Synthesis of potent and selective inhibitors of Candida albicans N-myristoyltransferase based on the benzothiazole structure

Article abstract of DOI:10.1016/j.bmc.2005.01.033

Two parallel synthetic methods using solid-supported reagents were established to examine the rapid optimization of weak hit compound 1. Several compounds showed high potency in the low nanomolar range against N-myristoyltransferase. The structure-activity relationship (SAR) and antifungal activities of a series of novel 2-aminobenzothiazole N-myristoyltransferase inhibitors are presented.

Full text of DOI:10.1016/j.bmc.2005.01.033

Bioorganic & Medicinal Chemistry 13 (2005) 2509–2522
Synthesis of potent and selective inhibitors of Candida albicans
N-myristoyltransferase based on the benzothiazole structure
Kazuo Yamazaki,* Yasushi Kaneko, Kie Suwa, Shinji Ebara,
Kyoko Nakazawa and Kazuhiro Yasuno
Central Research Laboratories, SSP Co., Ltd, 1143 Nanpeidai, Narita, Chiba 286-8511, Japan
Received 16 November 2004; revised 21 January 2005; accepted 21 January 2005
Abstract—Two parallel synthetic methods using solid-supported reagents were established to examine the rapid optimization of
weak hit compound 1. Several compounds showed high potency in the low nanomolar range against N-myristoyltransferase. The
structure–activity relationship (SAR) and antifungal activities of a series of novel 2-aminobenzothiazole N-myristoyltransferase
inhibitors are presented.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
(IC50: 1.5 lM) by virtual screening5 followed by CaNmt
screening of selected compounds.
Myristoylation by the myristoyl–CoA:protein N-myri-
stoyltransferase (Nmt) is an important lipid anchor
modification of eukaryotic and viral proteins. These en-
zymes catalyze the covalent attachment of the C14 fatty
acid to the N-terminal glycine on substrate proteins.
According to eukaryote genome analyses, the predicted
rate of substrate proteins is about 0.5% of all encoded
proteins.1 The incidence of systemic fungal infections
caused by Candida albicans has been observed during
the last two decades, particularly in immunocompro-
mised patients. Several classes of antifungal drugs have
been used, but for the best treatment of life-threatening
infections, new, effective drugs are urgently needed. Ge-
netic experiments have shown Nmt is an essential en-
zyme for survival of Candida. Therefore, Nmt is an
attractive target for new fungicidal drugs.2 Several
Nmt inhibitors have already been reported as shown
in Figure 1,3 but further investigations of in vivo efficacy
are necessary.4 In this paper, we describe a solution
phase parallel synthesis and SAR of a series of novel
benzothiazole N-myristoyltransferase inhibitors. Figure
1 also shows the identified weak hit compound 1
2. Chemistry
Scheme 1 shows the combinatorial synthetic route.
Diversity was initially introduced to carboxylic acid 3
using a commercially available set of N-Boc protected
diamines 4a–k. After deprotection of the Boc group,
reductive amination with aldehydes 6a–k using a macro-
porous triacetoxyborohydride MP-BH(OAc)3 was per-
formed.6 Then, a catch and release purification using
macroporous p-toluenesulfonic acid (MP-TsOH) affor-
ded the final products 7aa–7kk. (examples: 3 + 4a!5a,
5a + 6a!7aa; 3 + 4k!5k, 5k + 6k!7kk). The amide
junction modified compounds 10ai–10ck (examples:
9a + 6i!10ai; 9c + 6k!10ck) were prepared using a pro-
tocol Benzothiazole carboxamide regio-isomers 11a,b
(Table 4) were also prepared from benzothiazole 5- or
7-carboxylic acid7 by the above synthetic route (Table 1).
In order to optimize the effects of C2 substitution of
benzothiazole, the synthetic route was changed as
shown in Scheme 2. N-Boc protected amine 12 was re-
acted with 2-naphthaldehyde under general reductive
amination conditions, followed by N-protection with
an Alloc group and deprotection of the Boc group to
give 13. Condensation of 13 with 2-N-Boc-aminobenzo-
thiazole-6-carboxylic acid gave 14b. After the general
transformations, palladium-catalyzed deprotection of
Keywords: N-Myristoyltransferase; Antifungal; Parallel synthesis;
Solid-supported reagents.
*
Corresponding author. Tel.: +81 476 27 1571; fax: +81 476 26
7948; e-mail: kazuo.yamazaki@ssp.co.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.033

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K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
N
N
OH
O
O
H
N
N
H
N
H
O
SC-58272 IC50: 0.056 µM
HN
N
NH2
O
O
S
N
N
NH
O
O
O
F
NH2
F
F
RO-09-4879 IC50: 0.0057 µM
UK-370,485 IC50: 0.042 µM
O
N
O
S
N
NH
O
weak hit compound 1 IC50: 1.5 µM
Figure 1.
HOOC
S
N
EtOOC
a,b
c,d
S
N
NH
O
NH2
3
2
O
O
H2N
S
H
N
Y
e
R1
NH
S
N
Y
N
NH
O
HCl or TFA
Y=linker
O
5a-k
7aa-7kk
H
N
H2N
S
N
H2N-(CH2)n
f,d
S
N
NH
NH
O
O
O
9a, n=1, 9b, n=2, 9c, n=3
8
HCl or TFA
R1
H
N
HN-(CH2)n
S
N
g
NH
O
NEt3BH(OAc)3
O
MP-BH(OAc)3
10ai-10ck
O
O
same as above
H
N
S
HO
S
N
N
H
NH
O
NH
O
N
benzothiazlole 5- or 7-carboxylic acid
11a: 5-
11b: 7-
Scheme 1. Reagents and conditions: (a) cyclohexanecarboxylic acid, EDC, HOBt, DMF; (b) 1 N-NaOH, MeOH; (c) BocNH-linker-NHR (4a–k),
EDC, HOBt, DMF; (d) 4N-HCl/dioxane or 50% TFA–CH 2Cl2; (e) R1CHO (6a–k), MP-BH(OAc)3, DMF, then PS-CHO, catch and release
purification using MP-TsOH then 2 M-NH3/MeOH; (f) BocNH(CH2)nCOOH, EDC, HOBt, DMF; (g) R1CHO (6i–k), MP-BH(OAc)3, DMF.

K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
2511
Table 1. List of diverse sets 4 and 6
NHBoc
NHBoc
H2N
NHBoc
H2N
NHBoc
BocHN
NH
H2N
H2N
4e
4d
4b
4a
4f
4c
BocHN
BocHN
BocHN
BocHN
NH
BocHN
NH
NH
NH
BocHN
NH2
NH2
4g
4j
4h
4i
4k
CHO
CHO
CHO
CHO
CHO
CHO
CHO
CHO
CHO
F
CHO
CHO
N
CF3
NO2
Cl
Me
OMe
F
Ph
6g
6j
6a
6b
6c
6d
6e
6f
6h
6i
6k
a, b, c
Alloc
N
Alloc
N
O
d
H2N
NHBoc
S
N
NH2
N
H
R
12
13
R=H; 14a
R=NHBoc; 14b
c
e
e
14a
14b
16a
R=NH2; 15
O
16e
N
O
N
O
f, e, c
g, e
14b
15
16b-d
16f-t
Solid-Supported Barbituric Acid
Scheme 2. Reagents and conditions: (a) 2-naphthaldehyde, NaBH(OAc)3, DCE; (b) Alloc-Cl, K2CO3, THF–H2O; (c) 50% TFA–CH2Cl2; (d)
benzothiazole-6-carboxylic acid for 14a, 2-N-Boc-aminobenzothiazole-6-carboxylic acid for 14b, EDC, HOBt, DMF; (e) solid-supported barbituric
acid, Pd(PPh3)4, THF; (f) Aryl CH2Br or Alkyl CH2Br, K2CO3, DMF; (g) Ac2O, DMAP, CH2Cl2, or carboxylic acid, EDC, HOBt, DMF.
the Alloc group was conducted by the parallel mode
using solid-supported barbituric acid.8 This solid-sup-
ported reagent facilitated the isolation and purification
of the deprotected polar and basic secondary amines.
28g and 28e were synthesized from the known
(1S,3R)- or (1R,3S)-1,3-cyclohexanedicarboxylic acid
mono ester.11 Thus, enzyme-catalyzed desymmetrization
of a meso cis-cyclohexanecarboxylic acid diester 21 was
conducted using lipase AYS Amano or PS Amano to
give monocarboxylic acid 22a or 22b, respectively. The
Curtius rearrangement of 22a was performed under gen-
eral conditions, followed by hydrolysis to give (1R,3S)-
3-Boc-aminocyclohexanecarboxylic acid 24a. Condensa-
tion of 24a with 6-aminobenzothiazole 25 gave 26a.
Deprotection of the Boc group was performed, followed
by reductive amination to give (1R,3S)-enantiomer 28g
(98% ee). The (1S,3R)-enantiomer 28h (93% ee) was
synthesized by the same procedure from 22b
(Scheme 4).
Benzoxazole scaffolds (Table 4, 11c,d) were prepared
according to the literature.9 Thus, the reaction of 2-ami-
nophenol derivatives 17a,b with di-(imidazole-1-
yl)methanimine afforded methyl 2-aminobenzoxazole
5- and 6-carboxylate, respectively, which were converted
to compound 11c,d by the same procedure as shown in
Scheme 1. Benzimidazole scaffolds (Table 4, 11e,f) were
prepared from N-protected diamines derivatives 13.
Condensation of 13 with fluoronitrobenzoic acid gave
19a,b, which were treated with methylamine for the
nucleophilic aromatic substitution and followed by the
reduction of the nitro group to afford 20a,b. A cycliza-
tion reaction using a literature procedure10 followed by
the palladium-catalyzed deprotection of the Alloc group
gave 11e,f (Scheme 3).
3. Biology
The enzyme inhibitory activities of compounds against
C. albicans Nmt (CaNmt) and human Nmt (HsNmt)
were measured according to the literature.12–14 In vitro
antifungal assay against C. albicans (ATCC90028) was
Compounds 28a–k (Table 6) were prepared in a similar
manner to that described for 10ai–10ck. Enantiomers

2512
K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
same as scheme 1
a
OH
O
N
MeOOC
MeOOC
NH2
11c: 5-
11d: 6-
NH2
17a, b
18a: 5-
18b: 6-
b
Alloc
N
O
F
Alloc
N
c, d
N
H
NH2
NO2
19a, b
13
e, f, g
Me
N
Alloc
N
O
NHMe
NH2
O
H
N
NH
O
N
H
N
H
N
20a, b
11e: 5-
11f: 6-
Scheme 3. Reagents and conditions: (a) di-(imidazole-1-yl) methanimine, THF; (b) 4-fluoro-3-nitrobenzoic acid for 19a, 3-fluoro-4-nitrobenzoic acid
for 19b, EDC, HOBt, DMF; (c) MeNH2–THF; (d) SnCl2Æ2H2O, 6 N-HCl, EtOH; (e) cyclohexanecarbonyl isothiocyanate, CH2Cl2; (f) EDC, DIEA,
CH2Cl2; (g) solid-supported barbituric acid, Pd(PPh3)4, THF.
COOH
COOEt
COOEt
a
or
COOEt
22b (1R,3S)
COOEt
COOH
22a (1S,3R)
21 meso
COOEt
COOEt
COOH
c
b
COOH
22a
NHBoc
23a
NHBoc
24a (1R,3S)
NHBoc
H2N
S
N
d
H
N
NH
O
S
N
NH
O
O
25
26a
NH2
TFA
NH
f
e
H
N
H
N
S
S
N
NH
O
NH
O
O
N
O
28g (1R,3S)
27a
same as above
22b
28h (1S,3R)
Scheme 4. (a) Lipase AYS Amano for 22a or Lipase PS Amano for 22b, pH 7.2 phosphate buffer; (b) (PhO)2PON3, Et3N, toluene, then t-BuOH;
(c) 5 N-NaOH, THF; (d) 24a, EDC, HOBt, DMF; (e) TFA, CH2Cl2; (f) 2-naphthaldehyde, MP-BH(OAc)3, DMF.
performed by a broth microdilution method according
to the National Committee for Clinical Laboratory
Standards (NCCLS) method M27-A2. The minimum
inhibitory concentration (MIC) of a compound was de-
fined as the lowest concentration at which there was 80%
inhibition of growth compared to the growth of a drug-
free control.
4. Results and discussion
Based on the proposed reaction mechanism,15 we took
particular interest in the polar amine moiety of 1. To in-
crease enzymatic inhibitory activity, substitutes at C6 of
benzothiazole 1 were initially examined. Several cyclic
and acyclic amides that contain primary, secondary,

K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
Table 2. Matrix of CaNmt inhibitory activity (%) at 10 lM of 7aa–7kk
2513
From
4a
4b
4c
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
4d
4e
4f
4g
4h
4i
4j
4k
>90%
50-90%
0-49%
Table 3. SAR of the substitutes at position 6 of the benzothiazole
and tertiary amines as the C6 substituents were synthe-
sized using standard methods and it was determined that
the amides with secondary amines displayed the highest
potency (data not shown). Therefore, we focused on
synthesizing secondary amine utilizing solution phase
combinatorial chemistry. Table 2 summarizes the
CaNmt inhibitory activities at 10 lM of the first library
compounds.
X
S
N
N
H
NH
O
Compd
X
CaNmt
inhibition
IC50 (lM)
HsNmt
inhibition
(%) at 10 lM
7ai
7bi
7ci
7di
–(CH2)2NHCO–
–(CH2)3NHCO–
–(CH2)4NHCO–
–(CH2)5NHCO–
90.0344
0.11
From the matrix, hydrophobic groups such as 2-naph-
thyl (derived from 6i), 4-chrolophenyl (6c), and 4-biphen-
yl (6j) seem suitable for high potency, although the
matrix does not show accurate potencies. In most cases,
2-naphthyl derivatives showed high potency. Fluorine
compounds such as 3-trifluoromethylpheyl (derived
from 6h) and 2,4-difluorophenyl (6f) groups had poor
potency. Furthermore, shorter alkyl chain lengths
(4a,b) seem suitable. Table 3 summarizes the close-up
SAR of the 2-naphthyl derivatives.
61
30.0744
0.3
46
14
NCO-
NCO-
NCO-
7ei
7fi
0.065
0.19
36
27
32
7gi
7hi
0.5
NCO-
NCO-
0.026
Table 3 clearly shows that even-numbered alkyl chain
lengths (7ai, 7ci) are more potent than odd-numbered
ones (7bi, 7di). Reversed amide (10bi) resulted in a slight
loss of activity. The selectivity against the HsNmt is gen-
erally good. Based on the above results, compound 7ai
was selected as the starting point for further
optimization.
7ii
0.21
27
7ji
>10
1.9
N.T.
N.T.
NHCO-
7ki
NHCO-
–CH2CONH–
–(CH2)2CONH–
Next, the effects of modifying the carboxamide regio-
isomer and the core scaffold were investigated. Table 4
summarizes the results. The position of the amide group
that contains the secondary amine is crucial for potent
enzyme inhibitory activity (7ai vs 11a,b). Benzoxazole
(11c,d) and benzimidazole (11e,f) resulted in a significant
loss of activity.
10ai
10bi
2.3
0.18
8
32
N.T.: not tested.
or greater than 7ai, but inserting heteroatoms resulted
in a significant loss of activity (16k,l). Our experiments
indicated that replacing the cycloalkyl groups with aryl
groups decreased the potency.
In order to further optimize the potency, the effects of
C2 substitution of benzothiazole were examined. Table
5 summarizes the assay results. Activity was not ob-
served when the amide group was removed (compounds
15, 16a–e). Less lipophilic alkyl amides decreased the
potency (16f,g). In contrast, more lipophilic acyclic
and cyclic alkyl amides (16h–j) showed potency equal
Then all compounds were screened for the antifungal
activity against C. albicans (ATCC90028). Surprisingly,
all the compounds had weaker antifungal activities than

2514
K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
Table 4. SAR for core scaffolds
Linear alkyl series 28b showed greater enzyme inhibitory
activity than 10bj, but the antifungal activity was disap-
pointing. A correlation between the enzyme inhibitory
activity and the antifungal activity was not observed.
However, introducing a cycloalkyl linker, in particular,
a cyclohexyl linker improved both the CaNmt inhibitory
activity and antifungal activity. The antifungal activity
seems to be associated with CaNmt inhibitory activity
only in the cycloalkyl linker series (28c–m). The (1R,S)-
enantiomer 28g exhibited the most potent CaNmt inhib-
itory activity (IC50: 0.49 nM) with an excellent selectivity
(>10,000-fold) over the HsNmt (IC50: 5.4 lM) and anti-
fungal activity (MIC: 0.78 lM). Table 7 summarizes
more screening results of 28g and known inhibitors.
The MIC value of 28g was about 2–6 times lower than
RO-09-4879 and UK-370753.3c There was a large differ-
ence between the effective concentrations of 28g that
were required for enzyme inhibition and fungal growth
inhibition compared to RO-09-4879 and UK-370753.
One possible explanation is that 28g has a low ability
to enter the cell, but it is unclear how many Nmt mole-
cules must be deactivated before growth is inhibited.
The permeation of most drugs across the cell membrane
by passive diffusion or via a transporter is a critical step
in the interaction of a drug with its cellular target and can
influence its overall efficacy. Increased antifungal activity
requires drug interaction with a transporter to increase
the intracellular concentration. For example, caspofun-
gin acetate is taken up by a high-affinity transpoter.16
7
1
O
2
X
6
H
N
N
H
NH
N
3
5
O
4
Compd
X
Position
CaNmt inhibition IC50 (lM)
7ai
S
6
5
7
5
6
5
6
0.034
6.8
1.0
11a
11b
11c
11d
11e
11f
S
S
O
1.1
O
0.29
>10
>10
NMe
NMe
Table 5. SAR for C2 substitution of benzothiazole
O
H
N
S
N
N
H
R
Compd
R
CaNmt
inhibition
HsNmt
inhibition
(%) at 10 lM
IC50 (lM)
16a
15
H
NH2
>10
>10
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
4
16b
16c
16d
16e
16f
16g
16h
16i
16j
7ai
16k
NHCH2Ph
>10
>10
NHCH2–2-pyridyl
NHCH2–c-hexyl
NHCOO–t-Bu
NHCOMe
>1
>10
The serum effect of 28g was not observed in an in vitro
antifungal assay and 28g did not inhibit other strains
such as Candida krusei (ATCC 6258; MIC: >25 lM),
Candida glabrata (TIMM3171; MIC: >25 lM) and
Aspergillus fumigatus (IFM40808; MIC: >25 lM).
Unfortunately, the cytotoxicity of 28g was about ten
times stronger than RO-09-4879 and UK-370753, but
28g exhibited an excellent selectivity against the HsNmt,
which suggests that it may have an alternative mecha-
nism for cytotoxicity.17 Further investigations are neces-
sary to determine the usefulness of the Nmt inhibitors.
>10
NHCOEt
NHCO–i-Pr
0.13
0.046
0.028
0.023
09.0344
0.89
14
22
NHCO–c-butyl
NHCO–c-pentyl
NHCO–c-hexyl
NHCO–tetrahydro-
pyran-4-yl
32
5
16l
NHCO–piperidine-4-yl
NHCOPh
>10
0.16
>10
>10
0.73
>10
>10
>10
0.31
N.T.
32
37
16
11
8
16m
16n
16o
16p
16q
16r
16s
16t
NHCO–2-pyridyl
NHCO–2-Cl–3-pyridyl
NHCO–2-pyrazine
NHCOPh–4-Cl
NHCOPh–3,4-Cl
NHCOPh–4-CF3
NHCOPh–2,5-OMe
5. Conclusion
22
1
In summary, the synthesis and optimization of a series
of 2-aminobenzothiazole N-myristoyltransferase inhibi-
tors using solution phase combinatorial chemistry are
described. The present study demonstrates the effi-
ciency of parallel synthesis using solid-supported re-
agents in a rapid synthesis and SAR evaluation.
Members of this class of compounds exhibit potent en-
zyme inhibitory activity and antifungal activity. Studies
on further modifications of 28g (FTR1335) are cur-
rently underway.
71
N.T.: not tested.
expected from the potent enzyme inhibitory activity.
One of them, the reversed amide 10bj showed moderate
antifungal activity as shown in Figure 2, but 10bj
showed less potent enzyme inhibitory activity than 7aj.
Therefore, we returned to reversed amide 10bj to explore
the effect and the role of the reversed amide on the anti-
fungal activity.
6. Experimental
Several analogues, which were prepared by the same
procedure described above, and the reversed amide ser-
ies were screened for CaNmt inhibitory activities and
antifungal activities against C. albicans. Table 6 summa-
rizes the results.
6.1. General comments
All the solvents and reagents were purchased from com-
mercial sources and were used without further purifica-

K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
2515
Table 6. SAR for reversed amide series
H
linker
N
S
N
R2
N
H
NH
O
O
R1
Compd
Linker
R1
R2
CaNmt inhibition IC50 (lM)
MIC (lM) C. albicans ATCC90028
6.25
28a
–(CH2)2–
0.16
28b
28c
28d
–(CH2)3–
0.015
0.015
0.011
50
>12.5
3R
3S
1S
6.25
1R
28e
28f
28g
28h
28i
0.13
>12.5
1,3-cis
0.0012
0.78
1,3-cis
1R
0.00049
>0.01
0.78
3.13
3.13
3S
3R
1S
0.0033
1,3-cis
28j
0.00243.13
0.0023
1,3-cis
1,3-cis
1,3-cis
28k
28l
1.56
3.13
0.0081
28m
0.0346.25
1,4-trans
tion. MP-BH(OAc)3 (Part Number 800414), MP-TsOH
cartridge (Part Number 800477-0050-C) and PS-Benzal-
dehyde (Part Number 800360) were purchased from
Argonaut Technologies. Solid-supported barbituric acid
was prepared according to a literature procedure.8
Parallel syntheses were performed on a Quest 210
All the HPLC analyses were carried out using an Inertsil
ODS3 column, 1.5 mm · 150 mm (GL Sciences). A flow
rate of 0.12 mL/min and a gradient of (5–95)% B over
5 min were used (solvent A, water/0.05% formic acid;
solvent B, acetonitrile/0.05% formic acid). The enantio-
meric excess (ee) was determined by HPLC analysis.
HPLC was performed on a Hitachi HPLC L-4200
(detector), L-6000 (pump), L-7300 (column oven) using
a Daicel Chiralcel OD-H column; mobile phase hexane/
EtOH/Et2NH = 60:40:0.1; flow rate, 0.5 mL/min; tem-
perature, 25 ꢁC; detection, UV at 254nm. Optical reso-
lutions were recorded on a high sensitive polarimeter
SEPA-300 (Horiba).
1
(Argonaut) or on an SY-1000 (Shimadzu). H NMR
(400 MHz) spectra were recorded on JNM-EX400 using
tetramethylsilane as an internal standard. FAB mass
spectra were recorded on JEOL JMS-700 spectrometer.
The LC-LRMS system consisted of an SCL-10Avp ser-
ies (Shimadzu) coupled to an LCMS-QP8000a (Shima-
dzu, atmospheric pressure chemical ionization, APCI).

2516
K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
reversed amide
(m, 1H), 1.09–1.95 (m, 13H). MS (FAB): 333 (M+1).
HRMS (FAB) Calcd for C17H21N2O3S: 333.1273,
Found: 333.1281.
Ph
Ph
H
N
H
N
S
NH
O
N
The mixture of the above compound (33 g, 0.1 mol), 1 N-
NaOH (1 L), and MeOH (200 mL) was stirred at room
temperature for 4h and then the solvent was evaporated.
The resulting aqueous layer was washed with CHCl3.
The aqueous layer was separated and cooled to 5 ꢁC,
10% aqueous HCl was added to lower the pH 2. The
resulting solid was collected by filtration and washed
with water. The solid was dried to afford 25 g (84%) of
O
10bj
CaNmt IC50: 0.078 µM
C. albicans MIC: 4 µg/ml
O
H
N
S
N
N
H
NH
O
1
3. H NMR (CDCl3) d (ppm): 8.58 (d, J = 1.4Hz, 1H),
7aj
7.99 (dd, J = 8.3, 1.4Hz, 1H), 7.77 (d, J = 8.3 Hz, 1H),
3.26 (br s, 1H), 2.48–2.60 (m, 1H), 1.09–1.90 (m, 10H).
MS (FAB): 305 (M+1). HRMS (FAB) Calcd for
C15H17N2O3S: 305.0960, Found: 305.0965.
CaNmt IC50: 0.025 µM
C. albicans MIC: >8 µg/ml
Figure 2.
6.4. 2-[(Cyclohexanecarbonyl)amino]-N-(2-aminoethyl)-
benzothiazole-6-carboxamide hydrochloride (5a)
6.2. Ethyl 2-amino-benzothiazole-6-carboxylate (2)
A mixture of ethyl 4-aminobenzoate (16.5 g, 0.1 mol),
KSCN (97.18 g, 1 mol), and CuSO4 (80 g, 0.5 mol) in
MeOH (270 mL) was stirred for 3 h at 70 ꢁC. After cool-
ing, the suspension was filtered and the filtrate diluted
with water (400 mL) and heated to boiling. Ethanol
was added to the boiling filtrate until a clear slight yel-
low solution resulted. Cooling of this solution resulted
in crystallization of 2 (11 g, 50%) as a pale yellow solid.
To a solution of 3 (1 g, 3.48 mmol) and tert-butyl N-(2-
aminoethyl)carbamate (0.56 g, 3.48 mmol) in DMF
(15 mL) was added EDC (1 g, 5.22 mmol) and HOBt
(0.7 g, 5.22 mmol) at room temperature. The mixture
was stirred for 16 h at room temperature and then
poured into water (100 mL). The resulting solid was
collected by filtration and washed with water and then
the solid was dried. The crude product was purified by
chromatography on silica gel to afford 1.28 g (86%) of
2-[(cyclohexanecarbonyl)amino]-N-[(2-tert-butoxycar-
1H NMR (DMSO-d6) d (ppm): 8.34(d, J = 1.4Hz, 1H),
7.87 (dd, J = 1.4, 8.3 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H),
4.30 (q, J = 7.3 Hz, 2H), 1.32 (t, J = 7.3 Hz, 3H). MS
(FAB): 223 (M+1).
bonyl)aminoethyl]-benzothiazole-6-carboxamide as
a
1
pale yellow solid, which was used in the next step. H
NMR (DMSO-d6) d (ppm): 12.34(s, 1H), 8.42 (s, 1H),
7.90 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 6.83
(br s, 1H), 3.25–3.38 (m, 2H), 3.08–3.19 (m, 2H), 2.50–
2.62 (m, 1H), 1.16–1.91 (m, 19H). MS (FAB): 447
(M+1). HRMS (FAB) Calcd for C22H31N4O4S:
447.2066, Found: 447.2097.
6.3. 2-[(Cyclohexanecarbonyl)amino]-benzothiazole-6-
carboxylic acid (3)
To a solution of 2 (50 g, 0.225 mol) and cyclohexane-
carboxylic acid (35 g, 0.27 mol) in DMF (200 mL) was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
[EDC] (65 g, 0.338 mol) and 1-hydroxybenzotriazole
[HOBt] (46 g, 0.338 mol) at room temperature. The mix-
ture was stirred at 50 ꢁC for 16 h, and then poured into
water (1.5 L). The resulting solid was collected by filtra-
tion and washed with water. The solid was dried, then
washed with hot isopropyl ether to afford 33 g (41%)
of ethyl 2-[(cyclohexanecarbonyl)amino]-benzothiazole-
6-carboxylate, which was used in the next step without
further purification.
To the above compound (210 mg, 0.47 mmol) was
added 4N-HCl-dioxane (10 mL). The mixture was stir-
red at room temperature for 16 h and then the resulting
solid was collected by filtration and washed with diox-
ane. The solid was dried to afford 179 mg (quant.) of
5a as a colorless solid.
1H NMR (DMSO-d6) d (ppm): 12.38 (br s, 1H), 8.75–
8.87 (m, 1H), 8.56 (d, J = 1.4Hz, 1H), 8.05–8.30 (br s,
3H), 8.00 (dd, J = 8.3, 1.4Hz, 1H), 7.76 (d, J = 8.3 Hz,
1H), 7.17 (br s, 2H), 3.48–3.63 (m, 2H), 2.92–3.08 (m,
2H), 2.48–2.65 (m, 1H), 1.13–1.91 (m, 10H). MS
(FAB): 347 (M+1). HRMS (FAB) Calcd for
C17H23N4O2S: 347.1542, Found: 347.1585.
1H NMR (CDCl3) d (ppm): 10.87 (br s, 1H), 8.57 (d,
J = 1.4Hz, 1H), 8.14(dd, J = 8.3, 1.4Hz, 1H), 7.76 (d,
J = 8.3 Hz, 1H), 4.43 (q, J = 6.8 Hz, 2H), 2.28–2.45
Table 7. In vitro antifungal activity and cytotoxicity of selected compound 28g with the known inhibitors
Compd
CaNmt inhibition IC50 (lM) MIC (lM) C. albicans ATCC90028 MIC (lM) C. albicans
Cytotoxicity assay
ATCC90028 (with 10% serum) HeLa 229 CC50 (lM)
28g
0.00049
0.78
0.1
0.78
0.2
1.0
13.3
14.4
RO-09-4879 0.0029
UK-370753 0.01
0.39
0.39

K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
2517
6.5. Analogues 5b–k
6.9. Analogues 10ai–10ck
These compounds were prepared in a similar manner
to that described for 5a. The products were adequ-
ately characterized by 1H NMR and HRMS
spectrum.
These compounds were synthesized by the reductive
amination using MP-BH(OAc)3. The products were
analyzed by TLC and LC-LRMS (>90% pure as judged
by LC-LRMS analysis). The expected molecular ion was
confirmed by LC-LRMS (APCI).
6.6. 2-[(Cyclohexanecarbonyl)amino]-N-[(2-naphthyl-
methyl)amino]ethyl-benzothiazole-6-carboxamide (7ai)
6.10. tert-Butyl 2-[(naphthalene-2-ylmethyl)amino]ethyl-
carbamate (13)
A
DMF solution (1 mL) of 2-naphthaldehyde
To a stirred solution of tert-butyl N-(2-aminoethyl)car-
bamate (25.95 g, 162 mmol) and 2-naphthaldehyde
(21.09 g, 135 mmol) in DCE (540 mL) was added so-
dium triacetoxyborohydride (42.9 g, 202.5 mmol). The
mixture was stirred at room temperature for 4h, and
then saturated aqueous NaHCO3 was added (300 mL).
The organic phase was separated and aqueous phase
was extracted with CHCl3. The combined extracts were
washed with brine, dried over Na2SO4, and evaporated
to afford the crude product. The crude product was puri-
fied by chromatography on silica gel to afford 30.78 g
(76%) of tert-Butyl 2-[(naphthalene-2-ylmethyl)-
amino]ethyl-carbamate as pale yellow oil, which was
used in the next step. 1H NMR (CDCl3) d (ppm):
7.80–7.87 (3H, m), 7.74(1H, s), .495 (1H, br), 3.95
(2H, s), 3.22–3.32 (2H, m), 2.78–2.83 (2H, m), 1.44
(9H, s). MS (FAB): 301 (M+1). HRMS (FAB) Calcd
for C18H25N2O2: 301.1916, Found: 301.1899.
(0.24mmol) was added to a DMF solution of 5a
(1 mL, 0.3 mmol). The macroporous triethylammo-
nium methylpolystyrene triacetoxyborohydride [MP-
BH(OAc)3] (1.8 mmol/g, 430 mg, 0.9 mmol) was then
added and the mixture was agitated at room tempera-
ture for 16 h. After which PS-Benzaldehyde
(1.08 mmol/g, 185 mg, 0.2 mmol) and DMF (1 mL) were
added and the mixture was further agitated for a period
of 6 h. The solution was filtered and the resin was
washed with DMF. The combined filtrate was added
onto the macroporous p-toluenesulfonic acid (MP-
TsOH) cartridge, preconditioned by washing with
CH2Cl2, followed by washing with DMF (3 · 5 mL),
CH2Cl2 (3 · 5 mL), and MeOH (3 · 5 mL) to remove
any nonbasic impurities. The amine was released from
cartridge by the addition ammonia in MeOH (2 M,
5 mL). The resulting solution was concentrated to
afford crude product. The crude product was thrown a
short pad of silica gel (1 g) to afford 43 mg (37%) of
7ai.
To a THF solution of the above compound (140 mL,
23.2 g, 77.3 mmol) was added aqueous K2CO3 (2 M,
80 mL, 160 mmol) and then cooled to 0 ꢁC. To the stir-
red solution was added allyl chloroformate (9.95 g,
79.27 mmol). The mixture was stirred at room tempera-
ture for 2 h and then poured into water. The organic
phase was separated and aqueous phase was extracted
with AcOEt. The combined extracts were washed with
brine, dried over Na2SO4, and evaporated to afford
the crude product. The crude product was purified by
chromatography on silica gel to afford 29.7 g (quant.)
of allyl (2-tert-butoxycarbonylaminoethyl)-naphthal-
ene-2-ylmethyl-carbamate as pale yellow oil, which
1H NMR (CDCl3) d (ppm): 8.26 (d, J = 1.4Hz, 1H),
7.75–7.85 (m, 5H), 7.71 (d, J = 8.2 Hz, 1H), 7.41–7.47
(m, 3H), 6.95–7.04(m, 1H), 4.02 (s, 2H), 3.58–3.65 (m,
2H), 2.95–3.05 (m, 2H), 2.35–2.45 (m, 1H), 1.18–2.01
(m, 10H). LC-LRMS (APCI): 487 (M+1). HRMS
(FAB) Calcd for C28H31N4O2S: 487.2168, Found:
487.2162.
6.7. Analogues 7aa–7kk and 11a,b
These compounds were synthesized by the reductive
amination of 5a–k with a series of aromatic aldehyde
6a–k. The products were analyzed by TLC and LC-
LRMS (>90% pure as judged by LC-LRMS analysis).
The expected molecular ion was confirmed by LC-
LRMS (APCI).
1
was used in the next step. H NMR (CDCl3) d (ppm):
7.78–7.84(m, 3H), 7.60–7.73 (m, 1H), 7.26–7.50 (m,
3H), 5.91–6.02 (m, 1H), 5.22–5.36 (m, 2H), 4.69 (s,
4H), 3.27–3.43 (m, 4H), 1.42 (s, 9H). MS (FAB): 385
(M+1). HRMS (FAB) Calcd for C22H29N2O4:
385.2127, Found: 385.2133.
6.8. N-[6-(3-[(2-Naphthylmethyl)amino]propionylamino)-
benzothiazol-2-yl]cyclohexanecarboxamide (10bi)
To a CH2Cl2 solution (100 mL) of the above compound
(29 g, 77 mmol) was added TFA (100 mL). The mixture
was stirred at room temperature for 2 h and then evap-
orated to dryness. The residue was dissolved in CHCl3,
washed with saturated aqueous NaHCO3 and brine,
dried over K2CO3, and evaporated to afford the crude
product. The crude product was purified by chromatog-
raphy on aluminum oxide to afford 16.27 g (74%) of 13
as an yellow oil.
Titled compound 10bi was prepared in a similar manner
to that described for 7ai.
1H NMR (CDCl3) d (ppm): 10.70 (s, 1H), 8.27 (d,
J = 1.4Hz, 1H), 7.87–7.90 (m, 4H), 7.63 (d,
J = 8.3 Hz, 1H), 7.42–7.55 (m, 3H), 7.35 (d, J =
8.3 Hz, 1H), 4.06 (s, 2H), 3.08–3.15 (m, 2H), 2.54–2.60
(m, 2H), 2.30–2.39 (m, 1H), 1.20–1.97 (m, 10H). MS
(FAB): 487 (M+1). HRMS (FAB) Calcd for
C28H31N4O2S: 487.2168, Found: 487.2175.
1H NMR (CDCl3) d (ppm): 7.77–7.86 (m, 3H), 7.61–7.74
(m, 1H), 7.29–7.55 (m, 3H), 5.83–6.03 (m, 1H), 5.16–5.40
(m, 2H), 4.68 (s, 4H), 4.25 (br s, 2H) 3.39–3.54 (m, 2H),

2518
K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
2.91–3.07 (m, 2H). MS (FAB): 285 (M+1). HRMS (FAB)
Calcd for C17H21N2O2: 285.1603, Found: 285.1640.
added onto the MP-TsOH cartridge, preconditioned by
washing with CH2Cl2, followed by washing with DMF
(3 · 5 mL), CH2Cl2 (3 · 5 mL), and MeOH (3 · 5 mL)
to remove any nonbasic impurities. The amine was re-
leased from cartridge by the addition ammonia in MeOH
(2 M, 5 mL). The resulting solution was concentrated to
afford crude product. The crude product was thrown a
short pad of silica gel (1 g) to afford 33 mg (79%) of 16f
6.11. Allyl-N-{2-[(2-N-tert-butoxycarbonylamino-ben-
zothiazol-6-yl)carbonyl]-aminoethyl}-N-(2-naphthyl-
methyl) carbamate (14b)
To a solution of 13 (7.1 g, 25 mmol) and 2-N-tert-but-
oxycarbonylamino-benzothiazole-6-carboxylic
1
acid
as a colorless solid. H NMR (CDCl3) d (ppm): 8.24(s,
(6.85 g, 25 mmol) in DMF (100 mL) was added EDC
(7.17 g, 37.5 mmol) and HOBt (5.07 g, 37.5 mmol) at
room temperature. The mixture was stirred at room
temperature for 16 h, and then poured into water
(500 mL). The resulting solid was collected by filtration
and washed with water. The crude product was purified
by chromatography on silica gel to afford 11.2 g (80%)
1H), 7.65–7.84(m, 7H), 7.43–7.52 (m, 3H), 3.98–4.04
(m, 2H), 3.56–3.63 (m, 2H), 2.93–3.01 (m, 2H), 2.28 (s,
3H). MS (FAB): 419 (M+1). HRMS (FAB) Calcd for
C23H23N4O2S: 419.1541, Found: 419.1527.
6.14. N-{2-[(2-Naphtylmethyl)amino]ethyl}-2-[(tetra-
hydro-2H-pyran-4-ylcarbonyl)amino]-benzothiazole-6-
carboxamide 16k
1
of 14b as a colorless solid. H NMR (CDCl3) d (ppm):
8.26 (s, 1H), 7.77–7.89 (m, 5H), 7.62–7.71 (m, 1H),
7.32–7.49 (m, 3H), 5.88–5.98 (m, 1H), 5.14–5.33 (m,
2H), 4.64–4.75 (m, 4H), 3.54–3.68 (m, 4H), 1.61 (s,
9H). MS (FAB): 561 (M+1). HRMS (FAB) Calcd for
C30H33N4O5S: 561.2172, Found: 561.2129.
To a solution of 15 (92 mg, 0.2 mmol) and tetra-
hydorpyran-4-yl carboxylic acid (26 mg, 0.2 mmol) in
DMF (10 mL) was added EDC (57 mg, 0.3 mmol) and
HOBt (40 mg, 0.3 mmol) at room temperature. The mix-
ture was stirred at room temperature for 48 h, and then
poured into water. The resulting mixture was extracted
with AcOEt, washed with water and brine, dried over
Mg2SO4, and evaporated to afford the crude product.
The crude product was purified by chromatography on
silica gel to afford 75 mg (66%) of allyl carbamate as a
colorless solid, which was used in the next step. 1H
NMR (CDCl3) d (ppm): 8.30 (s, 1H), 7.35–7.84(m,
10H), 5.85–5.96 (m, 1H), 5.15–5.32 (m, 2H), 4.59–4.73
(m, 4H), 4.06–4.10 (m, 2H), 3.58–3.65 (4H, m), 3.44–
3.50 (m, 2H), 2.65–2.70 (m, 1H), 1.89–1.98 (m, 4H).
MS (FAB): 573 (M+1). HRMS (FAB) Calcd for
C31H33N4O5S: 573.2127, Found: 573.2125.
6.12. Allyl-N-{2-[(2-amino-benzothiazol-6-yl)carbonyl]-
aminoethyl}-N-(-2naphthylmethyl) carbamate (15)
To a CH2Cl2 solution (20 mL) of 14b (5.8 g, 9.8 mmol)
was added TFA (10 mL). The mixture was stirred at room
temperature for 2 h. The mixture was evaporated to dry-
ness and then dissolved in CHCl3. A saturated aqueous
NaHCO3 was added to neutralized. The resulting solid
was collected by filtration and washed with CHCl3 to af-
1
ford 4.5 g (99%) of 15 as a colorless solid. H NMR
(DMSO-d6) d (ppm): 8.10 (s, 1H), 7.82–7.91 (m, 3H),
7.66–7.78 (m, 3H), 7.30–7.53 (m, 3H), 5.89–5.97 (m,
1H), 5.10–5.36 (m, 2H), 4.54–4.71 (m, 4H), 3.37–3.49
(m, 4H). MS (FAB): 461 (M+1). HRMS (FAB) Calcd
for C25H25N4O3S: 461.1647, Found: 461.1645.
In a similar manner to that described for 16f, palladium-
catalyzed facile removal strategy was performed to
afford 16k in 92% yield.
6.13. 2-(Acetylamino)-N-2-[(2-naphthylmethyl)-
amino]ethyl-benzothiazole-6-carboxamide 16f
1H NMR (CDCl3) d (ppm): 8.25 (s, 1H), 7.44–7.83 (m,
10H), 3.92–4.05 (m, 4H), 3.57–3.63 (m, 2H), 3.45 (t,
J = 11 Hz, 2H), 2.96–3.06 (m, 2H), 2.57–2.67 (m, 1H),
1.78–1.92 (m, 4H). LC-LRMS (APCI): 489 (M+1).
HRMS (FAB) Calcd for C27H29N4O3S: 489.1961,
Found: 489.1951.
To a solution of 15 (138 mg, 0.3 mmol) and 4-dimethyl-
aminopyridine (37 mg, 0.3 mmol) in CH2Cl2 (10 mL) was
added Ac2O (92 mg, 0.9 mmol). The mixture was stirred
at room temperature for 4h and then washed with 3 N-
HCl, water and brine, dried over Mg2SO4, and evapo-
rated to afford the crude product. The crude product
was purified by chromatography on silica gel to afford
137 mg (91%) of allyl {2-[(2-acetylamino-benzothiaz-
ole-6-carbonyl)-amino]-ethyl}-naphthalene-2-ylmethyl
carbamate as a colorless solid, which was used in the
next step. 1H NMR (CDCl3) d (ppm): 8.30 (s, 1H),
7.35–7.84(m, 10H), 5.86–5.97 (m, 1H), 5.15–5.34(m,
2H), 4.66–4.75 (m, 4H), 3.62–3.69 (m, 4H), 2.30 (s,
3H). MS (FAB): 503 (M+1). HRMS (FAB) Calcd for
C27H27N4O4S: 503.1753, Found: 503.1736.
6.15. Analogues 16g–t
These compounds were synthesized by palladium-cata-
lyzed facile removal strategy using solid-supported bar-
bituric acid. The products were analyzed by TLC and
LC-LRMS (>90% pure as judged by LC-LRMS analy-
sis). The expected molecular ion was confirmed by LC-
LRMS (APCI).
6.16. Methyl 2-[(cyclohexylcarbonyl)amino]-benzoxazole-
5-carboxylate (18a)
A mixture of the above compound (50 mg, 0.1 mmol),
Pd(PPh3)4 (6 mg, 0.005 mmol), and solid-supported bar-
bituric acid (100 mg, 0.1 mmol) in THF (1 mL) were agi-
tated at 50 ꢁC for 12 h. The solution was filtered and the
resin was washed with THF. The combined filtrate was
To a solution of 3-amino-4-hydroxybenzoate (4.7 g,
2.8 mmol) and di-(imidazole-1-yl)methanimine (4.5 g,
2.8 mmol) in THF was stirred at 70 ꢁC for 4h. The reac-
tion mixture was evaporated to dryness. To the residue

K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
2519
was added water (100 mL) and the resulting solid
was collected by filtration, washed with water, and then
dried to afford 3.7 g (70%) of 18a as a colorless solid.
5.20–5.43 (m, 2H), 3.60 (br s, 4H). MS (FAB): 453
(M+1). HRMS (FAB) Calcd for C24H23FN3O5:
452.1622, Found: 452.1646.
1H NMR (DMSO-d6) d (ppm): 7.23 (d, J = 2.0 Hz, 1H),
7.66 (dd, J = 8.3, 2.0 Hz, 1H), 7.57 (br s, 2H), 7.42 (d,
J = 8.3 Hz, 1H), 3.84(s, 3H). MS (FAB): 193 (M+1).
HRMS (FAB) Calcd for C9H9N2O3: 193.0613, Found:
193.0621.
6.21. Allyl-N-2-[(3-fluoro-4-nitrobenzoyl)amino]ethyl-N-
(2-naphthylmethyl)carbamate (19b)
Titled compound 19b was prepared in a similar manner
to that described for 19a.
6.17. Methyl 2-[(cyclohexylcarbonyl)amino]-benzoxazole-
6-carboxylate (18b)
1H NMR (CDCl3) d (ppm): 8.00–8.18 (m, 1H), 7.30–
7.83 (m, 10H), 5.82–6.05 (m, 1H), 5.19–5.39 (m, 2H),
4.65–4.79 (m, 4H), 3.60 (br s, 4H). MS (FAB): 453
(M+1). HRMS (FAB) Calcd for C24H23FN3O5:
452.1622, Found: 452.1640.
Titled compound 18b was prepared in a similar manner
to that described for 18a.
1H NMR (CDCl3) d (ppm): 7.62–7.85 (m, 4H), 7.26 (d,
J = 7.8 Hz, 1H), 3.80 (s, 3H). MS (FAB): 193 (M+1).
HRMS (FAB) Calcd for C9H9N2O3: 193.0613, Found:
193.0606.
6.22. Allyl-N-(2-[3-amino-4-(methylamino)benzoyl]ami-
noethyl)-N-(2-naphthylmethyl)carbamate (20a)
A mixture of 19a (2.04g, 4.54mmol) and 2 M-MeNH
/
2
THF (4.5 mL, 9 mmol) was stirred for 1.5 h at room
temperature and then evaporated to dryness. To the res-
idue was added SnCl2Æ2H2O (4.7 g, 20.9 mmol) and dis-
solved in EtOH (10 mL). To the solution was added
6 N-HCl (16 mL), stirred for 16 h at room temperature.
The mixture was cooled to <10 ꢁC and basified to pH 11
with 10% aqueous NaOH. The mixture was extracted
with CHCl3, washed with water and brine, dried over
Mg2SO4, and evaporated to afford the crude product.
The crude product was purified by chromatography on
silica gel to afford 1.8 g (92%) of 20a as pale yellow oil.
6.18. 2-[(Cyclohexylcarbonyl)amino]-N-[2-(2-naphthyl-
amino)ethyl]-benzoxazole-5-carboxamide (11c)
Titled compound 11c was prepared in a similar manner
to that described for benzothiazole derivatives 7ai.
1H NMR (CDCl3) d (ppm): 7.99 (s, 1H), 7.62–7.85 (m,
5H), 7.38–7.50 (m, 4H), 6.90–7.02 (m, 1H), 4.01 (s,
2H), 3.56–3.41 (m, 2H), 2.80–3.08 (m, 2H), 2.58–2.70
(m, 1H), 1.22–2.08 (m, 10H). MS (FAB): 471 (M+1).
HRMS (FAB) Calcd for C28H31N4O3: 471.2397, Found:
471.2419.
1H NMR (CDCl3) d (ppm): 7.28–7.86 (m, 9H), 7.00–
7.22 (m, 1H), 6.56 (d, J = 7.8 Hz, 1H), 5.80–6.01 (m,
1H), 5.12–5.43 (m, 2H), 4.62–4.80 (m, 4H), 3.42–3.71
(m, 4H), 3.26 (br s, 2H), 2.91 (s, 3H). MS (FAB): 433
(M+1). HRMS (FAB) Calcd for C25H29N4O3:
433.2240, Found: 433.2195.
6.19. 2-[(Cyclohexylcarbonyl)amino]-N-[2-(2-naphthyl-
amino)ethyl]-benzoxazole-6-carboxamide (11d)
Titled compound 11d was prepared in a similar manner
to that described for benzothiazole derivatives 7ai.
6.23. Allyl-N-(2-[4-amino-3-(methylamino)benzoyl]ami-
noethyl)-N-(2-naphthylmethyl)carbamate (20b)
1H NMR (CDCl3) d (ppm): 8.05 (s, 1H), 7.70–7.85 (m,
4H), 7.40–7.63 (m, 5H), 6.90–7.05 (m, 1H), 4.02 (s,
2H), 3.48–3.61 (m, 2H), 2.90–3.01 (m, 2H), 2.58–2.72
(m, 1H), 1.20–2.05 (m, 10H). MS (FAB): 471 (M+1).
HRMS (FAB) Calcd for C28H31N4O3: 471.2397, Found:
471.2406.
Titled compound 20b was prepared in a similar manner
to that described for 20a.
1H NMR (CDCl3) d (ppm): 7.60–7.90 (m, 5H), 6.85–
7.56 (m, 5H), 6.64(d, J = 7.3 Hz, 1H), 5.80–6.18 (m,
1H), 5.10–5.39 (m, 2H), 4.60–4.75 (m, 4H), 3.50–3.70
(m, 6H), 2.89 (s, 3H). MS (FAB): 433 (M+1). HRMS
(FAB) Calcd for C25H29N4O3: 433.2240, Found:
433.2195.
6.20. Allyl-N-2-[(4-fluoro-3-nitrobenzoyl)amino]ethyl-N-
(2-naphthylmethyl)carbamate (19a)
To a solution of 13 (2.8 g, 10 mmol) and 4-fluoro-3-nit-
orobenzoic acid (1.8 g, 10 mmol) in DMF (20 mL) was
added EDC (2.9 g, 15 mmol) and HOBt (2.1 g,
15 mmol) at room temperature. The mixture was stirred
at room temperature for 1.5 h, and then poured into
water. The resulting mixture was extracted with AcOEt,
washed with water and brine, dried over Mg2SO4, and
evaporated to afford the crude product. The crude prod-
uct was purified by chromatography on silica gel to af-
ford 3.9 g (86%) of 19a as yellow oil.
6.24. 2-[(2-Cyclohexylcarbonyl)amino]-1-methyl-N-2-[(2-
naphthylmethyl)amino]ethyl-1H-benzimidazole-5-carbox-
amide (11e)
To a solution of 20a (879 mg, 2 mmol) in CH2Cl2
(10 mL) was added cyclohexanecarbonyl isothiocyanate
(378 mg, 2.2 mmol). The mixture was stirred at room
temperature. After 2 h, to the mixture was added EDC
(582 mg, 3 mmol) and DIPEA (1.7 mL, 10.15 mmol),
stirred for 16 h at room temperature. The mixture was
washed with water and brine, dried over Mg2SO4, and
1H NMR (CDCl3) d (ppm): 8.41–8.53 (m, 1H), 7.20–
8.08 (m, 10H), 5.85–6.08 (m, 1H), 4.60–4.80 (m, 4H),

2520
K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
evaporated to afford the crude product. The crude prod-
uct was purified by chromatography on silica gel to
afford 503 mg (44%) of allyl-N-2-[(2-[(cyclohexylcarbo-
nyl)amino]-1-methyl-1H-benzimidazol-5-ylcarbonyl)am-
ino]ethyl-N-(2-naphthylmethyl)carbamate as a colorless
solid, which was used in the next step. 1H NMR
(CDCl3) (ppm): 12.20 (br s, 1H), 7.60–7.83 (m, 5H),
7.26–7.53 (m, 5H), 7.16 (d, J = 8.3 Hz, 1H), 5.84–6.20
(m, 1H), 5.16–5.43 (m, 2H), 4.70 (br s, 4H), 3.50–3.70
(m, 7H), 2.38–2.50 (m, 1H), 1.20–2.11 (m, 10H). MS
(FAB): 568 (M+1). HRMS (FAB) Calcd for
C33H38N5O4: 568.2924, Found: 568.2891.
diphenylphosphoryl azide (4.8 mL, 22 mmol) at room
temperature. The mixture was stirred at 110 ꢁC for
1 h, and then cooled to 70 ꢁC. To the mixture was added
tert-BuOH (11 mL, 100 mmol) and then stirred at 95 ꢁC
for 12 h. The mixture was cooled to room temperature,
washed with water and brine, dried over Mg2SO4, and
evaporated to afford the crude product. The crude prod-
uct was purified by chromatography on silica gel to af-
ford 1.37 g (25%) of 23a as a colorless solid. The
product was recrystallized from isopropyl ether to pro-
duce colorless needles.
25
D
1
½aŠ À32.3 (c 0.5, CHCl3), H NMR (CDCl3) d (ppm):
The above compound was converted to 11e by palla-
dium-catalyzed facile removal strategy using solid-sup-
ported barbituric acid as described for 16f.
4.42 (br, 1H), 4.11 (q, J = 6.8 Hz, 2H), 3.45 (m, 1H),
2.30–2.43 (m, 1H), 2.20–2.28 (m, 1H), 1.80–2.00 (m,
3H), 1.44 (s, 9H), 1.18–1.42 (m, 6H), 0.98–1.15 (m,
1H). MS (FAB): 272 (M+1). HRMS (FAB) Calcd for
C14H26NO4: 272.1862, Found: 272.1850.
1H NMR (CDCl3) d (ppm): 7.60–7.90 (m, 8H), 7.42–
7.54(m, 3H), 7.15 (d, J = 8.3 Hz, 1H), 6.92 (br s, 1H),
4.00 (s, 2H), 3.62 (s, 3H), 3.50–3.60 (m, 2H), 2.90–3.00
(m, 2H), 2.38–2.51 (m, 1H), 1.21–2.01 (m, 10H). MS
(FAB): 484 (M+1). HRMS (FAB) Calcd for
C29H34N5O2: 484.2712, Found: 484.2725.
6.28. (1R,3S)-3-[(tert-Butoxycarbonyl)amino]cyclohex-
anecarboxylic acid (24a)
To a solution of 23a (1.35 g, 5 mmol) in THF (30 mL)
was added 5 N aqueous NaOH (6 mL, 30 mmol) at
room temperature. The mixture was stirred at 50 ꢁC
for 12 h, and then cooled to room temperature. The mix-
ture was washed with diethyl ether, and the aqueous
phase was cooled to 0 ꢁC. The mixture was acidified to
pH 1 with 3 N HCl water and extracted with CHCl3,
washed with water and brine, dried over Mg2SO4, and
evaporated to afford the 1.15 g (95%) of 24a as a color-
less solid.
6.25. 2-[(2-Cyclohexylcarbonyl)amino]-1-methyl-N-2-[(2-
naphthylmethyl)amino]ethyl-1H-benzimidazole-6-carbox-
amide (11f)
Titled compound 11f was prepared in a similar manner
to that described for 11e.
1H NMR (CDCl3) d (ppm): 7.70–7.83 (m, 5H), 7.41–
7.50 (m, 5H), 7.20 (br s, 1H), 6.90–6.98 (m, 1H), 4.00
(s, 2H), 3.62 (s, 3H), 3.50–3.61 (m, 2H), 2.90–3.02 (m,
2H), 2.30–2.50 (m, 1H), 1.18–2.01 (m, 10H). MS
(FAB): 484 (M+1). HRMS (FAB) Calcd for
C29H34N5O2: 484.2712, Found: 484.2664.
23
D
1
½aŠ À32.8 (c 0.5, CHCl3), H NMR (CDCl3) d (ppm):
4.45 (br, 1H), 3.47 (m, 1H), 2.20–2.50 (m, 2H), 1.82–
2.02 (m, 3H), 1.44 (s, 9H), 0.98–1.42 (m, 4H). MS
(FAB): 244 (M+1). HRMS (FAB) Calcd for
C12H22NO4: 244.1549, Found: 244.1533.
6.26. (1S,3R)-3-(Ethoxycarbonyl)cyclohexanecarboxylic
acid (22a)
6.29. tert-Butyl N-(1R,3S)-3-[(2-[(cyclopentylcar-
bonyl)amino]-benzothiazol-6-ylamino)carbonyl]cyclo-
hexyl carbamate (26a)
A suspension of diethyl cis-1,3-cyclohexanesicarboxyl-
ate (2.28 g, 10 mmol) and lipase AYS Amano (1 g) in
pH 7.2 phosphate buffer (100 mL) was stirred for 24h
at room temperature. A Celite (5.7 g) was added and
the mixture was stirred thoroughly. The mixture was fil-
tered a Celite pad, and the filtrate acidified to pH 1 with
3 N HCl. The resulting mixture was extracted with
AcOEt, washed with water and brine, dried over
Mg2SO4, and evaporated to afford 2 g (quant.) of 22a
as colorless oil.
Titled compound 26a was prepared in a similar manner
to that described for 5a.
25
D
½aŠ À22.8 (c 0.45, DMF), 1H NMR (DMSO-d6) d
(ppm): 12.15 (s, 1H), 9.93 (s, 1H), 8.29 (d, J = 2.0 Hz,
1H), 7.62 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8, 2.0 Hz,
1H), 6.72 (br, 1H), 3.28–3.40 (m, 1H), 2.90–3.02
(m, 1H), 2.40–2.50 (m, 1H), 1.52–2.00 (m, 12H), 1.38
(s, 9H), 1.05–1.37 (m, 4H). MS (FAB): 487 (M+1).
HRMS (FAB) Calcd for C25H35N4O4S: 487.2379,
Found: 487.2351.
25
D
1
½aŠ +4.2 (c 1.0, CHCl3), H NMR (CDCl3) d (ppm):
9.75 (br, 1H), 4.13 (q, J = 6.8 Hz, 2H), 2.20–2.40 (m,
3H), 1.57 (q, J = 12.7 Hz, 1H), 1.25–1.45 (m, 3H), 1.25
(t, J = 6.8 Hz, 3H). MS (FAB): 201 (M+1). HRMS
(FAB) Calcd for C10H17O4: 201.1127, Found: 201.1116.
6.30. (1R,3S)-3-Amino-N-2-[(cyclopentylcarbonyl)-
amino]-benzothiazol-6-ylcyclohexanecarboxamide
trifluoroacetate (27a)
6.27. Ethyl (1R,3S)-3-[(tert-butoxycarbonyl)amino]cyclo-
hexanecarboxylate (23a)
To a suspension of 26a (187 mg, 0.39 mmol) in CH2Cl2
(2 mL) was added TFA (2 mL). The mixture was stirred
for 1 h at room temperature then evaporated to dryness.
The residue was added diethyl ether (10 mL) and the
To a solution of 22a (4.0 g, 20 mmol) and triethylamine
(3.1 mL, 22 mmol) in toluene (100 mL) was added

K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
2521
resulting solid was collected, washed with diethyl ether,
dried to give 180 mg (94%) of 27a as a colorless solid.
The bacteria was then suspended in PBS, and sonicated.
The lysate was cleared by centrifugation and the super-
natant was applied to TALON metal affinity resin
(CLONTECH) to purify the poly-histidine tagged
CaNmt according to the manufacturer protocol. The
fraction containing the purified CaNmt or HsNmt1
was dialyzed overnight against the same buffer as for
Nmt reaction (10 mM Tris–HCl pH 7.4, 1 mM DTT,
5 mM MgCl2, 0.1 mM EGTA).
25
D
½aŠ +5.0 (c 0.45, DMF), 1H NMR (DMSO-d6) d (ppm):
12.27 (s, 1H), 10.13 (s, 1H), 8.31 (d, J = 2.0 Hz, 1H),
7.85 (br, 3H), 7.65 (d, J = 8.8 Hz, 1H), 7.49 (dd,
J = 8.8, 2.0 Hz, 1H), 2.90–3.18 (m, 2H), 1.20–2.08 (m,
17H). MS (FAB): 387 (M+1). HRMS (FAB) Calcd for
C20H27N4O2S: 387.1855, Found: 387.1842.
6.31. (1R,3S)-N-{2-[(Cyclopenthylcarbonyl)amino]-benzo-
thiazol-6-yl}-3-[(2-naphthylmethyl) amino]cyclohexane-
carboxamide (28g)
8. Measurement of inhibitory activities of compounds
against CaNmt and HsNmt1
Titled compound 28g was prepared in a similar manner
to that described for 7ai (98% ee).
In vitro Nmt assay was performed as described methods
previously4c,13,14 with slight modification. The reagents
were added in 100 lL to give a final concentration of
10 mM Tris–HCl pH 7.4, 1 mM DTT, 5 mM MgCl2,
0.1 mM EGTA, 0.01% Triton X-100, 0.5 lM peptide
substrate (GLTISKLFRR or GLTISKLFRRK-biotin
for CaNmt, GNAASARR for HsNmt1), 32 nM [3H]-
myristoyl-CoA (Amersham Biosciences), the appropri-
ate amount of enzyme and a compound at variable con-
centrations. After pre-incubation at 30 ꢁC for 10 min,
the reaction was initiated by the addition of enzyme
and performed at 30 ꢁC for 10 min. In the case of using
GLTISKLFRRK-biotin as peptide substrate, the reac-
tion was terminated with 0.1% phosphoric acid and
the reaction solution was transferred into streptavidin-
coated 96-well plate, followed by incubation at 30 ꢁC
with shaking for 30 min. After washes with PBS 5 times,
the amount of bound 3H in each well was measured to
quantify the reaction product [3H]-myristoylated
GLTISKLFRRK-biotin with the plate scintillation
counter 1450 MicroBeta (PerkinElmer). In the case of
using other peptides as peptide substrate, the reaction
was terminated by the addition of 100 lL ice-cold meth-
anol containing 2% tritonX-100 and 10% TCA. The
reaction mixture (100 lL) was analyzed by HPLC in
the conditions described previously13 to quantify the
[3H]-myristoylated peptide.
25
D
½aŠ À72.6 (c 0.25, CHCl3), 1H NMR (CDCl3) d (ppm):
8.25 (d, J = 1.0 Hz, 1H), 7.75–7.92 (m, 4H), 7.74 (s, 1H),
7.60 (d, J = 8.8 Hz, 1H), 7.40–7.50 (m, 3H), 7.31 (dd,
J = 8.3, 1.5 Hz, 1H), 4.01 (br s, 2H), 2.74–2.85 (m, 1H),
2.63–2.72 (m, 1H), 2.23–2.40 (m, 2H), 1.20–2.10 (m,
15H). LC-LRMS (APCI): 527 (M+1). HRMS (FAB)
Calcd for C31H35N4O2S: 527.2481, Found: 527.2495.
6.32. Analogues 28a–k
These compounds were synthesized by the reductive
amination using MP-BH(OAc)3. The products were
analyzed by TLC and LC-LRMS (>90% pure as judged
by LC-LRMS analysis). The expected molecular ion was
confirmed by LC-LRMS (APCI).
7. Expression in E. coli and purification of Nmts
CaNmt coding gene was obtained by PCR from C. albi-
cans (ATCC 90028) genomic library using the primers
50-NNNNNNNNCATATGTCGGGAGATAACACA-
GGGAATAAATC-30 and 50-NNNNNNGCGGCCG-
CTAATAAAACTACACCTATACCACTTGTTTGA-
TCTTCG-30 and using Advantage-HF PCR kit
(CLONTECH). The PCR product was inserted into E.
coli expression vector pET-15b (Novagen) between the
NdeI and the BamHI site, to obtain CaNmt protein with
the poly-histidine tag at N-terminus. In the same way,
HsNmt1 coding gene was obtained from human
lymphocyte cDNA library using the primers
50-NNNNNNCATATGGCGGACGAGAGTGAGA-
CAGC-30 and 50-NNNNNNCTCGAGTTATTGTAG-
CACCAGTCCAACCTTCTCTGC-30, and inserted
into pET-15b between the NdeI and the XhoI site. Both
expression vectors were confirmed the sequences with
PRISM 310 (ABI).
In order to measure the inhibitory activity of com-
pounds at each concentration, the quantity of [3H]-myr-
istoylated peptide in reaction with no compound was
defined as control (100%), and with no enzyme was de-
fined as background (0%). The effects of compounds at
each concentration were calculated as percentage of con-
trol. The 50% inhibitory concentration (IC50) of com-
pound was calculated by nonlinear regression analysis
using the Prism 3.0 software package (GraphPad Soft-
ware, San Diego, CA).
9. In vitro antifungal activity
E. coli BL21(DE3) containing the CaNmt or HsNmt1
expression vector was grown at 37 ꢁC in a medium
(10 g/L trypton, 5 g/L yeast extract, 10 g/L NaCl, and
100 g/mL ampicilin) until the absorbance at 600 nm
reached 0.8 at which time isopropyl b-D-thiogalactopyr-
anoside was added to give a final concentration of
1 mM. The cultures were maintained at 28 ꢁC for a fur-
ther 4h and the bacteria harvested by centrifugation.
The MICs of the compounds and reference compounds
for C. albicans were determined by a broth microdilu-
tion method according to National Committee for Clin-
ical Laboratory Standards (NCCLS) method M27-A2.18
Compounds were dissolved in DMSO. The strains were
cultured at 35 ꢁC for 48 h. The MICs were read spectro-
photometrically. The MICs of the compounds were

2522
K. Yamazaki et al. / Bioorg. Med. Chem. 13 (2005) 2509–2522
K.; Shindoh, H.; Shiratori, Y.; Aoki, Y.; Ohtsuka, T.;
Shimma, N. Bioorg. Med. Chem. Lett. 2002, 12, 607–610;
(b) Kawasaki, K.; Masubuchi, M.; Morikami, K.; Sogabe,
S.; Aoyama, T.; Ebiike, H.; Niizuma, S.; Hayase, M.;
Fujii, T.; Sakata, K.; Shindoh, H.; Shiratori, Y.; Aoki, Y.;
Ohtsuka, T.; Shimma, N. Bioorg. Med. Chem. Lett. 2003,
13, 87–91; (c) Masubuchi, M.; Ebiike, H.; Kawasaki, K.;
Sogabe, S.; Morikami, K.; Shiratori, Y.; Tsujii, S.; Fujii,
T.; Sakata, K.; Hayase, M.; Shindoh, H.; Aoki, Y.;
Ohtsuka, T.; Shimma, N. Bioorg. Med. Chem. 2003, 11,
4463–4478.
defined as the lowest concentration at which there was
80% inhibition of growth compared with the growth of
a drug-free control.
10. Cytotoxicity assay
Human cervix adenocarcinoma HeLa 229 (HeLa) cells
were purchased from Dainippon Pharmaceutical Co.
Ltd (Osaka, Japan), and maintained in MEM medium
containing 5% fetal bovine serum (FBS; GIBCO) at
37 ꢁC under 5% CO2 condition. Cytotoxicity of com-
pounds was measured in HeLa cells. Cells were pre-incu-
bated in 96-well plates at 5 · 103 cells/75 lL of medium
per well for 24h. Then, the serial dilutions of com-
pounds in DMSO were added to the wells, with the final
concentration of DMSO in all wells at 1%, in a total vol-
ume of 150 lL. After 2 days incubation, 50 lL of WST-
8 (Dojindo, Kumamoto, Japan) that was diluted with
medium into 5 times was added to each well, and the
plates returned to the incubator for 2 h, after which
the absorbance at 450 nm of each well was measured
(reference: 650 nm). The survival rates were determined
by comparing the absorbance of a well containing com-
pound to that of control wells. Cytotoxicity (CC50
value) is defined as the concentration of each compound
that produces a 50% reduction in the survival rate.
5. We utilized the virtual screening to identify the active
compounds. The initial database comprised about
2,000,000 entries. Pharmacophore model derived from
the crystal structure of the target protein and the flexible
docking served as sequential filters to reduce the initial set
to about 100 prospective entries. Catalyst was used for the
pharmacophore modeling (version 4.6, Molecular Simu-
lations, Inc. now Accelrys Inc., San Diego, CA, USA,
2000). Cerius2 was used for the flexible docking (Version
4.7; Accelrys Inc., San Diego, CA, 2002).
6. Bhattacharyya, S.; Rana, S.; Gooding, O. W.; Labadie, J.
Tetrahedron Lett. 2003, 44, 4957–4960.
7. Kunz, W.; Jau, B. U.S. Patent 5770758, 1998. Chem.
Abstr. 1998, 129, 81579.
8. Tsukamoto, H.; Suzuki, T.; Kondo, Y. Synlett 2003,
1105–1108.
9. Wu, Y.-Q.; Limburg, D. C.; Wilkinson, D. E.; Hamilton,
G. S. J. Heterocycl. Chem. 2003, 40, 191–193.
10. Seth, P. P.; Robinson, D. E.; Jefferson, E. A.; Swayze,
E. E. Tetrahedron Lett. 2002, 43, 7303–7306.
11. Boaz, N. W. Tetrahedron: Asymmetry 1999, 10, 813–816.
12. Raju, R. V. S.; Datla, R. S. S.; Sharma, R. K. Protein
Expr. Purif. 1996, 7, 431–437.
13. Towler, D.; Glaser, L. Proc. Natl. Acad. Sci. U.S.A. 1986,
83, 2812–2816.
14. French, K. J.; Zhuang, Y.; Schrecengost, R. S.; Copper, J.
E.; Xia, Z.; Smith, C. D. J. Pharmacol. Exp. Ther. 2004,
309, 340–347.
Acknowledgements
We thank the other Nmt project members for their help-
ful discussions and contributions to this project. We
thank Dr. Y. Nagao and Mr. T. Okada for the spectra
measurements.
15. Bhatnagar, R. S.; Futterer, K.; Farazi, T. A.; Korolev, S.;
¨
References and notes
Murray, C. L.; Jackson-Machelski, E.; Gokei, G. W.;
Gordon, J. I.; Waksman, G. Nat. Struct. Biol. 1998, 5,
1091–1097.
1. Maure-Stroh, S.; Eisenhaber, B.; Eisenhaber, F. J. Mol.
Biol. 2002, 317, 541–557.
2. Weinberg, R. A.; McWherter, C. A.; Freeman, S. K.;
Wood, D. C.; Gordon, J. I.; Lee, S. C. Mol. Microbiol.
1995, 16, 241–250.
3. (a) Ohtsuka, T.; Aoki, Y. Drugs Future 2003, 28, 143–152,
and references cited therein; (b) Armour, D. R.; Bell, A.
S.; Kemp, M. I.; Edwards, M. P.; Wood, A. Abstracts of
papers, 221st national meeting of the American Chemical
Society, San Diego, CA; American Chemical Society:
Washington, DC, 2001, Abstract MEDI-349; (c) Bell, A.
S.; Armour, D. R.; Edwards, M. P.; Kemp, M. I.; Wood,
A. Abstracts of papers, 221st national meeting of the
American Chemical Society, San Diego, CA; American
Chemical Society: Washington, DC, 2001, Abstract
MEDI-350.
16. Paderu, P.; Park, S.; Perlin, D. S. Antimicrob. Agents
Chemother. 2004, 48, 3845–3849.
17. For example, some benzothiazole based inhibitors of
p56lck have been reported in recent papers, see: (a) Das, J.;
Lin, J.; Moquin, R. V.; Shen, Z.; Spergel, S. H.; Wityak,
J.; Doweyko, A. M.; De Fex, H. F.; Fang, Q.; Pang, S.;
Pitt, S.; Shen, D. R.; Schieven, G. L.; Barrish, J. C. Bioorg.
Med. Chem. Lett. 2003, 13, 2145–2149; (b) Das, J.;
Moquin, R. V.; Lin, J.; Liu, C.; Doweyko, A. M.; De
Fex, H. F.; Pang, S.; Pitt, S.; Shen, D. R.; Schieven, G. L.;
Barrish, J. C.; Wityak, J. Bioorg. Med. Chem. Lett. 2003,
13, 2587–2590.
18. National Committee for Clinical Laboratory Standards;
Reference Methods for Broth Dilution Antifungal Sus-
ceptibility Testing of Yeasts. Approval standard—2nd ed.
Document M27-A2. National Committee for Clinical
Laboratory Standards, Wayne, Pa; 2002.
4. (a) Ebiike, H.; Masubuchi, M.; Liu, P.; Kawasaki, K.;
Morikami, K.; Sogabe, S.; Hayase, M.; Fujii, T.; Sakata,