Design and synthesis of diketopiperazine and acyclic analogs related to the caprazamycins and liposidomycins as potential antibacterial agents

Article abstract of DOI:10.1016/j.bmc.2007.09.022

A systematic simplification methodology of a class of 6′-N-alkyl-5′-O-aminoribosyl-glycyluridine antibiotics was shown to produce potential antibacterial agents having a novel mechanism of action. Diketopiperazines and acyclic analogs of the caprazamycins

Full text of DOI:10.1016/j.bmc.2007.09.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 428–436
Design and synthesis of diketopiperazine and acyclic analogs
related to the caprazamycins and liposidomycins as
potential antibacterial agents
Shinpei Hirano, Satoshi Ichikawa* and Akira Matsuda*
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
Received 4 August 2007; revised 11 September 2007; accepted 12 September 2007
Available online 15 September 2007
Abstract—A systematic simplification methodology of a class of 60-N-alkyl-50-O-aminoribosyl-glycyluridine antibiotics was shown
to produce potential antibacterial agents having a novel mechanism of action. Diketopiperazines and acyclic analogs of the capraz-
amycins (CPZs) and liposidomycins (LPMs) were efficiently synthesized, and their antibacterial activity was evaluated. The diketo-
piperazine analog 11a and the acyclic analogs 12a and 16a having a palmitoyl group as a lipophilic side chain exhibited moderate
antibacterial activities with MICs of 12.5–50 lg/mL. This approach could provide ready access to a range of analogs for the devel-
opment of potential antibacterial agents.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
I, Fig. 2). Peptide glycan biosynthesis consists of three
stages, including the formation of uridine diphosphate
N-acetylmuraminylpentapeptide (UDP-MurNAc-penta-
peptide) in cytoplasm, the membrane-anchored synthe-
sis of lipid I and lipid II, a precursor to the peptide
glycan, and polymerization of the resulting lipid II by
transpeptidation and transglycosidation. The second
and the third stages are involved in a lipid cycle, and
the MraY catalyzes the first step of the lipid-linked cycle
of reactions, where UDP-MurNAc-pentapeptide is at-
tacked by the undecaprenol monophosphate in the bac-
terial cell membrane providing lipid I. Lipid I anchored
to the cell membrane is further glycosylated by N-acetyl-
glucosamine to afford lipid II. MraY is one of the key
enzymes for peptidoglycan biosynthesis and appears to
be conserved in both Gram-negative and Gram-positive
bacteria. The enzyme is highly conserved across bacte-
rial species and is essential for the growth of the bacte-
ria.6 Attention therefore has been focused on MraY as
a new target enzyme for the development of antibacte-
The extensive use of antibiotics has raised a serious glo-
bal public health problem. Since bacterial pathogens
inevitably develop resistance to every new drugs
launched in the clinic,1 the need for new antibiotics is
critical. The caprazamycins (CPZs) (Fig. 1, 1) isolated
from a culture broth of the Actinomycete strain Strepto-
myces sp. MK730-62F2 in 20032 constitute the newest
member of the naturally occurring 60-N-alkyl-50-O-
aminoribosyl-glycyluridine structure, which also in-
cludes the liposidomycins (LPMs, 2),3 the muraymycins
(MRYs, 3)4, and FR-900493 (4).5 CPZs have shown
excellent anti-mycobacterial activity in vitro against
drug-susceptible and multi drug-resistant Mycobacte-
rium tuberculosis strains, and exhibit no significant tox-
icity in mice. With these excellent properties, CPZs are
expected to be a promising lead for developing anti-
tuberculosis agents. LPMs and MRYs are also known
to exhibit antibacterial activity similar to that of the
CPZs.4 LPMs and MRYs prevent formation of bacterial
cell wall peptidoglycan by strong inhibition of phospho-
MurNAc-pentapeptide translocase (MraY, translocase
7,8
rial agents.
CPZs and LPMs consist of a uridine, an aminoribose,
a fatty acyl side chain, and a characteristic diazepa-
none. It is suggested that their complex structures
resemble the transition state of a MraY-catalyzed
transfer reaction of a UDP-MurNAc-pentapeptide
onto an undecaprenol monophosphate inside the
bacterial cell membrane. Because of their molecular
Keywords: Antibacterial; Caprazamycin; Translocase I; MraY.
*
Corresponding authors. Tel.: +81 11 706 3763; fax: +81 11 706 4980
(S.I.); tel.: +81 11 706 3228; fax: +81 11 706 4980 (A.M.); e-mail
addresses:
hokudai.ac.jp
ichikawa@pharm.hokudai.ac.jp;
matuda@pharm.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.022

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
429
Figure 1. Structures of nucleoside antibiotics possessing a 60-N-alkyl-50-O-aminoribosyl-glycyluridine structures.
Figure 2. Formation of lipid/catalyzed by MraY (translocase I).
complexity, CPZs and LPMs are not suitable for use
in preparing analogs for the study of a structure–
activity relationship in a lead optimization process.
Dini et al., in a structure–activity relationship of
LPMs with quite simple analogs, found that the
30-hydroxy group, the amino group of the ribose
attached on the 50-hydroxyl group, and the uracil
moiety are crucial for MraY inhibitiocn.9 Thus, the
50-b-O-aminoribosyl-glycyluridine structure is predicted
to be the pharmacophore of this class of natural
products. Moreover, it suggests that the lipophilic
moiety is necessary in order to penetrate inside the
bacterial cell membrane to exhibit antibacterial activ-
ity. Considering these results, we speculated that the
characteristic diazepanone ring systems of the CPZs
and LPMs might play an important role as a scaffold
on which to hang each of the aminoribosyluridine
and the fatty acyl moieties. This would allow them
to be placed in the right orientation to interact with
the target MraY, and the diazepanone structure,
which constitutes a molecular complexity of this class
of natural products, could be replaced by a simpler
scaffold. We selected a diketopiperazine structure to
provide newly designed simple analogs of CPZs and
LPMs in order to connect the aminoribosyluridine
and the fatty acyl moieties with the simpler scaffold
(Fig. 3). A diketopiperazine is synthetically readily
accessible and is already used as a scaffold in medic-
inal chemistry.10 Herein we describe the synthesis of
simplified analogs, where the diazepanone was substi-
tuted by a diketopiperazine. Acyclic analogs, which
are regarded as acyclic analogs of CPZs and LPMs,
were also synthesized with the aim of better under-
standing the importance of the cyclic structure.

430
S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
Figure 3. Structures of diketopiperazine and acyclic analogs.
2. Results and discussion
may also be regarded as acyclic analogs of CPZs and
LPMs to understand the importance of the cyclic struc-
ture. These were obtained by simple treatment of the
intermediates 10a and 10b with 80% aqueous TFA
(11a: 90% in two steps from 9a, 11b: 96% in two steps
from 9b). In order to see the impact of the lipophilic side
chain, the deacyl derivative 13 was prepared by the cou-
pling of 7 with L-serine benzyl ester followed by global
deprotection (three steps from 7, 68%).
Recently, we have synthesized efficiently the key 50-b-O-
aminoribosyl-glycyluridine structure.11 Our strategy in-
cluded a sequential installation of L- or D-serine benzyl
ester and an acyl side chain to the carboxylic acid deriv-
ative 7, and a cyclization to set the diketopiperazine sys-
tem. The synthesis of these analogs is summarized in
Scheme 1. Initially, we attempted to prepare target mol-
ecules with the N3-benzyloxymethyl (BOM)-protected
derivative 6, which was obtained by hydrolysis of an
aminoriboside 511 with Ba(OH)2 in 50% yield. However,
we had difficulty removing the BOM group by hydrog-
enolysis at the final deprotection stage, as observed in
our recent FR-900493 synthesis.12 Therefore we decided
to synthesize the target compounds using substrates
without the BOM protection at the N3-position of the
uracil moiety throughout the synthesis. Consequently,
both the Cbz and the BOM groups of 6 were removed
by catalytic hydrogenation, and the resulting free amine
at the 60-position was re-protected with a Cbz group to
give 7 in 79% in two steps. Coupling of 7 with L-serine
benzyl ester using N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide (EDCI) and 1-hydroxybenzotriazole
(HOBt)13 in DMF gave the amide 8 without any racemi-
zation at the 60-position. The use of CH2Cl2 as a solvent
in the coupling reaction gave no desired compounds be-
cause 7 was insoluble in that solvent. The amide 8 was
then acylated with either palmitic acid or 4-phenylben-
zoic acid with EDCI in the presence of DMAP in
CH2Cl2 to give 9a (89% in two steps) or 9b (87% in
A variety of analogs could also be synthesized with this
strategy using a variation of the condensed amino acids.
By using D-serine benzyl ester in the first coupling reac-
tion with 7, the intermediates 14a and 14b were synthe-
sized as shown in Scheme 2. Compounds 15a and 15b
were prepared in a manner similar to the syntheses of
11a and 11b. However, the diketopiperazine derivatives
with the trans-substituents tend to be less stable com-
pared to those with the cis-substituents for promoting
b-elimination of the acyloxy group. Transformation of
14a and 14b did proceed to give in moderate yield the
corresponding protected diketopiperazine derivatives,
deprotection of which afforded 15b in moderate yield
(43% in three steps from 14a) or resulted in decomposi-
tion for 15a. On the other hand, the synthesis of the acy-
clic analogs 16a and 16b was accomplished without any
difficulty, and the same deprotection conditions as for
the preparation of 12 were applied to provide 16a
(64% in two steps) and 16b (80% in two steps),
respectively.
two steps), respectively. Deprotection of both the Cbz
The antibacterial activity of the synthesized compounds
was evaluated against a range of Gram-positive bacte-
rial strains, including Mycobacterium sp., and the mini-
mum inhibitory concentrations (MIC, lg/mL) are
summarized in Table 1. None of the newly synthesized
analogs exhibited antibacterial activity against Myco-
bacterium smegmatis. However, the diketopiperazine
analog 11a and the acyclic analogs 12a and 16a, which
possess a palmitoyl side chain, demonstrated moderate
antibacterial activity (MIC = 12.5–50 lg/mL) against
some strains including Micrococcus sp. or Corynebacte-
rium sp. Since the acyclic analogs retain the amino and
the carboxylic acid functionalities of the parent natural
products, it is assumed that their reduced activity is
due to the loss of conformational constraint, and the
cyclic structure is crucial for MraY inhibition. Although
the activity was moderate, the analogs still possess anti-
bacterial activity, and the optimization of the bridging
amino acid moiety might lead to better activity. On
the other hand, the diketopiperazine analog 11a exhib-
ited antibacterial activity to a lesser extent than the acy-
0
group at the N6 -position and the Bn group on the serine
moiety of 9 was conducted by hydrogenolysis catalyzed
by Pd(OH)2/C in MeOH to provide the free amino acid
10, the precursor for the cyclization reaction. Next, the
cyclization to construct the diketopiperazine
scaffold was examined. Treatment of 10 with either
N,N0-dicyclohexylcarbodiimide (DCC) and HOBt or
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOPCl) in CH2Cl2 resulted in a
complex mixture of products, and only a trace amount
of the desired diketopiperazine derivative was obtained.
However, the cyclization was successful when EDCI and
HOBt were used in CH2Cl2 to give smoothly the diketo-
piperazine derivative, which was sequentially treated
with 80% aqueous TFA to afford the target compound
11a (70% in three steps from 9a) or 11b (84% in three
steps from 9b). These compounds were obtained effi-
ciently in five steps with a minimum number of isola-
tions from the key intermediate 7. Compounds 12a
and 12b were also designed and synthesized since they

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
431
Scheme 1. Synthesis of diketopiperazine and acyclic analogs containing L-serine moiety.
Scheme 2. Synthesis of diketopiperazine and acyclic analogs containing D-serine moiety.
clic analogs 12a and 16a although 11a does not have the
amino and the carboxylic acid functionalities of the par-
ent CPZs and LPMs. The amino and the carboxylic acid
functionalities found in the parent natural products may
play an important role in the interaction with MraY.
Introduction of amino and carboxylic acid groups to
the diketopiperazine scaffold may be effective in increas-
ing the activity. Analogs 11b, 12b, 15b, and 16b possess-
ing a p-phenylbenzoyl group as a compact lipophilic
acyl side chain all showed no activity at >100 lg/mL
against bacterial strains tested in this study. The lipo-
philic acyl group at the hydroxyl group of the serine
moiety may be responsible for transporting the active
structure to the target enzyme in the membrane. This
was supported by a total loss of activity of the deacyl
derivative 13. In conjunction with the previous study,7
the antibacterial activity of this class of antibiotics
would be changed by the structural alteration of the acyl
side chains. Antibacterial activity of the newly synthe-
sized analogs against M. tuberculosis H37Rv was also

432
S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
Table 1. Antibacterial activity of synthesized analogs
Test organisms
MIC (lg/mL)
11a
11b
12a
12b
13
15b
16a
16b
Staphylococcus aureus Smith
25
>100
>100
>100
>100
100
25
50
>100
>100
>100
>100
50
>100
>100
>100
>100
> 100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25
50
>100
>100
>100
>100
>100
>100
>100
100
Staphylococcus aureus MS9610 (MDR)
Staphylococcus aureus MRSA No. 5 (MRSA)
Micrococcus luteus FDA16
100
100
25
50
50
50
50
Micrococcus luteus PCI 1001
Bacillus subtilis NRRL B-558
50
25
25
25
12.5
25
>100
>100
100
>100
100
100
Corynebacterium bovis 1810
Mycobacterium smegmatis ATCC607
Enterococcus faecalis NCTC 12201 (VRE)
50
12.5
100
50
12.5
50
100
100
>100
>100
>100
50
>100
evaluated (Table 2). The assay was performed by the
Alamar blue assay, and the inhibition rate of the M.
tuberculosis H37Rv at 6.25 lg/mL concentration is sum-
marized in Table 2. The acyclic analogs exhibited mod-
erate antibacterial activity. Different from the results
obtained in Table 1, the 4-phenylbenzoyl derivatives
showed similar potency to the corresponding palmitoyl
derivatives.
NMR spectra. Optical rotations were recorded on JAS-
CO DIP-370 digital polarimeter or JASCO P-1030
polarimeter. FAB-MS was obtained on a JEOL JMS-
HX101 or JEOL JMS-700TZ. Analytical thin layer
chromatography (TLC) was performed on Merck silica
gel 60F254 plates. Normal-phase column chromatogra-
phy was performed on Merck silica gel 5715 or Kanto
Chemical silica gel 60N (neutral). Flash column chroma-
tography was performed on Merck silica gel 60.
In conclusion, simplification of a class of 60-N-alkyl-50-
O-aminoribosyl-glycyluridine antibiotics, including
CPZs and LPMs, based on the construction of the pre-
dicted pharmacophore of MraY inhibitors, was shown
to produce potential antibacterial agents with a novel
mechanism of action. Diketopiperazine and acyclic ana-
logs of CPZs and LPMs were synthesized, and their
antibacterial activity was evaluated. Some analogs
exhibited moderate antibacterial activity against Micro-
coccus sp. or Corynebacterium sp. By altering the amino
acids and/or the acyl side chains, a variety of analogs
could be synthesized easily using our strategy. There-
fore, this approach would provide ready access to a
range of analogs for the development of antibacterial
agents, and this study is underway.
3.1.1. Carboxylic acid (7). A mixture of 611 (10 mg,
0.011 mmol),
2,2,2-trichloroacetic
acid
(18 mg,
0.1 mmol), and 10% Pd(OH)2/C (5 mg) in MeOH
(1.5 mL) was vigorously stirred for 1 h under H2 atmo-
sphere. The catalyst was filtered off through Celite
pad, and the filtrate was concentrated in vacuo. The res-
idue was dissolved in CH2Cl2 (10 mL), to which satu-
rated aqueous NaHCO3 (10 mL) was added. The
biphasic mixture was treated with CbzCl (6.3 lL,
0.044 mmol), and the whole mixture was vigorously stir-
red at room temperature for 30 min. The organic phase
was separated, washed with brine, dried (Na2SO4), fil-
trated, and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (1 · 5, 12%
MeOH/CHCl3) to afford 7 (6.9 mg, 79%) as a white
21
1
foam: ½aꢀ +21.6 (c 1.00, CHCl3); H NMR (CDCl3,
D
3. Experimental
500 MHz) d 7.64 (br d, 1H, H-6, J6,5 = 8.0 Hz), 7.38–
7.27 (m, 5H, phenyl), 5.74 (br s, 1H, H-10), 5.65 (br d,
1H, H-5, J5,6 = 8.0 Hz), 5.22 (s, 1H, H-100), 5.16 (d,
1H, benzyl, J = 12.5 Hz), 5.11 (m, 1H, H-20), 5.02 (d,
1H, benzyl, J = 12.5 Hz), 4.98 (m, 1H, H-30), 4.90 (m,
1H, H-200), 4.65 (m, 1H, H-60), 4.56 (d, 1H, H-300,
3.1. General experimental methods
NMR spectra were obtained on a JEOL EX270, JEOL
GX270, JEOL AL400, or Bruker ARX-500, and were
reported in parts per million (d) relative to tetramethyl-
silane (0.00 ppm) as internal standard otherwise noted.
Coupling constant (J) was reported in Hertz (Hz).
Abbreviations of multiplicity were as follows: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Data were presented as follows; chemical shift (multi-
plicity, integration, coupling constant). Assignment
was based on 1H–1H COSY, HMBC and HMQC
J3 ;2 ¼ 5:8 Hz), 4.50 (m, 1H, H-50), 4.18–4.11 (m, 2H,
00 00
H-40, H-400), 3.13 (m, 2H, H-500a, H-500b), 1.60 (m, 4H,
CH2CH3 · 2), 1.49 (s, 3H, acetonide), 1.48 (s, 9H, tert-
butyl), 1.31 (s, 3H, acetonide), 0.79 (m, 6H,
CH2CH3 · 2); 13C NMR (CDCl3, 125 MHz) d 177.5,
166.23, 158.54, 152.00, 138.25, 130.27, 129.68, 129.24,
128.70, 117.36, 115.63, 88.44, 87.87, 83.81, 83.38,
80.50, 57.39, 44.48, 30.53, 29.03, 28.59, 28.17, 27.78,
27.32, 25.78, 8.60, 7.89, 7.55; FABMS-LR (negative)
m/z 789 (MꢁH)ꢁ; FABMS-HR (NBA) calcd for
C37H49N4O15 789.3194, found 789.3194.
Table 2. Antibacterial activity (% inhibition) against Mycobacterium
tuberculosis at 6.25 lg/mL
3.1.2. O-Palmitoyl-L-serylamide (9a). A solution of 7
(44 mg, 0.056 mmol), L-Ser(OBn) (22.4 mg, 0.056 mmol),
and HOBt (30.8 mg, 0.17 mmol) in DMF (560 lL) was
treated with EDCI (30.8 mg, 0.17 mmol) at room temper-
11a
11b
12a
12b
15b
16a
16b
Mycobacterium
tuberculosis
35
48
51
52
29
59
27

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
433
ature for 12 h. The reaction mixture was partitioned be-
tween AcOEt and 0.3 N aqueous HCl, and the organic
layer was washed with saturated aqueous NaHCO3 and
brine, dried over Na2SO4, and concentrated in vacuo.
The residue in CH2Cl2 (1.4 mL) was treated with palmitic
acid (28 mg, 0.11 mmol), DMAP (2.8 mg, 0.017 mmol),
and EDCI (30.8 mg, 0.17 mmol) at room temperature
for 2 h. The reaction mixture was partitioned between
AcOEt and 0.3 N aqueous HCl, and the organic layer
was washed with saturated aqueous NaHCO3 and brine,
dried over Na2SO4, and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography
(2 · 15 cm, 33–50% AcOEt/hexane) to give 9a (61.5 mg,
89% in two steps) as a colorless glass: 1H NMR (CDCl3,
500 MHz) d 9.02 (br s, 1H, NH-3), 7.42–7.27 (m, 12H,
phenyl, NHCO, H-6), 5.87 (m, 1H, NHCbz), 5.69 (d,
1H, H-5, J5,6 = 8.0 Hz), 5.68 (s, 1H, H-10), 5.64 (br s,
1H, NHBoc), 5.24–5.09 (m, 5H, benzyl · 4, H-100), 4.84
(m, 3H, H-20, H-30, H-90), 4.58 (m, 3H, H-200, H-300, H-
60), 4.46 (m, 2H, H-100a, H-50), 4.34 (dd, 1H, H-100b,
MS-LR m/z 1170 (MNa+); FABMS-HR (NBA) calcd
for C60H69N5O18Na 1170.4536, found 1170.4534.
3.1.4. O-Palmitoyl diketopiperazine (11a). A mixture of
9a (13 mg, 0.011 mmol) and 10% Pd(OH)2/C (5 mg) in
MeOH (1 mL) was vigorously stirred for 1 h under H2
atmosphere. The catalyst was filtered off through Celite
pad, and the filtrate was concentrated in vacuo to give
the free amino acid 10a. The amino acid 10a in CH2Cl2
(1 mL) was treated with EDCI (8.6 mg, 0.043 mmol)
and HOBt (5.8 mg, 0.043 mmol) at room temperature
for 24 h. The reaction mixture was partitioned between
AcOEt and 0.3 N aqueous HCl, and the organic layer
was washed with saturated aqueous NaHCO3 and brine,
dried over Na2SO4, and concentrated in vacuo. The res-
idue was treated with aqueous 80% TFA at room tem-
perature for 1 h. The reaction mixture was
concentrated in vacuo, and the residue was purified by
C18 reverse phase column chromatography (1 · 10 cm,
0.5% aqueous TFA–MeOH) to afford 11a (6.6 mg,
1
J10 b;10 a ¼ 11:4, J10 b;9 ¼ 2:9 Hz), 4.26 (dd, 1H, H-40,
70% in three steps) as a TFA salt: H NMR (CD3OD,
0
0
0
0
J4 ;5 ¼ 4:2, J4 ;3 ¼ 6:4 Hz), 4.22 (m, 1H, H-400), 3.32 (m,
1H, H-500a), 3.07 (m, 1H, H-500b), 2.14 (m, 2H, H-1000),
1.62 (m, 2H, CH2CH3), 1.53 (m, 2H, CH2CH3), 1.48
(m, 5H, acetonide, H-2000a, H-2000b), 1.43 (s, 9H, tert-bu-
tyl), 1.31 (s, 3H, acetonide), 1.25 (m, 26H, palmitoyl),
0.85 (m, 9H, CH2CH3 · 2, palmitoyl terminal-Me); 13C
NMR (CDCl3, 125 MHz) d 173.30, 169.05, 162.71,
156.06, 150.08, 141.88, 136.07, 134.88, 128.59, 128.53,
128.49, 128.35, 128.18, 117.10, 114.78, 110.86, 102.83,
93.58, 86.62, 85.93, 84.06, 81.84, 79.49, 77.76, 67.73,
67.29, 63.44, 55.26, 52.39, 43.39, 33.69, 31.88, 29.65,
29.63, 29.61, 29.60, 29.46, 29.31, 29.25, 29.07, 28.97,
28.36, 27.02, 25.36, 24.65, 22.64, 14.07, 8.32, 7.41; FAB-
MS-LR m/z 1228 (MNa+); FABMS-HR (NBA) calcd
for C63H91N5O18Na 1228.6257, found 1228.6255.
500 MHz) d 7.77 (d, 1H, H-6, J6,5 = 8.0 Hz), 5.74 (s,
1H, H-10), 5.69 (d, 1H, H-5, J5,6 = 8.0 Hz), 5.29 (s, 1H,
H-100), 4.42–4.16 (m, 8H, H-20, H-30, H-40, H-50, H-60,
H-90, H-120a, H-120b), 4.02 (m, 2H, H-300, H-400), 3.94
(m, 1H, H-200), 3.18 (m, 2H, H-500a, H-500b), 2.42 (m,
2H, COCH2), 1.56 (m, 2H, COCH2CH2), 1.31 (m,
24H, palmitoyl), 0.91 (m, 3H, palmitoyl terminal-Me);
13C NMR (CD3OD, 125 MHz) d 175.08, 167.59,
167.24, 166.16, 151.96, 109.93, 102.59, 93.91, 83.87,
80.63, 79.82, 76.13, 74.88, 73.70, 71.14, 66.58, 57.41,
55.57, 43.96, 34.77, 33.06, 30.79, 30.75, 30.64, 30.52,
30.46, 30.22, 25.87, 23.72, 14.42; FABMS-LR m/z 756
(MH+); FABMS-HR (NBA) calcd for C35H58N5O13
756.4031, found 756.4037.
0
0
0
0
3.1.5. O-4-Phenylbenzoyl diketopiperazine (11b). Com-
pound 11b was prepared from 9b (41 mg, 0.036 mmol)
as described above for the synthesis of 11a. Purification
by C18 reverse phase column chromatography
(1 · 10 cm, 0.5% aqueous TFA–MeOH) afforded 11b
3.1.3. O-4-Phenylbenzoyl-L-serylamide (9b). Compound
9b was prepared from 7 (50 mg, 0.063 mmol) and 4-phe-
nylbenzoic acid instead of palmitic acid as described
above for the synthesis of 9a. Purification by silica gel
column chromatography (1 · 15 cm, 33-50% AcOEt/
hexane) afforded 9b (63.6 mg, 87% in two steps) as a col-
1
(9.4 mg, 84% in three steps) as a TFA salt: H NMR
(CD3OD, 500 MHz)
d
8.15 (d, 2H, o-phenyl,
1
orless glass: H NMR (CDCl3, 500 MHz) d 9.18 (br s,
J = 8.4 Hz), 7.78 (d, 2H, m-phenyl, J = 8.4 Hz), 7.73
(d, 1H, H-6, J6,5 = 8.1 Hz), 7.68 (d, 2H, o-phenyl,
J = 7.4 Hz), 7.46 (t, 2H, m-phenyl, J = 7.4 Hz), 7.39 (t,
1H, p-phenyl, J = 7.4 Hz), 5.77 (d, 1H, H-10,
1H, NH-3), 7.92-7.23 (m, 21H, phenyl, H-6, CONH),
5.87 (br s, 1H, CbzNH), 5.65 (d, 1H, H-5,
J5,6 = 8.0 Hz), 5.63 (s, 1H, H-10), 5.55 (m, 1H, BocNH),
5.28 (d, 1H, benzyl, J = 12.1 Hz), 5.23 (s, 1H, H-100),
5.15 (m, 2H, benzyl · 2), 5.04 (d, 1H, benzyl,
J = 12.2 Hz), 4.98 (m, 1H, H-90), 4.85 (m, 2H, H-20,
H-30), 4.67 (m, 2H, H-100a, H-100b), 4.62 (m, 1H, H-
60), 4.55 (m, 1H, H-200), 4.49 (m, 2H, H-300, H-50), 4.27
(m, 1H, H-40), 4.12 (m, 1H, H-400), 3.27 (m, 1H, H-
500a), 2.98 (m, 1H, H-500b), 1.59 (m, 2H, CH2CH3),
1.47 (m, 2H, CH2CH3), 1.40 (s, 12H, tert-butyl, aceto-
nide), 1.29 (s, 3H, acetonide), 0.79 (m, 6H,
CH2CH3 · 2); 13C NMR (CDCl3, 125 MHz) d 169.11,
165.78, 162.82, 156.05, 150.09, 146.01, 141.80, 139.79,
136.03, 134.80, 130.17, 128.93, 128.55, 128.47, 128.35,
128.26, 128.12, 127.86, 127.20, 127.06, 117.01, 114.80,
110.98, 102.80, 93.80, 86.56, 85.90, 83.99, 81.79, 80.81,
79.49, 77.67, 67.85, 67.28, 64.19, 55.36, 52.48, 43.32,
29.63, 29.39, 28.91, 28.33, 27.03, 25.36, 8.27, 7.39; FAB-
0
0
J1 ;2 ¼ 2:6 Hz), 5.67 (d, 1H, H-5, J5,6 = 8.1 Hz), 5.00
(d, 1H, H-100, J1 ;2 ¼ 2:0 Hz), 4.84 (m, 1H, H-120a),
00 00
4.60 (dd, 1H, H-120b, J12 b;9 ¼ 3:7, J12 b;12 a ¼ 11:2 Hz),
0
0
0
0
4.46 (m, 1H, H-90), 4.41 (d, 1H, H-60, J6 ;5 ¼ 5:7 Hz),
0
0
4.32 (dd, 1H, H-40, J4 ;5 ¼ 4:6, J4 ;3 ¼ 7:5 Hz), 4.20
0
0
0
0
(m, 2H, H-50, H-20), 4.10 (dd, 1H, H-30, J3 ;2 ¼ 5:7,
0
0
J3 ;4 ¼ 7:5 Hz), 4.00–3.95 (m, 3H, H-200, H-300, H-400),
3.21 (m, 2H, H-500a, H-500b); FABMS-LR m/z 698
(MH+); FABMS-HR (NBA) calcd for C32H36N5O13
698.2310, found 698.2304.
0
0
3.1.6. O-Palmitoyl-L-serylamide (12a). A mixture of 9a
(25 mg, 0.021 mmol) and 10% Pd(OH)2/C (15 mg) in
MeOH (1 mL) was vigorously stirred under H2 atmo-
sphere for 1 h. The catalyst was filtered off through Cel-
ite pad, and the filtrate was concentrated in vacuo. The

434
S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
1
residue was treated with aqueous 80% TFA (1 mL) at
room temperature for 50 min. The solution was concen-
trated in vacuo, and the residue was purified by C18 re-
verse phase column chromatography (1 · 10 cm, 0.5%
aqueous TFA–MeOH) to afford 11a (18.7 mg, 90% in
two steps) as a TFA salt: 1H NMR (CD3OD,
500 MHz) d 7.68 (d, 1H, H-6, J6,5 = 8.3 Hz), 5.81 (s,
1H, H-10), 5.73 (d, 1H, H-5, J5,6 = 8.3 Hz), 5.16 (s, 1H,
H-100), 4.61 (m, 1H, H-100a), 4.51 (m, 1 H, H-30), 4.42
(m, 1 H, H-300), 4.37 (m, 1 H, H-100b), 4.32 (m, 1 H,
H-90), 4.27 (m, 1 H, H-60), 4.24 (m, 1 H, H-50), 4.19
(m, 1 H, H-20), 4.04 (m, 3 H, H-40, H-200, H-400), 3.18
(m, 2 H, H-500a, H-500b), 2.32 (t, 2 H, COCH2,
J = 9.2 Hz), 1.59 (m, 2H, COCH2CH2), 1.28 (m, 24H,
palmitoyl), 0.89 (m, 3H, palmitoyl terminal-Me); 13C
NMR (CD3OD, 125 MHz) d 175.05, 173.63, 172.43,
168.43, 165.90, 155.01, 151.93, 142.50, 111.37, 111.24,
102.86, 93.23, 91.98, 85.48, 84.82, 81.02, 78.52, 78.27,
76.45, 76.36, 74.61, 73.48, 73.07, 73.00, 72.73, 71.15,
70.46, 65.16, 62.23, 56.36, 55.19, 43.46, 38.99, 34.84,
33.10, 31.87, 30.83, 30.79, 30.66, 30.50, 30.28, 25.93,
23.77, 14.49; FABMS-LR m/z 774 (MH+); FABMS-
HR (NBA) calcd for C35H60N5O14 774.4137, found
774.4128.
steps) as a TFA salt: H NMR (CD3OD, 500 MHz) d
7.68 (d, 1H, H-6, J6,5 = 8.0 Hz), 5.80 (s, 1H, H-10),
5.72 (d, 1H, H-5, J5,6 = 8.0 Hz), 5.16 (s, 1H, H-100),
4.48 (m, 2H, H-30, H-60), 4.35 (m, 1H, H-300), 4.31–
4.23 (m, 2H, H-100a, H-100b), 4.19 (m, 1H, H-20), 4.06
(m, 3H, H-200, H-400, H-90), 3.89 (m, 1H, H-40), 3.20
(m, 2H, H-500a, H-500b); 13C NMR (CDCl3, 125 MHz)
d 172.45, 168.36, 165.91, 154.97, 151.90, 142.46,
111.53, 111.40, 102.76, 93.00, 91.82, 84.93, 84.41,
80.92, 78.65, 78.44, 76.38, 76.22, 74.50, 73.35, 72.75,
71.02, 70.31, 62.81, 62.09, 57.36, 56.04, 43.20, 38.80,
31.68; FABMS-LR (negative) m/z 534 (MꢁH)ꢁ; FAB-
MS-HR (NBA) calcd for C19H28N5O13 534.1684, found
534.1680.
3.1.9. O-Palmitoyl-D-serylamide (14a). Compound 14a
was prepared from 7 (53 mg, 0.10 mmol) as described
above for the synthesis of 9a. Purification by silica gel
column chromatography (1 · 10 cm, 33–50% AcOEt/
hexane) afforded 14a (15.6 mg, 65% in two steps) as a
1
colorless glass: H NMR (CDCl3, 500 MHz) d 8.98 (br
s, 1H, NH-3), 7.36–7.33 (m, 11H, phenyl, NHCO),
7.25 (d, 1H, H-6, J6,5 = 8.2 Hz), 5.76 (br s, 1H, NHCbz),
5.70 (d, 1H, H-5, J5,6 = 8.2 Hz), 5.66 (br s, 1H, NHBoc),
5.60 (br s, 1H, H-10), 5.26-5.07 (m, 5H, benzy0l · 4, H-
100), 4.90 (m, 1H, H-90), 4.86 (m, 2H, H-2 , H-30),
4.57–4.45 (m, 5H, H-100a, H-100b, H-200, H-300, H-60),
4.35 (m, 1H, H-50), 4.18 (m, 2H, H-400, H-40), 3.30 (m,
1H, H-500a), 2.91 (m, 1H, H-500b), 2.15 (m, 2H, H-1000),
1.62 (m, 4H, CH2CH3 · 2), 1.52 (m, 2H, H-2000a, H-
2000b), 1.50 (s, 3H, acetonide), 1.48 (s, 9H, tert-butyl),
1.30 (s, 3H, acetonide), 1.25 (m, 26H, palmitoyl), 0.89–
0.79 (m, 9H, CH2CH3 · 2, palmitoyl terminal-Me); 13C
NMR (CDCl3, 125 MHz) d 178.07, 173.64, 169.37,
163.05, 156.39, 150.28, 142.30, 136.92, 135.19, 128.88,
128.83, 128.77, 128.58, 117.12, 115.05, 110.09, 103.08,
87.38, 86.36, 84.23, 82.28, 79.78, 68.03, 67.67, 63.49,
55.70, 52.56, 43.73, 34.07, 33.98, 32.15, 29.92, 29.88,
29.83, 29.75, 29.69, 29.58, 29.52, 29.49, 29.35, 28.65,
27.26, 25.00, 24.90, 22.91, 14.33, 8.57, 7.62; FABMS-
LR m/z 1228 (MNa+); FABMS-HR (NBA) calcd for
C63H91N5O18Na 1228.6257, found 1228.6255.
3.1.7. O-4-Phenylbenzoyl-L-serylamide (12b). Compound
12b was prepared from 9b (23 mg, 0.020 mmol) as de-
scribed above for the synthesis of 12a. Purification by
C18 reverse phase column chromatography (1 · 10 cm,
0.5% aqueous TFA–MeOH) afforded 12b (18.0 mg,
1
96% in two steps) as a TFA salt: H NMR (CD3OD,
500 MHz) d 8.10 (d, 2H, o-phenyl, J = 8.3 Hz), 7.72
(d, 2H, m-phenyl, J = 8.3 Hz), 7.66 (m, 3H, o0-phenyl,
H-6, J = 7.3, J6,5 = 8.1 Hz), 7.47 (t, 2H, m0-phenyl,
J = 7.2 Hz), 7.39 (t, 1H, p0-phenyl, J = 7.2 Hz), 5.80 (d,
1H, H-10, J1 ;2 ¼ 3:0 Hz), 5.77 (d, 1H, H-5,
0
0
J5,6 = 8.1 Hz), 5.18 (s, 1H, H-100), 4.78 (m, 1H, H-90),
4.71 (dd, 1H, H-100a, J10 a;10 b ¼ 11:7, J10 ;9 ¼ 2:0 Hz),
0
0
0
0
4.63 (dd, 1H, H-100b, J10 b;10 a ¼ 11:7, J10 b;9 ¼ 5:6 Hz),
0
0
0
0
4.55 (m, 1H, H-60), 4.34 (m, 1H, H-50), 4.26–4.21 (m,
2H, H-30, H-40), 4.19 (dd, 1H, H-20, J2 ;1 ¼ 3:0,
0
0
J2 ;3 ¼ 7:6 Hz), 4.07 (m, 3H, H-20, H-300, H-400), 3.20
(m, 2H, H-500a, H-500b); 13C NMR (CD3OD, 125 MHz)
d 168.72, 167.7, 166.69, 152.13, 147.54, 141.11, 131.48,
130.21, 129.79, 129.53, 128.32, 128.20, 111.57, 103.06,
93.51, 85.71, 81.26, 78.45, 76.58, 74.80, 73.71, 73.26,
70.67, 66.12, 62.45, 56.57, 43.75; FABMS-LR m/z 716
(MH+); FABMS-HR (NBA) calcd for C32H38N5O14
716.2416, found 716.2404.
0
0
3.1.10. O-4-Phenylbenzoyl-D-serylamide (14b). Com-
pound 14b was prepared from 7 (28 mg, 0.035 mmol)
and 4-phenylbenzoic acid instead of palmitic acid as de-
scribed above for the synthesis of 9a. Purification by sil-
ica gel column chromatography (1 · 15 cm, 33–50%
AcOEt/hexane) afforded 14b (36.8 mg, 91% in two steps)
1
as a colorless glass: H NMR (CDCl3, 500 MHz) d 8.58
3.1.8. L-Serylamide (13). A solution of 7 (16 mg,
0.02 mmol), L-Ser(Bn) (8 mg, 0.02 mmol), and HOBt
(11 mg, 0.06 mmol) in DMF (200 lL) was treated with
EDCI (11 mg, 0.06 mmol) at room temperature for
12 h. The reaction mixture was partitioned between
AcOEt and 0.3 N aqueous HCl, and the organic layer
was washed with saturated aqueous NaHCO3 and brine,
dried over Na2SO4, and concentrated in vacuo. The res-
idue was treated with 80% aqueous TFA (1 mL) at room
temperature for 30 min. The mixture was concentrated
in vacuo, and the residue was purified by C18 reverse
phase column chromatography (1 · 10 cm, 0.5% aque-
ous TFA–MeOH) to afford 13 (8.8 mg, 68% in three
(br s, 1H, NH-3), 7.96–7.22 (m, 20H, phenyl, H-6), 7.02
(m, 1H, NHCO), 5.80 (m, 1H, NHCbz), 5.79 (m, 1H,
NHBoc), 5.54 (d, 1H, H-5, J5,6 = 8.0 Hz), 5.28 (m, 2H,
benzyl · 2), 5.24 (s, 1H, H-10), 5.20 (s, 1H, H-100), 5.35
(d, 1H, benzyl, J = 15 Hz), 5.05 (d, 1H, benzyl,
J = 15 Hz), 4.99 (m, 1H, H-90), 4.86 (m, 1H, H-20),
4.82 (m, 2H, H-60, H-200), 4.52 (m, 2H, H-300, H-50),
4.22 (m, 1H, H-40), 4.11 (m, 1H, H-400), 3.38 (m, 1H,
H-500a), 2.96 (m, 1H, H-500b), 1.57 (m, 2H, CH2CH3),
1.44 (m, 14H, tert-butyl, CH2CH3, acetonide), 1.27 (s,
3H, acetonide), 0.80 (m, 6H, CH2CH3 · 2); 13C NMR
(CDCl3, 125 MHz) d 169.19, 165.66, 165.58, 156.08,
149.70, 145.86, 142.83, 139.62, 136.51, 134.81, 130.29,

S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
435
128.98, 128.58, 128.53, 128.47, 128.33, 128.13, 127.92,
127.15, 127.01, 116.82, 114.78, 102.66, 87.13, 86.11,
83.94, 82.04, 67.95, 67.30, 63.96, 55.46, 52.39, 29.47,
28.91, 28.39, 26.99, 25.27, 8.29, 7.32; FABMS-LR m/z
1170 (MNa+); FABMS-HR (NBA) calcd for
C60H69N5O18Na 1170.4535, found 1170.4531.
J6,5 = 8.1 Hz), 7.46 (t, 2H, m0-phenyl, J = 7.4 Hz), 7.39
(t, 1H, p0-phenyl, J = 7.4 Hz), 5.75 (s, 1H, H-10), 5.69
(d, 1H, H-5, J5,6 = 8.1 Hz), 5.03 (s, 1H, H-100), 4.82 (m,
1H, H-90), 4.74 (dd, 1H, H-100a, J10 a;9 ¼ 3:8,
0
0
J10 a;10 b ¼ 11:4 Hz), 4.69 (dd, 1H, H-100b, J10 b;10 a
¼
0
0
0
0
11:4, J10 b;9 ¼ 6:1 Hz), 4.36 (m, 2H, H-50, H-60), 4.23
(m, 2H, H-30, H-40), 4.16 (m, 1H, H-20), 4.05 (m, 3H,
H-20, H-300, H-400), 3.15 (m, 2H, H-500a, H-500b); 13C
NMR (CD3OD, 125 MHz) d 175.05, 173.63, 172.43,
168.43, 165.90, 155.01, 151.93, 142.50, 111.37, 111.24,
102.86, 93.23, 91.98, 85.48, 84.82, 81.02, 78.52, 78.27,
76.45, 76.36, 74.61, 73.48, 73.07, 73.00, 72.73, 71.15,
70.46, 65.16, 62.23, 56.36, 55.19, 43.46, 38.99, 34.84,
33.10, 31.87, 30.83, 30.79, 30.66, 30.50, 30.28, 25.93,
23.77, 14.49; FABMS-LR m/z 716 (MH+); FABMS-
HR (NBA) calcd for C32H38N5O14 716.2415, found
716.2411.
0
0
3.1.11. O-4-Phenylbenzoyl diketopiperazine (15b). Com-
pound 15b was prepared from 14b (13 mg, 0.012 mmol)
as described above for the synthesis of 11a. Purification
by C18 reverse phase column chromatography
(1 · 10 cm, 0.5% aqueous TFA–MeOH) afforded 15b
1
(3.7 mg, 43% in three steps) as a TFA salt: H NMR
(CD3OD, 500 MHz)
d
8.05 (d, 2H, o-phenyl,
J = 8.2 Hz), 7.73 (d, 2H, m-phenyl, J = 8.2 Hz), 7.66
(m, 3H, o-phenyl, H-6), 7.46 (t, 2H, m-phenyl,
J = 7.3 Hz), 7.39 (t, 1H, p-phenyl, J = 7.3 Hz), 5.72 (s,
1H, H-10), 5.69 (d, 1H, H-5, J5,6 = 8.0 Hz), 5.35 (s, 1H,
H-100), 4.74 (dd, 1H, H-120a, J12 a;12 b ¼ 11:6,
3.2. Antibacterial activity and determination of minimum
inhibitory concentration (MIC)
0
0
J12 a;9 ¼ 3:0 Hz), 4.67 (dd, 1H, H-120b, J12 b;12 a ¼ 11:6,
0
0
0
0
J12 b;9 ¼ 2:7 Hz), 4.47 (m, 2H, H-60, H-90), 4.38 (m,
1H, H-40), 4.21 (m, 3H, H-50, H-20, H-30), 4.07 (m,
1H, H-400), 4.00 (m, 1H, H-200), 3.95 (m, 1H, H-300),
3.24 (m, 1H, H-500a), 3.16 (m, 1H, H-500b); FABMS-
LR m/z 698 (MH+); FABMS-HR (NBA) calcd for
C32H36N5O13 698.2309, found 698.2324.
0
0
The minimum inhibitory concentrations (MIC) of syn-
thesized compounds were examined by serial agar
dilution method using Muller–Hinton (MH) agar (Dif-
co) containing 5% sheep blood for Enterococcus fae-
calis, MH agar containing 5% glycerol for M.
smegmatis, and MH agar for other bacteria. The
MIC was observed after 18 hours incubation at
37 ꢁC in 5% CO2 for E. faecalis, 42 h incubation at
37 ꢁC for M. smegmatis, and 18 h incubation at 37
ꢁC for other bacteria.
3.1.12. O-Palmitoyl-D-serylamide (16a). Compound 16a
was prepared from 14a (26 mg, 0.022 mmol) as de-
scribed above for the synthesis of 12a. Purification by
C18 reverse phase column chromatography (1 · 10 cm,
0.5% aqueous TFA–MeOH) afforded 13a (13.7 mg,
1
64% in two steps) as a TFA salt: H NMR (CD3OD,
3.3. Antibacterial activity against M. tuberculosis
500 MHz) d 7.62 (d, 1H, H-6, J6,5 = 8.1 Hz), 5.74 (d,
1H, H-10, J1 ;2 ¼ 1:9 Hz), 5.73 (d, 1H, H-5,
J5,6 = 8.1 Hz), 5.33 (s, 1H, H-100), 4.52 (m, 2H, H-100,
H-60), 4.40 (m, 2H, H-100b, H-90), 4.17 (dd, 1H, H-20,
The initial screen is conducted against M. tuberculosis
H37Rv (ATCC 27294) in BACTEC 12B medium using
the microplate Alamar blue assay (MABA). Com-
pounds are tested in 10 twofold dilutions, usually from
100 to 0.19 lg/mL. Compounds exhibiting autofluores-
cence are sent for testing in an alternative assay at the
University of Illinois at Chicago.
0
0
J2 ;1 ¼ 1:9, J2 ;3 ¼ 5:5 Hz), 4.13 (m, 1H, H-50), 4.07
0
0
0
0
(m, 2H, H-30, H-300), 4.01 (dd, 1H, H-200, J2 ;1 ¼ 2:2,
00 00
J2 ;3 ¼ 7:8 Hz), 3.98 (m, 1H, H-400), 3.68 (m, 1H, H-
00 00
40),
3.15
(dd,
1H,
H-500a,
J5 a;5 b ¼ 13:3,
00
00
J5 a;4 ¼ 3:2 Hz), 2.96 (dd, 1H, H-500b, J5 b;5 a ¼ 13:3,
00
00
00
00 00
00
J5 b;4 ¼ 5:9 Hz), 2.32 (t, 2H, COCH2, J = 7.5 Hz), 1.61
(m, 2H, COCH2CH2), 1.27 (m, 24 H, palmitoyl), 0.89
(m, 3H, palmitoyl terminal-Me); 13C NMR (CD3OD,
125 MHz) d 175.31, 175.34, 169.01, 165.89, 151.90,
142.55, 112.24, 112.19, 102.77, 93.33, 92.12, 85.28,
81.31, 79.46, 76.61, 76.48, 74.62, 73.49, 72.87, 72.79,
71.06, 70.37, 65.18, 62.24, 56.76, 55.10, 43.06, 34.92,
33.10, 31.87, 30.83, 30.80, 30.69, 30.51, 30.29, 26.00,
23.78, 14.49; FABMS-LR m/z 774 (MH+); FABMS-
HR (NBA) calcd for C35H60N5O14 774.4136, found
774.4144.
Acknowledgments
This work was supported financially by a Grant-in-Aid
from the Ministry of Education, Science, Sports, and
Culture. We thank Dr. Masayuki Igarashi (Bioresources
Unit, Bioactive Molecular Research Group, Microbial
Chemistry Research Center) for evaluating the antibac-
terial activities of the synthesized analogs. We thank Ms.
S. Oka (Center for Instrumental Analysis, Hokkaido
University) for measurement of mass spectra. This paper
constitutes Part 253 of Nucleosides and Nucleotides.
Part 252 is Ref. 14.
3.1.13. O-4-Phenylbenzoyl-D-serylamide (16b). Com-
pound 16b was prepared from 14b (17 mg, 0.015 mmol)
as described above for the synthesis of 12a. Purification
by C18 reverse phase column chromatography
(1 · 10 cm, 0.5% aqueous TFA–MeOH) afforded 16b
References and notes
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d
8.13 (d, 2H, o-phenyl,
(d, 2H, o0-phenyl, J = 7.4 Hz), 7.65 (d, 1H, H-6,

436
S. Hirano et al. / Bioorg. Med. Chem. 16 (2008) 428–436
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