 mm, 15
µm) and eluting with a linear gradient consisting of 5-70% of
B in 30 min. The flow rate was adjusted to 70 mL/min, and
the separtion was monitored by UV absorbance at 215 nm.
The appropriate fractions were pooled and freeze-dried, and
the products were isolated as TFA salts.
3-Iod o-N-(p h en ylm eth oxyca r bon yl)-L-tyr osin e Meth yl
Ester (4). To a suspension of 3-iodotyrosine methyl ester
hydrochloride (5.7 g, 0.156 mol) in dichloromethane (50 mL)
was added N-methylmorpholine (1.6 g, 0.016 mol). After 15
min, N-(benzyloxycarbonyl)oxysuccinimide (4.0 g, 0.016 mol)
was added, and the resulting mixture was stirred at 10 °C for
1 h and at room temperature for 2 h. The reaction mixture
was diluted with dichloromethane (25 mL), washed succes-
sively with 5% citric acid (2 × 20 mL) and water (3 × 25 mL),
and dried (Na2SO4). The solution was concentrated, and the
resulting substance was crystallized from 25% EtOAc in
hexane to afford 5.4 g (74%) of 4 as a white powder: Rf ) 0.58
(EtOAc:hexane, 1:1 v/v); 1H NMR (CDCl3) δ 7.35 (m, 5H), 7.28
(m, 1H), 6.96 (m, 1H), 6.87 (d, 1H, J ) 8.1 Hz), 5.1 (q, 2H),
4.61 (m, 1H), 3.73 (s, 3H), 3.0 (m, 2H); FAB-MS m/ z 462 (M
+ Li); HRMS calcd for C18H18NIO5Li (M + Li) 462.0389, found
462.0392.
The nonpeptidic analogues 16, 17, and 18 were
evaluated for inhibition of purified C. albicans and
human NMTs (Table 1). Like 2, the S and R isomers
16 and 18, are competitive Candida NMT inhibitors
with respect to the peptide substrate GNAASARR-NH2.
Compounds 16 and 18 have Kis of 20 and 9 µM,
respectively. Remarkably, these values are comparable
to that observed for 2 (15 µM). The modest 2-4-fold
selectivity3 of 2 for the fungal versus the human enzyme
was maintained by 16. These results indicate that none
of the amide bonds in 2 are necessary to generate an
inhibitor directed against the NMT peptide recognition
site.
The homologue 17 with an aminobutyl side chain is
also an inhibitor with a Ki of 9 ( 1 µM. It is evident
from these results that there is no clear chirality
preference within this nonpeptidic series. This differs
from previous results with either dipeptide6 or tripep-
tide3,6 inhibitors. The findings reported here suggest
that it may be possible to develop potent inhibitors of
NMT that are achiral as long as they maintain the three
critical enzyme recognition elements.
While the octapeptide 2 was devoid of biological
activity, compounds 17 and 18 were fungicidal against
C. albicans (Table 1), with MFCs of 150 and 250 µM,
respectively. In contrast, 16 is fungistatic only against
C. albicans. Compounds 17 and 18 are also fungicidal
against C. neoformans (e.g., MFC ) 200 µM for 18).
In order to probe the origin of the fungicidal activity
for 17, human NMT was expressed in C. neoformans in
place of the functional wild-type fungal enzyme. There
was a reproducible 2-fold reduction in the fungicidal
activity of 17 when tested against a strain containing
human NMT.10 The fungicidal effect was not due to cell
lysis but was accompanied by a rapid inhibition of
protein synthesis.10 The 2-fold difference in antifungal
activity of 17 against strains containing fungal versus
human NMT mirrors the observed differences in its Ki
against the human and fungal enzymes. Together,
these results indicate that the fungicidal effects of 17
involve inhibition of protein N-myristoylation in C.
neoformans.
O -(C y c lo h e x y le t h y l)-3-i o d o -N -(p h e n y lm e t h o x y -
ca r bon yl)-L-tyr osin e Meth yl Ester (5). A mixture of 4 (0.8
g, 0.00176 mol), 2-cyclohexylethyl bromide (0.5 g, 0.0026 mol),
and tetrabutylammonium fluoride (1.1 g) in dry THF (10.0 mL)
was heated to reflux under nitrogen for 1 h. Thereafter, the
solvent was distilled under reduced pressure, and the residue
was partitioned between cold 5% citric acid (25 mL) and
dichloromethane (30 mL). The organic phase was washed with
water (2 × 20 mL), dried (Na2SO4), and concentrated under
Con clu sion
A new class of nonpeptidic NMT inhibitors which
embody only one chiral center and exhibit fungicidal

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6 999
reduced pressure. The resulting syrup was purified by silica
gel flash chromatography using 20% EtOAc in hexane to give
0.6 g (60%) of 5 as a colorless syrup: Rf ) 0.42 (EtOAc:hexane,
3:7 v/v); 1H-NMR (CDCl3) δ 7.51 (s, 1H), 7.35 (m, 5H), 6.98
(dd, 1H, J ) 2.1, 8.4 Hz), 6.67 (d, 1H, J ) 8.4 Hz), 5.2 (br,
1H), 5.1 (d, 2H, J ) 3.0 Hz), 4.6 (m, 1H), 4.0 (t, 2H, J ) 6.6
Hz), 3.73 (t, 3H), 3.00 (m, 2H), 1.73 (m, 9H), 1.26 (m, 2H), 0.96
was heated at 70 °C for 3 h. DMF was distilled in vacuo, and
the residue was partitioned between water (10 mL) and EtOAc
(25 mL). The organic phase was washed with brine, dried
(Na2SO4), and concentrated to dryness under reduced pressure
to afford 0.58 g of the corresponding azide as a pale yellow
syrup which was used without further purification in the
following step.
(m, 2H); FAB-MS m/ z 572 (M + Li); HRMS calcd for C26H32
NIO5Li (M + Li) 572.1484, found 572.1475.
-
A solution of the azide (0.4 g, 0.00075 mol) in THF (6 mL)
and water (0.2 mL) was treated with
a solution of tri-
O -(C y c lo h e x y le t h y l)-3-(4-h y d r o x y -1-b u t y n y l)-N -
(p h en ylm eth oxyca r bon yl)-L-tyr osin e Meth yl Ester (6).
To a solution of 5 (1.4 g, 0.025 mol) and 1-butyn-4-ol (0.26 g,
0.0037 mol) in acetonitrile (8 mL) were added triethylamine
(0.36 g, 0.0036 mol), (Ph3)2PPdCl2 (0.1 g, 0.00014 mol), and
CuI, and the mixture was stirred at room temperature for 4 h
under argon atmosphere. The reaction mixture was then
concentrated under reduced pressure, and the residue was
partitioned between 5% citric acid (20 mL) and EtOAc (30 mL).
The organic phase was washed with water (2 × 20 mL), dried
(Na2SO4), and concentrated to dryness. The resulting dark
brown substance was purified by silica gel flash chromatog-
raphy using 25% EtOAc in hexane as the eluent to afford 1.2
g (96%) of 6 as a pale yellow syrup: Rf ) 0.16 (EtOAc:hexane,
phenylphosphine (0.3 g, 0.0015 mol) in THF (6 mL), and the
mixture was stirred at room temperature for 30 min. There-
after, the reaction mixture was heated at 60 °C for 2.5 h under
an atmosphere of nitrogen and concentrated in vacuo. The
residue was dissolved in dichloromethane (5 mL) and cooled
in an ice bath, (BOC)2O (0.16 g, 0.00075 mol) was added, and
the mixture was stirred at room temperature for 1.5 h. The
reaction mixture was then diluted with dichloromethane (20
mL), washed with saturated sodium bicarbonate (2 × 15 mL),
and brine (2 × 15 mL), dried (Na2SO4), and concentrated to
dryness under reduced pressure. The resulting material was
purified by silica gel flash chromatography using 25% EtOAc
in hexane containing 0.1% triethylamine to furnish 0.4 g (88%)
of 8 as a colorless syrup: Rf ) 0.41 (EtOAc:hexane, 3:7 v/v);
1H-NMR (CDCl3) δ 7.33 (s, 5H), 6.82 (m, 2H), 6.72 (d, 1H, J )
8.5 Hz), 5.21 (br, 1H), 5.09 (d, 2H, J ) 3.3 Hz), 4.6 (m, 1H),
3.94 (t, 2H, J ) 6.6 Hz), 3.72 (s, 3H), 3.12 (m, 2H), 3.05 (m,
2H), 2.56 (m, 2H), 1.8-1.6 (m, 8H), 1.43 (s over m, 10H), 1.27
(m, 4H), 0.95 (m, 4H); FAB-MS m/ z 617 (M + H); HRMS calcd
for C35H50N2O7Li (M + Li) 617.3778, found 617.3741.
4-(Cycloh exylet h oxy)-3-[4-[[(1,1-d im et h ylet h oxy)ca r -
bon yl]am in o]bu tyl]-â-[[(1,1-dim eth yleth yl)dim eth ylsilyl]-
m e t h oxy]-N -(p h e n ylm e t h oxyca r b on yl)-(S )-b e n ze n e -
eth a n a m in e (9). A mixture of 8 (0.44 g, 0.0007 mol) and 1
M LiOH (1.75 mL) in THF (1 mL) was stirred at room
temperature for 1.5 h. It was then diluted with 5% citric acid
(10 mL) and extracted with EtOAc (2 × 10 mL). The combined
organic extracts were washed successively with brine (2 × 10
mL), dried (Na2SO4), and concentrated to dryness. The
resulting residue was dried in a desiccator overnight over
NaOH pellets to afford 0.4 g of the corresponding acid, which
was subjected to reduction in the following step without
purification.
To a solution of the acid in THF (5 mL) was added dropwise
BH3-THF complex (1 M, 2 mL), and the mixture was stirred
at -5 °C for 1 h and at 10 C for 1 h. Thereafter, the reaction
was quenched by the addition MeOH (3 mL) containing acetic
acid (0.3 mL) and concentrated to dryness under reduced
pressure. The resulting residue was partitioned between
saturated sodium bicarbonate (10 mL) and dichloromethane
(25 mL). The organic phase was washed with brine (2 × 15
mL), dried (Na2SO4), and concentrated to dryness. The residue
was dried in a desiccator overnight to afford 0.3 g of the
corresponding primary alcohol, which was dissolved in aceto-
nitrile (3.0 mL). tert-Butyldimethylchlorosilane (0.11 g, 0.00073
mol), imidazole (0.03 g), and N-methylmorpholine (0.08 g,
0.0007 mol) were added. The resulting mixture was stirred
at 60 °C under a nitrogen atmosphere for 2.5 h and concen-
trated under a reduced pressure. The residue was partitioned
between 5% citric acid (10 mL) and dichloromethane (20 mL).
The organic phase was washed with brine, dried (Na2SO4), and
concentrated to dryness. The residue thus obtained was
purified by silica gel flash chromatography using 12% EtOAc
in hexane to furnish 0.22 g (47%) of 9 as a colorless syrup: 1H
NMR (CDCl3) δ 7.32 (s, 5H), 6.95 (m, 2H), 6.8 (d, 1H, J ) 8.2
Hz), 5.1 (s, 2H), 4.95 (br, 1H), 4.5 (m, 1H), 3.94 (t, 2H, J ) 6.6
Hz), 3.85 (br, 1H), 3.51 (d, 2H, J ) 3.3 Hz), 3.1 (m, 2H), 2.72
(d, 2H, J ) 6.9 Hz), 2.55 (m, 2H), 1.8-1.4 (m, 10H), 1.42 (s,
9H), 1.22 (m, 4H), 0.9 (s over m, 12H), 0.027 and 0.022 (2s,
6H); FAB-MS m/ z 697 (M + H); HRMS calcd for C40H65N2O6-
Si (M + H) 697.4612, found 697.4642.
1
3:7 v/v); H NMR (CDCl3) δ 7.34 (s, 5H), 7.1 (d, 1H, J ) 2.1
Hz), 6.95 (m, 1H), 6.74 (d, 1H, J ) 8.4 Hz), 5.2 (br, 1H), 5.1 (s,
2H), 4.6 (m, 1H), 4.01 (t, 2H, J ) 6.3 Hz), 3.8 (q, 2H, J ) 6.3
Hz), 3.72 (t, 3H), 3.0 (m, 2H), 2.71 (t, 2H, J ) 6.3 Hz), 2.08 (t,
1H, OH), 1.5-1.85 (m, 8H), 1.2-1.4 (m, 4H); FAB-MS m/ z
508 (M + H); HRMS calcd for C30H38NO6 (M + H) 508.2699,
found 508.2709.
O-(Cycloh e xyle t h yl)-3-(4-h yd r oxyb u t yl)-N -(p h e n yl-
m eth oxyca r bon yl)-L-tyr osin e Meth yl Ester (7a ). A solu-
tion of 6 (1.2 g, 0.0024 mol) in MeOH (25 mL) and acetic acid
(0.2 mL) was hydrogenated at 50 psi in the presence of 5%
Pd/C (1.00 g) for 16 h. The catalyst was removed by filtration,
and the filtrate was concentrated to dryness under reduced
pressure. The resulting residue was dissolved in dichlo-
romethane (10 mL), N-methylmorpholine (0.32 g, 0.0032 mol),
and (benzyloxycarbonyl)succinimide (0.50 g, 0.002 mol) were
added, and the mixture was stirred at room temperature for
16 h. The reaction mixture was then diluted with dichlo-
romethane (25 mL), washed successively with 5% citric acid
(2 × 10 mL), saturated sodium bicarbonate (2 × 15 mL), and
water, and dried (Na2SO4). After removal of the solvent under
reduced pressure, the residue was purified by silica gel flash
chromatography using 50% EtOAc in hexane to afford 0.81 g
(86%) of 7a as a colorless syrup: 1H NMR (CDCl3) δ 7.34 (s,
5H), 6.84 (s, over m, 2H), 6.71 (d, 1H, J ) 8.3 Hz), 5.22 (br,
1H), 5.1 (m, 1H), 3.95 (t, 2H, J ) 8.0 Hz), 3.73 (s, 3H), 3.62
(m, 2H), 3.0 (m, 2H), 2.55 (t, 2H), 1.8-1.5 (m, 7H), 1.4-1.2
(m, 6H), 1.1-0.8 (m, 4H); FAB-MS m/ z 512 (M + H); HRMS
calcd for C30H42NO6 (M + H) 512.3012, found 512.3049.
O-(Cycloh exylet h yl)-3-(4-iod ob u t yl)-N-(p h en ylm et h -
oxyca r bon yl)-L-tyr osin e Meth yl Ester (7b). To a solution
of 7a (0.8 g, 0.0016 mol) in acetonitrile (5 mL) was added
triphenoxymethylphosphonium iodide (1.00 g, 0.0022 mol), and
the mixture was stirred at room temperature for 3 h. The
reaction mixture was concentrated, and the residue was
partitioned between EtOAc (25 mL) and cold 0.25 N NaOH
(15 mL). The organic phase was washed successively with 0.25
N NaOH (2 × 15 mL) and water (3 × 15 mL), dried (Na2SO4),
and concentrated to dryness under reduced pressure. The
material thus obtained was purified by silica gel flash chro-
matography to furnish 0.75 g of 7b as a colorless liquid: 1H-
NMR (CDC13) δ 7.4 (s, 5H), 6.85 (m, 3H), 6.72 (d, 1H, J ) 8.5
Hz), 5.15 (br, 1H), 5.1 (d, 2H, J ) 2.4 Hz), 4.62 (m, 1H), 3.95
(t, 2H, J ) 6.6 Hz), 3.73 (s, 3H), 3.17 (t, 2H, J ) 7.7 Hz), 3.0
(m, 2H), 2.55 (t, 2H, J ) 7.7 Hz), 1.8-1.6 (m, 11H), 1.25 (m,
4H), 1.04 (m, 2H); FAB-MS m/ z 622 (M + H); HRMS calcd
for C30H41NIO5 (M + H) 622.2028, found 622.2000.
O-(Cycloh exylet h yl)-3-[4-[[(1,1-d im et h ylet h oxy)ca r b-
o n y l]a m i n o ]b u t y l]-N -(p h e n y lm e t h o x y c a r b o n y l)-L -
tyr osin e Meth yl Ester (8). A mixture of 7b (0.75 g, 0.0011
mol) and sodium azide (0.23 g, 0.0035 mol) in DMF (2.5 mL)
N-[1-[[3-(Am in ob u t yl)-4-(cycloh exylet h oxy)p h en yl]-
m eth yl]-2-h yd r oxyeth yl]-4-[(2-m eth yl-1H-im od a zol-l-yl]-
bu tyl]-(S)-ben zen eeth a n a m in e (17). A solution of 9 (0.2
g, 0.0003 mol) in EtOH (5.0 mL) was hydrogenated at 30 psi

1000 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Notes
in the presence of 10% Pd/C (0.05 g) for 1.5 h. The catalyst
was removed by filtration, the filtrate was concentrated under
reduced pressure, and the residue was dried in vacuo for 4 h
to afford the corresponding amine, which was used without
purification in the following step.
4-(Cyc loh e xyle t h oxy)-3-[[4-[(1,1-d im e t h yle t h oxy)-
ca r bon yl]a m in o]p r op yl]-â-[[(1,1-d im eth yleth yl)d im eth -
ylsilyl]m et h oxy]-N-(p h en ylm et h oxyca r b on yl)-(S)-b en -
1
zen eeth a n a m in e (12): H NMR (CDCl3) δ 7.33 (s, 5H), 6.98
(m, 2H), 6.70 (d, 1H, J ) 8.2 Hz), 5.1 (s, 2H), 4.98 (br, 1H), 4.7
(br, 1H), 3.96 (t, 2H, J ) 5.1 Hz), 3.85 (br, 1H), 3.50 (d, 2H, J
) 8.5 Hz), 3.18 (m, 2H), 2.75 (d, 2H, J ) 6.9 Hz), 2.58 (m, 2H),
1.74 (m, 9H), 1.4 (s over m, 9H), 1.25 (m, 4H), 0.91 (s over
m,11H), 0.037 and 0.03 (2s, 6H); FAB-MS m/ z 689 (M + Li);
HRMS calcd for C39H62N2O6SiLi (M + Li) 689.4537, found
689.4555.
To a solution of the acid 15 (0.14 g, 0.00045 mol) in
dimethylacetamide (2 mL) and dichloromethane (2.0 mL) were
added DCC (0.093 g, 0.00045 mol) and HOBt (0.075 g), and
the mixture was stirred at 0 °C for 1.5 h. The reaction mixture
was then added to a solution of the amine as prepared above,
in dimethylacetamide (0.5 mL), DMAP (0.018 g) and N-
methylmorpholine (0.046 g, 0.00045 mol) were added, and the
mixture was stirred at room temperature for 16 h. After the
removal of the solvents in vacuo, the residue was partitioned
between cold 0.25 N NaOH (5 mL) and EtOAc (20.0 mL). The
organic phase was washed with saturated sodium bicarbonate
(2 × 5 mL) and brine, dried (Na2SO4), and concentrated under
reduced pressure. The resulting substance (0.2 g) was stirred
with trifluoroacetic acid (2 mL) at room temperature for 3.5 h
and concentrated under reduced pressure. The residue was
then purified by reverse-phase HPLC using 5-70% acetonitrile
in water (30 min gradient) at a flow rate of 70 mL/min.
Appropriate fractions were pooled and freeze-dried to yield 17
as a hygroscopic white powder: tR ) 24.2 min; 1H NMR (CD3-
OD) δ 7.41 (d, 1H, J ) 2.1 Hz), 7.35 (d, 1H, J ) 2.1 Hz), 7.01
(d, 2H, J ) 7.8 Hz), 6.94 (m, 4H), 6.77 (d, 1H, J ) 8.1 Hz),
4.08 (t, 3H, J ) 7.2 Hz), 3.96 (m, 2H), 3.48 (d, 2H, J ) 1.2
Hz), 3.34 (q, 2H, J ) 5.1 Hz), 2.8-2.45 (s over m, 11H), 1.8-
1.3 (m, 15H), 1.3-1.15 (m, 2H), 0.95 (m, 2H); FAB-MS m/ z
603 (M + H); HRMS calcd for C37H55N4O3 (M + H) 603.4274,
found 603.4320.
Ack n ow led gm en t. The authors thank Mr. J ames
P. Doom and Charles A. Gloeckner of Monsanto Co. for
providing FAB mass spectral data.
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N-[1-[[3-(Am in op r op yl)-4-(cycloh exyleth oxy)p h en yl]-
m eth yl]-2-h yd r oxyeth yl]-4-[(2-m eth yl-1H-im od a zol-1-yl)-
bu tyl]-(S)-ben zen eeth a n a m in e (16): tR ) 23.6 min; 1H NMR
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7.02 (m, 6H), 6.79 (d, 1H, J ) 8.4 Hz), 4.1 (t, 3H, J ) 7.5 Hz),
4.01 (t, 2H, J ) 6.6 Hz), 3.52 (d, 2H, J ) 4.8 Hz), 3.41 (s, 2H),
2.81 (m, 4H), 2.66 (m, 7H), 2.0-1.4 (m, 14H), 1.27 (m, 3H),
1.05 (m, 2H); FAB-MS m/ z 589 (M + H); HRMS calcd for
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C
36H53N4O3 (M + H) 589.4118, found 589.4121.
N-[1-[[3-(Am in op r op yl)-4-(cycloh exyleth oxy)p h en yl]-
m eth yl]-2-h yd r oxyeth yl]-4-[(2-m eth yl-1H-im od a zol-l-yl]-
bu tyl]-(R)-ben zen eeth a n a m in e (18): tR ) 23.63 min; 1H
NMR (CD3OD) δ 7.44 (d, 1H, J ) 1.8 Hz), 7.39 (d, 1H, J ) 1.8
Hz), 7.02 (m, 6H), 6.78 (d, 1H, J ) 8.1 Hz), 4.12 (t, 3H, J )
7.4 Hz), 4.00 (t, 2H, J ) 6.6 Hz), 3.52 (d, 2H, J ) 5.1 Hz), 3.41
(s, 2H), 2.81 (m, 4H), 2.64 (s over m, 5H), 2.02-1.2 (m, 15H),
1.2-1.08 (m, 4H), 1.04 (m, 2H); FAB-MS m/ z 589 (M + H);
HRMS calcd for C36H53N4O3 (M + H) 589.4118, found 589.4130.
O -(C y c lo h e x y le t h y l)-3-(4-h y d r o x y -1-p r o p y n y l)-N -
(p h en ylm eth oxyca r bon yl)-L-tyr osin e Meth yl Ester (10):
yield 72%; pale yellow syrup; 1H NMR (CDCl3) δ 7.34 (s, 5H),
7.12 (d, 1H, J ) 2.1 Hz), 6.9 (m, 1H), 6.82 (d, 1H, J ) 8.4 Hz),
5.15 (br, 1H), 5.10 (s, 2H), 4.65 (br, 1H), 4.58 (m, 1H), 4.18 (d,
2H, J ) 7.1 Hz), 4.01 (t, 2H, J ) 6.6 Hz), 3.72 (s, 3H), 3.0 (m,
2H), 1.82-1.6 (m, 8H), 1.46 (s, 9H), 1.4-1.1 (m, 3H), 0.95 (m,
2H); FAB-MS m/ z 597 (M + H); HRMS calcd for C34H49N2O7
(M + H) 597.3534, found 597.3494.
O -(C y c lo h e x y le t h y l)-3-[[4-[(1,1-d im e t h y le t h o x y )-
ca r bon yl]a m in o]p r op yl]-N-(p h en ylm eth oxyca r bon yl)-L-
tyr osin e m eth ylester (11): yield 67%; Rf ) 0.25 (EtOAc:
hexane, 3:7 v/v); 1H NMR (CDCl3) δ 7.33(s, 5H), 6.85 (m, 2H),
6.71 (d, 1H, J ) 8.3 Hz), 5.4 (br, 1H), 5.10 (m, 2H), 4.7 (br,
1H), 4.6 (m, 1H), 3.95 (t, 2H, J ) 6.6 Hz), 3.72 (s, 3H), 3.05
(m, 4H), 2.58 (m, 2H), 1.8-1.6 (m, 10H), 1.44 (s, 9H), 1.25 (m,
3H), 1.05-0.8 (m, 2H); FAB-MS m/ z 603 (M + Li); HRMS
calcd for C34H48N2O7Li (M + Li) 603.3622, found 603.3651.
(8) McWherter, C. A.; Rocque, W. J .; Zupec, M. E.; Freeman, S. K.;
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of a Candida albicans and human myristoylCoA:protein N-
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(9) All new compounds exhibited spectral and analytical data
consistent with the structure.
(10) Lodge, J . K.; J ackson-Machelski, E.; Higgins, M.; McWherter,
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depeptidized inhibitor of Cryptococcus neoformans myristoylCoA:
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J M980001Q